WO2021079098A1

WO2021079098A1 - Method of diagnosis of a neurodegenerative disease

Info

Publication number: WO2021079098A1
Application number: PCT/GB2020/052632
Authority: WO
Inventors: Frances M. Platt; Penelope J. HALLETT; Ole Isacson
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 2019-10-21
Filing date: 2020-10-20
Publication date: 2021-04-29
Anticipated expiration: 2022-04-21
Also published as: EP4022316A1; GB201916838D0

Abstract

The present invention relates to the use of gangliosides and glycosphingolipids from cerebrospinal fluid, plasma or serum as biomarkers for obtaining an indication of the presence of a neurodegenerative disease or disorder, for example Parkinson's Disease or Dementia with Lewy Bodies, in a subject.

Description

METHOD OF DIAGNOSIS OF A NEURODEGENERATIVE DISEASE

The present invention relates to the use of gangliosides and glycosphingolipids from cerebrospinal fluid, plasma or serum as biomarkers for obtaining an indication of the presence of a neurodegenerative disease or disorder, for example Parkinson’s Disease or Dementia with Lewy Bodies, in a subject.

The invention also relates to methods for obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject; a method of classifying a subject into a neurodegenerative disease or disorder subgroup; a method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder; a method of treating a neurodegenerative disease or disorder in a subject; and a method of obtaining an indication of the risk of a REM sleep behaviour disorder (RBD) subject of developing a neurodegenerative disease or disorder.

Parkinson’s disease (PD) is the second most common, late-onset neurodegenerative disease after Alzheimer’s disease and is characterised by the degeneration of dopaminergic neurons within the substantia nigra (SN). Loss of dopaminergic neurons results in disrupted motor control, causing tremor, rigidity, bradykinesia and gait dysfunction (1). Only 5-10% of PD cases have been linked to a genetic cause, whilst 90% of PD cases are sporadic (2). Ageing is the greatest non-genetic risk factor for PD, with most PD patients being over the age of 60 (1). The brain is especially vulnerable to progressive age-related changes, as alterations in multiple biological pathways may impair dopaminergic and other vulnerable neurons, and therefore lower the threshold for developing PD (3).

Mutations in lysosomal genes are the cause of more than 70 rare lysosomal storage diseases (LSDs), which often have a relentless neurodegenerative clinical course (4, 5). Gaucher disease (GD) is one of the most common autosomal LSDs and is caused by mutations in GBA, which encodes the lysosomal glucocerebrosidase GBA. A significant reduction in GBA activity results in accumulation of its glycosphingolipid (GSL) substrates, glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Heterozygote mutations in GBA significantly increase the risk of PD (6-10). Reduced GBA activity has been reported in brain tissue from both PD-GBA patients and sporadic PD patients without GBA mutations, suggesting a broader role for the lysosome in PD (11-13). Furthermore, GBA activity has been shown to decline progressively with ageing in the substantia nigra (SN) and putamen of healthy controls, eventually becoming comparable to GBA activity found in PD patients (12).

Such studies have been based on analyses of post-mortem brain tissue. There remains a need, however, for accessible biomarkers which are indicative or prognostic of PD and which can be obtained pre-mortem, for example from blood serum or from cerebrospinal fluid (CSF).

Several studies have demonstrated altered activities of various lysosomal hydrolases in CSF from PD patients. For example, decreased GBA, b-hexosaminidase and b- galactosidase activities have been reported in CSF of PD-GBA patients, but also sporadic PD patients (14-16).

Gangliosides are complex glycosphingolipids. They are the most abundant glycosphingolipids in the central nervous system (CNS) in all mammals and they are essential for brain function (17-18). For example, ganglioside GM1 a is essential for myelination, neuritogenesis, synaptogenesis and signalling of the neurotrophic factor GDNF (19-21). A reduction in GM1a levels has previously been described in the substantia nigra and occipital cortex from PD patients (19, 22, 23).

No studies have previously been published regarding levels of glycosphingolipids in the CSF of PD patients.

Aging in mouse brain is associated with reduced GBA activity and accumulation of glucosylceramide, glucosylsphingosine, lactosylceramide and GM1a (24). Levels of the gangliosides GD1a, GD1b, and GT1b were reduced with age in the mouse brain. Although mouse is a relatively-close mammalian species to humans, the glycosphingolipid changes that occur in the mouse are known to be not necessarily the same as the changes which occur in humans; hence these changes cannot be considered to be predictive of changes in humans. For example, in the aging mouse, brain levels of GM1a were increased (24), whereas they are decreased in the brain in PD patients (19, 22, 23). Furthermore, the Hallett et al. (24) findings of GBA, glycosphingolipid and lysosomal changes in the brain did not include measurements of GM2 and GM3, and did not include any measurements in CSF or blood.

Significant changes in levels of some gangliosides of the a-series (GM3, GM2, GM1a, GD1a) and b-series (GD3, GD1b, GT1b) have now been found in PD patients’ serum and CSF compared to age-matched controls. In particular, reduced levels of the more complex gangliosides GM1a, GD1a, GD1b and GT1 b were detected in CSF. Consequently, alterations in ganglioside levels in ante-mortem CSF may serve as biomarkers for PD.

It is one object of the invention therefore to provide a method of obtaining an indication of the presence of a neurodegenerative disease or disorder in a subject. Other objects of the invention include the provision of methods for obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject; a method of classifying a subject into a neurodegenerative disease or disorder subgroup; a method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder in a subject; methods of treating a neurodegenerative disease or disorder in a subject; and a method of obtaining an indication of the risk of a REM sleep behaviour disorder (RBD) subject of developing a neurodegenerative disease or disorder.

In one embodiment, the invention provides a method of obtaining an indication of the presence of a neurodegenerative disease or disorder in a subject, the method comprising the steps: (a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject, and producing a first Ganglioside Fingerprint value using those levels;

(b) comparing the first Ganglioside Fingerprint value with a second Ganglioside Fingerprint value which has been produced using:

(i) levels of the corresponding biomarkers in corresponding biological samples obtained from a control subject, or

(ii) reference levels of the corresponding biomarkers from corresponding biological samples; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, and wherein a decrease in the first Ganglioside Fingerprint value compared to the second Ganglioside Fingerprint value is indicative of the subject having a neurodegenerative disease or disorder.

In another embodiment, the invention provides a method of obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(a) determining the levels of one or more biomarkers in one or more first biological samples obtained from the subject at a first time point and producing a first Ganglioside Fingerprint value using those levels; and

(b) determining the levels of the corresponding biomarkers in corresponding second biological samples obtained from the subject at a second (later) time point and producing a second Ganglioside Fingerprint value using those levels; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein a decrease in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of a decline in the prognosis of the subject, and wherein an increase in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of an improvement in the prognosis of the subject. In another embodiment, the invention provides a method of classifying a subject into a neurodegenerative disease or disorder subgroup, the method comprising the steps:

(a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject and producing a Ganglioside Fingerprint value using those levels, wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid; and

(b) classifying the subject as belonging to a neurodegenerative disease or disorder subgroup based on the Ganglioside Fingerprint value of the subject.

In another embodiment, the invention provides a method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(b) determining the levels of the corresponding biomarkers in corresponding second biological samples obtained from the subject at a second (later) time point and producing a second Ganglioside Fingerprint value using those levels; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein the drug has been administered to the subject in the interval between the first and second time points, and wherein a decrease in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of a lack of efficacy of the drug, and wherein an increase in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of the efficacy of the drug.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent which increases the levels of one or more a-series or b-series gangliosides in the central nervous system of the subject.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject and producing a Ganglioside Fingerprint value using those levels, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein the one or more biomarkers are a-series or b- series gangliosides;

(b) comparing the Ganglioside Fingerprint value with a reference level; and

(c) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject if the Ganglioside Fingerprint value is below the reference level, thereby treating the neurodegenerative disease or disorder in the subject.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of: (a) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject, wherein, prior to administration, a Ganglioside Fingerprint value which was produced using the levels of one or more biomarkers in one or more biological samples obtained from the subject, has been determined to be below a reference level, wherein the biological samples were samples of serum, plasma or of cerebrospinal fluid, and wherein the one or more biomarkers were a-series or b-series gangliosides.

In yet another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of: (a) receiving a Ganglioside Fingerprint value produced from the levels of one or more biomarkers in one or more biological samples obtained from the subject; wherein the biological samples were samples of serum, plasma or of cerebrospinal fluid, and wherein the one or more biomarkers were a-series or b-series gangliosides,

(b) identifying the subject as having a Ganglioside Fingerprint value below a reference level, thereby providing an indication of a neurodegenerative disease or disorder in the subject, and administering a treatment appropriate for treating the neurodegenerative disease or disorder to the subject.

In another embodiment, the invention provides a method of obtaining an indication of the risk of a REM sleep behaviour disorder (RBD) subject of developing a neurodegenerative disease or disorder, the method comprising the steps:

(a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject, and producing a first Ganglioside Fingerprint value using those levels;

(ii) reference levels of the corresponding biomarkers from corresponding biological samples; wherein the one or more biomarkers are a-series gangliosides or globo-series glycosphingolipids, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, and wherein a decrease in the first Ganglioside Fingerprint value compared to the second Ganglioside Fingerprint value is indicative of an increased risk of the subject developing a neurodegenerative disease or disorder, and wherein an increase in the first Ganglioside Fingerprint value compared to the second Ganglioside Fingerprint value is indicative of a reduced risk of the subject developing a neurodegenerative disease or disorder.

A method of screening for agents for preventing and/or treating a neurodegenerative disease or disorder, the method comprising the steps: (a) screening for agents which specifically promote or enhance expression of a- series gangliosides, b-series gangliosides or globo-series glycosphingolipids, wherein agents which are identified are ones for preventing and/or treating the neurodegenerative disease or disorder.

In some embodiments of the invention, the method is carried out in vitro or ex vivo.

As used herein, the term “a neurodegenerative disease or disorder” includes Parkinson’s Disease (PD), idiopathic Parkinson’s Disease, Vascular parkinsonism (arteriosclerotic parkinsonism), Drug-induced parkinsonism, Dementia with Lewy Bodies (DLB), Multiple system atrophy (MSA), Progressive supranuclear palsy (PSP),

Normal pressure hydrocephalus, and Various tremors (including essential tremor) and Wilson’s disease. Preferably, the term “a neurodegenerative disease or disorder” refers to Parkinson’s Disease (PD) or Dementia with Lewy Bodies (DLB). Parkinson’s Disease subjects may be classified as being sporadic PD, idiopathic PD, genetic PD or non-genetic PD.

As used herein, the term "is indicative of the subject having a neurodegenerative disease or disorder” means that there is a negative correlation between the presence of the Ganglioside Fingerprint value and the presence of a neurodegenerative disease or disorder in that subject.

Consequently, a decrease in the value of the Ganglioside Fingerprint of the subject (compared to a value from a control healthy subject or compared to a previous value from that subject) means an increased likelihood or stati sti ca I ly-si g n if i cant chance (where the increase is significant) of the subject having a neurodegenerative disease or disorder.

A Ganglioside Fingerprint value is produced by using the levels of one or more biomarkers or the Ganglioside Fingerprint value is derived from the levels of one or more biomarkers. In some embodiments, the Ganglioside Fingerprint may be defined as the output of a function combining the selected biomarkers. In one embodiment, the Ganglioside Fingerprint may be the level of one of the biomarkers. For example, the Ganglioside Fingerprint may be the determined level of GM1a, GD1a, GD1b or GT1b.

In some embodiments, the biomarkers are a-series or b-series gangliosides. In other embodiments, the biomarkers are globo-series glycosphingolipids.

Figure 1 illustrates the biosynthesis pathway of glycosphingolipids (GSLs) and the involvement of a-series and b-series gangliosides and globo-series glycosphingolipids in this pathway.

“a-series gangliosides” include GM3, GM2, GM1a and GD1a. Preferably, the a-series ganglioside is GM2, GM1a and/or GD1 a. Most preferably, the a-series ganglioside is GM1a and/or GD1a.

“b-series gangliosides” include GD3, GD2, GD1b, GT1b and GQ1b. Preferably, the b- series ganglioside is GD3, GD1b and/or GT1b. Most preferably, the b-series ganglioside is GD1 b and/or GT 1 b.

More preferably, the biomarkers are selected from the group consisting of GM1a, GD1a, GD1 b and GT 1 b. Most preferably, the a-series or a b-series ganglioside is GM1 a. Globo-series glycosphingolipids include Gb3 and GB4.

In some preferred embodiments, the biomarker is GM2 and the biological sample is CSF. In some preferred embodiments, the biomarker is GD1a and the biological sample is CSF.

In some preferred embodiments, the biomarker is GD3 and the biological sample is CSF. In some preferred embodiments, the biomarker is GD1b and the biological sample is CSF. In some preferred embodiments, the biomarker is GT1b and the biological sample is CSF. In some preferred embodiments, the biomarker is GM1a and the biological sample is serum or plasma. In some preferred embodiments, the biomarker is GD1a and the biological sample is serum or plasma.

In other embodiments, the Ganglioside Fingerprint is the sum or weighted sum of two or more (e.g. 3, 4, 5, or 6) of the biomarkers, e.g. (k1 x BM1) + (k2 x BM2), where k1 and k2 are independently positive numbers which may be the same or different, and BM1 and BM2 are the determined levels of two of the biomarkers. In other embodiments, the Ganglioside Fingerprint is n x BM1 x BM2, where BM1 and BM2 are the determined levels of two of the biomarkers, where n is a positive number. In other embodiments, the Ganglioside Fingerprint is n x BM1 / BM2, where BM1 and BM2 are the determined levels of two of the biomarkers, where n is a positive number.

In some embodiments, the Ganglioside Fingerprint is the sum or weighted sum of three of the biomarkers, e.g. (k1 x BM1) + (k2 x BM2) + (k3 x BM3), where k1 , k2 and k3 are independent positive numbers which may be the same or different, and BM1 , BM2 and BM3 are the determined levels of three of the biomarkers. In other embodiments, the Ganglioside Fingerprint is n x BM1 x BM2 x BM3, where BM1 , BM2 and BM3 are the determined levels of two of the biomarkers, where n is a positive number.

In some embodiments, the Ganglioside Fingerprint is the sum or weighted sum of four of the biomarkers, e.g. (k1 x BM1 ) + (k2 x BM2) + (k3 x BM3) + (k4 x BM4), where k1 , k2, k3 and k4 are independent positive numbers which may be the same or different, and BM1 , BM2, BM3 and BM4 are the determined levels of four of the biomarkers.

In other embodiments, the Ganglioside Fingerprint is n x BM1 x BM2 x BM3 x BM4, where BM1 , BM2, BM3 and BM4 are the determined levels of four of the biomarkers, where n is a positive number.

In embodiments of the invention wherein first and second Ganglioside Fingerprint values are compared, the same function (operation) is applied to both first and second Ganglioside Fingerprint values. In embodiments of the invention which refer to “corresponding biomarkers”, this is referring to the same biomarkers as the previously-mentioned biomarkers. For example, if the first Ganglioside Fingerprint is produced using the biomarkers GM1a and GD1a, then the second Ganglioside Fingerprint is also produced using the biomarkers GM1a and GD1a.

In embodiments of the invention which refer to “corresponding biological samples”, this is referring to the same biological samples as the previously-mentioned biological samples. For example, if the first Ganglioside Fingerprint is produced using biomarkers GM1a and GD1a from serum, then the second Ganglioside Fingerprint is also produced using the biomarkers GM1a and GD1a from serum.

In one preferred embodiment, the Ganglioside Fingerprint is the sum of the levels of GM1a, GD1a, GD1b and GT1b. Most preferably, the Ganglioside Fingerprint is the sum of the CSF levels of GM1 a, GD1a, GDIb and GT1b.

In other preferred embodiments, the Ganglioside Fingerprint is the sum of the serum or plasma levels of GM1a and GD1a.

The biological samples are samples of serum or plasma, or samples of cerebrospinal fluid. Serum and plasma may be obtained from a blood sample from the subject, wherein the blood cells have been removed. Cerebrospinal fluid (CSF) may be obtained via a lumbar puncture from the subject. The biological sample is a sample which is obtained or which has previously been obtained from the subject. In some embodiments, the method additionally comprises the step of obtaining one or more biological samples from the subject.

The levels of the biomarkers may be determined by any suitable means, for example using the method of Neville et al. (25). In summary, glycosphingolipids are analysed by NP-HPLC according to published methods. Samples are extracted in chloroform/methanol (C:M) (1 :2 v/v) overnight at 4°C. The mixture is centrifuged (3000rpm/10min) and 1ml_ chloroform and 1ml_ PBS were added to the supernatant and centrifuged again (3000rpm/10min). The lower phase is dried under N₂, resuspended in 50uL C:M 1 :3 v/v and combined with the upper phase. Subsequently, GSLs are recovered using C18 Isolute columns (100mg) (Biotage) pre-equilibrated with 4x1 mL MeOH and 2x1 mL H₂0. Samples are applied to the column which is then washed 3x1 mL H₂0 and eluted with 1 mL C:M 98:2, 2x1 mL C:M 1 :3, 1 mL MeOH. The column eluate is dried under N₂, resuspended in 100pL C:M 2:1 , dried again under N₂ and resuspended in ceramide glycanase (CGase) buffer (50mM sodium acetate pH 5.5, 1mg/mL sodium taurodeoxycholate). 50mU CGase was added, and samples incubated at 37°C overnight. Released oligosaccharides are then labelled with anthranilic acid (2- AA) and purified by mixing with 3mL Acetonitrile: H₂0 97:3 and added to Discovery DPA-6S columns (SUPELCO, # 52625-U) pre-equilibrated with 1mL acetonitrile, 2x1 mL H₂0, and 2x1 mL acetonitrile. Columns are washed with 2x1 mL Acetonitrile: H₂0 95:5 v/v, and eluted in 2x 0.75mL H₂0. Samples are then loaded 30:70 H₂0: MeCN (v/v) onto normal phase HPLC. Solvent A was acetonitrile, solvent B was H₂0, and solvent C was lOOmM NH₄OH (pH 3.85) in H₂0. Separation was carried out at 30°C using Waters Alliance 2695 separation module, excitation 360, emission 425 using Waters 2475 Fluorescence detector.

Other methods include mass spectroscopy, ELISA (with a suitable antibody), TLC and FACS of surface gangliosides using toxins or lectins.

The subject is preferably a mammal, e.g. a human, monkey, mouse, rat, horse, cow, pig, sheep or goat. Most preferably, the subject is a human.

In some embodiments, the subject is older than 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80 or 90 years old. In other embodiments, the subject is 5-100, 10-100, 20-100, 30-100, 40-100, 50-100, 60-100, 70-100, 80-100 or 90-100 years old. In other embodiments, the subject is 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90 or 90-100 years old. In some embodiments, the subject is not a foetus. In some preferred embodiments, the subject is one who has a neurodegenerative disease or disorder (preferably Parkinson’s Disease) which is characterised by the presence of one or more specific brain markers.

In some embodiments, the control may be defined as a non-diseased control, one without a cognitive disorder, one without a neurodegenerative disease or disorder (preferably without Parkinson’s Disease), a typically-developed control or a healthy- aged control.

As used herein, the term “control subject” relates to an individual or group of individuals of the same species as the subject being tested. For example, if the subject is a human, the control will be a human.

The reference level is the Ganglioside Fingerprint value obtained from a control subject or average Ganglioside Fingerprint value obtained from a group of control subjects.

In some embodiments, the Ganglioside Fingerprint value from the subject is significantly lower than the reference level. Significance may be measured by any suitable technique, e.g. Student’s t-test (p<0.01).

In one embodiment, the invention provides a method of obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(b) determining the levels of the corresponding biomarkers in corresponding second biological samples obtained from the subject at a second (later) time point and producing a second Ganglioside Fingerprint value using those levels; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein a decrease in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of a decline in the prognosis of the subject, and wherein an increase in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of an improvement in the prognosis of the subject.

The second time point is after the first time point. The first and second time points may be any suitable time intervals, e.g. at least one week, 1-12 months apart, or 1 , 2, 3, 4 or 5 years apart.

It is increasingly recognised that Parkinson’s Disease is not merely one disease but a general term for a number of associated disorders. The invention may therefore be used to stratify subjects into such associated disorders or Parkinson’s Disease subgroups.

In a further embodiment, therefore, the invention provides a method of classifying a subject into a neurodegenerative disease or disorder subgroup, the method comprising the steps:

The classification in Step (b) may be made using corresponding Ganglioside Fingerprint values from other subjects who have previously been identified as belonging to a specific subgroup.

For example, the neurodegenerative disease or disorder subgroup may be Parkinson’s Disease (PD), idiopathic Parkinson’s Disease, Vascular parkinsonism (arteriosclerotic parkinsonism), Drug-induced parkinsonism, Dementia with Lewy Bodies (DLB), Multiple system atrophy (MSA), Progressive supranuclear palsy (PSP), Normal pressure hydrocephalus, and Various tremors (including essential tremor) and Wilson’s disease. Parkinson’s Disease subjects may be classified as being sporadic PD, idiopathic PD, genetic PD or non-genetic PD. The classification of the subjects may also be used to select subjects for clinical trials.

The Ganglioside Fingerprint value of a subject provides a parameter which may be used to quantify the severity of a neurodegenerative disease or disorder in that subject. This value may therefore be used to determine whether or not a particular drug is having a beneficial effect on the treatment of the subject.

In a further embodiment, therefore, the invention provides a method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(b) determining the levels of the corresponding biomarkers in corresponding second biological samples obtained from the subject at a second time point and producing a second Ganglioside Fingerprint value using those levels; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein the drug has been administered to the subject in the interval between the first and second time points, and wherein a decrease in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of a lack of efficacy of the drug, and wherein an increase in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of the efficacy of the drug.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent which increases the levels of one or more a-series or b-series gangliosides in the central nervous system of the subject. Suitable agents for use in this regard include sialidases and various ganglioside synthase enzymes, e.g. GM1 synthase. Neither of these enzymes, on their own, are capable of crossing the blood-brain barrier. However, the enzymes could be introduced into the subject by gene therapy via the subject’s CNS.

In some embodiments, therefore, the agent comprises a nucleic acid vector encoding a sialidase and/or a ganglioside synthase, preferably wherein the agent is administered to the CNS of the subject.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject which comprises, inter alia, administering a treatment appropriate for treating the neurodegenerative disease or disorder to the subject. Many treatments of neurodegenerative diseases or disorders (e.g. PD) are known in the art. These include administering to the subject a medicament selected from the group consisting of Levodopa, COMT inhibitors (e.g. Tolcapone, entacapone), dopamine agonists (e.g. bromocriptine, pergolide, pramipexole, ropinirole, piribedil, cabergoline, apomorphine and lisuride), MAO-B inhibitors (e.g. safinamide, selegiline and rasagiline) and other drugs such as amantadine, anticholinergics, Doxepin and rasagline.

Rapid eye movement (REM) sleep behaviour disorder (RBD) is a parasomnia which involves acting out dreams and abnormal movements during REM sleep stage. RBD patients have an 80-90% risk of conversion to a synucleinopathy disorder (e.g. a neurodegenerative disease or disorder, particularly PD or dementia with Lewy bodies (DLB)) over 14 years from the time of RBD diagnosis (26-28). The inventors have found that levels of certain gangliosides and glycosphingolipids are significantly reduced in RBD subjects compared to control subjects and to PD subjects.

In a further aspect, therefore, the invention provides a method of obtaining an indication of the risk of a REM sleep behaviour disorder (RBD) subject of developing a neurodegenerative disease or disorder, the method comprising the steps: (a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject, and producing a first Ganglioside/Glycosphingolipid Fingerprint value using those levels;

(b) comparing the first Ganglioside/Glycosphingolipid Fingerprint value with a second Ganglioside/Glycosphingolipid Fingerprint value which has been produced using:

(ii) reference levels of the corresponding biomarkers from corresponding biological samples; wherein the one or more biomarkers are a-series gangliosides or globo-series glycosphingolipids, wherein the biological samples are samples of serum, plasma or cerebrospinal fluid, and wherein a decrease in the first Ganglioside/Glycosphingolipid Fingerprint value compared to the second Ganglioside/Glycosphingolipid Fingerprint value is indicative of an increased risk of the subject developing a neurodegenerative disease or disorder, and wherein an increase in the first Ganglioside/Glycosphingolipid Fingerprint value compared to the second Ganglioside/Glycosphingolipid Fingerprint value is indicative of a decreased risk of the subject developing a neurodegenerative disease or disorder.

In some embodiments, the biomarkers are preferably one or more a-series gangliosides selected from GM3, GM2, GM1a and GDIa.ln other embodiments, the biomarkers are preferably one or more a globo-series glycosphingolipids selected from Gb3 and Gb4.

The invention relates to the levels of certain biomarkers in biological samples and the association of these levels with neurodegenerative diseases or disorders, preferably Parkinson’s disease. This knowledge may therefore be used to screen for agents which - directly or indirectly - increase the levels of these biomarkers, thus providing agents which are useful in the treatment of neurodegenerative diseases or disorders, preferably, Parkinson’s disease. The invention therefore also provides a method of screening for agents for preventing and/or treating a neurodegenerative disease or disorder, the method comprising the steps:

(a) screening for agents which specifically promote or enhance expression of a- series gangliosides, b-series gangliosides or globo-series glycosphingolipids, wherein agents which are identified are ones for preventing and/or treating the neurodegenerative disease or disorder.

Most cultured cells express GM3 and GM2, and small amounts of the more complex gangliosides compared to nervous tissue. For example, screening Step (a) could be performed by contacting cells which normally express detectable amounts of a-series gangliosides, b-series gangliosides or globo-series glycosphingolipids with an agent, and determining whether the levels of one or more of those gangliosides or glycosphingolipids which are produced by those cells are increased.

Preferably, the screening method is carried out in vitro. In some embodiments, the screening method additionally comprises the step of combining an identified agent with one or more pharmaceutically-acceptable diluents, excipients or carriers.

The invention also provides an agent which is found using a screening method of the invention.

In yet a further embodiment, the invention provides a method of obtaining an indication of the presence of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(a) determining a first level of ganglioside GM3 in a sample of CSF obtained from the subject; and

(b) comparing the first level of ganglioside GM3 with a second level of ganglioside GM3 which has been obtained from:

(i) a sample of CSF from a control subject, or

(ii) a reference CSF level; wherein an increase in the first CSF level compared to the second CSF level is indicative of the subject having a neurodegenerative disease or disorder.

(a) determining a first level of ganglioside GM3 in a sample of CSF obtained from the subject at a first time point; and

(b) determining a second level of ganglioside GM3 in a sample of CSF obtained from the subject at a second time point; wherein an increase in the second level of ganglioside GM3 compared to the first level of ganglioside GM3 is indicative of a decline in the prognosis of the subject, and wherein a decrease in the second level of ganglioside GM3 compared to the first level of ganglioside GM3 is indicative of an improvement in the prognosis of the subject.

In another embodiment, the invention provides a method of classifying a subject into a neurodegenerative disease or disorder subgroup, the method comprising the steps:

(a) determining the level of ganglioside GM3 in a sample of CSF obtained from the subject; and

(b) classifying the subject as belonging to a neurodegenerative disease or disorder subgroup based on the level of ganglioside GM3 of the subject.

The classification in Step (b) may be made using corresponding GM3 levels from other subjects who have previously been identified as belonging to a specific neurodegenerative disease or disorder subgroup.

For example, the neurodegenerative disease or disorder subgroup may be Parkinson’s Disease (PD), idiopathic Parkinson’s Disease, Vascular parkinsonism (arteriosclerotic parkinsonism), Drug-induced parkinsonism, Dementia with Lewy Bodies (DLB), Multiple system atrophy (MSA), Progressive supranuclear palsy (PSP),

Normal pressure hydrocephalus, and Various tremors (including essential tremor) and Wilson’s disease. Parkinson’s Disease subjects may be further classified as being sporadic PD, idiopathic PD, genetic PD or non-genetic PD. The classification of the subjects may also be used to select subjects for clinical trials.

(a) determining the first level of ganglioside GM3 in a first sample of CSF obtained from the subject at a first time point; and

(b) determining the second level of ganglioside GM3 in a second sample obtained from the subject at a second time point; wherein the drug has been administered to the subject in the interval between the first and second time points, and wherein an increase in the second level of ganglioside GM3 compared to the first level of ganglioside GM3 is indicative of a lack of efficacy of the drug, and wherein a decrease in the second level of ganglioside GM3 compared to the first level of ganglioside GM3 is indicative of the efficacy of the drug.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent which decreases the level of ganglioside GM3 in the central nervous system of the subject.

In some embodiments, the agent is a biosynthesis inhibitor, e.g. miglustat or eliglustat.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of: (a) determining the level of ganglioside GM3 in a sample of CSF obtained from the subject; (b) comparing the level of ganglioside GM3 with a reference level; and

(c) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject if the level of ganglioside GM3 is above the reference level, thereby treating the neurodegenerative disease or disorder in the subject.

In another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of: (a) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject, wherein, prior to administration, the level of ganglioside GM3 in a sample of CSF obtained from the subject, has been determined to be above a reference level.

In yet another embodiment, the invention provides a method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) receiving a level of ganglioside GM3 from a sample of CSF obtained from the subject; and

(b) identifying the subject as having a level of ganglioside GM3 above a reference level, thereby providing an indication of a neurodegenerative disease or disorder in the subject, and administering a treatment appropriate for treating the neurodegenerative disease or disorder to the subject.

In yet another embodiment, the invention provides a method of screening for agents for preventing and/or treating a neurodegenerative disease or disorder, the method comprising the steps:

(a) screening for agents which specifically inhibit or reduce expression of ganglioside GM3, wherein agents which are identified are ones for preventing and/or treating the neurodegenerative disease or disorder.

For example, screening Step (a) could be performed by contacting cells which normally express detectable amounts of GM3 with an agent, and determining whether the levels of GM3 which are produced by those cells are decreased. In some embodiments, the screening method additionally comprises the step of combining an identified agent with one or more pharmaceutically-acceptable diluents, excipients or carriers.

The disclosure of each reference set forth herein is specifically incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Series of complex GSLs in the biosynthetic pathway of GSLs. GSLs are classified into lacto-series, globo-series and ganglio-series GSLs. Gangliosides are further divided into o-series, a-series and b-series gangliosides. Abbreviations: Cer: ceramide, GlcCer: glucosylceramide, LacCer: lactosylceramide.

Figure 2: Significant increase in LacCer and GM3 levels, but significant decrease in complex ganglioside levels in CSF of PD patients. Ante-mortem CSF from control subjects (n=15) and age-matched PD patients (n=28) was used to determine LacCer (A), GA2 (B), GM3 (C), GM2 (D), GD3 (E), GM1a (F), GD1a (G), GD1b (H), and GT1 b (I) levels with NP-HPLC (* = p<0.05, ** = p<0.01 , *** = p<0.001 , unpaired t-test). (J) Total ganglioside levels (sum of GM1a, GD1a, GD1b and GT1b) in ante-mortem CSF from control subjects and PD patients (** = p<0.01 , unpaired t-test). Data are presented as mean ± SD.

Figure 3: Receiver Operating Characteristic (ROC) curve assessment of the utility of ganglioside levels in CSF of PD patients as possible biomarkers. Comparison of PD patients (n=30) and age-matched controls (n=15) using GM1a (A), GD1a (B), GD1b (C), GT1 b (D) and total ganglioside (E) levels in CSF as biomarkers. The dashed line represents the line of no discrimination. AUC = Area under curve.

Figure 4: Significant reduction in GM1a and GD1a levels in serum from PD patients and significant reduction in all measured glycosphingolipids, except GlcCer, in serum from RBD patients. Levels of GlcCer (A), LacCer (B), Gb3 (C), Gb4 (D), GM3 (E), GM2 (F), GM1a (G) and GD1a (H) were determined in serum samples from control subjects (n=15), PD patients (n=30) and age-matched RBD patients (n=30) with NP-HPLC (* = p<0.05, ** = p<0.01 , *** = pO.001 , **** = p<0.0001 , one-way ANOVA). Data are presented as mean ± SD.

Figure 5: Receiver Operating Characteristic (ROC) curve assessment of the utility of ganglioside levels in serum of PD patients as possible biomarkers. Comparison of PD patients (n=30) and age-matched controls (n=15) using GM1a (F) and GD1a (G) levels in serum as biomarkers. The dashed line represents the line of no discrimination. AUC = Area under curve.

EXAMPLES

The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Materials and Methods

For biomarker studies, frozen ante-mortem cerebrospinal fluid (CSF) and serum samples from control subjects and PD patients were provided by the Oxford Parkinson’s Disease Centre (OPDC; Oxford, UK). Ante-mortem CSF of control subjects (n=15, mean age: 66 years) and age-matched PD subjects (n=28, mean age: 64 years) was used for GSL analysis (Table 1).

Serum samples from patients at risk of developing PD (prodromal PD phase), diagnosed with rapid eye movement (REM) sleep behaviour disorder (RBD), were provided. Serum of control subjects (n=15, mean age: 66 years), PD patients (n=30, mean age: 64 years) and RBD patients (n=30, mean age: 64 years) were used for GSL analysis (Table 1).

Table 1. Parkinson’s disease, RBD and control case information from ante-mortem CSF and serum received from OPDC.

RBD = REM sleep behaviour disorder. Only serum was available for RBD cases. Data summarised as mean ± SD.

GlcCer and GSL analysis with NP-HPLC

GlcCer and downstream GSLs were analysed essentially as described by Neville and co-workers (25). Lipids from tissue homogenates or body fluids were extracted with chloroform and methanol overnight at 4°C. The GSLs were then further purified using solid-phase C18 columns (Telos, Kinesis, UK). After elution, the GSL fractions were split in half, dried down under a stream of nitrogen at 42°C and treated with either Cerezyme® (Genzyme, Cambridge, MA, USA) to obtain glucose from GlcCer or recombinant ceramide glycanase (rEGCase, prepared by Genscript and provided by Orphazyme, Denmark) to obtain oligosaccharides from more complex GSLs. The liberated glucose and free glycans were then fluorescently-labelled with anthranillic acid (2AA). To remove excess 2AA label, labelled glycans were purified using DPA-6S SPE columns (Supelco, PA, USA). Purified 2AA-labelled glucose and 2AA-labelled oligosaccharides were separated and quantified by normal-phase high-performance liquid chromatography (NP-HPLC) as previously described (25). The NP-HPLC system consisted of a Waters Alliance 2695 separations module and an in-line Waters 2475 multi l-fluorescence detector set at Ex A360 nm and Em A425 nm. The solid phase used was a 4.6 ^c 250 mm TSK gel-Amide 80 column (Anachem, Luton, UK). A 2AA-labelled glucose homopolymer ladder (Ludger, UK) was included to determine the glucose unit values (GUs) for the HPLC peaks. Individual GSL species were identified by their GU values and quantified by comparison of integrated peak areas with a known amount of 2AA-labelled BioQuant chitotriose standard (Ludger, UK). Results for tissue homogenates were normalised to protein content, determined by the bicinchoninic acid (BCA) assay.

Statistical analysis

All statistical analyses were performed with GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA). Unpaired Student’s t-test was used to compare two groups and one way or two-way ANOVA followed by post-hoc tests (as appropriate) was used to compare multiple groups. Correlations were analysed with Pearson correlation analysis.

Example 2: GSL biomarkers in cerebrospinal fluid from PD patients

GSLs in cerebrospinal fluid (CSF) from control subjects and PD patients were quantified as potential biomarkers. Ante-mortem CSF samples from control subjects (n=15) and age-matched PD subjects (n=28) were provided by the Oxford Parkinson’s Disease Centre (OPDC; Oxford, UK). The pattern of GSLs in CSF was different and more complex than the GSL pattern in the brain.

Ante-mortem CSF displayed a large LacCer peak and GA2 peak (o-series), with prominent peaks of GM2, GM1 a, GD1 a, GD1 b and GT 1 b (a-series & b-series), but small peaks of GM3 and GD3 (precursors of a-series and b-series).

Owing to problems with the GlcCer digestion (inhibitory activity of CSF-derived lipids against Cerezyme), measurements of GlcCer in CSF were not possible with the NP- HPLC method. However, more complex GSLs could still be measured. LacCer levels in ante-mortem CSF of PD patients were significantly increased in comparison with age- matched control subjects (21.8% increase, Fig. 2k). There were no changes detected in GA2 levels in CSF of PD patients compared to control subjects (Fig. 2B). However, a significant increase in GM3 levels and a significant decrease in GM2 levels was found in ante-mortem CSF of PD patients compared to controls (GM3: 40.2% increase; GM2: 22.6% reduction; Figs. 2C, 2D). Furthermore, a significant decrease in GD3 levels was observed in CSF of PD patients compared to age-matched control subjects (33.0% reduction, Fig. 2E). We also analysed levels of more complex gangliosides of the a- series and b-series, GM1a, GD1a, GD1b and GT1b. A decrease in GM1a levels was observed in ante-mortem CSF of PD patients compared to age-matched control subjects, although it was not significant (17.4% reduction, Fig. 2F). However, a significant decrease in GD1a levels was found in CSF of PD patients compared to controls (37.6% reduction, Fig. 2G). In addition, gangliosides GD1b and GT1b were both significantly reduced in ante-mortem CSF of PD patients in comparison to control subjects (GD1b: 41.6% reduction; GT1b: 51.3% reduction; Figs. 2H, 2I). Consequently, in ante-mortem CSF of PD patients, complex ganglioside levels (sum of GM1a, GD1a, GD1b and GT1b) were significantly decreased to 61.4% of age-matched control subjects (Fig. 2J). Receiver Operating Characteristic (ROC) curve assessments to evaluate CSF ganglioside levels as PD biomarkers can be found in Fig. 3.

Example 3: GSL biomarkers in serum from PD patients and RBD patients

We analysed GSLs in serum from control subjects (n=16) and age-matched PD patients (n=30) (provided by OPDC, Oxford, UK) in the search for possible GSL biomarkers. The pattern of GSLs in serum was unique: GM3 (a precursor for a-series gangliosides) was the most prominent, with high levels of LacCer, Gb3 and Gb4 (globo-series), and low levels of GM2, GM1a, and GD1a (a-series).

No differences in levels of GlcCer and LacCer were detected in serum of PD patients compared to age-matched control subjects (Figs. 4A, 4B). No changes in downstream Gb3 or Gb4 levels (globo-series) were observed in serum of PD patients in comparison with control subjects (Figs. 4C, 4D). No significant change in GM3 levels was found, but a trend towards a reduction in GM2 levels (a-series) was observed in serum from PD patients compared to controls (GM3: 8.3% reduction; GM2: 15.3% decrease; Figs. 4E, 4F). Levels of more complex gangliosides GM1a and GD1a were significantly reduced in serum from PD patients in comparison to serum from age-matched control subjects (GM1a: 22.6% reduction; GD1a: 19.8% decrease; Figs. 4G, 4H). ROC curve assessments of the utility of serum ganglioside levels as possible PD biomarkers are given in Fig. 5.

RBD patients

GSLs in serum from patients at risk of developing PD (prodromal PD phase), diagnosed with REM sleep behaviour disorder (RBD, n=30, provided by the OPDC, Oxford, UK), were quantified. There were no differences in GlcCer levels (Fig. 4A), but a reduction in LacCer levels was detected in serum of RBD patients in comparison to serum from control subjects and PD patients (14.8% reduction, Fig. 4B).

Furthermore, a significant decrease in Gb3 and Gb4 levels (globo-series) was observed in serum of RBD patients in comparison to PD patients (Gb3: 25.8% decrease to controls, 22.8% decrease to PD; Gb4: 15.6% decrease to controls, 15.3% decrease to PD; Figs. 4C, 4D).

A significant reduction in GM3 and GM2 levels was found in serum of RBD patients compared to serum samples from controls and PD patients (GM3: 24.4% decrease to controls, 17.6% decrease to PD; GM2: 40.7% decrease to controls, 30.0% decrease to PD; Figs. 4E, 4F).

Levels of GM1a as well as GD1a were significantly reduced in serum from RBD patients in comparison to serum from age-matched control subjects, but less so compared to PD patients (GM1a: 36.4% decrease to controls, 17.8% decrease to PD; GD1a: 27.6% decrease to controls, 9.7% decrease to PD; Figs 4G, 4H).

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Claims

1 . A method of obtaining an indication of the presence of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(ii) reference levels of the corresponding biomarkers from corresponding biological samples; wherein the one or more biomarkers are a-series (preferably GM1a) or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid (preferably serum), and wherein a decrease in the first Ganglioside Fingerprint value compared to the second Ganglioside Fingerprint value is indicative of the subject having a neurodegenerative disease or disorder.

2. A method of obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(b) determining the levels of the corresponding biomarkers in corresponding second biological samples obtained from the subject at a second time point and producing a second Ganglioside Fingerprint value using those levels; wherein the one or more biomarkers are a-series or b-series gangliosides, wherein the biological samples are samples of serum, plasma or of cerebrospinal fluid, wherein a decrease in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of a decline in the prognosis of the subject, and wherein an increase in the second Ganglioside Fingerprint value compared to the first Ganglioside Fingerprint value is indicative of an improvement in the prognosis of the subject.

3. A method of classifying a subject into a neurodegenerative disease or disorder subgroup, the method comprising the steps:

4. A method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder in a subject, the method comprising the steps:

5. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent which increases the levels of one or more a-series or b-series gangliosides in the central nervous system of the subject.

6. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(b) comparing the Ganglioside Fingerprint value with a reference level; and

7. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject, wherein, prior to administration, a Ganglioside Fingerprint value which was produced using the levels of one or more biomarkers in one or more biological samples obtained from the subject, has been determined to be below a reference level, wherein the biological samples were samples of serum, plasma or of cerebrospinal fluid, and wherein the one or more biomarkers were a-series or b-series gangliosides.

8. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) receiving a Ganglioside Fingerprint value produced from the levels of one or more biomarkers in one or more biological samples obtained from the subject, wherein the biological samples were samples of serum, plasma or of cerebrospinal fluid, and wherein the one or more biomarkers were a-series or b- series gangliosides,

9. A method of obtaining an indication of the risk of a REM sleep behaviour disorder (RBD) subject of developing a neurodegenerative disease or disorder, the method comprising the steps: (a) determining the levels of one or more biomarkers in one or more biological samples obtained from the subject, and producing a first Ganglioside Fingerprint value using those levels;

10. A method of screening for agents for preventing and/or treating a neurodegenerative disease or disorder, the method comprising the steps:

11. A method as claimed in any one of the preceding claims, wherein the neurodegenerative disease or disorder is selected from the group consisting of Parkinson’s Disease (PD), idiopathic Parkinson’s Disease, Vascular parkinsonism (arteriosclerotic parkinsonism), Drug-induced parkinsonism, Dementia with Lewy Bodies (DLB), Multiple system atrophy (MSA), Progressive supranuclear palsy (PSP),

Normal pressure hydrocephalus, Various tremors (including essential tremor) and Wilson’s disease, preferably Parkinson’s Disease (PD) or Dementia with Lewy Bodies (DLB).

12. A method as claimed in any one of the preceding claims, wherein the a-series gangliosides are GM1a, GM3, GM2 and GD1a; preferably, GM2, GM1a and/or GD1a; and more preferably, GM1a and/or GD1a.

13. A method as claimed in any one of the preceding claims, wherein the b-series gangliosides are GD3, GD2, GD1b, GT1b and GQ1b; preferably, GD3, GD1b and/or GT1b; and more preferably, GD 1b and/or GT1b.

14. A method as claimed in any one of the preceding claims, wherein:

(i) the biomarker is GM2 and the biological sample is CSF;

(ii) the biomarker is GD1a and the biological sample is CSF;

(iii) the biomarker is GD3 and the biological sample is CSF;

(iv) the biomarker is GD1b and the biological sample is CSF;

(v) the biomarker is GT1b and the biological sample is CSF;

(vi) the biomarker is GM1a and the biological sample is serum or plasma; or

(vii) the biomarker is GD1a and the biological sample is serum or plasma.

15. A method as claimed in claim 9, wherein the biomarkers are one or more globo- series glycosphingolipids selected from Gb3 and Gb4.

16. A method of obtaining an indication of the presence of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

(a) determining a first level of ganglioside GM3 in a sample of CSF obtained from the subject; and (b) comparing the first level of ganglioside GM3 with a second level of ganglioside GM3 which has been obtained from:

(i) a sample of CSF from a control subject, or

17. A method of obtaining an indication of the prognosis of a neurodegenerative disease or disorder in a subject, the method comprising the steps:

18. A method of classifying a subject into a neurodegenerative disease or disorder subgroup, the method comprising the steps:

19. A method of obtaining an indication of the efficacy of a drug which is being used to treat a neurodegenerative disease or disorder in a subject, the method comprising the steps:

20. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising administering to a subject in need thereof an effective amount of an agent which decreases the level of ganglioside GM3 in the central nervous system of the subject.

21 . A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) determining the level of ganglioside GM3 in a sample of CSF obtained from the subject;

(b) comparing the level of ganglioside GM3 with a reference level; and

22. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

(a) administering a treatment appropriate for treating a neurodegenerative disease or disorder to the subject, wherein, prior to administration, the level of ganglioside GM3 in a sample of CSF obtained from the subject, has been determined to be above a reference level.

23. A method of treating a neurodegenerative disease or disorder in a subject, the method comprising the steps of:

24. A method of screening for agents for preventing and/or treating a neurodegenerative disease or disorder, the method comprising the steps: