WO2022033529A1 - Use of sema3d antagonist in preventing or treating neurodegenerative diseases and prolonging lifespan - Google Patents

Abstract

The present invention relates to a use of a composition in the preparation of a drug for preventing or treating neurodegenerative diseases, the composition comprising a therapeutically effective amount of a Sema3D antagonist. The present invention also relates to a use of a composition in the preparation of a drug for prolonging lifespan or promoting nerve regeneration, the composition comprising a therapeutically effective amount of a Sema3D antagonist.

Description

Use of SEMA3D antagonists in preventing or treating neurodegenerative diseases and prolonging lifespan technical field

The present invention relates to the use of a composition in the preparation of a medicament for preventing or treating neurodegeneration and prolonging lifespan, wherein the composition comprises a Sema3D antagonist.

Background technique

Aging affects the metabolism, function, and survival of neurons, which can lead to cognitive decline and neurodegenerative diseases. The hippocampus is critical for learning and memory consolidation; unfortunately, this brain region is one of the most vulnerable during aging. Neurodegeneration in the hippocampus has been reported to play a role in cognitive deficit and brain atrophy, implying that hippocampal neurons play a role in aging and age-associated cognitive decline ) importance. In addition, there is growing evidence that neurodegeneration shortens lifespan.

Autopsy and molecular studies have shown that abnormal protein metabolism, dendritic spine reduction and neuronal loss are characteristic features of neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal Frontotemporal lobar degeneration (FTLD). In addition, reduced neural regeneration resulting from the loss of neural stem cells (NSCs) may exacerbate functional decline in the aging brain. Given the growing size of the aging population and the complexities of neurodegeneration, there is a constant search for key factors that control the aging process in the brain.

Previous reports indicated that microRNA-195 (miR-195) could exert neuroprotective effects and improve functional recovery in acute stroke rats. Other studies have also demonstrated that miR-195 downregulates amyloid-beta (amyloid-beta), a core component of Alzheimer's plaques, and that miR-195 alleviates hypoperfusion-induced dementia. Therefore, it is reasonable to speculate that miR-195 may play a role in regulating neuronal function in the aging brain. Semaphorins 3 (Class III Semaphorins, Sema3) A-G have been previously reported as axon guidance molecules. Both semaphorin 3A (semaphorin 3A, Sema3A) and semaphorin 3D (semaphorin 3D, Sema3D) have been shown to stimulate peripheral branching of axons from the cell body (soma). Sema3 is a secreted protein that mainly binds to class A plexin receptors (PlexinA1–PlexinA4). Recent studies have shown that Sema3 family members are also involved in other functions related to the pathogenesis of various diseases, such as neurodegenerative diseases and diabetic retinopathy. Furthermore, recent studies have shown that Sema3A is a direct target of miR-195 and that Sema3A is involved in neuronal damage caused by acute stroke.

SUMMARY OF THE INVENTION

The present invention provides a method of preventing or treating a neurodegenerative disease in an individual comprising administering to the individual suffering from the neurodegenerative disease a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

The method according to the present invention is characterized by providing a method for prolonging lifespan in an individual, comprising administering to said individual in need of lifespan extension a medicament comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist combination.

Another feature of the method according to the present invention is that there is also provided a method for promoting nerve regeneration in an individual, comprising administering to the individual in need of nerve regeneration a therapeutically effective amount of semaphorin 3D (Sema3D) Antagonist pharmaceutical compositions.

In addition, according to the method of the present invention, another feature is that there is also provided a method for preventing or treating retinal neurodegenerative diseases in an individual, comprising administering to the individual suffering from retinal neurodegenerative diseases a method comprising a therapeutically effective A pharmaceutical composition for an amount of a semaphorin 3D (Sema3D) antagonist.

As used herein, the term "subject" refers to an animal, especially a mammal. In a preferred embodiment, the term "individual" refers to a human being.

Unless stated otherwise, "a" or "an" means one or more.

The present invention provides a method of preventing or treating a neurodegenerative disease in an individual comprising administering to the individual suffering from the neurodegenerative disease a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist .

The present invention also provides the use of a composition in the preparation of a medicament for preventing or treating neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

As used herein, the term "prevention" refers to preventing the onset, recurrence, or spread of a disease or disorder or one or more symptoms thereof. In certain embodiments, this term refers to the use of the drugs provided herein to treat a particular disorder described herein, with or without one or more other additional active agents, prior to the onset of symptoms or an individual at risk of a disorder, or to which a drug provided herein is administered.

As used herein, the term "treating" means alleviating symptoms or complications; delaying progression of a disease, disorder or condition; alleviating or alleviating symptoms and complications; and/or curing or eliminating a disease, disorder or condition.

In a specific embodiment, the individual suffers from a neurodegenerative disease of the central nervous system. The term "neurodegenerative diseases" as used herein is also used to describe an acute, progressive or chronic disease caused by damage to the central nervous system, which damage can be caused by the Sema3D antagonist treatment to reduce and/or alleviate. In a specific embodiment, the neurodegenerative disease comprises Alzheimer's disease (AD), Parkinson's disease (Parkinson's disease), frontotemporal dementia (FTLD), frontotemporal dementia with ubiquitin inclusion bodies ( frontotemporal lobar degeneration with ubiquitinated inclusions, FTLD-U), age-associated cognitive decline (age-associated cognitive decline), vascular dementia (Vascular dementia), cortico-basal ganglionic degeneration (CBD), progressive Progressive supranuclear palsy (PSP), Lewy body dementia (Lewy body dementia), Huntington's chorea, Alzheimer's disease, Pick's disease (PiD), argyrophilic grain dementia), Guam parkinsonism-dementia complex (Guam parkinsonism-dementia complex), Lytico-Bodig disease (Lytico-Bodig disease), Amyotrophic lateral sclerosis (ALS), spinocerebellar atrophy , Spinal and bulbar muscular atrophy (SBMA), Motor Neuron Disease (Motor Neuron Disease), Multiple Sclerosis (Multiple Sclerosis) or Traumatic Brain Injur (TBI). In a preferred embodiment, the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, frontotemporal dementia, frontotemporal dementia with ubiquitin inclusion bodies, or age-related cognitive decline.

In another specific embodiment, the individual is an elderly individual.

According to the method of the present invention, wherein the drug or pharmaceutical composition comprising the Sema3D antagonist can prevent/inhibit neurodegeneration by increasing the dendritic spine of the hippocampus and promoting nerve regeneration. Therefore, the Sema3D antagonist can be used to prevent or treat neurodegeneration. In a specific embodiment, the Sema3D antagonist prevents or treats the neurodegenerative disease by increasing dendritic spines in the hippocampus and promoting nerve regeneration.

In another specific embodiment, the Sema3D antagonist increases dendritic spine density in CA1 pyramidal neurons in the hippocampus. In a preferred embodiment, the Sema3D antagonist prevents or treats neurodegeneration by increasing the density of dendritic spines in CA1 pyramidal neurons in the hippocampus. In a more preferred embodiment, the Sema3D antagonist prevents or treats the neurodegenerative disease by increasing the density of dendritic spines of CA1 pyramidal neurons in the hippocampus.

According to the method of the present invention, the effect of the Sema3D antagonist in promoting nerve regeneration is achieved by increasing neural stem cells, up-regulating autophagy and promoting neuron proliferation. In a specific embodiment, the promoting nerve regeneration comprises increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation. With regard to increasing neural stem cells, the Sema3D antagonist can increase neural stem cells in the hippocampal dentate gyrus and subventricular zone (SVZ) to promote nerve regeneration. In another specific embodiment, the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote nerve regeneration to prevent or treat neurodegeneration. In a preferred embodiment, the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote nerve regeneration to prevent or treat the neurodegenerative disease.

As used herein, an "antagonist" includes, but is not limited to, a molecule that disrupts, prevents, inhibits, reduces or neutralizes a target activity or expression. "Sema3D antagonist" refers to any molecule that blocks, inhibits or reduces (including significantly affects) the biological activity or expression of Sema3D, the biological activity of which includes Sema3D downstream signaling pathways. The term "antagonism" does not imply a specific biological mechanism of activity, and its expression encompasses all possible direct or indirect pharmacological, physiological and biochemical effects on Sema3D. In a specific embodiment, the Sema3D antagonist comprises an aganist Sema3D conjugate, wherein the conjugate comprises a compound, polypeptide, antibody, antibody fragment or oligonucleotide. Examples of the Sema3D antagonists include anti-Sema3D antibodies or fragments thereof, anti-sense molecules corresponding to Sema3D (including antisense molecules encoded by nucleic acids corresponding to Sema3D), and small interfering RNAs (small interfering RNAs) corresponding to Sema3D. siRNA), short hairpin RNA (shRNA) corresponding to Sema3D, microRNA (microRNA, miRNA) corresponding to Sema3D, nucleic acid aptamer (aptamer) corresponding to Sema3D, and Sema3D inhibitory compounds, but the Sema3D of the present invention Antagonists are not limited to this. In a preferred embodiment, the Sema3D antagonist comprises an anti-Sema3D antibody, an anti-Sema3D antibody fragment, an antisense nucleic acid that inhibits the expression of Sema3D, an siRNA that inhibits the expression of Sema3D, an shRNA that inhibits the expression of Sema3D, and an antisense nucleic acid that inhibits the expression of Sema3D. miRNA or nucleic acid aptamer that inhibits Sema3D expression.

In a specific embodiment, the compound comprises a molecular compound. In a preferred embodiment, the molecular compounds include small molecular compounds and macromolecular compounds.

In another specific embodiment, the antibody or antibody fragment comprises a polyclonal antibody, monoclonal antibody, humanized antibody, diabody, antibody fragment Fab, Fv, F(ab') ₂ , single chain Fv (scFv ), Fv fragments or peptoids of antibodies.

In the present invention, the oligonucleotide comprises an antisense strand produced by the principle of nucleic acid complementation. In a specific embodiment, the oligonucleotide comprises a nucleotide sequence with sufficient complementarity to the Sema3D gene to directly bind Sema3D RNA to interfere with Sema3D RNA function. In a preferred embodiment, the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer. In a more preferred embodiment, the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. In another specific embodiment, the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In another specific embodiment, the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or mimics of miRNA-195. However, in some cases, the Sema3D antagonists described in the present invention do not contain molecules such as miRNA-195, modified miRNA-195 or mimetics of miRNA-195 to prevent or treat neurodegenerative diseases.

The present invention provides a method of extending lifespan in an individual comprising administering to said individual in need of lifespan extension a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

The present invention also provides the use of a composition in the manufacture of a life-extending medicament, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

According to the methods of the present invention, the Sema3D antagonist extends lifespan by delaying aging. In a preferred embodiment, the Sema3D antagonist has the effect of delaying aging. In a preferred embodiment, the Sema3D antagonist extends lifespan by delaying aging. In a more preferred embodiment, the Sema3D antagonist is used to delay aging and prolong lifespan.

According to the method of the present invention, the Sema3D antagonist delays aging by preventing/inhibiting neurodegeneration, thereby prolonging lifespan. In a specific embodiment, the Sema3D antagonist delays aging by preventing/inhibiting neurodegeneration, thereby extending lifespan. In a preferred embodiment, the Sema3D antagonist prevents/inhibits neurodegeneration by increasing dendritic spines in the hippocampus and promoting nerve regeneration.

In another specific embodiment, the Sema3D antagonist inhibits the expression of aging-related biomarkers. In a preferred embodiment, the aging-related biomarkers comprise senescence-associated β-galactosidase (senescence-associated β-galactosidase, SAβ-gal), p16 ^Ink4a and p19 ^Arf .

According to the method of the present invention, the Sema3D antagonist can delay brain aging to prolong lifespan. In a preferred embodiment, the Sema3D antagonist delays brain aging to prolong lifespan by promoting neurogenesis. In a preferred embodiment, the Sema3D antagonist increases neural stem cells in the dentate gyrus and subventricular zone of the hippocampus to promote neurogenesis. Therefore, the Sema3D antagonist has the effect of promoting neurogenesis, thereby prolonging lifespan.

In a specific embodiment, the individual is an elderly individual.

In another specific embodiment, the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide. In a preferred embodiment, the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer. In a more preferred embodiment, the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. In another specific embodiment, the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In a specific embodiment, the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or a mimetic of miRNA-195.

The present invention provides a method of promoting nerve regeneration in an individual comprising administering to said individual in need of nerve regeneration a pharmaceutical composition comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

The present invention also provides the use of a composition in the manufacture of a medicament for promoting nerve regeneration, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

In a specific embodiment, the Sema3D antagonist promotes nerve regeneration by increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation.

According to the method of the present invention, the Sema3D antagonist can upregulate the PI3K/AKT/mTOR pathway to upregulate autophagy to promote nerve regeneration to inhibit neurodegeneration. In a specific embodiment, the up-regulation of autophagy comprises up-regulation of the PI3K/Akt/mTOR signaling pathway.

In a specific embodiment, the individual is an elderly individual.

In another specific embodiment, the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide. In a preferred embodiment, the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer. In a more preferred embodiment, the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. In another specific embodiment, the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In a specific embodiment, the Sema3D antagonist comprises microRNA-195 (miRNA-195), modified miRNA-195 or a mimetic of miRNA-195. However, in some cases, the Sema3D antagonists described in the present invention do not comprise molecules such as miRNA-195, modified miRNA-195 or mimetics of miRNA-195 to promote nerve regeneration.

The present invention provides a method of preventing or treating retinal neurodegenerative disease in an individual comprising administering to said individual suffering from retinal neurodegenerative disease a medicament comprising a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist combination.

The present invention also provides use of a composition in the preparation of a medicament for preventing or treating retinal neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist.

In a specific embodiment, the retinal neurodegenerative disease comprises diabetic retinopathy (diabetic retinopathy), age-related macular degeneration (age-related macular degeneration), optic neuritis, myopia-induced retinopathy or Glaucoma-associated retinal disorders. In a preferred embodiment, the retinal neurodegenerative disease comprises diabetic retinopathy.

In a specific embodiment, the individual is an elderly individual.

In another specific embodiment, the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a molecular compound, polypeptide, antibody, antibody fragment or oligonucleotide. In a preferred embodiment, the oligonucleotide comprises antisense DNA, antisense RNA, siRNA, shRNA, miRNA or nucleic acid aptamer. In a more preferred embodiment, the sequence of the siRNA comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7. In another specific embodiment, the Sema3D antagonist comprises SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7.

In a specific embodiment, the medicament or the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier, adjuvant or excipient. In a preferred embodiment, the medicament or the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is determined by a particular combination of administration and a particular method of administration of the composition. The term "carrier" as used herein includes, but is not limited to, any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents such as osmotic and absorption delaying agents, buffers, carrier solutions, suspensions or colloid, etc. Such media and agents for pharmaceutically active substances are well known in the art. Unless any conventional medium or agent is incompatible with the active ingredient, its therapeutic combination needs to be considered. Supplementary active ingredients can also be incorporated into the compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a subject. The preparation of aqueous compositions with proteins as active substances is common knowledge in the art. Typically, such compositions are prepared as liquid solutions, troches, capsules, or injections as suspensions; solid forms that can be dissolved or suspended for injections can also be prepared.

In addition, the aforementioned pharmaceutically acceptable salts include, but are not limited to: inorganic cation salts, for example, alkali metal salts such as sodium, potassium or amine salts; alkaline earth metal salts such as magnesium or calcium salts; and salts containing divalent or tetravalent cations, such as zinc, aluminium or zirconium salts. In addition, organic salts such as dicyclohexylamine salt, methyl-D-glucamine, and amino acid salts such as arginine, lysine, histidine or glutamic acid amide.

Therefore, the medicine or pharmaceutical composition of the present invention can be prepared into a dosage form suitable for the present invention by using the techniques well known to those skilled in the art to prepare the above-mentioned Sema3D antagonist and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers thus include, but are not limited to, liposomes, water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and white petrolatum (white petrolatum), waxes (such as paraffin and yellow wax), preserving agents, antioxidants, solvents, emulsifiers, suspending agents (suspending agent), decomposer (decomposer), binder (binding agent), excipient (excipient), stabilizer (stabilizing agent), chelating agent (chelating agent), diluent (diluent), gelling agent ( gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants irritants), colorants, propellants, surfactants, or other similar or suitable carriers for use in the present invention.

In addition, the oligonucleotides or microRNA-195 (miRNA-195) of the present invention can be administered in combination with the following pharmaceutically acceptable carriers, including but not limited to: liposomes, micelles , metal particles, polymer particles, solvents, dispersion media, coatings, antibacterial and antifungal agents or isotonic and absorption delaying agents, etc. Compatible with pharmaceutical administration. For different modes of administration, pharmaceutical compositions can be formulated into different dosage forms using general methods. In one embodiment, the above-mentioned pharmaceutically acceptable carrier may be liposomes, micelles, metal particles or polymer particles. The particles described above can be prepared from various materials such as lipids, proteins, polysaccharides and synthetic polymers. Depending on the preparation method, nanoparticles, nanospheres, nanocapsules and the like can be obtained.

As used herein, the term "administering" refers to providing a drug or pharmaceutical composition to an individual. In another specific embodiment, the medicament or pharmaceutical composition comprising the Sema3D antagonist can be administered to the individual by any suitable route of enteral or parenteral administration. Suitable routes of enteral administration in the present invention include, for example, oral, rectal, or intranasal administration. Suitable routes of parenteral administration include, for example, intravascular administration (eg, intravenous bolus injection, intravenous infusion, arterial bolus, arterial infusion, and catheter instillation into blood vessels); peri- and intra-tissue injection (eg, intramuscular). injection, peritumoral and intratumoral injection, intravitreal injection or subretinal injection); subcutaneous injection or deposition, including subcutaneous infusion (eg, by osmotic pump); direct administration to the tissue of interest, such as by catheter or other placement Devices (eg, retinal pellets or suppositories or implants containing porous, non-porous, or gel-like substances); and inhalation. More preferred routes of administration are injection, infusion and direct injection to the target.

One of skill in the art can determine the amount of Sema3D to administer to a given individual by considering the following factors: the individual's size and weight; the severity of the disease; the individual's age, health, and gender; the route of administration; effective amount of antagonist. One of skill in the art can also readily determine the appropriate dosage regimen in administering the isolated said Sema3D antagonist to a given individual. For example, the Sema3D antagonist can be administered to the individual once or twice. When a dosage regimen comprises multiple administrations, it will be appreciated that the effective amount of the Sema3D antagonist administered to the individual may comprise the total amount of drug administered during the entire dosage regimen.

According to the present invention, the Sema3D antagonist in the form of an oligonucleotide can be delivered to the individual using a recombinant vector. In a specific embodiment, the recombinant vector comprises plastid DNA or a viral vector. In a preferred embodiment, the viral vector comprises an adenoviral vector, a lentiviral vector, an adeno-associated virus (AAV) vector, a retroviral vector, a poliovirus vector, a herpes simplex virus (HSV) vector or a A vector for the murine Maloney virus. Through the above method, the Sema3D antagonist can inhibit the expression of Sema3D in vivo to achieve the above effect.

In conclusion, the present invention confirms that the expression level of Sema3D is related to neurodegeneration and regeneration of the brain, reduction of life span and retinal degenerative diseases. Therefore, by inhibiting Sema3D and its related signaling pathways, symptoms such as neurodegeneration, shortened lifespan, retinal degenerative diseases, etc. in the brain can be improved, and nerve regeneration can be promoted.

Description of drawings

Figure 1 shows the original mice with two miR-195 genes, one of which was knocked out (Knockout, KO) called miR-195a KO mice. Figure 1A and Figure 1B show that miR-195a KO mice have lower amounts of miR-195 in the central nervous system and other organs compared to age-matched wild type (WT) mice, the number of per organ is 3. Figure 1C shows an age-dependent decrease in the amount of miR-195 in the total brain of wild-type (WT) mice (3/group). *p<0.05; **p<0.01 compared to 4-month-old mice.

Figure 2A shows the scheme of learning trials and memory trials. Figure 2B shows that longer escape latency to the hidden platform indicates lower learning ability. Figure 2C shows that longer escape latencies to reach the platform indicate poorer memory. Two-way analysis of variance (Two-way ANOVA) was used to assess overall differences in multiple days between knockout (KO) and wild-type (WT) mice. Figure 2D shows memory trials (the frequency in the platform quadrant) of miR-195a KO and WT mice.

Figure 3A shows the Y-maze test to assess working memory by measuring the time to reach the arm of a new toy; quantitative data are shown in the right panel. Figure 3B shows that locomotor function was measured by an open field test. Representative images (left panel) show the walking trajectories of WT and miR-195a KO mice in the open field; quantitative data are shown in the right panel. 3M: 3-5 month old mice; 6M: 6-9 month old mice; 12M: 10-12 month old mice; 18M: 15-18 month old mice; and 24M : 21-24 month old mice.

Figure 4A shows the analysis of the lifespan of miR-195a KO mice (number of 28). Historical data of WT mice served as a reference. Results for mean lifespan, median lifespan and percent reduction are shown in Table 3. Figure 4B shows staining of senescence-associated β-galactosidase (SAβ-gal) in the hippocampus. Representative images in the left panel show hippocampal senescent cells (green). Scale bar = 100 μm; quantitative data are shown in the lower panel, using 4-month-old WT mice as a reference group (number of 3 per group). Figure 4C shows the use of X-gal-based staining to measure senescence-associated beta-galactosidase (SA[beta]-gal) activity. The intensity of the green signal represents the activity of the SAβ-gal enzyme. Representative images show an age-dependent increase in SAβ-gal activity in the cortex and hippocampus of WT mice. Green signal intensity was similar between 4-month-old miR-195a KO mice and 12-month-old WT mice. Scale bar: 200 μm. Figure 4D shows p16 ^Ink4a (left panel) and p19 ^Arf (right panel) expression in the whole brain of 4-month-old miR-195a KO mice and age-matched WT mice (number 3/group) quantitative PCR analysis. Data are presented as mean ± standard error of the mean (SEM). *p<0.05;**p<0.01. 4M WT: 4 month old WT mouse; 12M WT: 12 month old WT mouse; 24M WT: 24 month old WT mouse; and 4M miR -195a KO: 4-month-old miR-195a KO mice.

Figure 5A shows that miR-195a KO mice have fewer neural stem cells (NSCs) populations compared to age-matched WT mice. Representative images show SOX2 ⁺ (red) neural stem cells in the hippocampal dentate gyrus (DG) of WT mice (top 3 panels on the left) and miR-195a KO mice (far right panels). The quantification of SOX2 ⁺ neural stem cells in the DG is shown on the right, using 4-month-old WT mice as a reference group (number of 3/group). Magnification: 20X. Scale bar = 200 μm. Figure 5B shows reduced dendritic spine density in miR-195a KO mice. Representative apical dendritic shafts of CA1 pyramidal neurons from 4-month-old miR-195a KO mice and age-matched WT mice (number 3 per group) image. Scale bar = 5 μm. Quantification of dendritic spine density from three independent experiments. *p<0.05, **p<0.01, ***p<0.001 compared to age-matched WT mice. Figure 5C shows representative images of SOX2 ⁺ (red) neural stem cells in the subventricular zone (SVZ) of WT mice (top 3 panels on the left) and miR-195a KO mice (far right panels) . Quantification of SOX2 ⁺ neural stem cells in the SVZ is shown on the right. Magnification: 20X. Scale bar = 200 μm. The quantity is 3/group. Data are presented as mean ± SEM. *p<0.05;**p<0.01 compared to 4 month old WT mice. Data are presented as mean±SEM from three independent experiments, *p<0.05 and **p<0.01. 4M WT: 4-month-old WT mice; 12M WT: 12-month-old WT mice; 21M WT: 21 month old WT mice; and 4M miR-195a KO: 4 month old miR-195a KO mice.

Figure 6A shows reporter plasmids carrying wild-type or mutant Sema3D 3'-UTRs were transfected into HEK293 cells. In cells transfected with a plasmid carrying the wild-type Sema3D 3'-UTR, miR-195 dose-dependently reduced luciferase activity. Figure 6B shows western blot and quantitative data (3/group) of Sema3A and Sema3D protein levels in the hippocampus of 4-month-old WT and miR-195a KO mice. Sema3A in the hippocampus of WT mice served as a reference group. Figure 6C shows the mRNA expression of Sema3A and Sema3D in the hippocampus of 4-month-old miR-195a KO mice and age-matched WT mice (number 3/group). WT mice served as the reference group. Figure 6D shows the mRNA expression of Sema3D and Sema3A in the human hippocampus according to RNA sequencing data from the Allen Brain Atlas (number 95). Data are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. NC: negative control group.

Figure 7A shows immunohistochemistry (IHC) staining of Sema3D protein in brain. According to the degree of expression of Sema3D, it showed an age-dependent increase in whole brain and hippocampus. Top panel: slice of whole brain; bottom panel: hippocampus. Figure 7B shows a flowchart for selection and analysis of neurodegenerative disease transcriptomes. Computational analyses consist of four main steps shown in oval boxes. 4M WT: 4 month old WT mouse; 13M WT: 13 month old WT mouse; and 21M WT: 21 month old WT mouse.

Figure 8A shows at day 7, Western blot data showing that after bilateral injection of a Sema3D-expressing lentivirus (Lv. Sema3D) into the hippocampus of 4-month-old WT mice, the Sema3D protein was present at higher levels, but not in the cortex. Lentivirus for control (Lv.Ctrl) was injected as a control group (the number was 3/group). Quantitative data from western blotting are shown in the right panel. Figure 8B shows the hippocampus from the same mouse shown in Figure 8A, stained with Golgi-cox stain. Dendritic spine density in the hippocampus was measured using Golgi-Cox staining. Representative images show apical dendritic axons of hippocampal CA1 pyramidal neurons. Scale bar = 5 μm. Quantification of dendritic spine density is shown in the right panel. Data are presented as mean ± standard error of the mean (SEM). *p<0.05. Figure 8C shows administration of Lv.Sema3D or Lv.Ctrl to WT mice; learning and memory tests were performed according to the protocol. Figures 8D to 8F show control and Sema3D overexpressing 4-month-old mice (4/group) tested for learning and spatial memory performance by Morris Water Maze test (MWM). Figure 8D shows a learning assay showing that mice overexpressing Sema3D took longer to reach the hidden platform, suggesting lower learning ability. Figures 8E and 8F show escape latency and frequency, respectively, as measured in memory trials. Mice overexpressing Sema3D had longer avoidance latencies and lower frequencies in the target quadrant. Figure 8G shows the effect of Sema3D on short-term memory assessed by the novel object recognition test (number of 4/group). Quantitative data are shown in the right panel. Data in Figures 8B to 8E are presented as mean ± SEM. *p<0.05 compared to Lv.Ctrl mice on the same experimental day. Two-way ANOVA was used to assess overall differences in multiple days between Sema3D-overexpressing and control mice. **p<0.01 and ***p<0.001. Figure 8H shows the memory performance of mice injected with Lv.Ctrl and Lv.Sema3D by Y-maze test (3/group). Quantitative data showed that mice overexpressing Sema3D took more time to reach the arm with the new toy, implying memory impairment (p=0.071). Data are presented as mean ± SEM. Figure 8I Measurement of Locomotor activity of mice injected with Lv.Ctrl and Lv.Sema3D by open field experiment (distance of Lv.Ctrl=116.4±4.01 cm, distance of Lv.Sema3D=100.1±11.35 cm; p= 0.248; the quantity is 3/group). Pre: Preprocessing.

Figure 9A shows representative images of immunofluorescence staining in the dentate gyrus (DG) of the hippocampus showing the efficiency of Sema3D silencing (knockdown). siRNA-Sema3D (si-Sema3D) or siRNA-Ctrl (si-Ctrl) were injected into the bilateral hippocampus of 12-month-old miR-195a KO mice. Immunofluorescence staining was performed on the 7th day after siRNA administration (the number was 3/group). The right panel shows quantitative data. Figures 9B to 9D show spatial working memory and motor function of miR-195a KO mice (12 months old) (number 7/group) receiving siRNA-Ctrl or siRNA-Sema3 injections measured by the Y-maze test. Figure 9B shows the protocol showing details of the date of administration of siRNA to miR-195a KO mice, testing and brain sample collection. Figure 9C shows that mice were allowed to freely explore the Y-maze for 15 minutes. A higher percentage of alternations indicates better spatial working memory. Figure 9D shows the use of total walking distance to determine motor function, with longer distance indicating better motor function. Testing of siRNA-Sema3D and siRNA-Ctrl mice was performed on the same experimental day. Two-way analysis of variance was used to assess overall differences in multi-day numbers between siRNA-Sema3D and siRNA-Ctrl mice. *p<0.05 compared to siRNA-Ctrl treated mice. **p<0.01 and ***p<0.001. Figure 9E shows representative images of dendritic spine density of CA1 pyramidal neurons in siRNA-Sema3D injected mice (number of 3 per group). siRNA-Sema3D or siRNA-Ctrl were injected into the bilateral hippocampus of 12-month-old miR-195a KO mice and sacrificed on day 15 after injection. Scale bar = 5 μm. The right panel shows quantification of dendritic spine density.

Figure 10A shows survival curves of Sema2A-overexpressing flies and control flies (300 per group). Drosophila Sema2A is the homologous gene of human and mouse Sema3D. The Gehan-Breslow-Wilcoxon test was used to compare the lifespan of Drosophila. Average lifespan, median lifespan, longest survival time and percentage of reduced lifespan are shown in Table 6. Figure 10B shows that adult Sema2A-overexpressing flies showed age-related decline in locomotion by negative geotaxis assay at 2, 3 and 4 weeks post eclosion (p=0.020, Mann-Whitter). Mann-Whitney test). Figure 1OC shows representative images of human neural stem cells forming neurospheres 48 hours after Sema3D treatment. Scale bar = 200 μm. Quantitative data are presented as mean ± SEM from three independent experiments. **p<0.01 and ***p<0.001. Figure 10D shows that Sema3D reduces neural stem cell populations in a dose-dependent manner. Representative images of SOX2 ⁺ (red) neural stem cells in the dentate gyrus (DG) of the hippocampus of mice receiving Sema3D injections (upper panel). Bottom panel shows the quantification of SOX2 ⁺ neural stem cells in DG. Magnification: 20X. Scale bar = 200 μm; number is 3/group. Quantitative data are presented as mean ± SEM from three independent experiments. *p<0.05 and **p<0.01 compared to data for Sham WT mice. Figure 10E shows the measurement of neural stem cell populations in 4-month-old WT mice 72 hours after intracerebroventricular (ICV) injection of Sema3D. Representative images of SOX2 ⁺ (red) neural stem cells in the subventricular zone (SVZ) of Sema3D-injected mice (top). Bottom panel shows the quantification of SOX2 ⁺ neural stem cells in the SVZ. Magnification: 20X. Scale bar = 200 μm. The quantity is 3/group. Data are presented as mean ± SEM. *p<0.05;**p<0.01 compared to sham group data. Naive: mice in the blank group.

Figure 11A shows injection of Lv.Sema3D or Lv.Ctrl into the bilateral hippocampus of 4-month-old WT mice. On day 14, Sema3D and autophagy-associated proteins in the hippocampus were measured by western blotting (number 3/group). Right panel shows quantitative western blot data. Figure 11B shows Western blotting of autophagy-related proteins p62, Beclin-1 and LC3-II/I in human neurons 72 hours after Sema3D treatment. Right panel shows quantitative western blot data. Figure 11C shows Western blot of the effect of Sema3D on phosphorylation of the PI3K/Akt/mTOR signaling pathway 24 hours after Sema3D treatment. Right panel shows quantitative western blot data. All quantitative data are presented as mean±SEM from three independent experiments, *p<0.05 and **p<0.01. Figure 1 ID shows that Rapamycin reverses the inhibitory effect of Sema3D on cell proliferation. Human neuronal cells (SY5Y) were treated with Sema3D and rapamycin simultaneously for 72 hours. Proliferation of neuronal cells was determined using Ki67 staining. Representative fluorescence images of Ki67 and DAPI staining. Scale bar = 50 μm. Quantitative data are presented as mean ± SEM from three independent experiments. *p<0.05 and **p<0.01 based on SY5Y cell data without Sema3D and rapamycin treatment. Figure 11E shows whether Lv.Sema3D was injected with rapamycin or Lv.Ctrl alone into the bilateral hippocampus of 4-month-old WT mice, and dendritic spine density was measured on day 14. Representative images of apical dendritic shafts of CA1 pyramidal neurons (number 3/group). Scale bar = 5 μm. Bottom panel shows quantification of dendritic spine density. **p<0.01.

Figure 12 shows that Sema3D is highly expressed in the eyes of diabetic (Diabetes Mellitus, DM) rats and mice. Figure 12A shows that Sema3D mRNA expression is increased 4 weeks after the onset of diabetes, and quantitative data show that Sema3D mRNA is expressed to a higher degree than Sema3A mRNA. *p<0.05 compared to the level of Sema3A of the rats in the control group; ##p<0.01 compared with the level of Sema3D of the rats of the control group, the number is 2/group. Data are presented as mean ± SEM from three independent experiments. Figure 12B shows representative IHC images showing that Sema3D protein in the retina increases with time to diabetes onset (brown; number 3/group).

detailed description

The following examples are non-limiting and represent only various aspects and features of the present invention.

Materials and Methods

reagent

MiR-195 and negative control microRNA (NC-miR) were purchased from Ambion Inc. (Austin, TX, USA). The sequence information is:

miR-195 mimic: 5'-UAGCAGCACAAGAAAUAUUGGC-3' (SEQ ID NO: 1);

anti-miR-195: 5'-GCCAATATTTCTGTGCTGCTA-3' (SEQ ID NO: 2); and

Negative control sequence: 5'-AGUACUGCUUACGAUACGG-3' (SEQ ID NO: 3).

Green PCR Master Mix、

Reverse Transcriptase Kit是从Applied Biosystems(Foster City,CA,USA)所购买。雷帕霉素(Rapamycin)是一种化学mTOR抑制剂，购自Sigma Aldrich(St.Louis,MO,USA)。重组小鼠Sema3D和人类Sema3D蛋白购自R&D Systems(Minneapolis,MN,USA)。除非另有说明，所有其他试剂均为分析等级(analytical grade)。初级抗体：抗SOX2(ab97959,Abcam；Cambridge,MA,USA)、抗Sema3D(ab180147,Abcam)、抗GAPDH(5174,Cell Signaling；Beverly,MA,USA)、抗Ki67(ab16667,Abcam)、抗Beclin-1(ab207612,Abcam)、抗LC3(ab48394,Abcam)、抗p62/SQSTM1(ab56416,Abcam)、抗PI3K(E-AB-32575,Elabscience；Houston,Texas,USA)、抗phospho-PI3K(E-AB-20966,Elabscience)、抗Akt(9272S,Cell Signaling)、 抗phospho-Akt(9271S,Cell Signaling)、抗mTOR(E-AB-32128,Elabscience)和抗phospho-mTOR(E-AB-20929，Elabscience)用于蛋白质印迹法(western blot)、免疫组织化学染色和免疫荧光实验。靶向人类Sema 3D的siRNA和非靶向(控制)的RNA的寡核苷酸池(oligonucleotide pools)是从Dharmacon(Lafayette,CO,USA)所购买。siRNA-Sema3D的序列为5'-CUGUGAUGUAUAAGUCCGU-3'(SEQ ID NO：4)、5'-GCAAUAUGAUGGAAGGAUA-3(SEQ ID NO：5)、5'-CUGCCAACUUAUAAUGUUU-3'(SEQ ID NO：6)和5'-GCUAUGUGCUUAAUGUUUC-3'(SEQ ID NO：7)。siRNA-Ctrl的序列为5'-UGGUUUACAUGUCGACUAA-3'(SEQ ID NO：8)、5'-UGGUUUACAUGUUUUCUGA-3'(SEQ ID NO：9)、5'-UGGUUUACAUGUUUUCCUA-3'(SEQ ID NO：10)和5'-UGGUUUACAUGUUGUGUGA-3'(SEQ ID NO：11)。

Green PCR Master Mix,

Reverse Transcriptase Kit was purchased from Applied Biosystems (Foster City, CA, USA). Rapamycin, a chemical mTOR inhibitor, was purchased from Sigma Aldrich (St. Louis, MO, USA). Recombinant mouse Sema3D and human Sema3D proteins were purchased from R&D Systems (Minneapolis, MN, USA). All other reagents were analytical grade unless otherwise stated. Primary antibodies: anti-SOX2 (ab97959, Abcam; Cambridge, MA, USA), anti-Sema3D (ab180147, Abcam), anti-GAPDH (5174, Cell Signaling; Beverly, MA, USA), anti-Ki67 (ab16667, Abcam), anti-Beclin -1 (ab207612, Abcam), anti-LC3 (ab48394, Abcam), anti-p62/SQSTM1 (ab56416, Abcam), anti-PI3K (E-AB-32575, Elabscience; Houston, Texas, USA), anti-phospho-PI3K (E-AB-32575, Elabscience; Houston, Texas, USA) - AB-20966, Elabscience), anti-Akt (9272S, Cell Signaling), anti-phospho-Akt (9271S, Cell Signaling), anti-mTOR (E-AB-32128, Elabscience) and anti-phospho-mTOR (E-AB-20929 , Elabscience) for western blot, immunohistochemical staining and immunofluorescence experiments. Oligonucleotide pools of siRNA targeting human Sema 3D and non-targeting (control) RNA were purchased from Dharmacon (Lafayette, CO, USA). The sequences of siRNA-Sema3D are 5'-CUGUGAUGUAUAAGUCCGU-3' (SEQ ID NO: 4), 5'-GCAAUAUGAUGGAAGGAUA-3 (SEQ ID NO: 5), 5'-CUGCCAACUUAUAAUGUUU-3' (SEQ ID NO: 6) and 5'-GCUAUGUGCUUAAUGUUUC-3' (SEQ ID NO: 7). The sequences of siRNA-Ctrl are 5'-UGGUUUACAUGUCGACUAA-3' (SEQ ID NO: 8), 5'-UGGUUUACAUGUUUUCUGA-3' (SEQ ID NO: 9), 5'-UGGUUUACAUGUUUUCCUA-3' (SEQ ID NO: 10) and 5'-UGGUUUACAUGUUGUGUGA-3' (SEQ ID NO: 11).

Cell Culture and Cell Research

The human neuronal cell line SY5Y (ATCC CRL-2266) was obtained from the American Type Culture Collection. HEK293 cells (BCRC90016) were obtained from Bioresource Collection and Research Center. SY5Y cells and HEK293 cells were maintained in a humidified incubator with 37°C and 5% _CO supplemented with 10% FBS (Invitrogen, Waltham, MA, USA), 1% penicillin and streptomycin (Biowest) , Loire Valley, France) and 1% L-glutamine (Invitrogen) in DMEM. Human neural stem cells (NSCs) are derived from human induced pluripotent stem cells (iPSCs). Briefly, human iPSCs were first cultured into embryoid bodies (EB) medium on matrigel-coated dishes (BD Biosciences; Franklin Lakes, NJ, USA), supplemented with With recombinant human Noggin protein (250ng/ml, R&D). On day 10, the medium was changed to supplemented with Sonic Hedgehog (SHH, 20ng/ml, R&D) and fibroblast growth factor 8 (FGF8) (100ng/ml, R&D) EB medium. Until rosettes appeared (day 14), the medium was changed to medium supplemented with BDNF, ascorbic acid, SHH and FGF8. On day 22, FGF8 was removed and cells were maintained in medium supplemented with BDNF, ascorbic acid and SHH. On day 29, cells were seeded on poly-L-ornithine/laminin coated dishes in complete StemPro NSC medium and expanded to passage 10.

Sema3D and neurodegeneration

To investigate whether Sema3D induces neurodegeneration via the PI3K/Akt/mTOR/autophagy pathway, SY5Y cells were treated with recombinant Sema3D protein for 72 hours, and cell lysates were collected for western blot analysis. For rescue experiments, 1 μM rapamycin and Sema3D were co-administered into cultured SY5Y cells for 72 hours, and cell viability was detected by Ki67 staining at 72 hours. CHOV20191024 (a novel Sema3D antagonist) and Sema3D were co-treated with SY5Y cells to demonstrate the biological activity of CHOV20191024. MTT analysis, Western blot and cell survival analysis were used to assess the rescue ability of CHOV20191024.

Sema3D affects neural stem cells

Sema3D was added to the culture medium of human neural stem cells. To assess whether Sema3D can affect neural stem cell properties (stemness). Sphere formation assay and counting of sphere numbers were performed on day 5 after Sema3D treatment.

Wild-type (WT), miR-195a knockout (KO) mice and aged mice

[Corrected 17.11.2021 in accordance with Rule 26]
In the present invention, WT C57BL/6 mice from C57BL/6 and miR-195a KO mice were used. MiR-195a KO mice were produced by the National Laboratory Animal Center of Taiwan. 4, 12 and 21 month old wild-type C57BL/6 mice were obtained from the National Laboratory Animal Center in Taiwan. Mice were acclimated for at least 2 weeks before behavioral experiments or sacrifice. The animal experiment protocol was approved by the Animal Care and Use Committee of China Medical University, Taiwan, which strictly complied with the Guidelines for the Care and Use of Laboratory Animals, 8th Edition (2011).

Sema3D overexpressing mice and lentiviral administration

4 month old C57BL/6 mice were used to generate Sema3D overexpressing mice. Mice were anesthetized by using a mixture of Zoletil and Rompun (ratio: 3:1, 1 mg kg ^-1 , intraperitoneal) for stereotaxic injection procedures. A solution containing Sema3D lentivirus (Lv. Sema3D; ^4.5 x 105 TU/ml) was injected into the bilateral hippocampus (-1.2 mm anterior-posterior, 1 mm medial-lateral, and -2 mm dorsal-ventral Relative to bregma; -3.6 mm anterior-posterior, 3.2 mm medial-lateral and -4 mm dorsal-ventral relative to bregma). On day 7 post-injection, Sema3D overexpressing mice were subjected to behavioral testing, Golgi-cox stain or signaling pathway analysis. Mice receiving control lentivirus (Lv.Ctrl) served as a control group.

animal research

First, miR-195a KO and WT mice were used to investigate the role of miR-195 in cognitive function, neurogenesis and brain senescence. The cognitive function of miR-195a KO and WT mice was assessed by Morris Water Maze (MWM) test, Y-maze test (Y-maze test) and open field test (OFT). Neurogenesis capacity was assessed by quantifying SOX2 ⁺ neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ). Senescence-associated β-galactosidase (SAβ-gal) activity and p16 ^INK4a /p19 ^Arf expression were used to assess brain aging.

Second, the role of Sema3D in cognitive function, neurodegeneration and neurogenesis was explored by using Sema3D overexpressing mice. Cognitive function was assessed through the MWM test, the novel object recognition test, the Y-maze test, and the open field test. Cognitive testing was performed on day 7 after lentivirus administration. To support the findings of the present invention in behavioral testing, Golgi-Cox staining was used to detect neurodegeneration in the same mice after behavioral testing. If Golgi-Cox staining shows a decrease in the density of the dendritic spines of neurons, there is neurodegeneration in the brain. To explore the effect of Sema3D on autophagy, hippocampi were collected from the same mice subjected to behavioral testing and analyzed for autophagy-related proteins using western blotting. To study the effect of Sema3D on neurogenesis, recombinant Sema3D protein was injected intracerebroventricularly (ICV) into 4-month-old WT mice. Neurogenesis was assessed by quantifying SOX2 ⁺ neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ).

Two in vivo rescue experiments were performed to confirm the deleterious effects of Sema3D. First, rapamycin was delivered into the hippocampus of Sema3D-overexpressing mice to rescue autophagy efficiency. Rapamycin and Lv.Sema3D were injected into the bilateral hippocampus of mice. Golgi-Cox staining was performed to visualize dendritic spine density to assess the severity of Sema3D-induced neurodegeneration. Second, Sema3D siRNA or control siRNA was delivered into the hippocampus of 12-month-old miR-195a KO mice. Spatial working memory and motor function obtained by the Y-maze test were used to evaluate the effect of Sema3D siRNA on cognitive function. Rescue was assessed again using Golgi-Cox staining.

Y maze

Spatial working memory was assessed by the Y-maze test. The Y maze is composed of three closed arms, which are 50 cm long, 11 cm wide, and 10 cm high, and are made of black plexiglass (Plexiglas); and the three arms are 120° to each other in a Y shape. There are two different Y-maze schemes used in the present invention.

The first protocol consisted of two separate experiments performed at 15 min intervals in miR-195a KO mice and Sema3D overexpressing mice. Briefly, in the first trial (acquisition trial), mice were placed at the end of a selected arm (initial arm) and allowed to close in one of the arms (noted as the new arm) Exploring the maze for 5 minutes. The mice were then returned to their home cages away from the testing room for 15 minutes. In the second trial (the retention trial), mice were allowed to freely explore all three arms of the maze for 5 minutes and the time taken to reach the new arm (previously closed in the first trial) was recorded. The longer it takes to reach the new arm, the worse the working memory performance.

The second protocol was performed with Sema3D/control siRNA-treated miR-195a KO mice. Briefly, each animal was placed in the center of the Y-maze and was free to explore the arena for 8 minutes. Mice tended to explore the least recently visited arm and therefore tended to alternate visits among the three arms. In order to alternate effectively, mice need to use working memory, keep a constant record of recently visited arms, and continually update those records. Access to the arm was scored when the mouse placed its four paws inside the arm. Spatial working memory was assessed by alternation percentage. Alternation is defined as successive selection into three different arms. Alternation percentage is calculated as the ratio of the actual number of alternations to the maximum number of alternations. The maximum number of possible alternations is defined as the total number of entries into the arm minus 2. A low percentage of alternation indicates impaired spatial working memory, as the mouse does not remember which arm it has just visited, and thus shows reduced spontaneous alternation.

Open field test (OFT)

Motor function was examined using the open field test (OFT). Briefly, mice were placed in an open-field device equipped with 16 beams for a 15-minute session. Motor function was judged by the total walking distance of mice.

Morris Water Maze (MWM)

Spatial learning and spatial memory are tested by the MWM test. Briefly, mice were trained to find a hidden platform in opaque water for 5 days, with 4 acquisition trials per day from a pseudorandomized starting position. During a 5-day acquisition trial, the latency to find the hidden platform was recorded as an indicator of spatial learning ability. Next, to assess spatial memory ability, probe trials (in which the hidden platform was removed) were performed on days 1, 7, and 14 after the acquisition trial, recording the total time spent finding the hidden platform. The latency to find a hidden platform and the frequency of reaching the platform quadrant were recorded as indices of spatial memory capacity. All MWM experiments were recorded and analyzed using the ANY-Maze Animal Behavior Analysis System (Stoelting, Chicago, IL, USA).

Novel Object Recognition test

Recognition memory was assessed by a novel object recognition test. The mice were first placed in the center of the arena with two identical objects for 10 minutes. The mice were then returned to their home cages away from the testing room for another 15 minutes. Next, a 5-minute recognition memory test in which a familiar object was replaced by a new one. The time each animal spent exploring each object during the test was recorded with a video tracking system (ViewPoint Behavior Technology; Lyon, France). Object memory ability is shown as the ratio of the time spent exploring new objects to the time spent exploring all objects (discrimination index).

Golgi-Cox staining and dendritic spine density measurements

Dendritic spine density of hippocampal neurons in the CA1 area is shown by Golgi-Cox staining. Brains were immersed in Golgi staining solution according to the manufacturer's protocol (FD Rapid GolgiStainTM Kit, FD Neuro Technologies Inc., MD, USA). The stained brains were then sectioned using a vibratome (Leica VT1000S) to obtain 100 μm thick coronal sections from the dorsal hippocampus. Sections were embedded on gelatin-coated glass slides, then placed in Kodak Film Fixer for 15 minutes and dehydrated in xylene-based media.

Bright field microscopy images were obtained using a confocal microscope (Leica SP2/SP8X). Hippocampal CA1 dendrites were tracked semi-automatically using Image J software. Pyramidal neurons least obscured by other cells and with undamaged dendritic trees will be selected for analysis.

Immunofluorescence staining and quantification of neural stem cells (NSCs)

To assess neurogenesis in vivo, neural stem cells were detected using immunofluorescence staining. Neural stem cells were confirmed and quantified by SOX2-positive signal with clearly distinguishable nuclei (DAPI-positive cells). Briefly, brains were fixed with 4% paraformaldehyde (PFA), cryopreserved in 30% sucrose solution at 4°C for 24 hours, and then embedded in OCT. 15 μm thick cryosections were collected and stored at −20 °C until use. For immunostaining, brain sections were incubated with SOX2 antibody in 5% BSA in PBS overnight at 4°C with secondary antibody conjugated to Alexa Fluor 647 (Invitrogen). Images were acquired by immunofluorescence confocal microscopy (Leica SP2/SP8X). The number of SOX2-positive cells located in the dentate gyrus (DG) and subventricular zone (SVZ) was quantified by Image J software.

Sphere formation assay

The spheroid formation assay was used to determine the effect of Sema3D on the properties of neural stem cells. Briefly, human neural stem cells were seeded in ultra-low attachment 24-well plates (Corning; NY, USA). The number of spheroids (>50 μm in diameter) was counted on day 5 of culture.

Selection and analysis of gene expression datasets

The Gene Expression Omnibus (GEO) database (as of December 2019) was queried for human hippocampal microarray gene expression datasets related to neurodegenerative diseases and aging. The specific search terms used were: "neurodegeneration", "dementia", "cognitive impairment" and "postmortem brain". The retrieved datasets were filtered according to the following criteria: (1) derived from available raw data from human hippocampal tissue; (2) for neurodegenerative disease datasets, there was at least one control group (normal individuals) and one disease group; and (3) Sema3D should be detected in the microarray results. Table 1 summarizes all retrieved datasets and the reasons for their inclusion or exclusion in the present analysis.

Table 1. GEO dataset of gene expression in selected hippocampus

Raw gene expression data and disease severity classifications obtained from the GEO database. The retrieved datasets were microarray datasets obtained from two platforms: the Affymetrix human gene body U133 and the Affymetrix human gene 1.0ST array (Table 1). After filtering, retention of six human hippocampal microarray datasets from dementia, aging, frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U), and Alzheimer's disease (AD) (GSE84422, GSE11882, GSE13162, GSE1297, GSE48350 and GSE36980) for further analysis.

For each transcriptome dataset, raw expression data for Sema3D and Sema3A were queried and converted to signal intensities by the log2 method. Next, the results of signal intensity were normalized by controlling for the average intensity of individuals and classified according to disease severity provided in the original article. Finally, fold changes in Sema3D/Sema3A expression were calculated by comparing the signal intensities between the disease and control groups.

Gene expression data obtained from the Allen Atlas of the Human Brain

To investigate the extent of Sema3A and Sema3D expression in the human hippocampus, RNA-sequencing data were downloaded from the Allen Brain Atlas (https://portal.brain-map.org/). In the present invention, RNA sequencing data of 94 donors aged 77 to 100+ years were analyzed for Sema3A and Sema3D gene expression.

Analysis of Aging-Associated β-Galactosidase (SAβ-gal) Activity

Senescence-associated β-galactosidase activity was determined using SPiDER-βGal assay panels and staining of X-gal substrates. Briefly, brain sections were first fixed in PBS containing 4% paraformaldehyde. After 3 washes with PBS, 0.5 mm brain sections were exposed to X-gal solution (1 mg/ml X-gal; 5 mM K ₃ Fe(CN) ₆ ; 5 mM K ₄ Fe(CN) ₆ ; 1 mM MgCl ₂ in PBS medium; pH=6.0) or SPiDER-βGal staining solution. Signals in the cortex and hippocampus were investigated using dissection or immunofluorescence confocal microscopy (Leica SP2/SP8X) embedded on glass slides. The signal intensity of SPiDER-βGal-positive cells was normalized by the DAPI signal and quantified by Image J software.

RNA isolation and measurement of mRNA extent

Total RNA was extracted from cells using Trizol reagent. Quantitative real-time PCR analysis of cDNA from cells and tissues was performed using the AB7900 Real-Time PCR System (Applied Biosystems) according to the manufacturer's instructions. Primers specific for mouse Sema3A, ^Sema3D , ^p16INK4a , p19Arf and GAPDH were used. The proportion of each gene was normalized as an internal control group (GAPDH), and the 2 ^-ΔΔCt relative quantification method was used to quantify the degree of expression.

Targeting site prediction and luciferase reporter gene analysis

Two algorithms (algorithms) were used to predict miR-195 target genes and binding sites: miRanda (http://microrna.sanger.ac.uk/targets/v5/) and TargetScan (http://targetscan.org) . The corresponding seed region in the Sema3D 3'-UTR was mutated to disrupt base pairing between Sema3D and miR-195. Luciferase activity was used to test whether Sema3D is a miR-195 target gene. Reporter plasmids containing predicted Sema3D binding sites or mutant binding sites were constructed and transfected into HEK293 cells. Twenty-four hours later, miR-195 or negative control microRNA (microRNA) was transfected into HEK-293 cells by HiPerFect transfection reagent (Invitrogen) to investigate whether miR-195 could directly interact with the target 3'-untranslated region ( 3'-untranslated region, 3'-UTR) sequence binding. Luciferase activity was compared between cells transfected with normal or mutant plasmids. If Sema3D is the target of miR-195, the luciferase activity should be higher in cells transfected with the mutant plasmid, since miR-195 is unable to exert its knockdown effect.

Western blot analysis

For western blot analysis, cell pellets or brain samples were homogenized and lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (Complete and Phosphostop, Roche). 20 μg of protein was subjected to SDS-PAGE, and the electrophoresed protein was transferred to polyvinylidene difluoride membrane (Millipore). Immunoblots were incubated with primary antibodies overnight at 4°C. After washing, immunoblots were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at 4°C. Immunoblot analysis was performed using ECL Western Blot Detection Reagent (GE) according to the manufacturer's instructions.

Sema2A overexpression in Drosophila, lifespan and motility analysis

Drosophila carrying elav-Gal4 or UAS-Sema2a-GFP were obtained from the Bloomington Drosophila stock center (Indiana University, Bloomington, IN, USA) and incubated at 25°C with a 12-hour light-dark cycle and grown on standard cornmeal medium at 60% relative humidity. To overexpress Sema2A in the nervous system, virgin female flies carrying the neuron-specific driver gene elav-Gal4 were crossed with male flies carrying UAS-Sema2a-GFP, and their F1 offspring would be Sema2A-overexpressing flies . F1 progeny of virgin female flies and male wild-type flies carrying elav-Gal4 served as control flies.

To study the effect of Sema2A on lifespan, Sema2A-overexpressing flies and control flies (300/group) were used, and surviving flies were counted every 7 days. The number of dead flies was recorded and a survival curve was drawn. The Gehan-Breslow-Wilcoxon test was used to determine statistical differences between Sema2A-overexpressing flies and control flies.

The locomotor activity of flies overexpressing Sema2 was determined using a negative Geotaxis assay. Briefly, groups of approximately 15 flies were placed in vertical cylinders (25 cm in length, 1.5 cm in diameter) with tapered bottom ends. On a light tap, the flies startle and climb up. After 30 s, flies above the midline were counted, and climbing scores were determined by calculating the ratio of flies above the midline to the total number of flies. The Mann-Whitney test was used to determine statistical differences between Sema2A-overexpressing flies and control flies.

Template identification and protein homology modeling

The structure of human Sema3D is not available in the RCSB Protein Data Bank (http://www.rcsb.org/). BLASTP was used to identify homologs in the RCSB protein database. The X-ray crystal structure of Sema3A from mouse (Mus musculus) (PDB code 4GZ8), with 60.04% sequence identity to Sema3D, was used as a template during the SWISS-MODEL tool. After optimization, the predicted model of Sema3D was confirmed via the VERIFY 3D and PROCHECK programs available in the Structural Analysis and Verification Server (SAVES) (http://nihserver.mbi.ucla.edu/SAVES). The VERIFY 3D program checks the compatibility of protein 3D models with any amino acid sequence (1D). The PROCHECK program is used to assess the stereochemical quality of protein secondary structures.

Study Approval

[Corrected 17.11.2021 in accordance with Rule 26]
The Animal Care and Use Committee of China Medical University, Taiwan approved the animal experimental protocol (Approval Number: CMUIACUC-2017-292), which strictly complies with the NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication Number: 85-23, revised in 1996) .

Statistical Analysis

All values in text and figures are expressed as mean ± standard error of the mean (SEM). Statistical differences were assessed using Student's t test. Between-group comparisons on cognitive tests were assessed by two-way analysis of variance (ANOVA). The Gehan-Breslow-Wilcoxon test was used for Drosophila longevity analysis, and the Mann-Whitney test was used to analyze Drosophila climbing scores. In all experiments, p-values less than 0.05 were considered statistically different. Data analysis and graphing were performed using Prism 7 software (GraphPad Software Inc., San Diego, CA, USA).

result

Low miR-195 expression levels lead to cognitive impairment

Systemic miR-195a knockout (KO) mice have been produced. Notably, one mouse had two miR-195 genes (miR-195a and miR-195b), whereas only the miR-195a gene was knocked out in the mouse model of the present invention. The level of miR-195 expression in the brain is higher than in several internal organs. The level of miR-195 expression was significantly reduced by 25-50% in the brain of the miR-195a KO mice of the present invention, and by 50-75% in other organs (FIGS. 1A-1B). In addition, the level of miR-195 in the brain of WT mice decreased with age (Fig. 1C).

To investigate whether low levels of miR-195 expression could affect cognitive function, learning, memory, and motor functions were compared between miR-195a KO and WT mice at different ages (Table 2).

Table 2. Behavioral test results of miR-195a KO and WT mice at different ages

Young: 3-5 months, adult: 6-12 months, old: 15-24 months. The effect of age on Sema3D-overexpressing mice and Sema3D siRNA-injected mice was not assessed. MWM: Morris Water Maze; OFT: Open Field Test. N.S.: No statistical difference.

Spatial learning and memory were assessed by the Morris water maze (MWM) test, working memory was measured by the Y-maze test, and motor activity was assessed by the open field test (OFT). MWM testing was performed according to the protocol described in Figure 2A.

For spatial learning, 3-5 month old miR-195a KO mice did not perform as well as WT mice, as evidenced by the longer time required to find hidden platforms (Fig. 2B). There were three memory measures, two from the MWM test and one from the Y-maze test (Table 2). When mice were 3 to 12 months old, miR-195a KO was judged by a longer escape latency in the MWM test (Fig. 2C) and a longer latency to reach the toy arm in the Y-maze test (Fig. 3A) Mice have poor memory. However, after 12 months of age, the performance of KO and WT mice on these two memory tests became indistinguishable. Another memory parameter in the MWM test was the entry frequency of the target quadrant, but did not show any difference between KO and WT mice regardless of age (Fig. 2D). Compared with WT mice aged 6-12 months, miR-195a KO mice exhibited poor motor activity (Fig. 3B). Similar to memory and spatial learning, aged KO and WT mice did not differ in motor activity. The above results suggest that miR-195a KO mice may accelerate the aging process of the brain, resulting in poorer cognitive function.

Low miR-195 expression accelerates aging and exacerbates neurodegeneration

Since cognitive dysfunction is an important feature of aging, it is speculated that miR-195 deficiency is also associated with other aging phenotypes, including lifespan, molecular biomarkers, neural stem cells (NSCs), and dendritic spines. Therefore, the results of the present invention comparing the lifespan, mean lifespan, median lifespan and percentage reduction of WT mice and miR-195a KO mice are shown in Table 3. The mean and median lifespan of miR-195a KO mice was reduced by approximately 25% compared to WT mice (Fig. 4A). Furthermore, the present invention examines two biomarkers of aging in the brain, aging-associated beta-galactosidase (SAbeta-gal) activity and p16 ^Ink4a /p19 ^Arf expression. Two different SAβ-gal staining methods were used, both showing a large increase in SAβ-gal activity, as observed in the hippocampus and cortex of 4-month-old miR-195a KO mice . In contrast, in WT mice, SA[beta]-gal activity did not increase until 12 months of age (Figures 4B-4C). In addition, the SAβ-gal staining data of the present invention in WT mice are consistent with previous reports. The expression of p16 ^Ink4a and p19 ^Arf was significantly elevated in the brains of 4-month-old miR-195a KO mice compared with WT mice (Fig. 4D).

Table 3. Lifespan analysis of WT mice and miR-195a KO mice

Decreased neural stem cell numbers or dendritic spine density are associated with aging. The present invention compares the number of Sox2 ⁺ neural stem cells in the dentate gyrus (DG) and subventricular zone (SVZ) between miR-195a KO and WT mice. As expected, neural stem cell numbers decreased with age in the DG (Fig. 5A) and SVZ (Fig. 5C) of WT mice. However, in 4-month-old miR-195a KO mice, the number of neural stem cells in the DG and SVZ was reduced by 40% and 50%, respectively, which was the same result observed in 12-month-old WT mice. Furthermore, histological analysis of Golgi-Cox staining revealed a significant 50% decrease in dendritic spine density in the hippocampus of miR-195a KO mice compared with age-matched WT mice (Fig. 5B).

All the above experiments consistently showed that decreased expression of miR-195 promotes brain aging and impaired cognitive function, which prompted the present invention to search for the key miR-195-regulated molecules involved in the promotion of brain aging process.

Regulation of Sema3 in the brain by miR-195

It has been previously reported that Sema3A is a direct target of miR-195 and that neuronal cells are overexpressed under stress to promote apoptosis. To explore whether other Sema3 members are involved in the brain aging process in miR-195a KO mice, bio-informatics algorithms (miRanda and TargetScan) were used to predict miR-195 target genes in the Sema3 family. Among the Sema3 family members, Sema3A and Sema3D were shown to be direct targets of miR-195. Therefore, the corresponding seed region in the Sema3D 3'-UTR was mutated to disrupt the base pairing between Sema3D and miR-195. The present invention mutates at positions 2772-2792 of Sema3D 3'-UTR, and the mutation positions are shown in Table 4.

Table 4. Positions 2772-2792 of Sema3D 3'-UTR

Wild type 5'...uuCAGCAAUU-UA UGCUGCUa ... (SEQ ID NO: 12) Has-miR-195 3'...cgGUUAUAAAGACACGACGAu...(SEQ ID NO: 13) mutant 5'...uuCAGCAAUU-UA GUTGUTG a... (SEQ ID NO: 14)

The underlined part is the mutation position.

Reporter plasmids carrying wild-type or mutant Sema3D 3'-UTR were transfected into HEK293 cells. Luciferase reporter assay confirmed that miR-195 binds directly to the Sema3D RNA 3'-UTR (Fig. 6A). The amounts of both Sema3A and Sema3D were increased in the hippocampus of miR-195a KO mice (Fig. 6B-6C), and the amount of Sema3D protein was 3-fold higher than that of Sema3A in WT mice (Fig. 6B). Likewise, RNA-seq analysis of the Allen Human Brain Atlas revealed that the amount of Sema3D mRNA in the human hippocampus was 50% higher than that of Sema3A (Fig. 6D). Since it has been reported that Sema3A is related to neurodegenerative diseases, and the role of Sema3D in brain function is still unclear, the present inventors decided to further study the role of Sema3D in this aspect.

Sema3D degree correlates with aging characteristics and neurodegenerative diseases

To explore the correlation between Sema3D and age-related neurodegeneration, brain Sema3D expression was examined in WT mice aged 4, 13 and 21 months. Histological staining showed an age-dependent increase in Sema3D (Fig. 7A). The present invention then uses the NCBI Gene Expression Omnibus (GEO) database to test potential correlations between Sema3D and human neurodegenerative diseases. According to the filtering criteria described in the Methods section and Figure 7B, there were six GEO datasets available containing human hippocampal gene expression profiles with Sema3D expression data (Table 5). These datasets include one dataset of individuals with dementia according to the Clinical Dementia Rating, three datasets of Alzheimer's disease (AD) patients, one patient with ubiquitin inclusion bodies A dataset of patients with temporal lobe dementia (FTLD-U) as well as a dataset of a normal elderly individual. In addition, Sema3A data were also analyzed and presented in Table 5.

In the three AD datasets, the degree of hippocampal Sema3D expression was significantly higher in individuals with more severe AD in two datasets (GSE1297 and GSE48350) (Table 5). Likewise, in the dementia (GSE84422) and FTLD-U (GSE13162) data, more severe individuals had higher levels of Sema3D expression in the hippocampus. Furthermore, in the dataset of normal individuals of different ages (GSE11882), the level of Sema3D expression in the hippocampus showed an age-dependent increase, which is consistent with the findings of the rodent brain samples of the present invention (Fig. 7A). Taken together, these data suggest that both dementia patients and normal individuals exhibit high levels of Sema3D expression in neurodegeneration, suggesting that Sema3D may directly contribute to neurodegeneration.

Table 5. List of hippocampal Sema3D and Sema3A expression in the neurodegenerative disease and aging dataset

For each dataset, all individuals were classified according to severity and phenotype provided in the original article. The expression levels of Sema3D and Sema3A in all severity groups were normalized to that of normal individuals in each corresponding dataset. Data are presented as mean ± SEM. *p<0.05, **p<0.01 using t-test. #p=0.080, ##p=0.1658 using one-way ANOVA.

Sema3D is a direct cause of neurodegeneration and cognitive impairment

To establish a causal relationship between hippocampal Sema3D protein and neurodegeneration and cognitive insufficiency, a Sema3D-expressing lentivirus (Lv. Sema3D) was administered to the bilateral hippocampus of 4-month-old WT mice. This injection avoids overexpression of Sema3D in the cortex. The efficiency of viral transfection was confirmed by measuring the degree of expression of Sema3D protein on day 7. Immunoblot data showed a 3-fold increase in Sema3D expression in the hippocampus, while only a slight increase in Sema3D expression in the cortex (Fig. 8A). Histological analysis of Golgi-Cox staining revealed a significant decrease in dendritic spine density in the hippocampus of Sema3D-overexpressing mice (Fig. 8B), supporting a negative effect of Sema3D on neuronal cell morphology.

Next, four tests were performed to examine the deleterious effects of Sema3D on cognitive function in Sema3D-overexpressing mice. In addition to the three identical tests performed in miR-195a KO mice, a novel object recognition test was also used in Sema3D-overexpressing mice to perform recognition memory. The MWM test scheme is shown in Figure 8C. Sema3D-overexpressing mice exhibited significant learning impairments compared to control mice (p=0.0015; Figure 8D). In the memory test of the MWM test, Sema3D-overexpressing mice were inferior to controls in both latency (p=0.0025; Figure 8E) and frequency (p<0.0001; Figure 8F) in the plateau quadrant. Consistently, Sema3D overexpressing mice exhibited impaired recognition memory, which was reflected in a lower discrimination index (p=0.0007; Figure 8G). Although Sema3D overexpressing mice had poorer working memory, this test was not statistically significant due to too much variation (p=0.071; Figure 8H). Because Sema3D was overexpressed only in the hippocampus, Sema3D overexpressing mice did not have reduced motor activity (p=0.248; Figure 8I).

To further confirm that Sema3D could explain the cognitive impairment in miR-195a KO mice, Sema3D in the hippocampus was knockdowned by a single injection of siRNA into 12-month-old miR-195a KO mice. The silencing efficiency of Sema3D was assessed by immunofluorescence analysis (Figure 9A), and spatial working memory and motor function were assessed by the Y-maze test (see protocol described in Figure 9B). The results show that mice that received siRNA-Sema3D injections exhibited better spatial working memory than mice that received siRNA-Ctrl, as demonstrated by a higher percentage of alterations and an optimal fitness with a p-value of 0.042. This is demonstrated by the steeper slope of the best-fit-line (Figure 9C). Similar to the effect of Sema3D overexpression, siRNA-Sema3D did not affect motor function (Fig. 9D) because siRNA was injected specifically into the hippocampus, which should not affect motor function. Decreased Sema3D in miR-195a KO mice, the neurodegenerative effects were also reflected in the reduced density of dendritic spines in hippocampal neurons (Fig. 9E).

Taken together, the histological and behavioral results imply a causal relationship between Sema3D and cognitive impairment and neurodegeneration. Furthermore, inhibition of Sema3D may be a major reason to explain the protective function of miR-195 in the brain.

Increased Sema3D expression shortens lifespan and impairs climbing activity in Drosophila

A growing body of evidence supports the idea that neurodegeneration can shorten lifespan. The Drosophila model was used to test the association between Sema3D extent and lifespan. Since the Sema2A gene in Drosophila is the homolog of the Sema3D gene in human and mouse, Sema2A is overexpressed in the nervous system of Drosophila (annotated as Sema2A overexpressed Drosophila). As shown in Figure 10A, the lifespan of flies overexpressing Sema2A was significantly shorter than that of control flies (300 per group, p<0.0001). As shown in Table 6, the mean lifespan of the Sema2A group was reduced by 26%, and the maximum lifespan was also significantly reduced from 75 days to 63 days. The reduction in Drosophila lifespan was consistent with lifespan data in miR-195a KO mice (Fig. 4A). Furthermore, the present invention uses climbing assays in adult flies to assess whether neural function is intact, since movement as a behavioral output requires fine coordination of neural morphologies and is sensitive to functional disruption. Sema2A-overexpressing flies showed reduced motor function, as indicated by a reduced climbing score (p=0.020; Figure 10B).

Table 6. Lifespan analysis of Drosophila overexpressing Sema2A

  control group Sema2A overexpression magnification change number of animals 46 34 26%↓ average lifespan 63 51 19%↓ median lifespan 75 63 16%↓

Mechanisms of Sema3D's Harmful Effects on the Brain

The present invention explores two possible mechanisms for the deleterious effects of Sema3D, the function of neural stem cells and autophagy.

Neuroregeneration

The function of neural stem cells is related to nerve regeneration. The present invention speculates whether Sema3D can disrupt neural stem cell function to explain its effect on neurodegeneration, and partially explain the low neural stem cell population density in miR-195a KO mice (Figure 5A and Figure 5C). Neurosphere formation assays were performed and the number of neural stem cells was quantified. The results showed that Sema3D dose-dependently inhibited neurosphere formation of human neural stem cells, suggesting that Sema3D impairs the stem cell properties of neural stem cells (Fig. 10C). When recombinant Sema3D was injected intraventricularly (ICV) into mice, Sema3D dose-dependently reduced neural stem cell population size in the DG and SVZ (DG results in Figure 10D and SVZ results in Figure 10E). Therefore, Sema3D reduces neural stem cell properties and causes neural stem cell loss, which may lead to neurodegeneration.

autophagy

Disruption of autophagy plays an important role in neurodegenerative diseases, and the PI3K/AKT/mTOR pathway has been reported to act as a master regulator of autophagy. Therefore, the present invention hypothesizes that Sema3D may induce neurodegeneration by regulating autophagy and the PI3K/AKT/mTOR pathway. To test the hypothesis of the present invention, the present invention first explored the effect of Sema3D on the efficiency of autophagy by overexpressing Sema3D in the hippocampus of mice and treating SY5Y cells with Sema3D. Compared with Lv.Ctrl-treated mice, Lv.Sema3D-treated mice had higher degrees of p-mTOR/mTOR and p62, and lower degrees of Beclin-1 and LC3-II/I ratios in the hippocampus (FIG. 11A). Consistently, Sema3D dose-dependently increased p62 and decreased the ratio of Beclin-1 and LC3-II/I in Sema3D-treated SY5Y cells (FIG. 11B). Thus, both in vitro and in vivo models suggest that Sema3D significantly disrupts autophagy.

Next, the present invention assessed whether the PI3K/AKT/mTOR pathway could be modulated by Sema3D and whether Sema3D-induced neurodegeneration could be rescued by rapamycin, an mTOR inhibitor. In fact, the results of the present invention show that Sema3D dose-dependently increased the phosphorylation of PI3K, Akt and mTOR in SY5Y cells (Fig. 11C). Furthermore, Sema3D inhibited the proliferation of SY5Y cells, which was reversed by rapamycin (Fig. 11D). To demonstrate the in vivo findings of the present invention, a single dose of rapamycin was injected into the bilateral hippocampus 5 minutes after the injection of Lv. Sema3D into the bilateral hippocampus. Histological images showed that rapamycin reversed Sema3D-induced neurodegeneration as evidenced by increased density of dendritic spines (FIG. 11E). Taken together, these data suggest that Sema3D impairs neuronal autophagy through an mTOR-dependent pathway that is rescued by rapamycin.

Sema3D is highly expressed in diabetic rats

The level of Sema3D expression was also increased in diabetic retina. Furthermore, Sema3D was expressed to a higher degree than Sema3A in the retina of diabetic animals. The amount of retinal Sema3D mRNA was significantly increased by 1.6-fold in rats at week 4 after diabetes onset (p=0.0014; the number in each group was 2, Figure 12A). IHC staining also showed that Sema3D protein increased in the retina of mice at week 8 after diabetes (Figure 12B, number 3).

The invention is suitably described as being capable of practice under requirements or limitations not specifically disclosed herein. The terms that have been used for description are not limiting. There is no difference in expression and description using these terms and any equivalent otherwise, but it should be recognized that the rights within the invention are modifiable. Therefore, while the present invention has described embodiments and other aspects, what is disclosed herein may be modified and varied by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention.

Claims (17)

Use of a composition in the preparation of a medicament for preventing or treating neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist, and the Sema3D antagonist does not comprise a microRNA- 195. The use of claim 1, wherein the neurodegenerative disease comprises Alzheimer's disease, Parkinson's disease, frontotemporal dementia, frontotemporal dementia with ubiquitin inclusion bodies, age-related cognitive decline, Vascular dementia, corticobasal degeneration, progressive supranuclear palsy, dementia with Lewy bodies, Huntington's disease, senile dementia, Pick's disease, arginophilic granular dementia, Guam-type parkinsonism-dementia complex, Lytico -Bodig disease, amyotrophic lateral sclerosis, spinocerebellar atrophy, spinobulbar muscular atrophy, motor neuron disease, multiple sclerosis, or traumatic brain injury. The use of claim 1, wherein the Sema3D antagonist prevents or treats the neurodegenerative disease by increasing dendritic spines in the hippocampus and promoting nerve regeneration. 4. The use of claim 3, wherein said increasing the dendritic spines of the hippocampus comprises increasing the density of dendritic spines of CA1 pyramidal neurons in the hippocampus. The use of claim 3, wherein the promoting nerve regeneration comprises increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation. The use of claim 5, wherein said increasing neural stem cells comprises increasing neural stem cells in the dentate gyrus of the hippocampus and the subventricular zone. Use of a composition in the manufacture of a life-extending medicament, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist. 7. The use of claim 7, wherein the Sema3D antagonist extends lifespan by delaying aging. The use of claim 7, wherein the Sema3D antagonist inhibits the expression of aging-related biomarkers. The use of claim 9, wherein the aging-related biomarkers comprise aging-related beta-galactosidase, p16 ^Ink4a , and p19 ^Arf . Use of a composition in the manufacture of a medicament for promoting nerve regeneration, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist, and the Sema3D antagonist does not comprise microRNA-195. The use of claim 11, wherein the Sema3D antagonist promotes nerve regeneration by increasing neural stem cells, upregulating autophagy, and promoting neuronal proliferation. The use of claim 12, wherein the up-regulation of autophagy comprises up-regulation of the PI3K/Akt/mTOR signaling pathway. A use of a composition in the preparation of a medicament for preventing or treating retinal neurodegenerative diseases, wherein the composition comprises a therapeutically effective amount of a semaphorin 3D (Sema3D) antagonist. The use of claim 14, wherein the retinal neurodegenerative disease comprises diabetic retinopathy, age-related macular degeneration, optic neuritis, myopic retinopathy or glaucoma-related retinal disease. The use of any one of claims 1, 7, 11 or 14, wherein the Sema3D antagonist comprises a conjugate against Sema3D, wherein the conjugate comprises a compound, polypeptide, antibody, antibody fragment or oligonucleotide acid. The use of claim 16, wherein the oligonucleotide comprises antisense DNA, antisense RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) or nucleic acid aptamer .

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