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WO2024260261A1 - BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用 - Google Patents

BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用 Download PDF

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WO2024260261A1
WO2024260261A1 PCT/CN2024/097792 CN2024097792W WO2024260261A1 WO 2024260261 A1 WO2024260261 A1 WO 2024260261A1 CN 2024097792 W CN2024097792 W CN 2024097792W WO 2024260261 A1 WO2024260261 A1 WO 2024260261A1
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cdkl5
mice
epilepsy
bdnf
signaling pathway
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French (fr)
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熊志奇
朱姊艾
李奕彦
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants

Definitions

  • the present invention relates to the field of biopharmaceuticals, and in particular, to the application of BDNF-TrkB signaling pathway in preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • CDKL5 deficiency disorder is a rare genetic disease characterized by seizures that begin in infancy and are accompanied by significant developmental delays in multiple systems. CDKL5 deficiency occurs in approximately 1 in 40,000 to 60,000 newborns and is one of the most common genetic causes of childhood epilepsy, of which approximately 90% occur in females. CDKL5 deficiency was previously classified as an atypical form of Rett syndrome, with which they share common features, including seizures, intellectual disability, and other developmental problems. However, patients with CDKL5 deficiency have a significantly different clinical course from Rett syndrome.
  • CDKL5 deficiency In Rett syndrome, seizures generally begin in adolescence and decrease in severity with age; whereas patients with CDKL5 deficiency develop seizures in the first few months of life and are severely resistant to antiepileptic drugs and cannot be cured. Therefore, CDKL5 deficiency is now considered a separate disease from Rett syndrome.
  • CDKL5 deficiency is caused by mutations in the CDKL5 gene. Mutations in the CDKL5 gene reduce the expression level of functional CDKL5 protein or change its activity in nerve cells, but it is not clear how these changes lead to the specific phenotypes of CDKL5 deficiency.
  • the CDKL5 gene is located on the X chromosome and is inherited in an X-linked dominant pattern. Since women have two X chromosomes, random inactivation of the X chromosome can lead to different severity of symptoms and signs in different CDD patients. Women with a higher proportion of mutated neurons have more severe signs and symptoms than women with a lower proportion of mutated neurons. Since men have only one X chromosome in each cell, mutations in the CDKL5 gene are active in all cells, and affected men do not have a normal copy of the gene.
  • Seizures are often the first manifestation of patients with CDKL5 deficiency, with a median time from birth to onset of 4-6 weeks, and more than 90% of patients experience seizures within 3 months of birth, which can occur as early as the first week after birth. Patients' seizures can develop into different types over time and may follow a certain pattern of seizures. The most common types are generalized tonic-clonic seizures, including loss of consciousness, muscle rigidity, and convulsions; tonic seizures, which are characterized by abnormal muscle contractions; and epileptic spasms, in which the EEG shows mild arrhythmias and short-term muscle twitches. Although there may be remission periods (a period of no seizures), most people with CDD may have seizures every day.
  • CDKL5 deficiency will have developmental impairments, most of whom have severe intellectual disabilities, with language skills being particularly affected.
  • the development of gross motor skills such as sitting, standing, and walking is delayed, and only about one-third of CDD patients can walk independently.
  • Fine motor skills (such as picking up small objects with fingers) are also affected, and about half of CDD patients have limited fine motor skills that last a lifetime.
  • Most patients All have vision problems, some of which present as cortical visual impairment (complete loss of binocular vision, normal pupillary light reflex, and normal fundus).
  • Other common features of CDD patients include repetitive hand movements such as clapping, licking, and sucking; teeth grinding; disrupted sleep; feeding difficulties, and gastrointestinal problems, including constipation and gastroesophageal reflux.
  • Some patients have episodic irregular breathing.
  • Other limb abnormalities may also occur, such as head abnormalities (microcephaly), lateral curvature of the spine, and thin fingers.
  • the current medical management of patients with CDKL5 deficiency is mainly symptomatic and supportive treatment, aimed at maximizing the patient's personal abilities and improving any skills that may arise.
  • Emphasis is placed on early intervention treatments such as physical therapy, occupational therapy, and speech and assistive communication therapy.
  • epilepsy control is the most challenging content.
  • the "International Consensus Recommendations for the Evaluation and Management of Patients with CDKL5 Deficiency" points out that existing drugs, including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide, can reduce the frequency of epileptic seizures when used alone in the early stage, but they will lose their efficacy after a certain period of time (median response time is 6 months), and even in some cases, they will aggravate the seizures.
  • drugs including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide
  • the consensus document does not make specific anti-seizure drug treatment recommendations for CDKL5 deficiency, but suggests alternatives, including a ketogenic diet; implantation of a vagus nerve stimulator (VNS, which delivers small pulses of electrical current to the vagus nerve along the neck to the brain); corpus callosotomy (severing the main fibers connecting the two hemispheres of the brain); and pharmaceutical-grade cannabidiol.
  • VNS vagus nerve stimulator
  • corpus callosotomy severed the main fibers connecting the two hemispheres of the brain
  • pharmaceutical-grade cannabidiol cannabidiol.
  • Epilepsy is a central nervous system disease in which patients suffer from recurrent, partial or whole-body seizures, sometimes accompanied by loss of consciousness. It is a catastrophic neurological disease.
  • the severity of seizures in epilepsy patients can range from brief distraction or muscle rigidity to prolonged severe convulsions.
  • Epileptic seizures are generally classified as focal or generalized, depending on how and where the seizure begins.
  • Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. About 60% of patients with focal epilepsy have temporal lobe epilepsy, which often begins in the hippocampus or limbic system.
  • Temporal lobe epilepsy is considered to be the most suitable for scientific research on epilepsy: as the most common type of epilepsy, temporal lobe epilepsy has a large amount of EEG data and case reports available for research, and because temporal lobectomy has a good therapeutic effect on temporal lobe refractory epilepsy, its pathological tissue is easier to obtain than other types of epilepsy.
  • the most common pathological feature of temporal lobe epilepsy is hippocampal sclerosis, which can be observed in epilepsy patients and animal models. It is mainly manifested by the loss of hippocampal neurons, including the loss of pyramidal neurons in CA1 and CA3, and the diffuse distribution of dentate gyrus cells.
  • the dentate gyrus is a key structure for transmitting excitation to the hippocampus. In epilepsy patients and animal models of epilepsy, the dentate gyrus undergoes a variety of changes, making the hypothesis that epilepsy originates from the hippocampus a hot topic.
  • CDKL5 deficiency is a severe epileptic encephalopathy caused by mutations in the CDKL5 gene. Since CDKL5 deficiency was studied as an independent disease, there have been many research advances on the properties and functions of CDKL5 protein, but these findings cannot clearly explain the symptoms caused by CDKL5 deficiency, and there is no animal model that can well reproduce the spontaneous epileptic phenotype of CDD patients. As the most core clinical symptom, the mechanism of CDD-related spontaneous epilepsy is still unclear.
  • the purpose of the present invention is to provide a new drug for treating epilepsy associated with CDKL5 deficiency.
  • Another object of the present invention is to provide evidence that targeting the BDNF-TrkB signaling pathway can be used to prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention provides a use of a BDNF-TrkB signaling pathway inhibitor for preparing a composition or preparation for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • a first aspect of the present invention provides a use of a BDNF-TrkB signaling pathway inhibitor for preparing a composition or preparation for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the CDKL5 deficiency-related epilepsy has one or more phenotypic characteristics selected from the following group:
  • the patient's epilepsy phenotype is resistant to antiepileptic drugs
  • EEG electroencephalogram
  • composition or preparation is also used for one or more purposes selected from the following group:
  • the BDNF-TrkB signaling pathway inhibitor includes a TrkB antagonist.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the following group: ANA-12, K252a, or a combination thereof.
  • composition or preparation further comprises other drugs that can prevent and/or treat epilepsy associated with CDKL5 deficiency.
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy include TAK-935/OV935 and Ganaxolone.
  • the composition comprises a pharmaceutical composition.
  • the pharmaceutical composition contains (a) a BDNF-TrkB signaling pathway inhibitor and (b) a pharmaceutically acceptable carrier.
  • the component (a) accounts for 0.1-99.9wt% of the total weight of the pharmaceutical composition, preferably 10-99.9wt%, and more preferably 70%-99.9wt%.
  • the component (a) accounts for 60.0%-99.5wt% of the total weight of the pharmaceutical composition, preferably 70.0-99.5wt%, more preferably 80.0%-99.5wt%.
  • the pharmaceutical composition is liquid, solid, or semisolid.
  • the dosage form of the pharmaceutical composition includes tablets, granules, capsules, oral solutions, or injections.
  • the composition is an oral preparation.
  • composition (such as a pharmaceutical composition) is administered to a mammal by the following method: Drugs: Oral, intravenous, or local injection.
  • the mammal includes a mammal suffering from CDKL5 deficiency-related epilepsy.
  • the mammal includes a human or a non-human mammal.
  • the non-human mammals include rodents, such as mice and rats.
  • the second aspect of the present invention provides a pharmaceutical composition for preventing and/or treating CDKL5 deficiency-related epilepsy, comprising:
  • a first pharmaceutical composition comprising (a) a first active ingredient, the first active ingredient being a BDNF-TrkB signaling pathway inhibitor;
  • the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions, or the same pharmaceutical composition.
  • the BDNF-TrkB signaling pathway inhibitor includes a TrkB antagonist.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the following group: ANA-12, K252a, or a combination thereof.
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy include TAK-935/OV935 and Ganaxolone.
  • the weight ratio of the first active ingredient to the second active ingredient is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the content of component (a1) is 1%-99%, preferably, 10%-90%, more preferably, 30%-70%.
  • the component (a1) and the component (a2) account for 0.01-99.99 wt %, preferably 0.1-90 wt %, and more preferably 1-80 wt % of the total weight of the product combination.
  • the dosage form of the pharmaceutical composition includes an injection form and an oral dosage form.
  • the oral dosage form includes tablets, capsules, films, and granules.
  • the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.
  • the weight ratio of component (a1) to component (a2) is 1:100 to 100:1, preferably 1:10 to 10:1.
  • the third aspect of the present invention provides a medicine kit, comprising:
  • first container and the second container are the same or different containers.
  • the drug in the first container is a single-ingredient preparation containing a BDNF-TrkB signaling pathway inhibitor.
  • the drug in the second container is a single-ingredient preparation containing other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • the dosage form of the drug is an oral dosage form or an injection dosage form.
  • kit further contains instructions.
  • the description records one or more instructions selected from the following group:
  • the fourth aspect of the present invention provides a use of a combination, wherein the combination comprises a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, for preparing a pharmaceutical composition or a kit for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the combination comprises a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, for preparing a pharmaceutical composition or a kit for preventing and/or treating CDKL5 deficiency-related epilepsy.
  • the pharmaceutical composition or kit comprises (a) a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy; and (b) a pharmaceutically acceptable carrier.
  • the BDNF-TrkB signaling pathway inhibitor and the drug for preventing and/or treating CDKL5 deficiency-related epilepsy respectively account for 0.01-99.99wt%, preferably 0.1-90wt%, and more preferably 1-80wt% of the total weight of the pharmaceutical composition or medicine kit.
  • a fifth aspect of the present invention provides a method for preventing and/or treating epilepsy associated with CDKL5 deficiency, comprising:
  • a BDNF-TrkB signaling pathway inhibitor and optionally other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy, or the pharmaceutical composition of the second aspect of the present invention or the drug kit of the third aspect of the present invention is administered to a subject in need thereof.
  • the subject includes a human or non-human mammal suffering from CDKL5 deficiency-related epilepsy.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.
  • Figure 1 shows the domains of CDKL5 protein and pathogenic mutations.
  • the functional domains of CDKL5 protein are marked with different colors, and the numbers refer to the type and position of amino acids. Red marks are pathogenic or potentially pathogenic mutations; black marks are benign or potentially benign mutations.
  • Figure 2 shows a neural circuit diagram of the rodent hippocampus.
  • A, B A neural circuit diagram of the rodent hippocampus and a schematic diagram of the corresponding neural network.
  • the solid arrows depict the neural circuit of the entorhinal cortex (EC)-dentate gyrus-CA3-CA1-EC.
  • Neurons in layer II of the entorhinal cortex travel to the dentate gyrus through the perforated path (PP).
  • the dentate gyrus projects axons, including the lateral perforant pathway (LPP) and the medial perforant pathway (MPP).
  • the dentate gyrus projects to the pyramidal cells of CA3 via mossy fibers.
  • CA3 pyramidal neurons transmit information to CA1 pyramidal neurons via Schaffer collaterals.
  • CA1 pyramidal neurons output to the deep neurons of the entorhinal cortex.
  • CA3 also receives direct projections from the entorhinal cortex via the perforant pathway.
  • CA1 receives direct input from the entorhinal cortex via the temporoamnion pathway (TA).
  • TA temporoamnion pathway
  • Dentate granule cells also project to the mossy cells in the hilus region and the interneurons in the hilar region, and project back to the granule cells, respectively.
  • Figure 3 shows the biological actions of BDNF.
  • Na + influx depolarizes the membrane, which triggers an influx of Ca2 + and the release of the excitatory neurotransmitter glutamate into the synaptic cleft.
  • Glutamate binds to AMPA and NMDA receptors on the postsynaptic membrane. Activation of the receptors leads to membrane depolarization and influx of Ca2 + via NMDA and VDCC.
  • Ca2 + binds to CaMKs that activate CREB and NF-kB, which in turn induce transcription of the Bdnf gene.
  • BDNF is released at the synaptic cleft and activates the TrkB receptor, leading to the activation of downstream signaling cascades including PLC ⁇ , PI3K, and MAPKs and the subsequent expression of genes that are critical for neuronal survival and plasticity.
  • BDNF signaling also has acute effects on membrane excitability and synaptic transmission by altering the kinetics of NMDA receptors and increasing the number of synaptic vesicles presynaptically.
  • FIG. 4 shows that Cdk15 fl/Y ;Emx1-Cre mice develop spontaneous epilepsy phenotype.
  • FIG. B Representative images of electroencephalogram (EEG) and electromyogram (EMG) recordings during spontaneous epileptic seizures in Cdkl5 fl/Y ; Emx1-Cre mice.
  • the target mice were 6 months old.
  • the upper and middle images show the changes in electroencephalogram (EEG) recordings during epileptic seizures in Cdkl5 fl/Y ;Emx1-Cre mice.
  • the top arrow represents the onset of epileptic seizures.
  • the amplitude of epileptic seizures increased significantly during epileptic seizures, and there was suppression after the end of the seizures.
  • the lower image shows the changes in electromyogram (EMG) recordings during epileptic seizures in Cdkl5 fl/Y ;Emx1-Cre mice.
  • C Statistical graph of spontaneous epileptic seizure rates in Cdkl5 fl/Y ;Emx1-Cre mice and their control group. There were 10 Cdkl5 fl/Y mice and 15 Cdkl5 fl/Y ;Emx1-Cre mice.
  • FIG. 5 shows that Cdk15 fl/Y ;CaMK2 ⁇ -iCre mice develop spontaneous epilepsy phenotype.
  • FIG. 1 Representative images of electroencephalogram (EEG) and electromyogram (EMG) recordings during spontaneous epileptic seizures in Cdkl5 fl /Y ;CaMK2 ⁇ -iCre mice.
  • the target mice were 6 months old.
  • the upper figure shows the changes in electroencephalogram (EEG) recordings during epileptic seizures in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • the top arrow represents the onset of epileptic seizures.
  • the amplitude of epileptic seizures increased significantly during epileptic seizures and was suppressed after the end.
  • the lower figure shows the changes in electromyogram (EMG) recordings during epileptic seizures in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • C Statistical graph of spontaneous epileptic seizure rates in Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice and their control group. There were 12 Cdkl5 fl/Y mice and 15 Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice.
  • FIG6 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;Emx1-Cre mice is enhanced.
  • FIG. 7 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;CaMK2 ⁇ -iCre mice is enhanced.
  • FIG8 shows that the amplitude of mIPSCs in the dentate gyrus of the hippocampus of Cdkl5 fl/Y ;Emx1-Cre mice is enhanced.
  • FIG. 9 shows that Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice develop spontaneous epilepsy.
  • FIG. 10 shows that excitatory transmission in the dentate gyrus of the hippocampus of Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice is enhanced.
  • the control group consisted of 3 mice in the Cdkl5 fl/Y + tamoxifen group, with 11 neurons recorded; the Cdkl5 fl /Y ;CaMK2 ⁇ -CreER + corn oil group consisted of 3 mice, with 14 neurons recorded; the experimental group consisted of 3 mice in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER + tamoxifen group, with 11 neurons recorded. ****p ⁇ 0.0001, unpaired two-tailed t-test.
  • FIG. 11 shows that the density and maturity of dendritic spines in granule cells of Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice are normal.
  • C Classification of dendritic spines of hippocampal dentate gyrus granule cells in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER+tamoxifen mice and their control group. Two-way ANOVA with Bonferroni post hoc test. Each group contained 41-52 neurons.
  • FIG12 shows that the BDNF expression level in the hippocampus of Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice is increased, and the BDNF-TrkB signaling pathway is abnormally activated.
  • C qPCR analysis of BDNF mRNA levels in the hippocampus of Cdkl5 fl/Y ; CaMK2 ⁇ -CreER mice and their control group.
  • the control group consisted of 11 mice in the Cdkl5 fl/Y + tamoxifen group; 5 mice in the Cdkl5 fl / Y ; CaMK2 ⁇ -CreER + corn oil group; and the experimental group consisted of 10 mice in the Cdkl5 fl/Y ; CaMK2 ⁇ -CreER + tamoxifen group.
  • the control group consisted of 14 mice in the Cdkl5 fl/Y + tamoxifen group, 8 mice in the Cdkl5 fl /Y ; CaMK2 ⁇ -CreER + corn oil group, and the experimental group consisted of 12 mice in the Cdkl5 fl/Y ; CaMK2 ⁇ -CreER + tamoxifen group.
  • FIG. 13 shows that the Trk inhibitor K252a can improve the defect of enhanced excitatory synaptic transmission in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • the control group consisted of 4 mice in the Cdkl5 fl/Y + tamoxifen group, from which 11 neurons were recorded; the Cdkl5 fl/Y ;CaMK2 ⁇ CreER+corn oil group consisted of 3 mice, from which 10 neurons were recorded; the experimental group consisted of 4 mice in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER+tamoxifen group, from which 13 neurons were recorded.
  • FIG. 14 shows that the TrkB antagonist ANA-12 can improve the defect of enhanced excitatory synaptic transmission in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • FIG. 15 shows that knocking down TrkB receptor can improve spontaneous epileptic activity caused by knocking out Cdk15 in adulthood.
  • FIG. 16 shows that inhibition of TrkB receptor can improve spontaneous epileptic activity in Cdk15 fl/Y ;CaMK2 ⁇ -CreER mice.
  • BDNF-TrkB signaling pathway inhibitors such as TrkB antagonists
  • TrkB antagonists can effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • ANA-12 has the structural formula:
  • K252a has the structural formula:
  • Ganaxolone As used herein, the Chinese name of the term “Ganaxolone” is Ganaxolone, and the structural formula is:
  • CDKL5 deficiency disorder is a rare genetic disease characterized by seizures that begin in infancy and are accompanied by significant developmental delays in multiple systems. CDKL5 deficiency occurs in approximately 1 in 40,000 to 60,000 newborns and is one of the most common genetic causes of childhood epilepsy, of which approximately 90% occur in females. CDKL5 deficiency was previously classified as an atypical form of Rett syndrome, with which they share common features, including seizures, intellectual disability, and other developmental problems. However, patients with CDKL5 deficiency have a significantly different clinical course from Rett syndrome.
  • CDKL5 deficiency In Rett syndrome, seizures generally begin in adolescence and decrease in severity with age; whereas patients with CDKL5 deficiency develop seizures in the first few months of life and are severely resistant to antiepileptic drugs and cannot be cured. Therefore, CDKL5 deficiency is now considered a separate disease from Rett syndrome.
  • CDKL5 deficiency is caused by mutations in the CDKL5 gene. Mutations in the CDKL5 gene reduce the expression level of functional CDKL5 protein or change its activity in nerve cells, but it is not clear how these changes lead to the specific phenotypes of CDKL5 deficiency.
  • the CDKL5 gene is located on the X chromosome and is inherited in an X-linked dominant pattern. Since women have two X chromosomes, random inactivation of the X chromosome can lead to different severity of symptoms and signs in different CDD patients. Women with a higher proportion of mutated neurons have more severe symptoms than women with a lower proportion of mutated neurons. Because males have only one X chromosome in each cell, mutations in the CDKL5 gene are active in all cells, and affected males do not have a normal copy of the gene.
  • Seizures are often the first manifestation of patients with CDKL5 deficiency, with a median time from birth to onset of 4-6 weeks, and more than 90% of patients experience seizures within 3 months of birth, which can occur as early as the first week after birth. Patients' seizures can develop into different types over time and may follow a certain pattern of seizures. The most common types are generalized tonic-clonic seizures, including loss of consciousness, muscle rigidity, and convulsions; tonic seizures, which are characterized by abnormal muscle contractions; and epileptic spasms, in which the EEG shows mild arrhythmias and short-term muscle twitches. Although there may be remission periods (a period of no seizures), most people with CDD may have seizures every day.
  • CDKL5 deficiency will have developmental impairments, most of whom have severe intellectual disabilities, with language skills being particularly affected.
  • the development of gross motor skills such as sitting, standing, and walking is delayed, and only about one-third of CDD patients can walk independently.
  • Fine motor skills (such as picking up small objects with fingers) are also affected, and about half of CDD patients have limited fine motor skills and will accompany them for life.
  • Most patients have vision problems, some of which manifest as cortical visual impairment (complete loss of binocular vision, normal pupil light reflex, and normal fundus).
  • Other common features of CDD patients include repetitive hand movements such as clapping, licking, and sucking; teeth grinding; interrupted sleep; feeding difficulties and gastrointestinal problems, including constipation and gastroesophageal reflux.
  • Some patients have paroxysmal irregular breathing.
  • Other limb abnormalities may also occur, such as head abnormalities (microcephaly), lateral curvature of the spine, and thin fingers.
  • the current medical management of patients with CDKL5 deficiency is mainly symptomatic and supportive treatment, aimed at maximizing the patient's personal abilities and improving any skills that may arise.
  • Emphasis is placed on early intervention treatments such as physical therapy, occupational therapy, and speech and assistive communication therapy.
  • epilepsy control is the most challenging content.
  • the "International Consensus Recommendations for the Evaluation and Management of Patients with CDKL5 Deficiency" points out that existing drugs, including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide, can reduce the frequency of epileptic seizures when used alone in the early stage, but they will lose their efficacy after a certain period of time (median response time is 6 months), and even in some cases, they will aggravate the seizures.
  • drugs including corticosteroids, vigabatrin, valproic acid, phenytoin, felbamate, carbamazepine, clonazepam, oxcarbazepine, and lacosamide
  • the consensus document does not make specific anti-seizure drug treatment recommendations for CDKL5 deficiency, but suggests alternatives, including a ketogenic diet; implantation of a vagus nerve stimulator (VNS, which delivers small pulses of electrical current to the vagus nerve along the neck to the brain); corpus callosotomy (severing the main fibers connecting the two hemispheres of the brain); and pharmaceutical-grade cannabidiol.
  • VNS vagus nerve stimulator
  • corpus callosotomy severed the main fibers connecting the two hemispheres of the brain
  • pharmaceutical-grade cannabidiol cannabidiol.
  • CDKL5 Cyclin-dependent Kinase Like 5
  • STK9 Cyclin-dependent Kinase Like 5
  • the CDKL5 gene is located in region 22 of the short arm of chromosome X (Xp22).
  • the human CDKL5 gene has 24 exons.
  • the N-terminus of CDKL5 protein is a serine/threonine kinase domain.
  • the pathogenic mutations are most common in the catalytic region ( Figure 1). Exons 2-21 are the coding region.
  • CDKL5 protein belongs to the cell cycle dependent kinase family and is highly homological with the kinase domains of cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinases (GSKs).
  • CDKs cyclin-dependent kinases
  • MAPKs mitogen-activated protein kinases
  • GSKs glycogen synthase kinases
  • CDKL5 protein is expressed in large quantities in the central nervous system of humans and rodents, among which the highest expression levels are in the cerebral cortex, hippocampus, thalamus, striatum and olfactory bulb. CDKL5 is expressed at a low level in the embryonic period, and the expression level increases significantly after birth, reaching a peak in the first few weeks, and maintaining stable expression in adulthood. Neurons are the main cell type expressing CDKL5, and glial cells only express a small amount. CDKL5 is expressed in various subcellular structures, among which the cytoplasm is the main expression area, but nuclear aggregation gradually appears as the brain develops.
  • CDKL5 Some potential substrates of CDKL5 have been identified, and some of the substrates related to physiological functions have been verified in cells.
  • substrates include MAP1S, EB2, CEP131, a regulator of microtubule and centrosome function, and ELOA involved in DNA damage response, etc.
  • CDKL5 is mainly enriched in the nucleus in the area of RNA splicing and processing. CDKL5 also accumulates in different subcellular regions at different developmental stages: in the early stages of cultured neurons, CDKL5 was found to accumulate in growth cones; in mature neurons, CDKL5 accumulates in dendritic spines, especially in the postsynaptic density; some studies have also found that CDKL5 exists in centrosomes. These expression patterns indicate that CDKL5 is involved in the regulation of neuronal development and plays a role in the maturation of adult neurons.
  • CDKL5 plays different functions at different stages of neuronal development. In early neuronal development, CDKL5 regulates cell proliferation and neuronal migration. According to previous studies, CDKL5 has a negative regulatory function in regulating cell proliferation. Upregulating CDKL5 expression inhibits the proliferation of human neuroblastoma cells; while knocking out mouse Cdkl5 increases the proliferation rate of dentate gyrus granule cells. In addition, CDKL5 was found to be present in the centrosomes of dividing cells and post-mitotic neurons, indicating that it may regulate cell proliferation and division by affecting the centrosome. CDKL5 has a regulatory effect on neuronal migration, and it was found to interact with IQGAP1, an important regulator related to cell migration and polarity.
  • IQGAP1 an important regulator related to cell migration and polarity.
  • CDKL5 In rodent model animals, reducing the expression of CDKL5 in neural progenitor cells leads to delayed migration of layer 2-3 pyramidal neurons. In the process of neuronal maturation, CDKL5 is closely related to the growth and development of dendrites and axons, and is one of the key proteins for synapse formation. CDKL5 interacts with Shootin1, a key protein for axon growth, in growth cones.
  • CDKL5 Silencing Cdkl5 by RNAi or overexpressing CDKL5 leads to an increase in the number of neurons with multiple axons, but downregulating Cdkl5 does not hinder axon formation, indicating that CDKL5 has a regulatory function in axon growth, but does not play a decisive role in axon formation.
  • CDKL5 also regulates dendrite development. When Cdkl5 is silenced in cultured neurons, the dendritic branching of neurons is severely impaired. Cdkl5 knockout mice can be observed to have a significant reduction in the total length of dendrites in the cerebral cortex and hippocampal CA1 pyramidal neurons, as well as dentate gyrus granule cells.
  • CDKL5 Overexpression of CDKL5 in cultured neurons results in a kinase activity-dependent increase in total dendrite length. In addition, CDKL5 is also involved in synapse formation. CDKL5 has been observed to interact with palmitoylated PSD95 and the adhesion molecule NGL1. PSD is a major scaffolding protein that regulates the localization of synaptic proteins and synaptic strength; NGL1 is a key adhesion molecule for synapse formation. In dendritic spines, CDKL5 is highly enriched in the postsynaptic density, a dense protein complex composed of key proteins for synaptic transmission, signal transduction, and cell adhesion. Synaptic localization strongly suggests that this protein is involved in synaptic development and function.
  • CDKL5 The function of CDKL5 is not limited to neuronal development, and its expression persists in the adult brain. In adult mice lacking CDKL5, the stability and long-term potentiation of mature dendritic spines are impaired, and these animals have defects in hippocampal-dependent memory. Notably, these defects can be reversed by restoring CDKL5 protein levels in adult mice, including abnormal morphology of dendritic spines, hindlimb grasping, and learning and memory abilities. The defects of CDKL5 knockout mice can also be improved by activating the downstream signaling pathways of CDKL5. For example, the application of IGF-1 can restore the stability of dendritic spines, indicating that CDKL5 also plays an important role in the neural maturation period after adulthood.
  • CDKL5 itself are also regulated by neural activity, such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL5.
  • neural activity such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL5.
  • neural activity such as DYRK1A phosphorylation of serine residue 308 promotes the cytoplasmic localization of CDKL5 in Neuro-2a cells; in cultured neurons, BDNF induces transient phosphorylation of CDKL5; neuronal activity prompts PP1 to dephosphorylate CDKL
  • Cdkl5 exon knockout mice are the most commonly used Cdkl5 knockout mouse model. In this strain of Cdkl5 knockout mice, researchers observed impairment of limb coordination, motor skills, learning and memory abilities, autism-like symptoms, abnormal eye movements, abnormal breathing and sleep patterns, and atypical behavioral responses to whisker-mediated tactile stimulation. Most of these phenotypes can be mutually confirmed with the symptoms of patients with CDKL5 deficiency.
  • CDKL5 deficiency mouse model confirmed the importance of this gene for normal brain development and nervous system function, and also provided an in vivo environment to discover and validate various protein-protein interactions and signaling pathways involving CDKL5, and to test newly discovered treatments.
  • CDKL5 the endogenous function of CDKL5 and its role in the development, maintenance, and pathogenesis of the nervous system remain to be elucidated, and the core spontaneous epilepsy phenotype of CDKL5 deficiency has not been well reproduced, and there is still a lack of a new animal model to simulate CDD-related epilepsy.
  • Epilepsy is a central nervous system disease in which patients suffer from recurrent seizures involving part or all of the body, sometimes accompanied by loss of consciousness. It is a catastrophic neurological disease. The severity of seizures in epilepsy patients can range from brief distractions or muscle rigidity to prolonged severe convulsions. Epileptic seizures are generally classified as focal or generalized, depending on how and where the seizure begins. Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy. Approximately 60% of patients with focal epilepsy have temporal lobe epilepsy, which often begins in the hippocampus or limbic system.
  • Temporal lobe epilepsy is considered the most suitable for scientific research on epilepsy: as the most common type of epilepsy, temporal lobe epilepsy has a large amount of EEG data and case reports available for research, and because temporal lobectomy has a good therapeutic effect on refractory temporal lobe epilepsy, it ... Pathological tissue is easier to obtain than other types of epilepsy.
  • the most common pathological feature of temporal lobe epilepsy is hippocampal sclerosis, which can be observed in both epilepsy patients and animal models. It is mainly manifested by the loss of hippocampal neurons, including the loss of pyramidal neurons in CA1 and CA3, and the diffuse distribution of dentate gyrus cells.
  • the dentate gyrus is a key structure for the transmission of excitement to the hippocampus. In epilepsy patients and animal models of epilepsy, the dentate gyrus undergoes a variety of changes, making the hypothesis that epilepsy originates from the hippocampus a research hotspot.
  • the hippocampus is located in the medial region of the temporal lobe and plays an important role in emotion regulation, consolidation of information from short-term to long-term memory, and spatial processing. Understanding the anatomy of the hippocampus is crucial to its cognitive function.
  • the hippocampus consists of subregions such as Cornu ammonis (CA, including CA1-CA4), dentate gyrus and subthalamic region. Axons of neurons in layer II of the entorhinal cortex project to the dentate gyrus through the perforant pathway (PP), including the lateral perforant pathway (LPP) and the medial perforant pathway (MPP).
  • PP perforant pathway
  • LPP lateral perforant pathway
  • MPP medial perforant pathway
  • the dentate gyrus projects to the pyramidal cells of CA3 through mossy fibers, which are then transmitted to CA1 pyramidal neurons through Schaffer collaterals, and finally output to the deep neurons of EC through CA1 pyramidal neurons.
  • CA3 also receives direct projections from the entorhinal cortex through the perforant pathway; CA1 receives direct input from the entorhinal cortex through the temporo-ammonic pathway (TA).
  • TA temporo-ammonic pathway
  • the dentate granule cells project to interneurons and mossy cells, and receive their inhibitory and excitatory feedback respectively ( Figure 2, A-B).
  • the dentate gyrus consists of three layers from outside to inside: the molecular layer, the granule cell layer, and the polymorphic layer (also called the Hilus layer).
  • Granule cells as the main neurons of the dentate gyrus, are densely arranged in the granule cell layer, and their axons project to CA3 to form excitatory synapses with pyramidal neurons.
  • the molecular layer is a cell-free layer occupied by the dendrites of granule cells in the granule cell layer and the perforated path fibers from the entorhinal cortex.
  • mossy cells are one of the most important neurons.
  • the granular layer and the polymorphic layer there are several types of neurons, such as pyramidal basket cells, which are inhibitory interneurons.
  • neurons such as pyramidal basket cells, which are inhibitory interneurons.
  • the mossy fibers projected by granule cells terminate above the CA3 pyramidal cell layer, which is called the clear layer.
  • Mossy fibers also project to the polymorphic layer to form synaptic connections with interneurons.
  • the activity of granule cells in the dentate gyrus is very low, which is related to both the intrinsic characteristics of their own cells and the circuit network environment in which they are located.
  • granule cells exhibit a hyperpolarized resting membrane potential, which makes them more difficult to induce action potentials.
  • granule cell dendrites show significant voltage-dependent linear integration and strong attenuation of synaptic inputs to the entorhinal cortex.
  • dentate gyrus granule cells are also in a circuit network that is biased towards inhibition.
  • feedforward inhibition is provided by a variety of inhibitory neurons such as fast-spiking interneurons, hilar interneurons, and basket cells.
  • Granule cell axonal projections are also connected to a variety of GABAergic interneurons, which form dendritic targeted feedback regulation on granule cells.
  • the intrinsic properties of granule cells and the inhibitory circuit network environment make it exist as a shock absorber between the entorhinal cortex and CA3. Therefore, when abnormalities occur in the dentate gyrus, it is considered to be the "detonator/detonator" of the epileptogenic process, and together with CA3, it becomes the key to the formation of epileptic seizures, which is also called the gating hypothesis of the dentate gyrus.
  • epilepsy studies that support this theory and explain the gating hypothesis of the dentate gyrus, including mossy fiber sprouting (abnormal synapses formed on mossy fibers and other granule cells); abnormal formation and persistence of basal dendrites of granule cells; ectopic dispersion and migration of granule cells; changes in the intrinsic properties of granule cell membranes and synaptic receptor expression; downregulation of inhibitory GBAB synaptic transmission and abnormal regulation of various molecular signaling pathways.
  • the use of optogenetics to depolarize or hyperpolarize granule cells in animal models can effectively inhibit/induce the occurrence of spontaneous epilepsy, proving that the dentate gyrus is a key node in the temporal lobe epilepsy seizure network.
  • Optogenetic activation of inhibitory interneurons in the dentate gyrus can prevent the spread of epilepsy in the hippocampus and cortex, further highlighting the important role of the dentate gyrus in regulating cortical input.
  • CDD patients with CDKL5 deficiency will show different types of epileptic seizures at different stages of the disease course, but CDD patients can be observed to have high-intensity temporal lobe activity in MRI, and multiple case reports have shown temporal lobe atrophy in CDD patients; common pathological features of temporal lobe epilepsy such as mossy fiber sprouting can also be seen in CDD animal models, and these evidences indicate that CDD-related epilepsy is closely related to temporal lobe epilepsy. In CDD mouse models, various changes in the dentate gyrus of the hippocampus can also be observed.
  • the molecular layer of the dentate gyrus has reduced immunoreactivity to presynaptic markers synaptophysin and VGLUT1, indicating impaired synaptic maintenance;
  • GluN2B subunits accumulate ectopically at the synaptic junctions of the dentate gyrus granule cells-CA3 layer, causing synaptic hyperexcitation and increased susceptibility to epilepsy;
  • the death of postmitotic granule neuron precursors increases, the total number of granule cells decreases, and the newly generated granule cells show severe dendritic atrophy, which impairs hippocampal-dependent behaviors;
  • the density of dendritic spines and the number of mature dendritic spines of dentate gyrus granule cells and CA1 pyramidal neurons are significantly reduced.
  • Brain derived neurotrophic factor belongs to the neurotrophic factor family and is related to the typical nerve growth factor (NGF). The family also includes NT-3 and NT-4/NT5. BDNF acts on certain neurons in the central and peripheral nervous systems. In the brain, it is active in the hippocampus, cortex, and basal forebrain regions. It is also expressed in the retina, kidney, prostate, motor neurons, and skeletal muscle. BDNF mainly binds to the TrkB receptor with high affinity. The low-affinity nerve growth factor receptor LNGFR (also known as p75) can also respond to BDNF. BDNF plays different roles in neurons at different time and space stages. The existence of complex multi-level regulation proves the importance and diversity of BDNF functions.
  • NGF nerve growth factor
  • BDNF plays a vital role in the survival and differentiation of neuronal populations, promoting the growth and differentiation of newborn neurons by changing cell survival and proliferation; BDNF plays a key role in regulating plastic changes in the adult brain, including regulating protein transport, receptor phosphorylation, and glutamate receptor expression levels; BDNF can promote changes in the morphology of dendritic spines, increase the number, size, and complexity of dendritic spines, thereby stabilizing long-term potentiation (LTP), and plays a key role in memory formation and maintenance.
  • LTP long-term potentiation
  • BDNF-TrkB signaling pathway is abnormally upregulated during epilepsy, and this upregulation is closely related to the excitatory/inhibitory homeostasis of the neural circuit.
  • the gyrus is a key structure in the epileptogenic effect of BDNF. Increased BDNF expression levels may not only lead to structural reorganization of the hippocampal circuit (mossy fiber sprouting), but also affect synaptic transmission in multiple regions of the hippocampus (including enhanced glutamate-mediated excitatory synaptic transmission or GABA-mediated inhibitory synaptic transmission). These changes can lead to a hyperexcitable state of the hippocampal circuit, further promoting abnormal activation of BDNF, thereby forming status epilepticus.
  • CDKL5 deficiency-associated epilepsy disorder refers to an epilepsy phenotype caused by CDKL5 deficiency.
  • the CDKL5 deficiency-related epilepsy disease has one or more phenotypic characteristics selected from the following group:
  • the patient's epilepsy phenotype is resistant to antiepileptic drugs.
  • EEG electroencephalogram
  • the present invention focuses on exploring the pathogenesis of epilepsy associated with CDKL5 deficiency. We found that knocking out Cdkl5 in forebrain excitatory neurons can cause mice to have spontaneous epileptic phenotypes, and the excitatory synaptic transmission in the dentate gyrus of the hippocampus is enhanced, whether in the developmental period or in adulthood.
  • the density and maturity of dendritic spines of granule cells in the dentate gyrus were normal, and the electrophysiological properties mediated by AMPA receptors were not affected, but the BDNF-TrkB signaling pathway in the hippocampus was abnormally upregulated.
  • BDNF-TrkB signaling pathway By inhibiting the BDNF-TrkB signaling pathway, abnormal synaptic transmission in the dentate gyrus can be rescued and epileptic activity in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice can be reduced, suggesting that abnormal upregulation of the BDNF-TrkB signaling pathway promotes epilepsy in genetic epilepsy mouse models, and targeting this pathway may be an effective strategy for treating CDD-related epilepsy.
  • the BDNF-TrkB signaling pathway inhibitor refers to a small molecule that can inhibit the TrkB activity induced by BDNF in a competitive or non-competitive manner, including TrkB antagonists.
  • the BDNF-TrkB signaling pathway inhibitor is selected from the group consisting of ANA-12, K252a, or a combination thereof.
  • the present invention finds for the first time that BDNF-TrkB signaling pathway inhibitors can effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention provides a composition comprising active ingredients (a) a BDNF-TrkB signaling pathway inhibitor; optionally (b) a drug for preventing and/or treating epilepsy associated with CDKL5 deficiency; and (c) a pharmaceutically acceptable carrier.
  • Compound pharmaceutical composition include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, powders, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the pharmaceutical combination of the present invention can also be prepared in powder form for aerosol inhalation.
  • a preferred dosage form is an injection preparation.
  • the pharmaceutical composition of the present invention can also be used with other therapeutic agents.
  • the present invention also provides a drug kit for preventing and/or treating CDKL5 deficiency-related epilepsy, the drug kit comprising:
  • (b1) an optional second container, and other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency located in the second container, or containing other drugs for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • compositions and kit of the present invention are suitable for preventing and/or treating epilepsy associated with CDKL5 deficiency.
  • the preparation of the present invention can be taken three times a day to once every ten days, or once every ten days in a sustained-release manner.
  • the preferred mode is to take it once a day, because it is convenient for patients to adhere to it, thereby significantly improving the compliance of patients to take the medicine.
  • the total daily dose in most cases should be lower than (or equal to or slightly higher than) the daily dose of each single drug in a few cases.
  • the effective dose of the active ingredient used may vary depending on the mode of administration and the severity of the disease to be treated.
  • the present invention also provides a method for preventing and/or treating CDKL5 deficiency-related epilepsy using the above-mentioned active ingredients or corresponding drugs of the present invention, which comprises administering to a mammal an effective amount of the active ingredient (a) a BDNF-TrkB signaling pathway inhibitor; optionally (b) other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy (such as TAK-935/OV935 and Ganaxolone), or administering a pharmaceutical composition containing the active ingredient (a) and the optional active ingredient (b).
  • a BDNF-TrkB signaling pathway inhibitor optionally
  • other drugs for preventing and/or treating CDKL5 deficiency-related epilepsy such as TAK-935/OV935 and Ganaxolone
  • the active ingredient of the present invention can be mixed with one or more pharmaceutically acceptable carriers or excipients, such as solvents, diluents, etc., and can be administered orally in the form of tablets, pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspending agents), syrups (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or parenterally in the form of sterile injectable solutions or suspensions (containing about 0.05-5% suspending agents in isotonic media).
  • these pharmaceutical preparations can contain about 0.01-99%, more preferably about 0.1%-90% (weight) of the active ingredient mixed with a carrier.
  • the active ingredients or pharmaceutical compositions of the present invention can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration.
  • routes of administration include oral administration, intramuscular administration or intravenous administration.
  • the preferred pharmaceutical composition is a liquid composition, especially an injection.
  • BDNF-TrkB signaling pathway inhibitors such as TrkB antagonists
  • TrkB antagonists can Effectively prevent and/or treat CDKL5 deficiency-related epilepsy.
  • the present invention focuses on exploring the mechanism of epilepsy associated with CDKL5 deficiency. We found that knocking out Cdkl5 in forebrain excitatory neurons can cause mice to have spontaneous epilepsy phenotypes, and the excitatory synaptic transmission in the dentate gyrus of the hippocampus is enhanced , whether in the developmental period or in adulthood.
  • BDNF-TrkB signaling pathway By inhibiting the BDNF-TrkB signaling pathway, abnormal synaptic transmission in the dentate gyrus can be rescued and epileptic activity in Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice can be reduced, suggesting that abnormal upregulation of the BDNF-TrkB signaling pathway promotes epilepsy in genetic epilepsy mouse models, and targeting this pathway may be an effective strategy for treating CDD-related epilepsy.
  • the present invention first discovered that previous CDD animal models could not well replicate the core epilepsy phenotype of CDD.
  • the present invention constructed a mouse model with a spontaneous epilepsy phenotype by knocking out Cdkl5 in the excitatory neurons of the forebrain.
  • CDKL5 maintains the stability of the hippocampal neural network in adulthood, illustrating that CDKL5 deficiency is not only a neurodevelopmental disorder but also a neurofunctional disorder.
  • the present invention observed for the first time that the BDNF-TrkB signaling pathway was abnormally upregulated in adult Cdkl5 knockout mice, and that inhibition of the BDNF-TrkB signaling pathway could reduce spontaneous epileptic seizure activity, thus proposing a molecular mechanism for the occurrence of CDD-related epilepsy.
  • the research of the present invention illustrates the important role of CDKL5 in the balance of excitatory/inhibitory synaptic transmission, and provides a new model and new target for the occurrence of CDD-related epilepsy, laying a more solid foundation for the clinical treatment of CDD-related epilepsy.
  • the reagents related to mouse experiments are as follows:
  • Tamoxifen formulation (final concentration: 100 mg/kg): 0.5 g of tamoxifen (MedChemExpress, 1052910) was dissolved in a mixture of 2.5 ml of anhydrous ethanol (Sinopharm, 10009228) and 47.5 ml of corn oil (ABCONE, C67366), mixed in a 40°C water bath until completely dissolved, and stored in aliquots at -20°C. 0.1 ml was injected for every 10 g of mouse body weight.
  • ANA-12 formulation (final concentration: 6 mg/kg): Dissolve 1.2 mg ANA12 (MedChemExpress, HY-12497) in 100 ⁇ l dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, D2447), add 900 ⁇ l corn oil (ABCONE, C67366), shake until no precipitation, store at -20°C, and use within one week. Inject 0.1 ml for every 20 g of mouse body weight.
  • DMSO dimethyl sulfoxide
  • VPA formula final concentration: 50 mg/kg: 10 mg valproic acid (Valproic Acid, TargetMol, T7064) was dissolved in 100 ⁇ l dimethyl sulfoxide (DMSO) solution (Sigma-Aldrich, D2447), and then 900 ⁇ l corn oil (ABCONE, C67366) was added. The mixture was shaken until there was no precipitation, and stored at -20°C. It was used within one week. 0.1 ml was injected for every 20 g of mouse body weight.
  • DMSO dimethyl sulfoxide
  • pY816 formulation (final concentration: 20 mg/kg): 100 mg pY816 or control peptide (customized by GenScript) was added to 12.5 ml phosphate buffered saline (cellgro, 21-040-CVCa), shaken until no precipitation, stored at -20°C, and used within one week. 0.1 ml was injected for every 20 g of mouse body weight.
  • mice tail lysis buffer 200 ⁇ l of mouse tail lysis buffer and 2 ⁇ l of Proteinase K (20mg/mL) to about 2mm of mouse tissue (toe or tail), mix thoroughly, and place in a 56°C oven for digestion for 6 hours or overnight. Then, place in a metal bath at 95°C to inactivate proteinase K and store at 4°C.
  • 2X Taq PCR Master Mix kit (Tiangen, KT211): 7 ⁇ l 2X Taq PCR Master Mix; 0.5 ⁇ l each of F-end and R-end primers; 5 ⁇ l ddH2O; 1 ⁇ l template; total volume 14 ⁇ l.
  • Primer information is as follows:
  • Emx1-F GCAAGAACCTGATGGACATGTTCAG
  • Emx1-R GCAATTTCGGCTATACGTAACAGGG
  • TrkB-F ATGTCGCCCTGGCTGAAGTG
  • TrkB-R ACTGACATCCGTAAGCCAGT
  • Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice were monitored by video from adulthood, and Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice were monitored by video from the time of tamoxifen injection.
  • the observations were made under single-blind conditions and the observed seizure numbers, times, and seizure levels were recorded. The monitoring was performed for 1 hour every day (5 days a week). After tamoxifen injection, Cdkl5 fl/Y ;TrkB ;Cam2 ⁇ -CreER mice and their control groups were video recorded for 24 hours every week.
  • mice were graded according to the Racine standard: Grade 1, with shaking of the whiskers and face; Grade 2, with obvious nodding movements; Grade 3, unilateral forelimb spasm, with the tail erect; Grade 4, forelimb spasm, obvious myotonia; Grade 5, loss of limb control, forced clonus, and convulsions. Because first- and second-degree seizures are mild and can be easily missed during video recording, only third-degree or higher seizures were considered spontaneous seizures in our study (Racine, 1972).
  • the mouse head was fixed to a stereotaxic apparatus, the skull was exposed, two small holes were polished at the electrode implantation point, the EEG electrode was screwed into the small hole, and fixed to the skull with dental cement.
  • the EMG electrode was inserted into the trapezius muscle with forceps. Three days after the surgery, the adaptation was recorded for two consecutive days. EEG and EMG were recorded for freely moving mice, and the data were collected at a frequency of 1 kHz using Spike2 software (CED Ltd., Micro1401mkII).
  • Protein content was determined using the BCA protein quantification kit (Tiangen, PA115). Prepare BCA working solution, mix reagents A and B at a volume ratio of 50:1; dilute the BSA standard with a solution of the same buffer system as the sample, and dilute the sample 5-10 times; use a 96-well plate, with each well containing 25 ⁇ l of diluted sample or BSA standard and 200 ⁇ l of BCA working solution, mix thoroughly and cover Place in a 37°C oven for 30 minutes; after 30 minutes, use an ELISA reader (Molecular Devices) to detect the absorbance at 562 nm; calculate the protein concentration of the sample based on the standard curve.
  • BCA protein quantification kit Triangen, PA115.
  • the sample concentration was adjusted to level using protein loading buffer and heated at 80°C for 7 minutes.
  • An electrophoresis device equipped with SDS-PAGE gel plates (VE-180 vertical electrophoresis tank, Shanghai Tianneng Technology Co., Ltd., China) was prepared in advance and 1X electrophoresis buffer was added.
  • the cooked protein samples were then added to the lanes of the gel plates in sequence using a pipette.
  • the parameters for electrophoresis were: constant voltage 80V for 30 minutes in the first stage; constant voltage 110V for 100 minutes in the second stage.
  • PageRuler TM Plus Prestained Protein Ladder (Thermo Scientific, 26619) was used as the electrophoresis reference standard.
  • the protein was transferred to the PVDF membrane using a constant current mode of 350mA for 3 hours.
  • skim milk powder Yili
  • BSA aladdin, B265991
  • a 5% blocking solution with TBST buffer was used to prepare a 5% blocking solution with TBST buffer, and the solution was blocked at room temperature for 1 hour.
  • the corresponding protein primary antibody was diluted in the blocking solution and incubated overnight at 4°C.
  • the membrane was washed three times at room temperature using TBST buffer on a bed for 5-7 minutes each time.
  • the corresponding protein secondary antibody was dissolved in blocking solution and incubated at room temperature for 1-2 hours. After incubation, the membrane was washed three times and exposed using Pro-Light HRP chemiluminescent detection reagent (Tiangen, PA112) in a chemiluminescent imager (Analytik Jena AG).
  • RNA Master SYBR Green I kit (Roche, 03064760001) was used for RT-PCR reaction.
  • the reaction system was 10 ⁇ L SYBR Green Mix, 1 ⁇ l primer, 2 ⁇ l cDNA template, 6 ⁇ l ddH2O, and the total volume was 20 ⁇ l.
  • the reaction procedure was 95°C, 5 min; 95°C, 10 s, 60°C, 20 s, 72°C, 20 s, and the second to fourth steps were cycled 40 times; 95°C, 5 s; 65°C, 1 min.
  • Primer information is as follows:
  • GAPDH-R TTGGGGGTAGGAACACGGAAGG
  • Golgi staining was performed using the FD Rapid Golgi Stain TM Kit (FD Neuro Technologies, PK401). After anesthetizing the mouse, open the chest cavity and expose the heart. Use a syringe to inject the PBS solution from the left ventricle and out of the right atrium until the lungs and liver turn white. Cut off the head with scissors to expose the skull, and use the tip of the scissors to gently slide along the exposed inner surface of the bone from the cerebellum to the olfactory bulb, peel off the brain, and quickly remove the complete brain tissue.
  • FD Rapid Golgi Stain TM Kit FD Neuro Technologies, PK401
  • the slicing parameters were as follows: speed, 0.5-0.7 mm/s; amplitude, 1 mm; brain slice thickness, 150 ⁇ m; continuous collection.
  • the brain slices were attached to gelatin-coated slides and dried in the dark for more than 3 days before staining. First, the slides were placed in double distilled water twice, each for 4 minutes; then the slides were placed in a staining mixture for 10 minutes.
  • the staining mixture contained Solution D, Solution E and double distilled water in a ratio of 1:1:1; the slides were placed in double distilled water for two 4-minute washes before entering the dehydration process.
  • mice were anesthetized, the chest cavity was opened to expose the heart.
  • the perfusion needle infused the perfusion solution from the left ventricle and out of the right atrium.
  • PBS was perfused until the lungs and liver turned white, and then 4% paraformaldehyde (Acmec, P35120) was changed to continue the perfusion.
  • the mice were visibly in a rigid state. Cut off the head with scissors, cut the skin to expose the skull, and use the tip of the scissors to gently slide along the exposed inner surface of the bone from the cerebellum to the olfactory bulb to peel off the brain.
  • the brain was taken out and soaked in 10ml 4% paraformaldehyde fixative for 24h, and placed in a gradient dehydration of 10%, 20%, and 30% sucrose solution. Cool the slicer to a suitable temperature, flatten the bottom of the brain, absorb excess liquid with filter paper, place it in an embedding tank, add an appropriate amount of OCT embedding agent (Servicebio, G6059) to submerge the tissue, place it on a quick freezing table, and freeze it for 30 minutes. After removal, apply embedding agent to the slice base, fix the brain tissue, and place it in a quick freezer for 30 minutes. Place the frozen brain tissue on the slicer holder and flatten the tissue. Adjust the anti-roll plate to the appropriate position and start slicing. Set the thickness of the brain slice to 40 ⁇ m and collect the brain slice in PBS solution. Wash it twice with PBS, 10 minutes each time, attach the brain slice to a gelatin-coated slide, and dry it.
  • OCT embedding agent Servicebio, G6059
  • Dendritic spine density was calculated by dividing the total number of dendritic spines by the length of the dendritic shaft. The proportion of each dendritic spine subtype was calculated as the percentage of the total dendritic spines on the dendritic segment.
  • mice were anesthetized with isoflurane (Sigma, 1349003). After opening the chest, a small incision was made in the right atrium, and perfusion was performed with a syringe from the left ventricle near the apex of the heart. The perfusion fluid used was pre-cooled oxygenated (95% O2 + 5% CO2) slicing fluid until the lungs and liver turned white. The brain tissue was quickly peeled off and placed in pre-oxygenated pre-cooled slicing fluid for cooling. The brain tissue was cut into 300 ⁇ m coronal slices using a vibrating slicer (Leica, VT1200S) with continuous oxygenated pre-cooled slicing fluid, mainly retaining the hippocampus.
  • a vibrating slicer Leica, VT1200S
  • the cut brain slices were transferred to 32°C continuously oxygenated slicing fluid for incubation for 12 minutes. At this time, the brain slices can be divided into two halves along the sagittal plane. Then they were transferred to 25°C repair HEPES artificial cerebrospinal fluid for incubation for at least one hour. Electrophysiological recordings can be performed after one hour. The brain slices can be maintained for 7-8 hours and must always be in oxygen-saturated repair HEPES artificial cerebrospinal fluid. During the recording process, the brain slices were perfused with oxygen-saturated artificial cerebrospinal fluid at a rate greater than or equal to 2 ml/min.
  • Example 1 Spontaneous epilepsy phenotype can be observed in mice with CDKL5 knockout in forebrain excitatory neurons
  • CDKL5 deficiency In order to simulate the symptoms of patients with CDKL5 deficiency and conduct basic research on CDKL5 deficiency, researchers have constructed a variety of CDD mouse models since the discovery of the CDKL5 gene, mainly including Cdkl5 knockout mice, point mutation mice that simulate patients, and conditional knockout mice. These mouse models have proven that CDKL5 is essential in neurological functions such as movement, learning and memory, and social interaction, but the core clinical symptom of CDD patients, spontaneous epilepsy, has not been reproduced.
  • the conditional knockout mouse model (Conditional knockout, referred to as cKO), can knock out the target gene at a specific time or tissue-specifically to meet the specific needs of researchers and conduct more targeted research.
  • Emx1-ires-Cre and CaMK2 ⁇ -iCre tool mice have flox sequences inserted in the same direction on both sides of exon 6 of Cdkl5;
  • Emx1-cre tool mice express Cre recombinase in excitatory neurons in the cortex and hippocampus, and glial cells in the globus pallidus from embryonic stage E10.5;
  • CaMK2 ⁇ -iCre tool mice express Cre recombinase in glutamatergic excitatory neurons in the forebrain after birth.
  • mice of the Cdkl5 fl/Y ;Emx1-Cre strain will have spontaneous grand mal seizures; at 3 months of age, about 50% of the mice can be observed to have epileptic seizures; while some mice of the Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice have spontaneous grand mal seizures starting from 7 weeks of age, and about 50% of the mice can be observed to have epileptic seizures at 3 months of age.
  • this spontaneous epilepsy starts with mild to moderate clonus and forelimb tremor in the early stage, and gradually develops into a high-grade generalized seizure (Figure 4C, Figure 5C).
  • mice were overly sensitive to vibration, touch, and sound, which is very close to the irritable clinical signs of CDD patients.
  • EEG electroencephalogram
  • EMG electromyography
  • mice Both strains of mice can be observed to have sudden death caused by severe spontaneous epileptic seizures, which may occur in the early life of CDD model mice (2-3 months old). Some mice also have physical dysfunction due to frequent spontaneous epilepsy, and are unable to move and eat until they die. At 6-7 months of age, approximately 70% of Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice died ( FIG. 4D , FIG. 5D ).
  • Example 2 Excitation-inhibition imbalance in the hippocampal dentate gyrus of forebrain excitatory neurons in Cdk15 knockout mice
  • the dentate gyrus receives its main input from the entorhinal cortex through the perforant path with glutamate synapses, and its molecular layer granule cell proximal dendrites form synapses with perforant path axons. Subsequently, granule cells project to the CA3 region through mossy fibers, which terminate on pyramidal neurons of CA3. Under physiological conditions, there are few direct interconnections between granule cells. In addition, granule cells themselves have a high resting membrane potential and strong GABA receptor-mediated inhibition, which makes them considered to be shock absorbers between the entorhinal cortex and CA3.
  • the hippocampal circuit presents an excitatory/inhibitory imbalance phenotype, which has appeared in many epilepsy models and also exists in other Cdkl5 knockout models. This suggests that forebrain excitatory neurons, as a key cell type for CDD-related epilepsy, will enhance excitatory synaptic transmission in the dentate gyrus and disrupt the balance of excitatory/inhibitory synaptic transmission in the hippocampal circuit, which may be the circuit basis for the spontaneous epileptic seizures in these two Cdkl5 conditional knockout mouse strains.
  • Example 3 Spontaneous epilepsy phenotype can be observed in tamoxifen-induced Cdkl5 fl/Y ; CaMK2 ⁇ -CreER mice
  • CDKL5 not only plays an important role in the development of the nervous system, but also maintains a high expression level in adulthood. Studies have shown that restoring knocked-out Cdkl5 in adulthood can greatly improve the impaired cognitive function of mouse models, indicating that CDKL5 also plays an important role in neural maturation. But is CDKL5 protein in neural maturation also critical for its spontaneous epilepsy phenotype?
  • Cdkl5 fl/Y ;Emx1-Cre and Cdkl5 fl/Y ;CaMK2 ⁇ -iCre mice the main causes of death in Cdkl5 fl/Y ;CaMK2 ⁇ CreER mice include sudden death from epilepsy and physical dysfunction.
  • the control group i.e., the Cdkl5 fl/Y mice injected with tamoxifen and the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER mice injected with corn oil, no abnormal behavior, synchronized discharges or abnormal deaths were observed.
  • CDD spontaneous epilepsy associated with CDD is closely related to the functional defect of CDKL5 during neural maturation.
  • CDD has been considered a simple neurodevelopmental disease, but our results prove that CDKL5 protein is critical to circuit excitability during neural maturation.
  • the lack of CDKL5 protein during neural maturation can also induce circuit hyperexcitability and lead to epilepsy. Therefore, the study of CDD-related epilepsy should not only consider its neurodevelopmental stage, but also the dysfunction during its neuromaturation stage.
  • the tamoxifen-induced adult Cdkl5 knockout mouse model has the advantages of short onset latency (two weeks of action) and high spontaneous epileptic seizure rate (100% seizure rate), so it can well simulate the patient's seizure pattern and help study the mechanism of CDD-related epilepsy.
  • forebrain knockout of Cdkl5 will enhance the excitatory transmission of the hippocampal circuit, but it is still unknown whether knockout during neural maturation still has similar hyperexcitatory reactions.
  • Cdkl5 fl/Y CaMK2 ⁇ -CreER mice as the subject and performed whole-cell recordings on them one week after tamoxifen injection. At this time, CDKL5 in the forebrain tissue has been knocked out, but spontaneous epilepsy has not been observed.
  • Example 5 The morphology of dentate gyrus granule cells in tamoxifen-induced Cdk15 fl/Y ; CaMK2 ⁇ -CreER mice is normal
  • dendritic spines are closely related to the excitability of the circuit. Their morphology is affected by many factors and is in a constant adjustment process.
  • the morphological abnormalities of neuronal dendritic spines are an important part of epilepsy research. In different animal models, the morphological abnormalities of dendritic spines in multiple brain regions of the forebrain can be observed, including the cerebral cortex, the CA1 region of the hippocampus, and the dentate gyrus.
  • Dendritic spines are the transmission sites of excitatory synapses, which can receive information and form synaptic connections, and are considered to be the basis of synaptic plasticity.
  • Dendritic spines include three morphologies: thin, mushroom, and stubby. The thin type is considered to be an immature state, while the stubby and mushroom types are mature types.
  • Dendritic spine density analysis is widely used in neuroscience research and is an important means to evaluate the functional plasticity of synapses in the central nervous system. Through the Golgi staining experiment, using the silver-loving properties of nerve cells, we can The morphology of neurons and the various types of dendritic spines are well documented. Although the number of dendritic spines is not directly equivalent to the amount of synaptic activity, the analysis of dendritic spine density and type can be used to assess changes in synaptic connection strength and synaptic plasticity.
  • Example 6 Abnormal activation of the hippocampal BDNF-TrkB signaling pathway is the key to enhancing excitatory synaptic transmission in the dentate gyrus of the hippocampus
  • BDNF Brain-derived neurotrophic factor
  • TrkB Brain-derived neurotrophic factor
  • BDNF affects synaptic transmission and changes the morphological characteristics of neurons, leading to excessive excitation of the hippocampal circuit, and this abnormal excitation in turn promotes the increase in BDNF expression levels, forming a pathological positive feedback.
  • Trk receptor inhibitor K252 was used to verify the blocking Can the BDNF-TrkB signaling pathway rescue the enhanced excitatory synaptic transmission of the circuit caused by tamoxifen-induced Cdkl5 knockout?
  • sEPSC spontaneous excitatory postsynaptic current
  • mEPSC miniature excitatory postsynaptic current
  • Trk receptor inhibitor K252a can well rescue the increase in sEPSC/mEPSC frequency caused by Cdkl5 knockout without affecting the excitatory synaptic transmission of the control group, and improve the enhanced excitatory synaptic transmission of dentate gyrus granule cells.
  • K252a is a broad-spectrum Trk inhibitor that may interfere with the function of other neurotrophic factors and affect subsequent experimental designs
  • ANA-12 a specific antagonist of TrkB.
  • sEPSCs spontaneous excitatory postsynaptic currents
  • mEPSCs miniature excitatory postsynaptic currents
  • Example 7 Inhibition of the BDNF-TrkB signaling pathway reduces epileptic activity in Cdkl5fl/Y;CaMK2 ⁇ -CreER mice
  • TrkB receptor antagonist ANA12 intervenes in the abnormal activation of the BDNF-TrkB signaling pathway by inhibiting the TrkB receptor.
  • the frequency of epileptic activity in the Cdkl5 fl/Y ;CaMK2 ⁇ -CreER tamoxifen-injected mice was significantly reduced compared with 12 hours before injection, and its anti-epileptic effect was comparable to that of the classic anti-epileptic drug valproic acid (Figure 16A-C), indicating that inhibiting TrkB receptors after spontaneous epilepsy can also effectively reduce CDD-related epileptic activity, suggesting that the BDNF-TrkB signaling pathway can be an excellent target for the treatment of CDD-related epilepsy.

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Abstract

本发明涉及BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用，具体地，本发明提供一种BDNF-TrkB信号通路抑制剂的用途，用于制备一组合物或制剂，所述组合物或制剂用于预防和/或治疗CDKL5缺乏症相关癫痫，本发明首次发现，BDNF-TrkB信号通路抑制剂可预防和/或治疗CDKL5缺乏症相关癫痫。

Description

BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用 技术领域
本发明涉及生物制药领域，具体地，本发明涉及BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用。
背景技术
CDKL5缺乏症(CDKL5 deficiency disorder，CDD)是一种罕见的遗传性疾病，其特征是从婴儿期开始出现的癫痫发作，伴随多系统的发育显著延迟。大约4万至6万新生儿中会有一例CDKL5缺乏症患者，是儿童癫痫最常见的遗传原因之一，其中大约90％是女性。CDKL5缺乏症以前被归类为Rett综合征的一个非典型形式，它们有共同的特征，包括癫痫发作、智力障碍和其他发育问题。然而，CDKL5缺乏症患者与Rett综合征有明显不同的临床过程，Rett综合征的患者癫痫发作一般在青春期开始出现，随着年龄增长严重程度会下降；而CDKL5缺乏症患者在出生后的前几个月就会出现癫痫发作，且存在严重抗癫痫药耐药性，无法治愈。因此CDKL5缺乏症现在被认为是一种与Rett综合征不同的独立疾病。
CDKL5缺乏症是由CDKL5基因突变引起的，CDKL5基因的突变减少了功能CDKL5蛋白的表达水平或改变其在神经细胞中的活性，但目前尚不清楚这些变化是如何导致CDKL5缺乏症的具体表型。CDKL5基因位于X染色体上，是X连锁的显性模式遗传，由于女性有两条X染色体，X染色体随机失活现象可以导致不同CDD患者症状和体征的严重程度不同，具有突变的神经元比例较高的女性比具有突变的神经元比例较低的女性有更严重的体征和症状。由于男性每个细胞中只有一条X染色体，因此CDKL5基因的突变在所有细胞中都是活跃的，受影响的男性没有该基因的正常拷贝。
癫痫发作往往是CDKL5缺乏症患者最初的表现，出生到发病的中位时间为4-6周，90％以上的患者在出生后3个月内出现癫痫发作，最早可在出生后第一周出现。患者的癫痫发作随着时间的推移可以发展为不同的类型，可能遵循一定的发作模式。最常见的类型是全身强直-阵挛性发作，包括意识丧失、肌肉僵硬和抽搐；强直性发作，其特点是肌肉异常收缩；癫痫性痉挛，脑电图显示为轻度心律失常，出现短时发作的肌肉抽搐。尽管可能存在缓解期(一段时间内没有癫痫发作)，但大多数CDD患者可能每天都有癫痫发作。大约三分之一的人会有多个阶段的癫痫发作。阵挛、无张力和失神发作也可在CDD患者中被观察到，但不属于常见类型。起初，癫痫发作发生在睡眠中，但随着时间的推移，会经常出现在清醒的时候。第一次癫痫发作在脑电图上通常没有显著性特征，然而这并不意味着CDD相关的癫痫发作活动不存在。
患有CDKL5缺乏症的儿童发育会受到影响，其中大多数有严重的智力障碍，且着重影响语言能力。坐、站、走等粗大运动技能的发展被推迟，只有约有三分之一的CDD患者能够独立行走。精细运动技能(如用手指拿起小物品等)也受影响，约有一半的CDD患者精细运动受限且伴随终生。大多数患者 都有视力问题，部分表现为皮质性视觉障碍(双眼视觉完全丧失，瞳孔光反射正常，眼底正常)。CDD患者的其他常见特征包括重复的手部动作，如拍手、舔手和吸手；磨牙；睡眠中断；喂养困难以及胃肠道问题，包括便秘和胃食道反流。部分患者有发作性的不规则呼吸。其他肢体异常也可能发生，如头部异常(小头症)，脊柱侧向弯曲和手指变细。
目前对CDKL5缺乏症患者的医疗管理主要是对症治疗和支持性治疗，旨在最大限度地提高患者的个人能力和增进任何可能出现的技能。重点放在早期干预治疗上，如物理治疗、职业治疗、语言和辅助性交流治疗。其中，癫痫控制是最具挑战性的内容。《评估和管理CDKL5缺乏症患者的国际共识建议》指出，现有的药物，包括皮质类固醇(corticosteroids)、氨己烯酸(vigabatrin)、丙戊酸(valproic acid)、苯妥英(phenytoin)、非尔氨酯(felbamate)、卡马西平(carbamazepine)、氯硝西泮(clonazepam)、奥卡西平(oxcarbazepine)和拉考酰胺(lacosamide)等，虽然单独使用初期可以减少癫痫发作频率，但用药一定时间后会失去疗效(反应中位时间6个月)，甚至在某些情况下还会使发作加剧。该共识文件没有对CDKL5缺乏症进行具体的抗癫痫药物治疗建议，但提出替代方案，包括生酮饮食；植入迷走神经刺激器(VNS，向沿颈部到大脑的迷走神经输送小脉冲电流)；胼胝体切开术(切断连接大脑两半球的主要纤维)和药物级大麻二酚。患有CDKL5缺乏症的儿童通常不适合做局部病灶切除手术，因为CDKL5缺失广泛影响大脑，而不是在一个特定的位置。CDD患者往往需要多种抗癫痫药来控制癫痫发作。选择的依据是发作类型和药物的作用机制、潜在的副作用、以及与其他药物相互作用的可能性。
癫痫是一种中枢神经系统疾病，患者患有反复发作的、涉及部分或全身的癫痫发作，有时还伴随着意识丧失，是一种灾难性的神经系统疾病。癫痫患者的发作程度可以从短暂的注意力分散或肌肉僵硬到长时间的严重抽搐。一般将癫痫发作分为局灶性或全身性，这取决于发作开始的方式和位置。颞叶癫痫(TLE)是局灶性癫痫的最常见形式，大约60％的局灶性癫痫患者属于颞叶癫痫，往往开始于海马体或边缘系统。颞叶癫痫被认为最适合进行癫痫的科学研究：作为最常见的癫痫类型，颞叶癫痫有大量的脑电数据和病例报告可供研究，且因颞叶切除术对颞叶性难治性癫痫有很好的治疗效果，其病理组织相较于其他类型的癫痫更为易得。颞叶癫痫最常见的病理特征是海马硬化，在癫痫患者和动物模型中都可以被观察到，主要表现为海马神经元丢失，包括CA1和CA3的锥体神经元丢失、齿状回细胞的弥散分布，其中最为显著的莫过于颗粒细胞的丢失。齿状回是兴奋传递到海马的关键结构。在癫痫患者和癫痫动物模型中，齿状回发生了多种变化，这使得癫痫发生来自海马的假说成为研究热点。
CDKL5缺乏症是一种由CDKL5基因突变引起的严重癫痫性脑病。自CDKL5缺乏症作为独立疾病进行研究以来，已有许多关于CDKL5蛋白性质与功能的研究进展，但是这些发现还不能明确解释CDKL5缺失所导致的症状，也没有动物模型可以很好的复现CDD患者的自发癫痫表型。作为最核心的临床症状，CDD相关自发癫痫的发生机制依然不明确。
因此，本领域迫切需要开发一种治疗CDKL5缺乏症相关癫痫疾病的新药 物。
发明内容
本发明的目的就是提供一种治疗CDKL5缺乏症相关癫痫疾病的新药物。
本发明的另一目的在于提供了靶向BDNF-TrkB信号通路可用于预防和/或治疗CDKL5缺乏症相关癫痫的证据，具体地，本发明提供一种BDNF-TrkB信号通路抑制剂的用途，用于制备一组合物或制剂，所述组合物或制剂用于预防和/或治疗CDKL5缺乏症相关癫痫。
本发明的第一方面提供了一种BDNF-TrkB信号通路抑制剂的用途，用于制备一组合物或制剂，所述组合物或制剂用于预防和/或治疗CDKL5缺乏症相关癫痫。
在另一优选例中，所述CDKL5缺乏症相关癫痫具有选自下组的一种或多种表型特征：
(a)自发癫痫表型；
(b)海马齿状回兴奋性突触传递增强；
(c)海马区BDNF-TrkB信号通路异常上调；
(d)患者癫痫发作随着时间的推移可以发展为不同的类型；
(e)患者癫痫表型具有抗癫痫药耐药性；
(f)患者癫痫发作期间的脑电图显示双侧同步低平电位，随后反复出现尖波和棘波，典型的脑电图表现随着时间的推移而发展，在幼儿中不明显。
在另一优选例中，所述组合物或制剂还用于选自下组的一种或多种用途：
(i)减少CDD相关的癫痫活动。
在另一优选例中，所述BDNF-TrkB信号通路抑制剂包括TrkB拮抗剂。
在另一优选例中，所述BDNF-TrkB信号通路抑制剂选自下组：ANA-12、K252a、或其组合。
在另一优选例中，所述组合物或制剂还包括其他可预防和/或治疗CDKL5缺乏症相关癫痫药物。
在另一优选例中，其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物包括TAK-935/OV935和Ganaxolone。
在另一优选例中，所述的组合物包括药物组合物。
在另一优选例中，所述的药物组合物含有(a)BDNF-TrkB信号通路抑制剂以及(b)药学上可接受的载体。
在另一优选例中，所述组分(a)占所述药物组合物总重量的0.1-99.9wt％,较佳地10-99.9wt％，更佳地70％-99.9wt％。
在另一优选例中，所述组分(a)占所述药物组合物总重量的60.0％-99.5wt％，较佳地70.0-99.5wt％，更佳地80.0％-99.5wt％。
在另一优选例中，所述药物组合物为液态、固体、或半固体。
在另一优选例中，所述药物组合物的剂型包括片剂、颗粒剂、胶囊、口服液、或注射剂。
在另一优选例中，所述组合物为口服制剂。
在另一优选例中，所述的组合物(如药物组合物)通过以下方式施用于哺乳动 物：口服、静脉注射、或局部注射。
在另一优选例中，所述哺乳动物包括患有CDKL5缺乏症相关癫痫的哺乳动物。
在另一优选例中，所述哺乳动物包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物，如小鼠、大鼠。
本发明第二方面提供了一种用于预防和/或治疗CDKL5缺乏症相关癫痫的药物组合物，包括：
(a1)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为BDNF-TrkB信号通路抑制剂；和
(a2)任选的第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物；
(b)药学上可接受的载体。
在另一优选例中，所述的第一药物组合物和第二药物组合物为不同的药物组合物，或同一药物组合物。
在另一优选例中，所述BDNF-TrkB信号通路抑制剂包括TrkB拮抗剂。
在另一优选例中，所述BDNF-TrkB信号通路抑制剂选自下组：ANA-12、K252a、或其组合。
在另一优选例中，其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物包括TAK-935/OV935和Ganaxolone。
在另一优选例中，所述第一活性成分和第二活性成分的重量比为1:100至100：1，较佳地为1:10至10：1。
在另一优选例中，所述药物组合物中，所述组分(a1)含量为1％－99％，较佳地，10％－90％，更佳地，30％－70％。
在另一优选例中，所述药物组合物中，所述组分(a1)和组分(a2)占所述产品组合总重的0.01-99.99wt％，较佳地0.1-90wt％，更佳地1-80wt％。
在另一优选例中，所述的药物组合物的剂型包括注射剂型、和口服剂型。
在另一优选例中，所述口服剂型包括片剂、胶囊剂、膜剂、和颗粒剂。
在另一优选例中，所述的药物组合物的剂型包括缓释型剂型、和非缓释型剂型。
在另一优选例中，所述组分(a1)与组分(a2)的重量比为1:100至100：1，较佳地为1:10至10：1。
本发明第三方面提供了一种药盒，包括：
(a1)第一容器，以及位于所述第一容器中的BDNF-TrkB信号通路抑制剂，或含有BDNF-TrkB信号通路抑制剂的药物；
(a2)任选的，第二容器，以及位于所述第二容器中的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，或含其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物的药物。
在另一优选例中，所述的第一容器和第二容器是相同或不同的容器。
在另一优选例中，所述的第一容器的药物是含BDNF-TrkB信号通路抑制剂的单方制剂。
在另一优选例中，所述的第二容器的药物是含其他预防和/或治疗CDKL5缺乏症相关癫痫的药物的单方制剂。
在另一优选例中，所述药物的剂型为口服剂型或注射剂型。
在另一优选例中，所述的试剂盒还含有说明书。
在另一优选例中，所述说明书记载了选自下组的一个或多个说明：
(a)将BDNF-TrkB信号通路抑制剂用于预防和/或治疗CDKL5缺乏症相关癫痫的方法；
(b)将BDNF-TrkB信号通路抑制剂和其他预防和/或治疗CDKL5缺乏症相关癫痫的药物联用于预防和/或治疗CDKL5缺乏症相关癫痫的方法。
本发明第四方面提供了一种组合的用途，所述组合包括BDNF-TrkB信号通路抑制剂和任选的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，用于制备一药物组合物或药盒，所述药物组合物或药盒用于预防和/或治疗CDKL5缺乏症相关癫痫。
在另一优选例中，所述药物组合物或药盒包括(a)BDNF-TrkB信号通路抑制剂和任选的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物；和(b)药学上可接受的载体。
在另一优选例中，所述药物组合物或药盒中，所述BDNF-TrkB信号通路抑制剂和用于预防和/或治疗CDKL5缺乏症相关癫痫的药物分别占所述药物组合物或药盒总重的0.01-99.99wt％,较佳地0.1-90wt％，更佳地1-80wt％。
本发明第五方面提供了一种预防和/或治疗CDKL5缺乏症相关癫痫的方法，包括：
给需要的对象施用BDNF-TrkB信号通路抑制剂和任选的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，或本发明第二方面所述的药物组合物或本发明第三方面所述的药盒。
在另一优选例中，所述对象包括患有CDKL5缺乏症相关癫痫的人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了CDKL5蛋白的结构域以及致病突变。CDKL5蛋白的功能结构域用不同颜色标注，数字是指氨基酸的种类和位置。红色标记的是致病的或可能致病的突变；黑色标记的是良性或可能良性的突变。NES＝出核序列；NLS＝核定位信号；ST＝丝氨酸-苏氨酸激酶活性位点；TEY＝保守Thr-Glu-Tyr基序。
图2显示了啮齿动物海马区神经环路图。(A，B)啮齿动物海马区神经环路图以及对应的神经网络示意图。实心箭头描绘了内嗅皮层(EC)-齿状回-CA3-CA1-EC的神经环路。内嗅皮层II层的神经元通过穿孔通路(PP)向齿 状回投射轴突，包括外侧穿孔通路(LPP)和内侧穿孔通路(MPP)两部分。齿状回通过苔藓纤维向CA3的锥体细胞投射。CA3锥体神经元通过Schaffer侧支将信息传递给CA1锥体神经元。CA1锥体神经元向内嗅皮层的深层神经元输出。CA3也通过穿孔通路接受内嗅皮层的直接投射。CA1通过颞氨通路(TA)接受内嗅皮层的直接输入。齿状颗粒细胞也投射到hilus区的苔藓细胞和hilar区的中间神经元，并分别投射回颗粒细胞。
图3显示了BDNF的生物学作用。当轴突电位到达突触前时，Na+流入使膜去极化，这引发了Ca2+的流入和兴奋性神经递质谷氨酸释放到突触裂隙。谷氨酸与突触后膜上的AMPA和NMDA受体结合。受体的激活导致膜去极化，Ca2+通过NMDA和VDCC流入。Ca2+与激活CREB和NF-kB的CaMKs结合，后者反过来诱导Bdnf基因的转录。BDNF在突触裂隙释放并激活TrkB受体，导致下游信号级联的激活，包括PLCγ、PI3K和MAPKs以及随后对神经元的生存和可塑性至关重要的基因表达。BDNF信号还通过改变NMDA受体的动力学和增加突触前突触囊泡的数量，对膜兴奋性和突触传递产生急性影响。
图4显示了Cdkl5fl/Y；Emx1-Cre小鼠出现自发癫痫表型。
(A)Cdkl5fl/Y；Emx1-Cre小鼠及其对照组的CDKL5蛋白在大脑皮层(Cortex)、海马(Hippocampus)和纹状体(Striatum)的表达水平。共四对小鼠，Tubulin为内参蛋白。
(B)Cdkl5fl/Y；Emx1-Cre小鼠自发癫痫发作时脑电图(EEG)和肌电图(EMG)记录的代表图。目标小鼠处于6月龄，上中两图表示Cdkl5fl/Y；Emx1-Cre小鼠癫痫发作时的脑电图(EEG)记录的变化，顶部箭头代表癫痫发作开始，癫痫发作时的波幅明显增大，并在结束后有存在抑制；下图表示Cdkl5fl/Y；Emx1-Cre小鼠癫痫发作时肌电图(EMG)记录的变化。
(C)Cdkl5fl/Y；Emx1-Cre小鼠及其对照组自发癫痫发作率统计图，Cdkl5fl/Y小鼠共10只，Cdkl5fl/Y；Emx1-Cre小鼠共15只。
(D)Cdkl5fl/Y；Emx1-Cre小鼠及其对照组存活率统计图，Cdkl5fl/Y小鼠共10只，Cdkl5fl/Y；Emx1-Cre小鼠共15只。
图5显示了Cdkl5fl/Y；CaMK2α-iCre小鼠出现自发癫痫表型。
(A)Cdkl5fl/Y；CaMK2α-iCre小鼠及其对照组的CDKL5蛋白在大脑皮层(Cortex)、海马(Hippocampus)和纹状体(Striatum)的表达水平。共四对小鼠，Tubulin为内参蛋白。
(B)Cdkl5fl/Y；CaMK2α-iCre小鼠自发癫痫发作时脑电图(EEG)和肌电图(EMG)记录的代表图。目标小鼠处于6月龄，上图表示Cdkl5fl/Y；CaMK2α-iCre小鼠癫痫发作时的脑电图(EEG)记录的变化，顶部箭头代表癫痫发作开始，癫痫发作时的波幅明显增大，并在结束后存在抑制；下图表示Cdkl5fl/Y；CaMK2α-iCre小鼠癫痫发作时肌电图(EMG)记录的变化。
(C)Cdkl5fl/Y；CaMK2α-iCre小鼠及其对照组自发癫痫发作率统计图，Cdkl5fl/Y小鼠共12只，Cdkl5fl/Y；CaMK2α-iCre小鼠共15只。
(D)Cdkl5fl/Y；CaMK2α-iCre小鼠及其对照组存活率统计图，Cdkl5fl/Y小鼠共12只，Cdkl5fl/Y；CaMK2α-iCre小鼠共15只。
图6显示了Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回兴奋性传递增强。
(A)Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sEPSC的代表图。
(B)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sEPSC的频率与对照组相比升高。
(C)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sEPSC的振幅与对照组相比没有显著差异。(B)和(C)对照组为Cdkl5fl/Y小鼠，共5只，记录了8个神经元；实验组为Cdkl5fl/Y；Emx1-Cre小鼠，共4只，记录了16个神经元。*p<0.05，未配对双尾t检验。
(D)Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mEPSC的代表图。
(E)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mEPSC的频率与对照组相比升高。
(F)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mEPSC的振幅与对照组相比没有显著差异。(E)和(F)对照组为Cdkl5fl/Y小鼠，共4只，记录了14个神经元；实验组为Cdkl5fl/Y；Emx1-Cre小鼠，共3只，记录了10个神经元。**p<0.01，未配对双尾t检验。
图7显示了Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回兴奋性传递增强。
(A)Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回sEPSC的代表图。
(B)全细胞记录显示，Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回sEPSC的频率与对照组相比升高。
(C)全细胞记录显示，Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回sEPSC的振幅与对照组相比没有显著差异。(B)和(C)对照组为Cdkl5fl/Y小鼠，共3只，记录了11个神经元；实验组为Cdkl5fl/Y；CaMK2α-iCre小鼠，共4只，记录了11个神经元。***p<0.001，未配对双尾t检验。
(D)Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回mEPSC的代表图。
(E)全细胞记录显示，Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回mEPSC的频率与对照组相比升高。全细胞记录显示，Cdkl5fl/Y；CaMK2α-iCre小鼠海马齿状回mEPSC的振幅与对照组相比没有显著差异。(E)和(F)对照组为Cdkl5fl/Y小鼠，共4只，记录了13个神经元；实验组为Cdkl5fl/Y；CaMK2α-iCre小鼠，共4只，记录了15个神经元。****p<0.0001，未配对双尾t检验。
图8显示了Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mIPSC振幅增强。
(A)Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sIPSC的代表图。
(B)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sIPSC的频率与对照组相比没有显著差异。
(C)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回sIPSC的振幅与对照组相比没有显著差异。(B)和(C)对照组为Cdkl5fl/Y小鼠，共5只，记录了10个神经元；实验组为Cdkl5fl/Y；Emx1-Cre小鼠，共3只，记录了9个神经元。未配对双尾t检验。
(D)Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mIPSC的代表图。
(E)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mIPSC的频率与对照组相比没有显著差异。
(F)全细胞记录显示，Cdkl5fl/Y；Emx1-Cre小鼠海马齿状回mEPSC的振幅与对照组相比增强。(E)和(F)对照组为Cdkl5fl/Y小鼠，共6只，记录了13个神经元；实验组为Cdkl5fl/Y；Emx1-Cre小鼠，共4只，记录了13个神经元。**p<0.01，未配对双尾t检验。
图9显示了Cdkl5fl/Y；CaMK2α-CreER小鼠产生自发癫痫。
(A)Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照组的CDKL5蛋白在大脑皮层(Cortex)、海马(Hippocampus)、纹状体(Striatum)和小脑(Cerebellum)的表达水平。每组3只小鼠，GAPDH为内参蛋白。
(B)Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照小鼠自发癫痫发作时脑电图(EEG)和肌电图(EMG)记录的代表图。
(B)Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照组自发癫痫发作率统计图。
(D)Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照组存活率统计图，Cdkl5fl/Y+他莫昔芬组小鼠共10只；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠共5只；Cdkl5fl/Y；CaMK2αCreER+他莫昔芬组小鼠共6只。
图10显示了Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回兴奋性传递增强。
(A)Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回sEPSC的代表图。
(B)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回sEPSC的频率与对照组相比升高。
(C)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回sEPSC的振幅与对照组相比没有显著差异。(B)和(C)对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共3只，记录了11个神经元；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共3只，记录了14个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共3只，记录了11个神经元。****p<0.0001，未配对双尾t检验。
(D)Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回mEPSC的代表图。
(E)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回mEPSC的频率与对照组相比升高。
(F)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER小鼠海马齿状回mEPSC的振幅与对照组相比没有显著差异。(E)和(F)对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共3只，记录了10个神经元；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共3只，记录了15个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共3只，记录了11个神经元。****p<0.0001，未配对双尾t检验。
图11显示了Cdkl5fl/Y；CaMK2α-CreER小鼠颗粒细胞树突棘密度和成熟度正常。
(A)Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠及其对照组的海马齿状回颗粒细胞树突的代表片段。比例尺：5μm。
(B)Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠及其对照组的海马齿状回颗粒细胞树突棘密度的定量分析。单因素方差分析与Tukey的多重比较测试。
(C)Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠及其对照组的海马齿状回颗粒细胞树突棘的分类。双向方差分析与Bonferroni事后检验。每组包含41-52个神经元。
图12显示了Cdkl5fl/Y；CaMK2α-CreER小鼠海马区BDNF表达水平增加，BDNF-TrkB信号通路异常激活。
(A)免疫印迹实验检测Cdkl5fl/Y；CaMK2α-CreER小鼠海马区BDNF和CDKL5的蛋白水平，内参为GAPDH。
(B)对Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照组海马区BDNF的蛋白水平进行量化分析。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共25只；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共9只；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共23只。****p<0.0001，未配对双尾t检验。
(C)Cdkl5fl/Y；CaMK2α-CreER小鼠及其对照组海马区BDNF的mRNA水平进行qPCR分析。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共11只；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共5只；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共10只。*p<0.05，未配对双尾t检验。
(D)免疫印迹实验检测Cdkl5fl/Y；CaMK2α-CreER小鼠海马区p-TrkB(Tyr 516)、p-TrkB(Tyr 707)、p-TrkB(Tyr 816)、p-ERK1/2、p-Akt(Ser473)、p-GSK3β和p-mTOR(Ser 2448)的蛋白水平。
(E)对(D)中所示的蛋白质的磷酸化水平进行量化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共14只；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共8只；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共12只。*p<0.05，未配对双尾t检验。
图13显示了Trk抑制剂K252a可以改善Cdkl5fl/Y；CaMK2α-CreER小鼠兴奋性突触传递增强的缺陷。
(A)Cdkl5fl/Y；CaMK2α-CreER小鼠使用K252a前后sEPSC的代表。
(B和C)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠在孵育K252a后，升高的sEPSC频率恢复到对照组的正常水平，而振幅没有变化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共4只，记录了11个神经元；Cdkl5fl/Y；CaMK2αCreER+玉米油组小鼠，共3只，记录了10个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共4只，记录了13个神经元。
(D)Cdkl5fl/Y；CaMK2α-CreER小鼠使用K252a前后mEPSC的代表图。
(E和F)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠在孵育K252a后，升高的mEPSC频率恢复到对照组的正常水平，而振幅没有变化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共3只，记录了11个神经元；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共3只，记录了10个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共3只，记录了11个神经元。**p<0.01，***p<0.001，****p<0.0001，给药前后配对双尾t检验，不同组间非配对双尾t检验。
图14显示了TrkB拮抗剂ANA-12可以改善Cdkl5fl/Y；CaMK2α-CreER小鼠兴奋性突触传递增强的缺陷。
(A)Cdkl5fl/Y；CaMK2α-CreER小鼠使用ANA-12前后sEPSC的代表图。
(B和C)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠在孵育ANA-12后，升高的sEPSC频率恢复到对照组的正常水平，而振幅没有变化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共3只，记录了15个神经元；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共3只，记录了11个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共3只，记录了19个神经元。***p<0.001，****p<0.0001。
(D)Cdkl5fl/Y；CaMK2α-CreER小鼠使用ANA-12前后mEPSC的代表图。
(E和F)全细胞记录显示，Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠在孵育ANA-12后，升高的mEPSC频率恢复到对照组的正常水平，而振幅没有变化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共3只，记录了10个神经元；Cdkl5fl/Y；CaMK2α-CreER+玉米油组小鼠，共3只，记录了12个神经元；实验组为Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬小鼠，共3只，记录了13个神经元。**p<0.01，****p<0.0001，给药前后配对双尾t检验，不同组间非配对双尾t检验。
图15显示了敲低TrkB受体可以改善成年期敲除Cdkl5导致的自发癫痫活动。
(A)免疫印迹实验检测Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠大脑皮层和海马区CDKL5和TrkB的蛋白水平，内参为GAPDH。
(B)对Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠及其对照组大脑皮层和海马区CDKL5和TrkB的蛋白水平进行量化。对照组为Cdkl5fl/Y+他莫昔芬组小鼠，共4只；
Cdkl5fl/Y；CaMK2α-CreER+他莫昔芬组小鼠，共4只；实验组为Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER+他莫昔芬小鼠，共4只。**p<0.001，未配对双尾t检验。
(C)Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠的录像观察实验示意图。
(D)Cdkl5fl/Y；CaMK2α-CreER小鼠和Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠自发癫痫发作率统计图。对照组为Cdkl5fl/Y；CaMK2α-CreER小鼠，共11只；实验组为Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠，共7只。
图16显示了抑制TrkB受体可以改善Cdkl5fl/Y；CaMK2α-CreER小鼠的自发癫痫活动。
(A)Cdkl5fl/Y；CaMK2α-CreER小鼠的腹腔注射实验示意图。
(B，C)Cdkl5fl/Y；CaMK2α-CreER小鼠腹腔注射TrkB拮抗剂ANA12(B)或VPA(C)前后12小时自发癫痫活动频率变化。数量为6只。**p<0.01，*p<0.05，配对双尾t检验。
(D)单次注射ANA-12不会造成Cdkl5fl/Y小鼠和Cdkl5fl/Y； CaMK2α-CreER小鼠海马区的细胞凋亡。阳性对照组为Cdkl5fl/Y小鼠+DNase；实验组为Cdkl5fl/Y小鼠和Cdkl5fl/Y；CaMK2α-CreER小鼠+ANA12，每组3只小鼠。比例尺：200μm。
具体实施方式
经过广泛而深入的研究，本发明人意外发现，BDNF-TrkB信号通路抑制剂(比如TrkB拮抗剂)可有效预防和/或治疗CDKL5缺乏症相关癫痫。在此基础上，本发明人完成了本发明。
如本文所用，术语“ANA-12”的结构式为：
如本文所用，术语“K252a”的结构式为：
如本文所用，术语“TAK-935/OV935”的中文名称为索替司他，结构式为：
如本文所用，术语“Ganaxolone”的中文名称为加奈索酮，结构式为：
CDKL5缺乏症
CDKL5缺乏症(CDKL5 deficiency disorder，CDD)是一种罕见的遗传性疾病，其特征是从婴儿期开始出现的癫痫发作，伴随多系统的发育显著延迟。大约4万至6万新生儿中会有一例CDKL5缺乏症患者，是儿童癫痫最常见的遗传原因之一，其中大约90％是女性。CDKL5缺乏症以前被归类为Rett综合征的一个非典型形式，它们有共同的特征，包括癫痫发作、智力障碍和其他发育问题。然而，CDKL5缺乏症患者与Rett综合征有明显不同的临床过程，Rett综合征的患者癫痫发作一般在青春期开始出现，随着年龄增长严重程度会下降；而CDKL5缺乏症患者在出生后的前几个月就会出现癫痫发作，且存在严重抗癫痫药耐药性，无法治愈。因此CDKL5缺乏症现在被认为是一种与Rett综合征不同的独立疾病。
CDKL5缺乏症是由CDKL5基因突变引起的，CDKL5基因的突变减少了功能CDKL5蛋白的表达水平或改变其在神经细胞中的活性，但目前尚不清楚这些变化是如何导致CDKL5缺乏症的具体表型。CDKL5基因位于X染色体上，是X连锁的显性模式遗传，由于女性有两条X染色体，X染色体随机失活现象可以导致不同CDD患者症状和体征的严重程度不同，具有突变的神经元比例较高的女性比具有突变的神经元比例较低的女性有更严重 的体征和症状。由于男性每个细胞中只有一条X染色体，因此CDKL5基因的突变在所有细胞中都是活跃的，受影响的男性没有该基因的正常拷贝。
癫痫发作往往是CDKL5缺乏症患者最初的表现，出生到发病的中位时间为4-6周，90％以上的患者在出生后3个月内出现癫痫发作，最早可在出生后第一周出现。患者的癫痫发作随着时间的推移可以发展为不同的类型，可能遵循一定的发作模式。最常见的类型是全身强直-阵挛性发作，包括意识丧失、肌肉僵硬和抽搐；强直性发作，其特点是肌肉异常收缩；癫痫性痉挛，脑电图显示为轻度心律失常，出现短时发作的肌肉抽搐。尽管可能存在缓解期(一段时间内没有癫痫发作)，但大多数CDD患者可能每天都有癫痫发作。大约三分之一的人会有多个阶段的癫痫发作。阵挛、无张力和失神发作也可在CDD患者中被观察到，但不属于常见类型。起初，癫痫发作发生在睡眠中，但随着时间的推移，会经常出现在清醒的时候。第一次癫痫发作在脑电图上通常没有显著性特征，然而这并不意味着CDD相关的癫痫发作活动不存在。
患有CDKL5缺乏症的儿童发育会受到影响，其中大多数有严重的智力障碍，且着重影响语言能力。坐、站、走等粗大运动技能的发展被推迟，只有约有三分之一的CDD患者能够独立行走。精细运动技能(如用手指拿起小物品等)也受影响，约有一半的CDD患者精细运动受限且伴随终生。大多数患者都有视力问题，部分表现为皮质性视觉障碍(双眼视觉完全丧失，瞳孔光反射正常，眼底正常)。CDD患者的其他常见特征包括重复的手部动作，如拍手、舔手和吸手；磨牙；睡眠中断；喂养困难以及胃肠道问题，包括便秘和胃食道反流。部分患者有发作性的不规则呼吸。其他肢体异常也可能发生，如头部异常(小头症)，脊柱侧向弯曲和手指变细。
目前对CDKL5缺乏症患者的医疗管理主要是对症治疗和支持性治疗，旨在最大限度地提高患者的个人能力和增进任何可能出现的技能。重点放在早期干预治疗上，如物理治疗、职业治疗、语言和辅助性交流治疗。其中，癫痫控制是最具挑战性的内容。《评估和管理CDKL5缺乏症患者的国际共识建议》指出，现有的药物，包括皮质类固醇(corticosteroids)、氨己烯酸(vigabatrin)、丙戊酸(valproic acid)、苯妥英(phenytoin)、非尔氨酯(felbamate)、卡马西平(carbamazepine)、氯硝西泮(clonazepam)、奥卡西平(oxcarbazepine)和拉考酰胺(lacosamide)等，虽然单独使用初期可以减少癫痫发作频率，但用药一定时间后会失去疗效(反应中位时间6个月)，甚至在某些情况下还会使发作加剧。该共识文件没有对CDKL5缺乏症进行具体的抗癫痫药物治疗建议，但提出替代方案，包括生酮饮食；植入迷走神经刺激器(VNS，向沿颈部到大脑的迷走神经输送小脉冲电流)；胼胝体切开术(切断连接大脑两半球的主要纤维)和药物级大麻二酚。患有CDKL5缺乏症的儿童通常不适合做局部病灶切除手术，因为CDKL5缺失广泛影响大脑，而不是在一个特定的位置。CDD患者往往需要多种抗癫痫药来控制癫痫发作。选择的依据是发作类型和药物的作用机制、潜在的副作用、以及与其他药物相互作用的可能性。
CDKL5蛋白
细胞周期激酶样蛋白5(Cyclin-dependent Kinase Like 5，CDKL5)是由CDKL5基因编码，最初被命名为STK9，即丝氨酸/苏氨酸激酶9。CDKL5基因位于X染色体短臂22号区域(Xp22)。人的CDKL5基因有24个外显子，CDKL5蛋白N端为丝氨酸/苏氨酸激酶结构域，其致病突变多见于催化区域(图1)，2-21号外显子为编码区。CDKL5蛋白属于细胞周期依赖激酶家族，与细胞周期依赖激酶(cyclin-dependent kinase，CDKs)、有丝分裂原激活蛋白激酶(mitogen-activated protein kinases，MAPKs)、糖原合成激酶(glycogen synthase kinases，GSKs)的激酶结构域高度同源。
人类和啮齿动物的中枢神经系统中大量表达CDKL5蛋白，其中，大脑皮层、海马、丘脑、纹状体和嗅球中表达水平最高。CDKL5在胚胎期表达含量较低，在出生后表达水平有明显上升，在最初几周内达到高峰，并在成年期维持稳定的表达。神经元是CDKL5主要表达的细胞类型，胶质细胞仅有少量表达。各个亚细胞结构都有CDKL5的表达，其中细胞质是主要表达的区域，但随着大脑发育开始逐渐出现核聚集的现象。部分CDKL5的潜在底物已经被鉴定，其中一些与生理功能相关的底物已在细胞中得到验证。目前已知的底物包括MAP1S、EB2、微管和中心体功能的调节因子CEP131和涉及DNA损伤反应的ELOA等等。CDKL5在核中主要富集于RNA剪接和加工的区域。不同发育时期CDKL5也在不同亚细胞区域聚集：在培养神经元的早期，CDKL5被发现聚集于生长锥；在成熟神经元中，CDKL5聚集于树突棘，特别是突触后致密区；也有研究发现CDKL5存在于中心体。这些表达模式表明，CDKL5既参与了神经元发育的调控，又在成年的神经成熟期发挥着功能。、
CDKL5在神经元发育各个阶段发挥着不同的功能。在早期神经元发育中，CDKL5调控细胞增殖与神经元的迁移。据之前研究表明，CDKL5在调控细胞增殖中呈现负调控功能，上调CDKL5表达量会抑制人类神经母细胞瘤细胞增殖；而敲除小鼠Cdkl5会增加齿状回颗粒细胞增殖率。此外，CDKL5被发现存在于分裂细胞和有丝分裂后神经元的中心体，表明其可能是通过影响中心体对细胞增殖分裂进行调控。CDKL5对神经元的迁移有调节作用，其被发现与细胞迁移和极性相关的重要调节因子——IQGAP1相互作用。在啮齿类模型动物中，降低神经祖细胞中CDKL5的表达会导致2-3层锥体神经元的迁移延迟。而在神经元成熟的过程中，CDKL5与树突和轴突生长发育紧密相关，是突触形成的关键蛋白之一。CDKL5与轴突生长的关键蛋白Shootin1在生长锥中相互作用。通过RNAi沉默Cdkl5，或过表达CDKL5会导致具有多个轴突的神经元数量增加，但下调Cdkl5不阻碍轴突形成，表明CDKL5在轴突生长中有调控功能，但对轴突形成不起决定性作用。CDKL5也调控树突的发育，在培养的神经元中沉默Cdkl5，神经元的树突分支化受到严重损害。Cdkl5敲除的小鼠可以被观察到大脑皮层和海马CA1的锥体神经元，以及齿状回颗粒细胞的树突总长度显著减少。而在培养的神经元中过量表达CDKL5会出现激酶活性依赖的树突总长度增加。此外，CDKL5也参与了突触的形成。CDKL5被观察到与棕榈酰化的PSD95以及粘附分子NGL1相互作用。PSD是一种主要的支架蛋白，调节突触蛋白的定位和突触强度；NGL1是突触形成的关键粘附分子。在树突棘中，CDKL5在突触后致密区中高度富集，突触后致密区是由突触传递、信号转导和细胞粘附的关键蛋白组成的密集蛋白复合物，CDKL5的 突触定位强烈表明该蛋白参与了突触的发育和功能。在全身Cdkl5敲除小鼠中，齿状回颗粒细胞、海马CA1锥体神经元和皮质V层锥体神经元的树突棘密度和成熟树突棘的数量显著减少。在条件性敲除前脑兴奋性神经元Cdkl5的小鼠中，反而被观察到树突棘密度和复杂度增加的趋势。这种差异的原因尚不清楚，抑制性中间神经元的Cdkl5缺失可能引发补偿机制。
CDKL5的功能不止于神经元发育的范围，其表达在成人大脑中持续存在。在CDKL5缺乏的成年小鼠中，成熟树突棘的稳定和长时程增强受损，这些动物在海马依赖的记忆方面有缺陷。值得注意的是，这些缺陷可以通过在成年小鼠中恢复CDKL5蛋白水平得到逆转，包括树突棘的形态异常、后肢抓握以及学习记忆能力。CDKL5敲除小鼠的缺陷也可以通过激活CDKL5的下游信号通路而得到改善。例如，应用IGF-1可以恢复树突棘的稳定性，这表明CDKL5在成年后的神经成熟期也发挥着重要功能。此外，CDKL5本身的功能和活性也受神经活动的调控，如DYRK1A对308位丝氨酸残基的磷酸化促进CDKL5在Neuro-2a细胞中的细胞质的定位；在培养的神经元中，BDNF诱导CDKL5的瞬时磷酸化；神经元活动促使PP1对CDKL5进行去磷酸化。神经活动与CDKL5的相互调控，表明CDKL5在调控神经元及神经环路稳态中有其关键和独特的作用。
CDKL5缺乏症小鼠模型
Cdkl5外显子敲除小鼠是最常用的Cdkl5敲除小鼠模型，在这个品系的Cdkl5敲除小鼠中，研究人员观察到肢体协调性、运动能力、学习记忆能力的受损，还出现了自闭症样症状、异常眼动、呼吸和睡眠模式的异常，以及对胡须介导的触觉刺激的非典型行为反应。其中大部分表型都可以与CDKL5缺乏症患者的症状互相印证。
CDKL5缺乏症小鼠模型的建立证实了该基因对大脑正常发育和神经系统功能的重要性，还提供了一个体内环境来发现和验证涉及CDKL5的各种蛋白质-蛋白质相互作用和信号通路，并测试新发现的治疗方法。尽管对各种CDD小鼠模型进行了大量实验探索，但CDKL5的内源功能及其在神经系统发育、维持和发病机制中的作用仍未阐明，CDKL5缺乏症最核心的自发癫痫表型也没有得到很好的复现，仍缺乏一种新的动物模型来对CDD相关癫痫进行模拟。
海马齿状回与癫痫发生
癫痫是一种中枢神经系统疾病，患者患有反复发作的、涉及部分或全身的癫痫发作，有时还伴随着意识丧失，是一种灾难性的神经系统疾病。癫痫患者的发作程度可以从短暂的注意力分散或肌肉僵硬到长时间的严重抽搐。一般将癫痫发作分为局灶性或全身性，这取决于发作开始的方式和位置。颞叶癫痫(TLE)是局灶性癫痫的最常见形式，大约60％的局灶性癫痫患者属于颞叶癫痫，往往开始于海马体或边缘系统。颞叶癫痫被认为最适合进行癫痫的科学研究：作为最常见的癫痫类型，颞叶癫痫有大量的脑电数据和病例报告可供研究，且因颞叶切除术对颞叶性难治性癫痫有很好的治疗效果，其 病理组织相较于其他类型的癫痫更为易得。颞叶癫痫最常见的病理特征是海马硬化(Ammon's horn sclerosis)，在癫痫患者和动物模型中都可以被观察到，主要表现为海马神经元丢失，包括CA1和CA3的锥体神经元丢失、齿状回细胞的弥散分布，其中最为显著的莫过于颗粒细胞的丢失。齿状回是兴奋传递到海马的关键结构。在癫痫患者和癫痫动物模型中，齿状回发生了多种变化，这使得癫痫发生来自海马的假说成为研究热点。
海马位于颞叶的内侧区域，在情绪调节、从短期记忆到长期记忆的信息巩固以及空间处理方面起着重要作用。了解海马的解剖结构对其功能认知至关重要。
海马由阿蒙角(Cornu ammonis，CA，包括CA1-CA4)、齿状回和丘下区等亚区组成。内嗅皮层II层神经元轴突通过穿孔通路(Perforant pathway,PP)向齿状回投射，包括外侧穿孔通路(Lateral perforant pathway,LPP)和内侧穿孔通路(Medial perforant pathway,MPP)。齿状回通过苔藓纤维向CA3的锥体细胞发出投射，再通过Schaffer侧支传递给CA1锥体神经元，最后通过CA1锥体神经元向EC的深层神经元输出。此外，CA3也通过穿孔通路接受内嗅皮层的直接投射；CA1通过颞氨通路(Temporo-ammonic pathway,TA)接受内嗅皮层的直接输入。而齿状颗粒细胞会投射到中间神经元和苔藓细胞，并分别接受其抑制性和兴奋性的反馈(图2，A-B)。
齿状回从外到内由三层组成：分子层、颗粒细胞层和多形态层(也称Hilus层)。作为齿状回的主要神经元，颗粒细胞在颗粒细胞层中紧密排列，其轴突投射到CA3，与锥体神经元形成兴奋性突触。分子层是一个无细胞层，由颗粒细胞层中的颗粒细胞的树突和来自内嗅皮层的穿孔通路纤维占据。多形层中有许多细胞类型，苔藓细胞是最重要神经元之一。在颗粒层和多形态层的边缘，有几种类型的神经元，如锥体篮状细胞，这是一种抑制性中间神经元。关于齿状回的神经发生是否持续到成年存在很大争议，但普遍认为成年动物的颗粒细胞总数相对稳定。颗粒细胞投射的苔藓纤维终止于CA3锥体细胞层之上，该层被称为透明层。苔藓纤维还投射到多形态层，与中间神经元形成突触连接。在生理条件下，齿状回颗粒细胞活跃程度很低，这与其本身的细胞固有特性和其所处的环路网络环境都有关系。首先，与大多数兴奋性海马神经元相比，颗粒细胞表现出超极化的静息膜电位，这使得它们更难以被诱发动作电位。此外，颗粒细胞树突显示出显著的电压依赖的线性整合和对内嗅皮层突触输入的强衰减。除了相对低兴奋的膜固有特性外，齿状回颗粒细胞也处于一个偏向抑制的环路网络中。其中，前馈抑制由快闪中间神经元(fast-spiking interneurons)、门区中间神经元(hilar interneuron)和篮状细胞(basket cell)等多种抑制性神经元提供。而颗粒细胞轴突投射也会与多种GABA能中间神经元相连接，由这些抑制性神经元对颗粒细胞形成树突靶向的反馈调节。颗粒细胞的固有特性和抑制性的环路网络环境使它作为内嗅皮层和CA3之间的减震器而存在。因此，当齿状回出现异常时，它被认为是癫痫发生过程的“雷管/引爆器”，并与CA3一起成为形成癫痫发作的关键，这也被称为齿状回的门控假说。门控假 说认为，齿状回保护海马环路不受异常兴奋的影响，而该区域的破坏会导致癫痫。有大量支持这一理论的癫痫研究对齿状回的门控假说进行阐述，包括苔藓纤维发芽(苔藓纤维与其他颗粒细胞上形成异常的突触)；颗粒细胞基底树突的异常形成和持续存在；颗粒细胞异位分散和迁移；颗粒细胞膜固有特性和突触受体表达的改变；抑制性GBAB能突触传递的下调和各种分子信号通路的异常调控等。而且，利用光遗传技术在动物模型中对齿状回颗粒细胞进行去极化或超极化的调控，可以很好的抑制/诱发自发癫痫的发生，证明了齿状回是颞叶癫痫发作网络中的一个关键节点。而光遗传学激活齿状回抑制性中间神经元可以阻止癫痫在海马和皮层的扩散，进一步突出了齿状回在调节皮层输入方面的重要作用。
CDKL5缺乏症患者会在不同病程阶段表现出不同类型的癫痫发作，但CDD患者在MRI中可被观察到颞叶高强度活动，且多个病例报告显示CDD患者颞叶萎缩；在CDD动物模型中也可见苔藓纤维发芽等颞叶癫痫常见病理特征，这些证据表明CDD相关癫痫与颞叶癫痫密切相关。而在CDD小鼠模型中，也可以观察到海马齿状回的多种变化。在Cdkl5敲除小鼠中，齿状回的分子层对突触前标记物突触蛋白和VGLUT1的免疫反应性降低，表明突触维持受损；GluN2B亚基在齿状回颗粒细胞-CA3层突触连接处的异位积累，使突触过度兴奋，导致癫痫易感性增加；有丝分裂后的颗粒神经元前体死亡增加，颗粒细胞总数减少，新生成的颗粒细胞表现出严重的树突萎缩，损害了海马依赖的相关行为；齿状回颗粒细胞和CA1锥体神经元的树突棘密度和成熟树突棘数量都明显减少。这些实验数据都暗示海马齿状回在CDKL5缺乏症的病理性发展中占据着关键作用。
BDNF-TrkB信号通路与癫痫发生
脑源性神经营养因子(Brain derived neurotrophic factor,BDNF)属于神经营养因子家族，与典型神经生长因子(NGF)相关，该家族还包括NT-3和NT-4/NT5。BDNF作用于中枢神经系统和外周神经系统的某些神经元，在大脑中，它活跃在海马体、皮层和基底前脑区域，在视网膜、肾脏、前列腺、运动神经元和骨骼肌中也有表达。BDNF主要与TrkB受体高亲和力结合，低亲和力神经生长因子受体LNGFR(也称p75)也能够对BDNF做出反应。不同时空阶段里，BDNF在神经元中发挥着不同的作用，复杂的多层次调控的存在证明了BDNF功能的重要性和多样性。在发育过程中，BDNF对神经元种群的生存和分化起着至关重要的作用，通过改变细胞存活和增殖，促进新生神经元的生长和分化；BDNF在调节成人大脑的可塑性变化中起着关键作用，包括调节蛋白转运、受体磷酸化和谷氨酸受体表达水平；BDNF可以促进树突棘形态的变化，增加树突棘的数量、大小和复杂性，从而稳定长时程增强(Long-term potentiation，LTP)，在记忆形成和维持中起着关键作用。
BDNF对兴奋性突触传递起增强作用(图3)。
研究表明，癫痫发生过程中BDNF-TrkB信号通路会出现异常的上调，这种上调与神经环路的兴奋性/抑制性稳态平衡紧密相关，而海马，特别是海马齿 状回在BDNF的促癫痫作用中是一个关键的结构。BDNF表达水平上升，不仅可能导致海马环路的结构重组(苔藓纤维萌发)，还会影响海马多区域的突触传递(包括增强谷氨酸介导的兴奋性突触传递或GABA介导的抑制性突触传递)。这些变化会导致海马环路的超兴奋状态，进一步促进BDNF的异常激活，从而形成癫痫持续状态。
CDKL5缺乏症相关癫痫疾病
如本文所述，“CDKL5缺乏症相关癫痫疾病”是指由CDKL5缺乏症引发的癫痫表型。
在本发明中，CDKL5缺乏症相关癫痫疾病具有选自下组的一种或多种表型特征：
(a)小鼠模型的自发癫痫表型；
(b)小鼠模型的海马齿状回兴奋性突触传递增强；
(c)小鼠模型的海马区BDNF-TrkB信号通路异常上调；
(d)患者癫痫发作随着时间的推移可以发展为不同的类型。
(e)患者癫痫表型具有抗癫痫药耐药性。
(f)患者癫痫发作期间的脑电图显示双侧同步低平电位，随后反复出现尖波和棘波，典型的脑电图表现随着时间的推移而发展，在幼儿中不明显。
本发明聚焦于探索CDKL5缺乏症相关癫痫疾病的发生机制。我们发现，不管是在发育期还是成年期，在前脑兴奋性神经元敲除Cdkl5可以使得小鼠出现自发癫痫表型，且海马齿状回兴奋性突触传递增强。使用他莫昔芬在成年期敲除Cdkl5fl/Y；CaMK2α-CreER小鼠前脑兴奋性神经元的Cdkl5除了可以被观察到海马齿状回兴奋性突触传递增强外，齿状回颗粒细胞树突棘的密度和成熟度正常，AMPA受体介导的电生理特性不受影响，但海马区BDNF-TrkB信号通路异常上调。通过抑制BDNF-TrkB信号通路，可以挽救齿状回异常的突触传递并减少Cdkl5fl/Y；CaMK2α-CreER小鼠的癫痫活动，提示BDNF-TrkB信号通路的异常上调促进遗传性癫痫小鼠模型的癫痫发生，靶向这一通路可能是治疗CDD相关癫痫的一种有效策略。
BDNF-TrkB信号通路抑制剂
在本发明中，BDNF-TrkB信号通路抑制剂是指能通过竞争或非竞争的方式抑制BDNF诱导的TrkB活性的小分子，包括TrkB拮抗剂。
在一优选实施方式中，BDNF-TrkB信号通路抑制剂选自下组：ANA-12、K252a、或其组合。
本发明首次发现，BDNF-TrkB信号通路抑制剂可有效预防和/或治疗CDKL5缺乏症相关癫痫。
复方药物组合物和药盒
本发明提供了含有活性成分(a)BDNF-TrkB信号通路抑制剂；任选的(b)预防和/或治疗CDKL5缺乏症相关癫痫的药物；以及(c)药学上可接受的载体的 复方药物组合物。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、粉剂、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。本发明的药物组合也可以被制成粉剂用于雾化吸入。一种优选的剂型是注射制剂。此外，本发明药物组合物还可与其他治疗剂一起使用。
本发明还提供了一种可用于预防和/或治疗CDKL5缺乏症相关癫痫的药盒，该药盒含有：
(a1)第一容器，以及位于所述第一容器中的BDNF-TrkB信号通路抑制剂，或含有BDNF-TrkB信号通路抑制剂的药物；
(b1)任选的第二容器，以及位于所述第二容器中的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，或含有其他预防和/或治疗CDKL5缺乏症相关癫痫的药物。
本发明的药物组合物和药盒适用于预防和/或治疗CDKL5缺乏症相关癫痫。
本发明制剂可以每一天服用三次到每十天服用一次，或者以缓释方式每十天服用一次。优选的方式是每天服用一次，因为这样便于病人坚持，从而显著提高病人服药的顺应性。
服用时，极大多数病例一般每天应用的总剂量应低于(或少数病例等于或略大于)各个单药的每天常用剂量，当然，所用的活性成分的有效剂量可随给药的模式和待治疗的疾病的严重程度等而有所变化。
治疗方法
本发明还提供了用本发明的上述活性成分或相应的药物预防和/或治疗CDKL5缺乏症相关癫痫的方法，它包括给哺乳动物施用有效量的活性成分(a)BDNF-TrkB信号通路抑制剂；任选的(b)其他预防和/或治疗CDKL5缺乏症相关癫痫的药物(比如TAK-935/OV935和Ganaxolone)，或者施用含有所述活性成分(a)和任选的活性成分(b)的药物组合物。
当本发明的活性成分被用于上述用途时，可与一种或多种药学上可接受的载体或赋形剂混合，如溶剂、稀释剂等，而且可以用如下形式口服给药：片剂、丸剂、胶囊、可分散的粉末、颗粒或悬浮液(含有如约0.05-5％悬浮剂)、糖浆(含有如约10-50％糖)、和酏剂(含有约20-50％乙醇)，或者以无菌可注射溶液或悬浮液形式(在等渗介质中含有约0.05-5％悬浮剂)进行非肠胃给药。例如，这些药物制剂可含有与载体混合的约0.01-99％，更佳地约为0.1％-90％(重量)的活性成分。
本发明的活性成分或药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：肌内、腹膜内、静脉内、皮下、皮内、口服、瘤内或局部给药。优选的给药途径包括口服给药、肌内给药或静脉内给药。
从易于给药的立场看，优选的药物组合物是液态组合物，尤其是注射剂。
本发明的主要优点包括：
(1)本发明首次发现BDNF-TrkB信号通路抑制剂(比如TrkB拮抗剂)可 有效预防和/或治疗CDKL5缺乏症相关癫痫。
(2)本发明聚焦于探索CDKL5缺乏症相关癫痫的发生机制。我们发现，不管是在发育期还是成年期，在前脑兴奋性神经元敲除Cdkl5可以使得小鼠出现自发癫痫表型，且海马齿状回兴奋性突触传递增强。使用他莫昔芬在成年期敲除Cdkl5fl/Y；CaMK2α-CreER小鼠前脑兴奋性神经元的Cdkl5除了可以被观察到海马齿状回兴奋性突触传递增强外，齿状回颗粒细胞树突棘的密度和成熟度正常，AMPA受体介导的电生理特性不受影响，但海马区BDNF-TrkB信号通路异常上调。通过抑制BDNF-TrkB信号通路，可以挽救齿状回异常的突触传递并减少Cdkl5fl/Y；CaMK2α-CreER小鼠的癫痫活动，提示BDNF-TrkB信号通路的异常上调促进遗传性癫痫小鼠模型的癫痫发生，靶向这一通路可能是治疗CDD相关癫痫的一种有效策略。
(3)本发明首次发现，以往的CDD动物模型无法很好的复刻CDD的核心癫痫表型，本发明通过在前脑兴奋性神经元敲除Cdkl5，构建具有自发癫痫表型的小鼠模型。
(4)本发明首次发现，CDKL5在成年期维持着海马环路神经网络稳定性，阐明了CDKL5缺乏症也不仅是一种神经系统发育障碍疾病，也是神经系统功能障碍疾病。
(5)本发明首次观察到成年期Cdkl5敲除小鼠BDNF-TrkB信号通路异常上调，且抑制BDNF-TrkB信号通路可以减少自发癫痫发作活动，提出了CDD相关癫痫发生的分子机制。
(6)本发明的研究阐述了CDKL5在兴奋性/抑制性突触传递平衡中的重要作用，并提供了CDD相关癫痫发生的新模型和新靶点，为CDD相关癫痫的临床治疗打下更为坚实的基础。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非有特别说明，否则实施例中所用的材料和试剂均为市售产品。
通用方法
1.实验材料
1.1实验动物
所有实验动物都源于C57BL/6J背景，Cdkl5flox/flox由宾夕法尼亚大学周兆兰教授提供；Emx1-Cre小鼠(JAX 005628)购买自The Jackson Laboratory；CaMK2α-iCre小鼠和CaMK2α-CreER小鼠来自德国癌症研究中心的Schutz教授；TrkBflox/flox小鼠来自中国科学院脑科学与智能技术卓越创新中心杜久林教授的馈赠。所有动物均饲养于SPF(Specific Pathogen Free)级别屏障系统环境内，自由进水进食，生长温度21-24℃，12小时光照/黑暗环境交替。本文涉及的动 物实验及操作满足中国科学院脑科学与智能技术卓越创新中心动物伦理委员会的相关要求。
1.2实验抗体
1.3.实验试剂


生化实验相关的试剂配方如下：

小鼠实验相关的试剂如下：
(1)他莫昔芬配方(终浓度：100mg/kg)：0.5g他莫昔芬(MedChemExpress，1052910)溶解于2.5ml无水乙醇(国药，10009228)和47.5ml玉米油(ABCONE，C67366)的混合溶液后于40度水浴锅中混匀直至完全溶解，分装保存于-20℃。小鼠体重每10g注射0.1ml。
(2)ANA-12配方(终浓度：6mg/kg)：将1.2mg ANA12(MedChemExpress，HY-12497)溶解于100μl二甲基亚砜(DMSO)溶液(Sigma-Aldrich，D2447)，再加入900μl玉米油(ABCONE，C67366)，摇匀至无沉淀，保存于-20℃，一周内用完。小鼠体重每20g注射0.1ml。
(3)VPA配方(终浓度：50mg/kg)：10mg丙戊酸(Valproic Acid，TargetMol，T7064)溶于100μl二甲基亚砜(DMSO)溶液(Sigma-Aldrich，D2447)，再加入900μl玉米油(ABCONE，C67366)，摇匀至无沉淀，保存于-20℃，一周内用完。小鼠体重每20g注射0.1ml。
(4)pY816配方(终浓度：20mg/kg)：100mg pY816或对照多肽(金斯瑞定制)加入12.5ml磷酸盐缓冲液(Phosphate-Buffered Saline，cellgro，21-040-CVCa)，摇匀至无沉淀，保存于-20℃，一周内用完。小鼠体重每20g注射0.1ml。
电生理实验相关的试剂配方如下：

1.4.实验仪器和软件

2.实验方法
2.1小鼠基因鉴定
使用碱裂解法，将约2mm小鼠组织(脚趾或鼠尾)，加入200μl鼠尾裂解液和2μl Proteinase K(20mg/mL)，充分混匀后置于56℃烘箱消化6小时或消化过夜。之后金属浴95℃，使蛋白酶K失活，保存至4℃。
使用2X Taq PCR Master Mix试剂盒(Tiangen，KT211)7μl 2X Taq PCR Master Mix；F端和R端引物各0.5μl；5μl ddH2O；模版1μl；总体积14μl。
鉴定程序：95℃，3min；95℃，30s，58℃，30s，72℃，30s，第2-4步循环35次；72℃，7min；10℃，∞。
引物信息如下：
Cdkl5-F:CCACCCTCTCAGTAAGGCAG
Cdkl5-R:GTCCTTTGCCACTCAATTCCATCC
Emx1-F:GCAAGAACCTGATGGACATGTTCAG
Emx1-R:GCAATTTCGGCTATACGTAACAGGG
iCre-F:TGCCCAAGAAGAAGAGGAA
iCre-R:TTGCAGGTACAGGAGGTAGTC
creER-F:GACAGGCAGGCCTTCTCTGAA
creER-R:CTTCTCCACACCAGCTGTGGA
TrkB-F:ATGTCGCCCTGGCTGAAGTG
TrkB-R:ACTGACATCCGTAAGCCAGT
2.2.录像观察
Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠从成年期开始，Cdkl5fl/Y；CaMK2α-CreER小鼠从注射他莫昔芬后开始录像监控，在单盲条件进行观察并录像，记录观察到的发作编号、时间以及发作等级，每天监测1小时(每周5天)。在注射他莫昔芬后，每周对Cdkl5fl/Y；TrkB；Cam2α-CreER小鼠及其对照组进行24小时连续视频录像。对小鼠的癫痫发作按照Racine标准进行分级：一级，有胡须和面部的抖动；等级二，有明显点头运动；等级三，单侧前肢痉挛，尾巴竖直；等级四，前肢痉挛，肌强直明显；等级五，失去肢体控制，强制阵挛，抽搐。因为一级和二级癫痫发作很轻微，在录像过程中很容易被忽略，所以在我们的研究中，只有三级或更高级别的癫痫发作才被认为是自发性癫痫发作(Racine，1972)
当Cdkl5fl/Y；CaMK2α-CreER小鼠癫痫发作进入稳定期，发作频率稳定(24小时录像中每三小时一次发作以上，且发作强度、间隔相似)，可进行药理学实验。在注射对照用玉米油(ABCONE，C67366)、TrkB拮抗剂ANA12(MedChemExpress，HY-12497)或经典抗癫痫药物VPA(TargetMol，T7064)前后12小时进行视频录像，通过计算注射前后全身发作事件的次数来评估抗癫痫效果，每只小鼠大于一次给药实验并取平均值，每次药理实验间隔2天以上。
2.3脑电(EEG)和肌电(EMG)记录
将小鼠头部固定至立体定位仪上，暴露头骨，在电极植入点打磨两个小孔，将EEG电极拧入小孔，用牙科水泥固定在头骨。用镊子将EMG电极插入斜方肌。在手术3天后，进行连续2天记录适应。对自由移动的小鼠进行EEG和EMG的记录，使用Spike2软件(CED Ltd.，Micro1401mkⅡ)以1kHz的频率进行了采集。
2.4蛋白质提取和免疫印迹实验
将小鼠用异氟烷(Sigma，1349003)麻醉后，取下头部，分离头骨暴露出脑组织。用镊子取下大脑，在提前预冷的生理盐水中进行清洗。将组织用刀片对半切后，用海马勺分离出大脑、海马、纹状体和小脑等组织，放入2ml的离心管中，并加入磁珠和相应体积的RIPA强蛋白裂解液。使用60Hz的频率于快速研磨仪(上海净信实验设备科技部，JX-FSTPRP)中研磨一分钟，再放置于冰块中裂解1小时进行充分裂解。之后将EP管转入提前预冷的4℃离心机中以14,000g离心25分钟，吸出上清至新的离心管。
使用BCA蛋白定量试剂盒(Tiangen，PA115)进行蛋白含量测定。配制BCA工作液，按体积比＝50:1的比例对试剂A和B进行混匀；使用与样品相同缓冲体系的溶液对BSA标准品进行稀释，同时对样品进行5-10倍的稀释；使用96孔板，每孔含25μl稀释后样品或者BSA标准品和200μl BCA工作液，充分混匀后加盖 放入37℃烘箱30分钟；30分钟后使用酶标仪(Molecular Devices)于562nm处检测吸光值；根据标准品曲线计算出样品的蛋白浓度。
使用蛋白上样缓冲液将样品浓度调平，80℃加热7分钟。提前准备好装有SDS-PAGE胶板的电泳装置(VE-180型垂直电泳槽，中国上海天能科技有限公司)，并加入1X电泳液。然后把煮好的蛋白样品用移液枪依次加入到胶板的泳道中。电泳的参数为：第一阶段恒压80V，30分钟；第二阶段恒压110V，100分钟。电泳参照标准品使用PageRulerTMPlus Prestained Protein Ladder(Thermo Scientific，26619)。电泳结束后，使用恒流模式350mA将蛋白转入PVDF膜上，用时3小时。根据蛋白性质使用脱脂奶粉(伊利)或BSA(aladdin，B265991)加上TBST缓冲液配制5％封闭液，室温封闭1小时，封闭后将相应蛋白一抗稀释于封闭液中，4℃过夜孵育。在室温使用TBST缓冲液于要床上洗膜三次，每次5-7分钟。洗膜完成后将对应蛋白二抗溶解于封闭液中，室温孵育1-2小时。孵育结束后洗膜三次，并使用Pro-Light HRP化学发光检测试剂(Tiangen，PA112)在化学发光成像仪(Analytik Jena AG)中进行曝光。
2.5组织RNA提取和反转录
使用DEPC(Acmec，D21980)处理的手术器械对相应脑组织进行解剖后放入2ml离心管。在离心管中加入1ml的Trizol(Invitrogen，15596026)，使用快速研磨仪中以60Hz的频率研磨一分钟。加入200μl氯仿(Sigma，1601383)，剧烈摇晃15次，室温静置10分钟，四度离心12,000g 15分钟，可得三分层样品。取上层透明相至新离心管，加入500μl异丙醇(Sigma，W292907)，上下摇晃后静置10分钟，四度离心12,000g 10分钟，弃上清，得白色沉淀。用1ml 75％无水乙醇清洗，四度离心7,500g 5分钟，去上清后倒置离心管干燥1小时。然后，加入20-50μl DEPC水，56℃加热溶解，用Nanodrop 2000超微量分光光度计(Thermo Scientific)进行核酸定量。定量后使用iScript cDNA Synthesis kit试剂盒(Bio-Rad，1708891)进行反转录，取1μg的mRNA将其定量到15μl，加入4μl的5x iScript Reaction Mix Buffer和1μl的iScript逆转录酶，混匀后反转录程序为25℃，5分钟；42℃，30分钟；85℃，5分钟；之后维持在10℃。反转录结束后，将样品保存至-80℃。
2.6实时荧光定量PCR
使用RNA Master SYBR Green I kit试剂盒(Roche，03064760001)进行RT-PCR反应。反应体系为10μL SYBR Green Mix，1μl引物，2μl cDNA template，6μl ddH2O，总体积20μl。反应程序为95℃，5min；95℃，10s，60℃，20s，72℃，20s，第二至四步循环40次；95℃，5s；65℃，1min。
引物信息如下：
BDNF-F:AGGTCTGACGACGACATCACT
BDNF-R:CTTCGTTGGGCCGAACCTT
GAPDH-F:ATCCCAGAGCTGAACGGGAAGC
GAPDH-R:TTGGGGGTAGGAACACGGAAGG
反应结束后确认RT-PCR的扩增曲线和溶解曲线，进行分析和计算。
2.7高尔基染色
使用FD快速高尔基染色试剂盒FD Rapid GolgiStainTMKit(FD Neuro Technologies，PK401)进行高尔基染色。将小鼠麻醉后打开胸腔暴露出心脏。使用注射器将PBS溶液从左心室灌入，右心房流出，直到待肺和肝脏颜色变白。剪刀剪下头部，暴露出颅骨，用剪刀的尖端沿着露骨内表面从小脑处到嗅球轻轻滑动，将大脑剥离，快速取出完整脑组织。放入24小时前预先混匀的AB混合液，室温遮光孵育一天，再更换一次AB混合液，继续室温遮光孵育14天。14天后更换为C液，孵育1天后更换一次C液，继续孵育3天。3天后进行切片处理。将大脑以冠状切的方式用3％的琼脂(ASONE，CC-5166-03)包埋，放入冰箱进行冷却，用502粘合剂固定于振动切片机(Leica，VT1200s)进行切片，切片参数如下：速度，0.5-0.7mm/s；振幅，1mm；脑片厚度为150μm；连续收片。将脑片贴在明胶包被的载玻片上，避光晾干3天以上，之后对脑片进行染色。先将玻片放入双蒸水两次，每次4分钟；再将玻片放入染色混合液中10min，染色混合液包含Solution D、Solution E和双蒸水，比例1:1:1；将玻片放入双蒸水中进行两次4分钟的清洗后，进入脱水流程。将玻片放入50％乙醇、75％乙醇、95％乙醇、无水乙醇溶液中逐级进行脱水，每次4分钟，无水乙醇溶液需重复四次，再将玻片放入二甲苯溶液(Acmec，X31920)固定4分钟，固定步骤重复三次。使用中性树脂封片，避光保存。
2.8 TUNEL染色
将小鼠麻醉后打开胸腔暴露出心脏。灌流针将灌流溶液从左心室灌入，右心房流出。先灌流PBS，直到待肺和肝脏颜色变白，再换4％多聚甲醛(Acmec，P35120)继续灌注，灌注结束后可见小鼠呈僵硬状态。剪刀剪下头部，剪开皮肤暴露出颅骨，用剪刀的尖端沿着露骨内表面从小脑处到嗅球轻轻滑动，将大脑剥离。大脑取出浸泡于10ml 4％多聚甲醛固定液24h，置于10％、20％、30％蔗糖溶液梯度脱水。将切片机降温到合适的温度，将大脑底部修平，滤纸吸干多余液体，放置于包埋槽中，加入适量OCT包埋剂(Servicebio，G6059)淹没组织，放置于速冻台上，冰冻30分钟。取出后，将切片底托涂上包埋剂，固定脑组织，放置于速冻台冰冻30分钟将冷冻好的脑组织，放置于切片机持承器上，将组织修平。调整防卷板至适当的位置，开始切片。脑片厚度设置为40μm，收集脑片于PBS溶液中。用PBS清洗两次，每次10分钟，将脑片贴附于明胶包被的载玻片中，晾干。
用PBS清洗两次，每次10分钟，加入含0.1％Triton X-100的PBS，冰浴孵育2分钟。用甲醇配制的0.3％过氧化氢溶液(Sigma，SML0790)室温孵育20分钟，目的是灭活切片内源的过氧化物酶，PBS清洗3次，每次10分钟。使用显色法TUNEL细胞凋亡检测试剂盒(Colorimetric TUNEL Apoptosis Assay Kit，碧云天，C1098)进行染色，按TdT酶：Biotin-dUTP：生物素标记液＝2:48:50的比例配制生物素标记工作液，充分混匀。在脑片上加50μl生物素标记工作液，37℃孵育一小时，用PBS清洗一次，孵育时需注意保持湿润，尽量减少工作液的蒸 发。加入0.3ml标记反应终止液，室温孵育10分钟，PBS清洗一次。在样品上加50μl Streptavidin-HRP工作液，室温孵育30分钟，用PBS清洗三次。滴加0.5ml DAB显色液，室温孵育30分钟，用PBS清洗三次。用苏木素染色液进行细胞核染色，PBS清洗三次。用95％乙醇对脑片脱水，换100％乙醇脱水两次，最后使用二甲苯两次，所有操作每次5分钟，随后封片观察。阳性样品使用TUNEL检测阳性对照制备试剂盒(碧云天，C1082)制备，在100μl 1X Reaction Buffer中加入1μl DNase I，混匀，滴加到样品上，室温孵育10分钟，PBS清洗三次。通过Olympus VS120显微镜观察细胞凋亡程度。
2.9图像采集和处理
高尔基染色图像数据是通过Nikon A1激光扫描显微镜或Nikon A1R激光扫描显微镜以60x油镜采集，像素分辨率为1024x 1024，以1μm的间隔进行采集。图像处理使用ImageJ软件进行，所有数据分析均采用“单盲”方式。树突棘密度由总树突棘数除以树突轴的长度来计算。每个树突棘亚型的比例计算为树突段上总树突棘的百分比。
2.10电生理脑片制备
小鼠使用异氟烷(Sigma，1349003)进行麻醉，开胸后先在右心房剪一小口，从左心室接近心尖处使用注射器进行灌流，灌流液使用提前预冷充氧(95％O2+5％ CO2)的切片液，直至肺部和肝脏变白。将大脑组织进行快速剥离，放入提前充氧预冷的切片液中进行冷却，使用带持续充氧预冷切片液的振动切片机(Leica，VT1200S)将大脑组织切成300μm的冠状切脑片，主要保留海马区域。将切好的脑片转至32℃的持续充氧的切片液孵育12分钟，此时可以将脑片沿矢状面分割成两半。之后转入25℃修复用HEPES人工脑脊液孵育至少一小时，一小时后可以进行电生理记录。脑片可以维持7-8小时，需一直处于氧饱和修复用HEPES人工脑脊液中。在记录过程中脑片一直处于氧饱和人工脑脊液灌流，灌流速度大于等于2ml/min。
2.11全细胞记录
全细胞记录实验在300μm冠状切脑片中进行。使用电极拉制仪(Sutter，P-97)拉制带纤维丝的玻璃电极(Sutter,BF150-86)，电阻为4-8MΩ，玻璃电极尖端根据需要加入铯内液或钾内液，保证电极尖端无气泡。用FN1显微镜(Nikon)和40倍水浸物镜观察细胞。玻璃电极与神经元之间形成高阻封接后等待1-2分钟使漏电流降至20pA以内，吸破细胞，在Membrane test模式下记录破膜参数。使用MultiClamp 700B放大器(Axon CNS,Molecular Devices)的Rs compensation模块进行系列电阻补偿。信号用Digidata 1440A数模转换器(Molecular Devices)进行采集，采样频率为10kHz，2kHz过滤。
记录sEPSC期间，使用含有木防己苦毒素(Picrotoxin，Tocris，1128)的人工脑脊液进行灌流；记录mEPSC期间，使用含有木防己苦毒素(Picrotoxin，Tocris，1128)和河豚毒素(Tetrodotoxin，Tocris，1078)的人工脑脊液进行灌 流；记录sIPSC期间，使用含有D-AP5(Tocris，0106)和CNQX(Tocris，1045)的人工脑脊液进行灌流；记录mIPSC期间，使用含有D-AP5(Tocris，0106)、CNQX(Tocris，1045)和河豚毒素(Tetrodotoxin，Tocris，1078)的人工脑脊液进行灌流；记录静息膜电位和基电流期间，使用含有木防己苦毒素(Picrotoxin，Tocris，1128)的人工脑脊液进行灌流；进行内源性大麻素受体敏感性期间，使用含有木防己苦毒素(Picrotoxin，Tocris，1128)的人工脑脊液进行灌流；进行AMPA受体介导的EPSC电流-电压相关性实验时，使用含有木防己苦毒素(Picrotoxin，Tocris，1128)的人工脑脊液进行灌流；进行BDNF-TrkB信号通路抑制实验时，在原有基础上加入K252a(CST，12754S)或ANA-12(MedChemExpress，HY-12497)的人工脑脊液进行灌流
2.12数据分析和统计
图片和文字中的所有统计值均以平均值±标准差(mean±SEM)表示。除特别注明外，N为小鼠数量。使用GraphPad Prism 7进行统计检验。电生理数据分析均在Clampfit 10.4或Mini Analysis Program中进行。未配对t-检验(Unpaired Student's t-test)或配对t-检验(Paired Student's t-test)被用于检验两组之间差异的显著性；单因素方差分析(One-way ANOVA)和Tukey多重比较检验(Tukey'spost-test)被用于分析多组间差异的显著性；双因素方差分析(two-way ANOVA)及邦弗朗尼事后检验法(Bonferroni post-test)被用于多组别树突棘的形态类别分析。具有统计学差异的统计数据均注明：*p<0.05，**p<0.01，***p<0.001，****p<0.0001。
实施例1前脑兴奋性神经元CDKL5敲除小鼠可被观察到自发癫痫表型
为了模拟CDKL5缺乏症患者的症状，对CDKL5缺乏症进行基础研究，从CDKL5基因被发现以来，研究者们构建了多种CDD小鼠模型，主要包括Cdkl5全敲小鼠、模拟病人的点突变小鼠和条件性敲除小鼠。这些小鼠模型证明了CDKL5在运动，学习记忆，社交等神经功能中至关重要，但CDD患者最核心的临床症状——自发癫痫还未被复现。条件性敲除小鼠模型(Conditional knockout，简称cKO)，可以在特定时间或组织特异性敲除目的基因，满足研究者的特定需求，进行更有针对性的研究，其中最常见的是Cre/Loxp系统。我们将Emx1-ires-Cre和CaMK2α-iCre工具鼠和Cdkl5-flox小鼠进行杂交。其中，Cdkl5-flox小鼠是在Cdkl5的第6号外显子两侧插入同向的flox序列；Emx1-cre工具鼠在皮层和海马的兴奋性神经元，以及苍白球的胶质细胞自胚胎期E10.5开始表达Cre重组酶；CaMK2α-iCre工具鼠在出生之后，在前脑谷氨酸能兴奋性神经元中表达Cre重组酶。通过免疫印迹实验，我们发现Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠的大脑皮层和海马的CDKL5表达水平显著下降，但只有在Cdkl5fl/Y；CaMK2α-iCre小鼠纹状体中能观察到CDKL5表达水平的下降(图4A，图5A)。
为了观察这两种条件性敲除小鼠品系是否存在自发癫痫，我们对Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠进行录像。我们根据Racine癫痫分级规则观察癫痫活动。因为1级和2级癫痫发作很轻微，在录像过程中很容易被忽略，所以在我们的研究中，只有3级或更高级别的癫痫发作才被 认为是自发性癫痫发作。我们发现，从2月龄开始，Cdkl5fl/Y；Emx1-Cre品系会有部分小鼠产生自发癫痫大发作的现象；在3月龄，有约50％的小鼠可被观察到癫痫发作；而Cdkl5fl/Y；CaMK2α-iCre小鼠从7周龄开始就有部分小鼠出现自发癫痫大发作，在3月龄时约50％的小鼠可被观察到癫痫发作。与CDD患者的临床表型相似的是，这种自发癫痫由早期的轻中等级的阵挛、前肢颤抖开始，逐渐发展为高等级的全身大发作(图4C，图5C)。
同时，我们还观察到部分小鼠会出现对震动、触碰、声音的过度敏感，这与CDD患者的易激惹临床体征非常接近。我们也通过记录小鼠的脑电图(Eletroencephalo-graph，EEG)和肌电图(Electromyography，EMG)的方式，对其自发癫痫时的生理指标进行观测。我们可以观测到Cdkl5fl/Y；Emx1-Cre小鼠和Cdkl5fl/Y；CaMK2α-iCre小鼠在癫痫发作时都有异于对照组和正常生理情况下的异常发电(图4B，图5B)。与此同时，与CDD患者的境况相似的是，自发的癫痫发作会严重影响小鼠的寿命。两种品系的小鼠都可被观察到严重自发癫痫大发作导致的癫痫猝死现象，这种现象在CDD模型小鼠的生命早期(2-3月龄)就可能出现，也有部分小鼠由于频繁的自发癫痫导致其身体机能紊乱，无法运动和进食，直至死亡。在6-7月龄，Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠约有70％的小鼠死亡(图4D，图5D)。
上述结果表明，在小鼠前脑兴奋性神经元中敲除Cdkl5，会造成该品系的小鼠出现自发癫痫发作。与此同时，一些与临床CDD患者类似的其他症状，如易激惹、癫痫猝死，影响寿命等都可以被观察到。这些现象说明了前脑兴奋性神经元在CDD相关癫痫发生中起着关键作用，在前脑兴奋性神经元对Cdkl5进行敲除的条件性敲除小鼠模型可以很好的模拟CDD患者的癫痫表型。
实施例2前脑兴奋性神经元Cdkl5敲除小鼠海马齿状回出现兴奋抑制失衡
齿状回通过具有谷氨酸突触的穿孔通路接收来自内嗅皮层的主要输入，其分子层颗粒细胞近端树突与穿孔路径轴突形成突触。随后，颗粒细胞通过苔藓纤维投射到CA3区，苔藓纤维终止于CA3的锥体神经元上。在生理条件下，颗粒细胞之间的直接互连很少，此外，颗粒细胞本身具有较高的静息膜电位和较强的GABA受体介导的抑制能力，使其被认为是内嗅皮层与CA3之间的减震器。。当齿状回异常时，会使得海马环路兴奋性/抑制性失衡，引发癫痫，因此也被称为癫痫发生的“雷管”，在癫痫发生中有着重要意义。为了检测Cdkl5条件性敲除小鼠的海马齿状回突触传递是否存在异常，我们将3月龄的Cdkl5fl/Y；Emx1-Cre小鼠的大脑进行振动切片，对脑片进行全细胞记录。我们记录了小鼠的自发兴奋性突触后电流(spontaneous Excitatory Post Synaptic Current，sEPSC)和微小兴奋性突触后电流(miniature Excitatory Post Synaptic Current，mEPSC)，发现其自发兴奋性突触后电流和微小兴奋性突触后电流的频率对比对照组有显著的增高，而振幅没有差异(图6，A-F)，这表明在前脑兴奋性神经元中敲除CDKL5蛋白会导致海马齿状回颗粒细胞兴奋性突触传递增强。而海马区环路的超兴奋在多种癫痫模型中被认为与癫痫发生紧密相关。
我们也对3月龄的Cdkl5fl/Y；CaMK2α-iCre小鼠进行了全细胞记录，我们同样发现了该品系小鼠的自发兴奋性突触后电流(sEPSC)和微小兴奋性突触后电流(mEPSC)的频率的增高，而振幅没有差异，表现出齿状回兴奋性突触传递的增强(图7，A-F)。
除此之外，海马环路抑制性神经元的异常也会影响环路的兴奋性/抑制性平衡，因此我们也对齿状回抑制性突触传递进行了分析。我们观察到该品系小鼠的微小抑制性突触后电流(miniature inhibitory Post Synaptic Current，mIPSC)的振幅相比对照组有显著上升，但其频率，还有自发抑制性突触后电流(spontaneous inhibitory Post Synaptic Current，sIPSC)的频率和振幅都没有改变(图8，A-F)，这表明Cdkl5在前脑兴奋性神经元中的敲除也影响了其抑制性突触传递，且可能是对增强的兴奋性突触传递的一种补偿现象。海马环路呈现出一种兴奋/抑制失衡的表型，这一表型在多种癫痫模型上都有出现，且在其他的Cdkl5敲除模型中也存在。这表明前脑兴奋性神经元作为CDD相关癫痫的关键细胞类型，其Cdkl5敲除会增强齿状回兴奋性突触传递，打破海马环路的兴奋性/抑制性突触传递的平衡，可能是这两种Cdkl5条件性敲除小鼠品系产生自发癫痫发作的环路基础。
实施例3他莫昔芬诱导的Cdkl5fl/Y；CaMK2α-CreER小鼠可被观察到自发癫痫表型
CDKL5不仅对神经系统的发育起着重要作用，其在成年期依然维持着很高的表达水平。研究显示，在成年期恢复已敲除的Cdkl5，可以极大改善小鼠模型受损的认知功能，表明CDKL5在神经成熟期也发挥着重要作用。但神经成熟期的CDKL5蛋白对于其自发癫痫表型是否也同样关键呢。为此，我们利用他莫昔芬诱导表达的creER/loxp系统，将Cdkl5flox/flox雌鼠与Camk2a-CreER雄鼠结合，得到Cdkl5fl/Y；CaMK2α-CreER小鼠，并在其2月龄连续注射他莫昔芬5天(对照组使用玉米油)，并在注射完成后7天进行敲除验证。利用该系统，我们得以在成年期对前脑的兴奋性神经元进行Cdkl5的敲除。如图所示，他莫昔芬注射完成后7天，大脑皮层和海马中的CDKL5蛋白表达水平与对照组相比显著下降，纹状体和小脑的CDKL5蛋白水平和对照组一致。两个对照组，包括注射了他莫昔芬的Cdkl5flox/Y小鼠组和注射了玉米油的Cdkl5fl/Y；CaMK2α-CreER小鼠组，其大脑皮层、海马区、纹状体区和小脑的CDKL5蛋白水平没有差异(图9A)。
为了观察成年期敲除前脑兴奋性神经元CDKL5蛋白是否会造成小鼠自发癫痫，我们对该品系小鼠进行录像观察。我们发现，在注射完成后两周，就可以观察到50％的Cdkl5fl/Y；CaMK2α-CreER小鼠出现自发癫痫表型，且在8周后，100％的小鼠都出现了自发癫痫表型(图9C)。而在前人的非特异性杂合模型和条件性敲除模型中，都需要在敲除2个月以上才被观察到自发癫痫的产生。我们通过EEG和EMG的记录，观察到与Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠自发癫痫发作时类似的脑电和肌电的同步化放电信号(图9B)。同时，Cdkl5fl/Y；CaMK2α-CreER小鼠也出现了较高的致死率，在注射完成后3个月内，已有80％的小鼠致死(图9D)， 而相近的致死率在条件性敲除小鼠中需要6个月以上。与Cdkl5fl/Y；Emx1-Cre和Cdkl5fl/Y；CaMK2α-iCre小鼠相似，Cdkl5fl/Y；CaMK2αCreER小鼠的死亡原因主要包括癫痫猝死和身体机能减退。而对照组，即注射了他莫昔芬的Cdkl5flox/Y小鼠组和注射了玉米油的Cdkl5fl/Y；CaMK2α-CreER小鼠组，没有被观察到行为异常、同步化放电或者非正常死亡现象。以上结果说明，CDD相关的自发癫痫与神经成熟期CDKL5的功能缺陷息息相关。在此之前，CDD一直被认为单纯是一个神经发育性疾病，而我们的结果证明了CDKL5蛋白在神经成熟期对环路兴奋性的关键性，在神经成熟期缺乏CDKL5蛋白一样会诱发环路的超兴奋，导致癫痫发生。因此，对CDD相关癫痫的研究不应只考虑其神经发育阶段，也应考虑其神经成熟阶段的功能障碍。
实施例4他莫昔芬诱导的Cdkl5fl/Y；CaMK2α-CreER小鼠兴奋性突触传递增强
他莫昔芬诱导的成年期敲除Cdkl5小鼠模型有发作潜伏期短(两周作用)、自发癫痫发作率高(100％发作率)等优势，因此能很好的模拟患者发作模式，有助于研究CDD相关癫痫发生机制。如前所示，前脑敲除Cdkl5会增强海马环路兴奋性传递，但在神经成熟期进行敲除是否仍有类似的超兴奋反应仍不得而知。为了进一步验证这一可能，我们以Cdkl5fl/Y；CaMK2α-CreER小鼠为对象，对其在注射他莫昔芬后一周这一时间点进行了全细胞记录，此时前脑组织的CDKL5已被敲除，但自发癫痫的现象还未被观察到。
我们发现，小鼠的自发兴奋性突触后电流(sEPSC)和微小兴奋性突触后电流(mEPSC)的频率显著高于注射他莫昔芬的野生型小鼠和注射了玉米油的敲除对照型小鼠，但三者的振幅都没有差异(图10，A-F)，表明在神经成熟期对前脑兴奋性神经元进行Cdkl5敲除会增强海马齿状回颗粒细胞兴奋性突触传递。这一结果与从发育期敲除Cdkl5的自发癫痫小鼠模型一致，上述实验结果支持了海马齿状回环路的超兴奋性也许是CDD相关癫痫发生的共同基础。
实施例5他莫昔芬诱导的Cdkl5fl/Y；CaMK2α-CreER小鼠齿状回颗粒细胞形态学正常
树突棘的密度和形态与环路的兴奋性紧密相关，其形态受很多因素的影响，处于不断的调整过程中，神经元树突棘的形态学异常是癫痫研究的重要内容，在不同的动物模型中都可以被观察到前脑多个脑区的树突棘形态学异常，包括大脑皮层、海马CA1区和齿状回。树突棘是兴奋性突触的传递位点，能够接收信息并形成突触联系，被认为是突触可塑性的基础。药物诱导的癫痫发作模型可被观察到海马区树突棘总密度的增加和不同类型树突棘密度的变化。树突棘包括三种形态：瘦长型(thin)、蘑菇型(mushroom)和短粗型(stubby)，其中瘦长型被认为是不成熟状态，短粗型与蘑菇型为成熟型。树突棘密度分析广泛应用于神经科学研究，是评估中枢神经系统突触功能可塑性的重要手段。通过高尔基染色实验(Golgi staining)，利用神经细胞的嗜银特性，我们可以 很好的记录神经元的形态和树突棘的各种类型。虽然树突棘的数量于突触活动的数量不直接等同，但通过树突棘密度和类型分析，可以评估突触连接强度和突触可塑性变化。
为了探究CDD相关的癫痫与海马齿状回树突棘密度和形态之间可能存在的相互关系，我们对成年期敲除Cdkl5小鼠进行了高尔基染色和树突棘密度分析。结果表明，Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组的树突棘密度和Cdkl5fl/Y；CaMK2α-CreER玉米油注射组、Cdkl5fl/Y他莫昔芬注射组都没有差异(图11A，B)。同时，Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组的类型比例与对照组相比也不存在差异(图11A，C)，这一结论表明，在成年期敲除前脑兴奋性神经元Cdkl5不会造成海马齿状回树突棘密度和成熟度的显著变动，从而确认了CDD相关的癫痫与树突棘形态学变化之间没有直接的联系。
实施例6海马BDNF-TrkB信号通路异常激活是导致海马齿状回兴奋性突触传递增强的关键
脑源性神经营养因子(BDNF)及其受体TrkB在中枢神经系统中广泛表达，其信号转导对神经元的生存、形态发生和可塑性至关重要。BDNF不仅介导突触前递质的释放，而且还介导突触后受体的插入、树突和突触的分布等。BDNF-TrkB信号通路被认为与癫痫、抑郁等多种疾病相关，当不同的病理因素促进BDNF上调时，BDNF通过影响突触传递、改变神经元的形态特征，导致海马环路过度兴奋，而这一异常兴奋又转而促进BDNF表达水平提高，形成病理性正反馈。。
这些原因促使我们探究BDNF-TrkB信号通路是否与他莫昔芬诱导的Cdkl5敲除小鼠的环路超兴奋相关。首先，我们检查了Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组和对照组小鼠海马中的BDNF表达水平。通过免疫印迹实验和实时荧光定量PCR(qRT-PCR)分析实验，我们发现Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组海马区BDNF蛋白水平和BDNF mRNA水平显著高于Cdkl5fl/Y；CaMK2αCreER玉米油注射组和Cdkl5fl/Y他莫昔芬注射组(图12A-C)，提示成年期敲除Cdkl5会导致BDNF表达水平升高。我们又对BDNF-TrkB下游通路的磷酸化水平进行免疫印迹实验。我们发现p-TrkB 707位点(Tyr707)相较对照组小鼠显著增加，p-TrkB 816位点(Tyr 816)和p-ERK1/2也有增加的趋势。与此同时，p-TrkB 516位点(Tyr 516)、p-Akt(Ser 473)、p-GSK3β、p-CREB、p-mTOR(Ser 2448)和p-4EBP没有被观察到与对照组的差异(图12D-E)。这些数据证明了他莫昔芬诱导Cdkl5敲除使得海马BDNF/TrkB信号通路异常激活，但这种异常激活是否与海马环路的超兴奋性相关呢？
为了验证这一假说，我们使用了Trk受体抑制剂K252，来验证阻断 BDNF-TrkB信号通路是否能挽救他莫昔芬诱导Cdkl5敲除导致的环路兴奋性突触传递增强。通过全细胞记录实验的方式，我们发现孵育Trk受体抑制剂K252a可以使Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组海马齿状回颗粒细胞的自发兴奋性突触后电流(sEPSC)和微小兴奋性突触后电流(mEPSC)的频率下降到正常水平，而其振幅与Cdkl5fl/Y他莫昔芬注射组和Cdkl5fl/Y；CaMK2αCreER玉米油注射组两个对照组没有差异(图13，A-F)。以上实验结果表明，Trk受体抑制剂K252a在不影响对照组兴奋性突触传递的情况下，可以很好的挽救因Cdkl5敲除带来的sEPSC/mEPSC频率上升，改善被增强的齿状回颗粒细胞的兴奋性突触传递。
鉴于K252a是一种广谱的Trk抑制剂，可能会干扰其他神经营养因子的功能，影响随后的实验设计，我们测试了TrkB的特异性拮抗剂ANA-12。我们观察到，ANA-12与K252a的效果类似，可以引起Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组海马齿状回颗粒细胞的自发兴奋性突触后电流(sEPSC)和微小兴奋性突触后电流(mEPSC)的频率下降，在10μM浓度下可以将Cdkl5敲除引起的sEPSC/mEPSC频率上升挽回到正常频率，而同等浓度不影响对照组的兴奋性突触传递(图14，A-F)。在以上实验中，所有组别的振幅均不受K252a或ANA-12的影响。鉴于我们的取材时间点早于被观察到的癫痫发作时间，我们认为Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组BDNF表达水平的上升及其信号通路的过度激活，是导致海马齿状回兴奋性突触传递增强的关键因素，可能是CDD相关癫痫发生的关键机制。
实施例7抑制BDNF-TrkB信号通路可减少Cdkl5fl/Y；CaMK2α-CreER小鼠的癫痫活动
我们之前的研究发现，成年期Cdkl5敲除导致BDNF-TrkB信号通路异常激活，且这一异常激活介导了海马齿状回兴奋性突触活动的增强，这一系列实验结果为靶向BDNF-TrkB信号通路减少CDD相关的癫痫活动的假说提供了足够的背书。
为了验证这一假说，我们将Cdkl5flox/flox；TrkBflox/+雌鼠与CaMK2α-CreER雄鼠交配，得到Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER小鼠，并在其2月龄连续注射他莫昔芬5天。免疫印迹实验表明，该品系小鼠大脑皮层和海马区的CDKL5蛋白表达水平相比对照组显著下降，与Cdkl5fl/Y；CaMK2α-CreER小鼠相似；而TrkB受体表达水平在大脑皮层和海马区相比对照组下降约50％(图15A，B)。通过录像进行连续监测，我们发现，所有Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组小鼠在注射完成8周后，都被观察到自发癫痫的产生，然而与之形成鲜明对比的是，Cdkl5fl/Y；TrkBflox/+；CaMK2α-CreER他莫昔芬注射组只有一只小鼠出现过自发癫痫现象(图15C，D)，这表明减少TrkB受体的表达可以很好的干预CDD相关的癫痫活动。
与基础研究不同，临床上许多CDD的患者是在癫痫发生后才清楚自己的患病情况。因此，探究在自发癫痫发作后干预BDNF-TrkB信号通路是否能减少CDD相关的癫痫活动对临床的意义更为重要。前人的研究显示，ANA-12的连续注射会导致海马区细胞凋亡，因此我们选择使用单次注射ANA12并 通过TUNEL染色判断海马区细胞的凋亡情况。实验显示，单次注射ANA-12不会导致海马区细胞凋亡(图16D)。我们监测Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组小鼠的癫痫活动，在其癫痫活动频率趋于稳定时，对其腹腔注射
TrkB受体拮抗剂ANA12，通过抑制TrkB受体来干预BDNF-TrkB信号通路的异常激活。我们发现，注射后12小时内Cdkl5fl/Y；CaMK2α-CreER他莫昔芬注射组小鼠的癫痫活动频率相比注射前12小时显著下降，其抗癫痫作用与经典的抗癫痫药丙戊酸相当(图16A-C)，表明在自发癫痫后抑制TrkB受体也可有效减少CDD相关的癫痫活动，提示BDNF-TrkB信号通路可以成为CDD相关癫痫治疗的优秀靶点。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种BDNF-TrkB信号通路抑制剂的用途，其特征在于，用于制备一组合物或制剂，所述组合物或制剂用于预防和/或治疗CDKL5缺乏症相关癫痫。
  2. 如权利要求1所述的用途，其特征在于，所述CDKL5缺乏症相关癫痫具有选自下组的一种或多种表型特征：
    (a)自发癫痫表型；
    (b)海马齿状回兴奋性突触传递增强；
    (c)海马区BDNF-TrkB信号通路异常上调；
    (d)患者癫痫发作随着时间的推移可以发展为不同的类型；
    (e)患者癫痫表型具有抗癫痫药耐药性；
    (f)患者癫痫发作期间的脑电图显示双侧同步低平电位，随后反复出现尖波和棘波，典型的脑电图表现随着时间的推移而发展，在幼儿中不明显。
  3. 如权利要求1所述的用途，其特征在于，所述组合物或制剂还用于选自下组的一种或多种用途：
    (i)减少CDD相关的癫痫活动。
  4. 如权利要求1所述的用途，其特征在于，所述BDNF-TrkB信号通路抑制剂包括TrkB拮抗剂。
  5. 如权利要求1所述用途，其特征在于，所述BDNF-TrkB信号通路抑制剂选自下组：ANA-12、K252a、或其组合。
  6. 一种用于预防和/或治疗CDKL5缺乏症相关癫痫的药物组合物，其特征在于，包括：
    (a1)第一药物组合物，所述第一药物组合物含有(a)第一活性成分，所述第一活性成分为BDNF-TrkB信号通路抑制剂；和
    (a2)任选的第二药物组合物，所述第二药物组合物含有(b)第二活性成分，所述第二活性成分为其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物；
    (b)药学上可接受的载体。
  7. 如权利要求6所述的药物组合物，其特征在于，所述BDNF-TrkB信号通路抑制剂包括TrkB拮抗剂。
  8. 如权利要求6所述的药物组合物，其特征在于，所述BDNF-TrkB信号通路抑制剂选自下组：ANA-12、K252a、或其组合。
  9. 一种药盒，其特征在于，包括：
    (a1)第一容器，以及位于所述第一容器中的BDNF-TrkB信号通路抑制剂，或含有BDNF-TrkB信号通路抑制剂的药物；
    (a2)任选的，第二容器，以及位于所述第二容器中的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，或含其他可预防和/或治疗CDKL5缺乏症相关癫痫的药物的药物。
  10. 一种组合的用途，其特征在于，所述组合包括BDNF-TrkB信号通路抑制剂和任选的其他预防和/或治疗CDKL5缺乏症相关癫痫的药物，用于制备一药物组合物或药盒，所述药物组合物或药盒用于预防和/或治疗CDKL5缺乏症相关癫痫。
PCT/CN2024/097792 2023-06-20 2024-06-06 BDNF-TrkB信号通路在预防和/或治疗CDKL5缺乏症相关癫痫中的应用 Pending WO2024260261A1 (zh)

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US10525024B2 (en) * 2014-08-15 2020-01-07 The Johns Hopkins University Methods for rescuing phenobarbital-resistance of seizures by ANA-12 or ANA-12 in combination with CLP290

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10525024B2 (en) * 2014-08-15 2020-01-07 The Johns Hopkins University Methods for rescuing phenobarbital-resistance of seizures by ANA-12 or ANA-12 in combination with CLP290

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LIU, HUI ET AL.: "The Brain Derived Neurotrophic Factor and Epilepsy", LIN CHUANG YU BING LI ZA ZHI = FOREIGN MEDICAL SCIENCES (PATHOPHYSIOLOGY AND CLINICAL MEDICINE), CN, vol. 22, no. 6, 31 December 2002 (2002-12-31), CN , pages 618 - 620, XP009559945, ISSN: 1001-1773 *
MIAO, LU: "k252a Inhibits BDNF-TrkB Signaling Pathway to Effect the Expression of Arc in Electrical Stimulation Kindled Rats and Epilepsy", WANFANG MASTER'S THESES, 28 November 2012 (2012-11-28), XP093251821 *

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