WO2019175379A1

WO2019175379A1 - Markers of synaptopathy in neurodegenerative disease

Info

Publication number: WO2019175379A1
Application number: PCT/EP2019/056535
Authority: WO
Inventors: Olivia BELBIN; Alberto LLEÓ BISA; Alejandro BAYÉS PUIG; Juan FORTEA ORMAECHEA; Daniel ALCOLEA RODRÍGUEZ
Original assignee: Fundació Institut De Recerca De L'hospital De La Santa Creu I Sant Pau
Priority date: 2018-03-16
Filing date: 2019-03-15
Publication date: 2019-09-19
Also published as: EP3765854A1

Abstract

The present invention provides in vitro methods for the diagnosis and/or prognosis of synaptopathy and/or neurodegeneration in an individual, for deciding whether to initiate a therapeutic intervention, and for monitoring the efficacy of the therapeutic intervention, all based on the determination of the level of product expression of VAMP-2and/or Syntaxin-1.Advantageously, these markers allow the early detection of synaptopathy in diseases such as Alzheimer's disease.

Description

Markers of synaptopathy in neurodegenerative disease.

Technical Field This application claims the benefit of European Patent Application EP18382175.0 filed March 16^th, 2018.

The invention relates to the diagnosis and prognosis of synaptopathy and

neurodegeneration in neurodegenerative and neuropsychiatric disorders. Background Art

The pathological alteration of the structure or function of the synapse, also so-called synaptopathy, is a fundamental process underlying many neurodegenerative and neuropsychiatric disorders.

Neurodegenerative diseases have in common a progressive loss of neurons and synaptic connections, thus resulting in a synapse loss function, which usually occurrs in later life. Neurodegenerative diseases include progressive dementing conditions, such as

Alzheimer's disease (representing the 60-70% of cases) or frontotemporal dementia; movement disorders, exemplified by Parkinson's disease; and a range of other neurological disorders such as Huntington’s disease, and Lewy body diseases.

Alzheimer’s disease mouse models have shown that the synapse is the direct target of the core pathological proteins, b-amyloid (Ab₄₂) peptide and hyper-phosphorylated tau protein (Spires-Jones & Hyman,“The intersection of amyloid beta and tau at synapses in

Alzheimer's disease”, Neuron, 2014, vol. 82, pp. 756-771 ). Furthermore, it has been found that in Alzheimer’s disease patients, synapse loss is directly related to dementia progression (McKhann et al.,“The diagnosis of dementia due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease” Alzheimers Dement, 201 1 , vol 7, pp. 263-269).

Likewise, alpha-synuclein pathology, a hallmark of Parkinson’s disease and Lewy Body diseases, aggregates at the pre-synapse, resulting in a reduced functional connectivity, and, hence, with a loss of synapse function, which correlates with disease progression in Parkinson’s disease patients (Bellucci et al.,“Review: Parkinson’s disease: from synaptic loss to connectome dysfunction”, 2016, Neuropathology and Applied Neurobiology, vol.

42, pp. 77-94). In addition, neuropsychiatric disorders such as schizophrenia and depression are also characterized by synaptopathy. Synaptopathy has been detected in subjects before the onset of depression. In schizophrenia, paradigms used to generate animal models of psychiatric diseases with a development component, such as schizophrenia, result in synaptopathy, and synaptic dysfunctions are detected in preclinical and clinical studies of schizophrenia in human subjects (Calabrese et al.,” Synaptic alterations associated with psychiatric disorders: potential as therapeutic target”, 2016 Expert Opinion on Therapeutic Targets, vol. 20(10), pp.1 195-207)

Taken together, these data strongly support the concept that synaptic dysfunction, here defined as synaptopathy, is an early fundamental event in neurodegenerative and neuropsychiatric disorders, which precedes neuronal death. Neurodegenerative and neuropsychiatric disorders are usually non-symptomatic in the first stages of the diseases and, even after symptoms appear, the severity of symptoms and disease duration can differ substantially between patients, ranging from a few months to several years. As a drug that can slow or stop the progression of the disease would likely be more effective if given prior to the onset of symptoms, identification of the synaptopathy in the first stages of the disease is crucial. Consequently, there is the need of diagnostic tools that allow an early detection of synaptopathy and/or neurodegeneration in individuals suffering, or being suspected of suffering from neurodegenerative or neuropsychiatric disorders and which, at the same time, can be useful in monitoring the patient’s evolution

In addition to the above, there is also the need of a tangible measure of disease progression to modify the medical treatment accordingly, allowing patients, relatives and caregivers to adapt the patients' lifestyle. Up to now, several methods are known in the prior art for determining synaptic function. One of them is based on measuring brain glucose metabolism in living subjects by ¹⁸F- fluoro-D-glucose positron emission tomography as an indirect measure of synaptic activity. In the search for a more direct measure, many researchers have turned to methods based on detecting particular biological markers in the cerebrospinal fluid (CSF). In Alzheimer’s disease, for example, both Ab and tau, either quantified in the CSF or in situ by neuroimaging, can complement a clinical diagnosis of dementia. However, Ab and Tau have limited prognostic value regarding future cognitive decline, and plaques and tangles show poor correlation with cognitive impairment. Although they are excellent diagnostic biomarkers for AD, they provide little information about disease progression. In recent years, some proteins with a synaptic function such as neurogranin (Thorsell et al.,“Neurogranin in cerebrospinal fluid as a marker of synaptic degeneration in

Alzheimer's disease”. Brain Research, 2010, vol. 1362, pp. 13-22) and SNAP-25

(Brinkmalm et al.,“SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer's disease”, Mol Neurodegener, 2014, vol. 9, pp 53) have been detected increased in CSF from Alzheimer’s disease patients in symptomatic stages (i.e., in advance stages of the disease). In spite of the efforts made, however, there is still the need for specific biomarkers that allow the early diagnosis and monitoring of the progression of synaptopathy, in neurodegenerative and neuropsychiatric diseases.

Summary of Invention

The inventors have identified a panel of proteins that is expressed at the human synapse and that is altered in two of the most common neurodegenerative diseases, that are characterized by early synaptopathy and widespread neurodegeneration, as depicted in Examples 1 and 2. In paticular, when cerebroespinal fluid (CSF) samples taken from patients with Alzheimer’s disease (AD) and frontotemporal dementia (FTD) were analysed, Neurexin-2 (Uniprot code: Q9P2S2, NRX2A), Neurexin-3 (Uniprot code:

Q9Y4C0, NRX3A), Thy-1 membrane glycoprotein (Uniprot code: P04216 THY1 ), Vesicle- associated membrane protein 2 (Uniprot code: P63027, VAMP-2), Tenascin-R (Uniprot code: Q92752, TNR), Glutamate receptor 4 (Uniprot code: P48058, GRIA4), Neuroligin-2 (Uniprot code: Q8NFZ4, NLGN2), Glutamate receptor 2 (Uniprot code: P42262, GRIA2), Syntaxin-1 (Uniprot code: P61266, STX1 B), and Calsyntenin-1 (Uniprot code: 094985, CLSTN1 ) were altered compared to matched healthy controls in each condition.

In Alzheimer’s disease (AD), the CSF level of all the markers of the invention were quantified in two independent clinical cohorts comprising all stages of the AD clinical spectrum; pre-clinical AD stage 1 (Stagel ), pre-clinical AD stage 2 (Stage2), prodromal AD, and dementia, Alzheimer’s-type (DAT). Controls were cognitively normal without underlying amyloidosis or neurodegeneration. As it is shown in Table 1 , the proteins were altered across the AD continuum in a biphasic manner.

On the one hand, the markers of the invention were decreased in preclinical AD stage 1 subjects compared with controls. These individuals were non-symptomatic without widespread neurodegeneration. Therefore, it can be concluded that the markers of the invention reflect underlying active synaptic pathological changes (synaptopathy event) at very early stages before widespread neurodegeneration has occurred. Identification of synaptopathy in asymptomatic individuals for therapeutic intervention increases the probability that a therapeutic intervention will be effective.

On the other hand, it was also found that these markers increased with the progress of the disease to the symptomatic stages (prodromal and DAT), compared to controls (see Table 1 and FIG. 1 ). Without being bound to the theory, it is believed that this remarkable increase at symptomatic stages is due to the activation of neurodegenerative

mechanisms (i.e., the neurodegeneration process has started). Once the patient is at this stage, monitoring the disease progression can be informative for assessing the prognosis of the patient. .

Therefore, these markers are highly sensitive to detect a synaptopathy event in an early stage (decreased level vs control) but also the appearance and progression of neurodegeneration (increased level vs control).

Furthermore, as it is shown in Example 2, the markers of the invention were increased in patients with the neurogenerative disease frontotemporal dementia (FTD), who already have advanced synaptopathy and widespread neurodegeneration compared to controls (see Table 3).

Altogether, the inventors have identified a panel of proteins informative of the

synaptopathy but also of the neurodegeneration, being useful in the diagnostic and prognosis of neurodegenerative diseases such as Alzheimer disease and frontotemporal dementia, which are also characterized by an early and profound synaptopathy.

Thus, in a first aspect, the present invention provides an in vitro method of diagnosis and/or prognosis of synaptopathy in an individual, the method comprising:

(i) determining the level of the expression product of:

(a) Neurexin-2A and/or Neurexin-3A in an isolated test sample from the individual; or, alternatively,

(b) one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated CSF sample from the individual; or alternatively,

(c) Neurexin-2A and/or Neurexin-3A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated test sample from the individual; and

(ii) comparing it with a reference value, or alternatively (ibis) determining the level of the expression product of one or more of Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and/or Calsyntenin-1 in an isolated test sample from the individual;

(iibis) determining the level of expression product of a neurodegenerative protein marker other than the markers referred in step ibis;

(iiibis) normalizing the level of expression product(s) determined in step (ibis) with respect to the marker level determined in step (iibis); and

(ivbis) comparing the value obtained in (iiibis) with a reference value, wherein if the level determined in step (i) or (iiibis) is lower than the reference value, this is indicative that the individual suffers synaptopathy. In an embodiment, it is determined, in step (i), the level of expression product of Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood.

Additionally, the inventors found that the markers of the invention were expressed in human synapsis. As shown below, the expression of the markers at the human synapse was assessed in human autopsy tissue taken from the frontal cortex, and it was concluded that markers were found co-localized with established markers of the pre- synapse (Synaptophysin-38) and the post-synapse (PSD-95) compartments. Moreover, the expression of the markers was elevated in synapse-enriched fractions compared with the whole tissue homogenate.

In addition, in a second aspect, the invention provides an in vitro method of diagnosis and/or prognosis of neurodegeneration in an individual, the method comprising:

(i) determining the level of expression product of:

(ii) comparing it with a reference value, wherein if the level in the test sample is higher than the reference value, this is indicative that the individual suffers neurodegeneration. In an embodiment, in step (i) it is determined the level of expression product of Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood.

Since the CSF levels of the markers reflect the extension (i.e., severity) of

neurodegeneration, determining the marker level in a test sample could inform the clinican on the most appropriate therapeutic strategy (evaluation undertaken to assess the results or consequences of management and procedures used in combatting disease in order to determine the efficacy, effectiveness, safety, practicability, etc., of these interventions in individual cases or series).

Thus, in a third aspect, the present invention provides an in vitro method of deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, wherein the method comprises the steps of:

(1 ) determining the level of the expression product:

(2) comparing it with a reference value, or alternatively

(I bis) determining the level of the expression product of Neurexin-2A, Neurexin-3A Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and/or Calsyntenin-1 , in an isolated test sample from the individual;

(2bis) determining the level of expression product of a neurodegenerative protein marker other than the markers referred in step 1 bis;

(3bis) normalizing the level of expression product(s) determined in step (I bis) with respect to the marker level determined in step (2bis); and

(4bis) comparing the value obtained in (3bis) with a reference value, wherein, if the level in the test sample is different from the reference value, this is indicative that the individual has to start a therapeutic intervention. In one embodiment, in step (1 ) it is determined the level of expression product of Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood.

Furthermore, the markers Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R,

GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and/or Calsyntenin-1 could be used to determine drug efficacy and efficiency in clinical trials targeted towards neurodegenerative and neuropsychiatric disorders. Accordingly, when it is decided that an individual has to initiate a therapeutic intervention because he suffers from, or is suspicious of having, a neurodegenerative or neuropsychiatric disorder, it can be monitored how efficient is the therapeutic intervention using Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and/or Calsyntenin-1 as marker(s): a return to a normal level of these marker(s) (i.e., to the level of a control subject) can indicate that the patient has favourably reacted to the therapeutic intervention and, therefore, said intervention is effective; if the level of the marker(s) does not significantly change, this can indicate that the intervention is not effective.

Therefore, in a fourth aspect, the invention provides an in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy, the method comprising performing step (i), or alternatively steps (ibis) to (iiibis), as defined in the first aspect of the invention before and after starting the therapeutic intervention, wherein:

if the level measured once started the intervention is equal or higher than the level measured before starting the intervention, and lower than the reference value it is indicative that the therapeutic intervention is effective in the treatment of the

synaptopathy. In an embodiment, in step (i) it is determined the level of expression product of Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood.

In addition, in a fifth aspect, the invention also provides an in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering neurodegeneration, the method comprising performing step (i) as defined in the second aspect of the invention before and after starting the therapeutic intervention, wherein: if the level measured once started the intervention is lower than the level measured before starting the intervention, it is indicative that the therapeutic intervention is effective in the treatment of the neurodegeneration.

In a sixth aspect, the present invention provides the use of: - Neurexin-2A and/or Neurexin-3A as: diagnostic or prognostic marker(s) of synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or

neurodegeneration; or alternatively,

- one or more of Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 in a CSF sample as: diagnostic or prognostic marker(s) of

synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or neurodegeneration in a CSF sample isolated from the individual; or alternatively,

- Neurexin-2A and/or Neurexin-3A in combination with one or more of the markers: Thy-1 ,

VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, and Syntaxin-1 , Calsyntenin-1 , as: diagnostic or prognostic marker(s) of synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or neurodegeneration. Preferably, it is provided the use of Syntaxin-1 , VAMP-2 or both as diagnostic or prognostic marker(s) of synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or neurodegeneration in a CSF sample isolated from the individual.

Importantly, the level of the protein markers forming part of the methods and uses provided by the present invention can be quantified by easy and low cost methods, such as immunochemistry, chemiluminescent assay, or ELISA, platforms which are widely available in hospitals. Consequently, these protein biomarkers can be easily implemented as routine clinical diagnostic and/or prognostic kits with reduced costs for the health system.

Therefore, in a seventh aspect, the invention provides a kit for the diagnosis and/or prognosis of synaptopathy and/or neurodegeneration in an individual, or for deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, the kit comprising a solid support and means for detecting the level of expression of one or more of the following proteins Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 and optionally means for detecting the level of expression of Nf-L. In an embodiment, the kit further comprising means for detecting the level of expression of one or more of Neurexin-2A, Neurexin-3A, Thy-1 , Tenascin-R, GluR4, Neuroligin-2, GluR2, and Calsyntenin-1.

The invention further provides the use of a kit for the diagnosis and/or prognosis of synaptopathy and/or neurodegeneration in an individual, or for deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, the kit comprising a solid support and means for detecting the level of expression of one or more of VAMP-2 Syntaxin-1 , and means for detecting the level of expression of Nf-L.

Finally, in an eighth aspect, the invention provides an algorithm for carrying out any of the methods as defined above, in which after the determination of the level of expression of one or more of the proteins for the diagnosis and/or for the prognosis of synapse loss condition, said level(s) are given a value and/or a score, and optionally are computed in a mathematical formula to obtain a computed value; wherein in function of the said level(s), score(s) and or computed value(s), a decision is taken regarding the diagnosis of an underlying synaptopathy, the appropriate therapeutic intervention, efficacy of the treatment, and whether the synaptopathy is progressing or has halted.

Brief Description of Drawings

FIG. 1 Synaptic Panel protein levels in the CSF of FTD patients. The log2 fold-change (+/- standard error; SE) in CSF levels of the 10 synaptic Panel proteins (A) and for the synaptic protein:Nf-L ratios (B) are plotted for frontotemporal dementia patients versus cognitively normal controls. For ease of interpretation, the natural values are labelled on the y-axis on a log2 scale. The linestyle of the error bars were determined by p-value cut- offs for pair-wise group comparisons.

Fig. 2. Expression of the panel proteins at the human cortical synapse. Mean fold- enrichment plotted for each panel protein in homogenate (H), synaptosome-enriched (S) and PSD-enriched (PSD) fractions taken from post-mortem human cortex (n=6). S/H and PSD/H; intensity in S or PSD fractions relative to H for the same sample. H/H; intensity in the H fraction for each sample relative to the mean intensity in the H fraction across all samples. Enrichment of the pre- (synaptophysin) and post (PSD-95) markers are also shown. Degrees of freedom (df), F statistic and p-values for the ANOVA are shown at the top of each plot and Dunnett’s pvalues are shown for significant pair-wise comparisons (a=0.05).

Detailed description of the invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

The present invention provides in vitro methods for the diagnosis and prognosis of synaptopathy and/or neurodegeneration, for deciding whether to initiate a therapeutic intervention, and for monitoring the efficacy of the therapeutic intervention, all based on the determination of the level of product expression of Neurexin-2A, and/or Neurexin-3A in an isolated test sample from the individual, or, alternatively, determining one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated CSF sample from the individual, or alternatively, determining Neurexin-2A, and/or Neurexin-3A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated test sample from the individual. In a preferred embodiment of the methods and uses provided by the present invention, step (i) or (1 ) is performed determining the level of expression product of Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood. .

In the present invention, the term“synaptopathy” refers to any neurological condition that involves an alteration of synaptic structure and/or function or loss of synapses. The synapse is a structure that permits a neuron (or nerve cell) to pass an electrical or chemical signal to another neuron or to the target efferent cell. At a synapse, the plasma membrane of the signal-passing neuron (the presynaptic neuron) comes into close apposition with the membrane of the target (postsynaptic) cell. Both the presynaptic and postsynaptic sites contain extensive arrays of a molecular machinery that link the two membranes together and carry out the signalling process. That is, synapses are structures that contain the molecular machinery to release transmitter and detect it using cognate postsynaptic receptors that produce downstream ionic or metabotropic signalling. The highly organized synaptic structure arises as a consequence of protracted

developmental programs in the course of which synapses are assembled and stabilized in the mature brain while retaining sufficient flexibility to exhibit plasticity of structure and function. Thus, in the present invention, synaptopathy is understood as the malfunction in any of the processes or structures that sustain the synapse function or a reduction in synapse number. In one preferred embodiment, the marker providing valuable information of synaptopathy is Syntaxin-1.

As shown in Examples 1 , the inventors have identified a panel of markers which provide valuable information of dementia progression, thus becoming useful for monitoring the disease. Thus, in a particular embodiment of the methods of the invention, optionally in combination with any of the embodiments provided below, the individual is suspected of suffering or suffers from a condition which includes neurodegenerative and

neuropsychiatric disorders and their pre-clinical stages. In a further embodiment, optionally in combination with any embodiment provided below, the individual is suspected of suffering or suffers from a condition which is a neurodegenerative disease or a preclinical stage thereof. In a further embodiment, optionally in combination with any of the embodiments provided below, the individual is suspected of suffering or suffers from a condition selected from the group consisting of Alzheimer disease, frontotemporal dementia, Huntington's disease, Parkinson's diseases; vascular dementia, Lewy Body diseases, Creutzfeldt-Jakob disease, mixed dementia, normal pressure hydrocephalus, schizophrenia, depression and a preclinical stage thereof. In another embodiment of the invention, optionally in combination with any embodiment provided below, the individual is suspected of suffering or suffers from a condition which is Alzheimer disease or a preclinical stage thereof selected from the group consisting of preclinical AD stage 1 (cognitively normal with underlying amyloidosis; PC1 ), preclinical AD stage 2 (cognitively normal with underlying amyloidosis and neurodegeneration; PC2), prodromal AD (patients with amnesic mild cognitive impairment and underlying amyloidosis and

neurodegeneration; PROD), and dementia Alzheimer-type (patients with dementia and underlying amyloidosis and neurodegeneration; DAT) , or, alternatively, is a

frontotemporal dementia. In one preferred embodiment, the marker providing valuable information of dementia progression is VAMP-2.

Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. As the most common cause of dementia, AD represents 60% to 80% of dementia cases. The most common early symptom is difficulty in remembering recent events (short-term memory loss). As the disease advances, symptoms can include language difficulties, disorientation (including easily getting lost), mood swings, loss of motivation, neglect of self-care, and behavioural issues. As a person's condition declines, they often withdraw from family and society. Gradually, bodily functions are lost, ultimately leading to death. Although the speed of progression can vary, the average life expectancy following diagnosis is three to nine years. Its cause is poorly understood. About 70% of the risk is believed to be genetic with many genes usually involved. Other risk factors include a history of head injuries, depression, or hypertension. The disease is characterized by extracellular (amyloid plaques) and intracellular (neurofibrillary tangles) protein inclusions in the brain. A probable diagnosis is based on family history of the illness and a combination of cognitive testing, medical imaging and blood tests to rule out other possible causes. There are currently no treatments capable of stopping, reversing or slowing disease progression, though some may temporarily improve symptoms. Affected people increasingly rely on others for assistance, often placing a burden on the caregiver; the pressures can include social, psychological, physical, and economic elements. In 2015, there were approximately 29.8 million people worldwide with AD.

Frontotemporal dementia (FTD) is the clinical presentation of frontotemporal lobar degeneration, which is characterized by progressive neuronal loss predominantly involving the frontal or temporal lobes, and typical loss of over 70% of spindle neurons, while other neuron types remain intact. FTD is the most prevalent early-onset dementia, accounting for 20% of early-onset dementia cases. Signs and symptoms typically manifest in late adulthood, more commonly between the ages of 55 and 65, affecting men and women, equally. Common signs and symptoms include significant changes in social and personal behaviour, apathy, blunting of emotions, and deficits in both expressive and receptive language. Currently, there is no cure for FTD, but there are treatments that help alleviate symptoms.

Huntington's disease (HD), also known as Huntington's chorea, is an inherited disorder that results in death of brain cells. The earliest symptoms are often subtle problems with mood or mental abilities. A general lack of coordination and an unsteady gait often follow. As the disease advances, uncoordinated, jerky body movements become more apparent. Physical abilities gradually worsen until coordinated movement becomes difficult and the person is unable to talk. Mental abilities generally decline into dementia. The specific symptoms vary somewhat between people. Symptoms usually begin between 30 and 50 years of age, but can start at any age. The disease may develop earlier in life in each successive generation. HD affects about 4 to 15 in 100,000 people of European descent. The disease affects men and women equally. There is no cure for HD. Full-time care is required in the later stages of the disease. Treatments can relieve some symptoms and in some improve quality of life.

Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. The symptoms generally come on slowly over time. Early in the disease, the most obvious signs are shaking, rigidity, slowness of movement, and difficulty with walking. Cognitive and behavioural problems may also occur. Dementia is common at advanced stages of the disease. Depression and anxiety are also common occurring in more than a third of people with PD. Other symptoms include sensory, sleep, and emotional problems. Its cause is generally unknown, but believed to involve both genetic and environmental factors. Those with a family member affected are more likely to get the disease themselves. There is also an increased risk in people exposed to certain pesticides and among those who have had prior head injuries. Diagnosis of typical cases is mainly based on symptoms, with tests such as neuroimaging being used to rule out other diseases. PD typically occurs in people over the age of 60, and males are more often affected than females. In 2015, PD affected 6.2 million people and resulted in about 117,400 deaths globally.

Vascular dementia, also known as multi-infarct dementia (MID) and vascular cognitive impairment (VCI), is dementia caused by problems in the supply of blood to the brain, typically a series of minor strokes, leading to progressive cognitive decline. The term refers to a syndrome consisting of a complex interaction of cerebrovascular disease and risk factors that lead to changes in the brain structures due to strokes and lesions, and resulting changes in cognition.

Dementia with Lewy bodies (DLB) is a neurodegenerative disorder characterized by dementia that progresses over a long time-course. Symptoms may include fluctuations in alertness, visual hallucinations, and slowness of movement, trouble walking, and rigidity. The cause is unknown. Typically, no family history of the disease exists among those affected. The underlying mechanism involves the aggregation of phosphorylated alpha- synuclein protein at the neuronal pre-synapse into inclusions termed Lewy bodies. A diagnosis may be suspected based on neurological and neuropsychological evaluation, and blood tests and medical imaging to rule out other possible causes. At present there is no cure. Treatments are supportive and attempt to relieve some of the motor and psychological symptoms associated with the disease. DLB is the most common cause of dementia after Alzheimer's disease and vascular dementia. It typically begins after the age of 50 and men appear to be more commonly affected than women.

Creutzfeldt-Jakob disease (CJD) is a rapidly degenerative brain disorder. Early symptoms include memory problems, behavioural changes, poor coordination, and visual disturbances. Later dementia, involuntary movements, blindness, weakness, and coma occur. About 90% of people die within a year of diagnosis. Onset is typically around 60 years of age. There is no specific treatment for CJD. It affects about one per million people per year.

Mixed dementia (MX) is a condition in which abnormalities characteristic of more than one type of dementia occur simultaneously. In the most common form of mixed dementia, the abnormal protein deposits associated with Alzheimer's disease coexist with blood vessel disturbances linked to vascular dementia. Alzheimer's pathology often co-exist with Lewy bodies. In some cases, a person may have pathology linked to all three conditions— Alzheimer's disease, vascular dementia and dementia with Lewy bodies. Mixed dementia symptoms may vary, depending on the types of pathology involved and the brain regions affected. In many cases, symptoms may be similar to or even indistinguishable from those of Alzheimer's or another type of dementia. In other cases, a person's symptoms may suggest that more than one type of dementia is present.

A definitive diagnosis of mixed dementia is achieved at autopsy. Most individuals whose autopsies show they had mixed dementia were diagnosed with one specific type of dementia during life, most commonly with Alzheimer's disease.

Normal pressure hydrocephalus (NPH) is a brain disorder in which excess cerebrospinal fluid accumulates in the brain's ventricle, causing cognitive and reasoning problems, difficulty walking and loss of bladder control. NPH primarily affects people in their 60s and 70s. There is no single test to determine if someone has normal pressure hydrocephalus. However, brain imaging to detect enlargement of the ventricles, often with magnetic resonance imaging (MRI), plays a key role in diagnosing NPH.

Wernicke-Korsakoff syndrome (WKS) is the combined presence of Wernicke's

encephalopathy (WE) and Korsakoff's syndrome. Due to the close relationship between these two disorders, people with either are usually diagnosed with WKS, as a single syndrome. Korsakoff syndrome is a chronic memory disorder caused by severe deficiency of thiamine (vitamin B-1 ). Korsakoff syndrome is most commonly caused by alcohol misuse, but certain other conditions also can cause the syndrome. Korsakoff syndrome causes problems learning new information, inability to remember recent events and long- term memory gaps. Memory problems may be strikingly severe while other cognitive and social skills are relatively unaffected. For example, individuals may seem able to carry on a coherent conversation, but moments later, be unable to recall that the conversation took place or to whom they were speaking.

Schizophrenia is a mental disorder characterized by abnormal social behaviour and failure to understand what is real. Common symptoms include false beliefs, unclear or confused thinking, hearing voices that others do not, reduced social engagement and emotional expression, and a lack of motivation. Symptoms typically come on gradually, begin in young adulthood, and last a long time. The causes of schizophrenia include environmental and genetic factors. Diagnosis is based on observed behaviour, the person's reported experiences and reports of others familiar with the person. The mainstay of treatment is antipsychotic medication, along with counselling, job training and social rehabilitation. About 0.3-0.7% of people are affected by schizophrenia during their lifetimes. In 2013 there were an estimated 23.6 million cases globally. Males are more often affected, and on average experience more severe symptoms. The average life expectancy of people with the disorder is ten to twenty-five years less than for the general population.

Depression (major depressive disorder or clinical depression) is a common but serious mood disorder. It causes severe symptoms that affect how you feel, think, and handle daily activities, such as sleeping, eating, or working. To be diagnosed with depression, the symptoms must be present for at least two weeks. Some forms of depression are slightly different, or they may develop under unique circumstances, such as: persistent depressive disorder (also called dysthymia), perinatal depression, psychotic depression, seasonal affective disorder, disruptive mood dysregulation disorder (diagnosed in children and adolescents) and premenstrual dysphoric disorder (PMDD). Depression is one of the most common mental disorders in the U.S. Current research suggests that depression is caused by a combination of genetic, biological, environmental, and psychological factors. Depression can happen at any age, but often begins in adulthood. Depression, even the most severe cases, can be treated. The earlier that treatment can begin, the more effective it is. Depression is usually treated with medications, psychotherapy, or a combination of the two. If these treatments do not reduce symptoms, electroconvulsive therapy (ECT) and other brain stimulation therapies may be options to explore.

In the present invention, the term "individual" is intended to include animals which are capable of suffering from or afflicted with a neurodegenerative or neuropsychiatric disorder, including neurological diseases such as Alzheimer's disease, frontotemporal dementia, Huntington's disease, Parkinson's diseases; prion-related disease, vascular dementia, Dementia with Lewy Bodies, mixed dementia, normal pressure hydrocephalus, Wernicke-Korsakoff syndrome, schizophrenia, depression or any disorder involving, directly or indirectly, any of them. Examples of individuals include mammals, e.g., humans, non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In one embodiment, optionally in combination with any embodiment provided above or below, the individual is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from Alzheimer's disease, Alzheimer's disease preclinical stage, Alzheimer's disease- associated dementia or frontotemporal dementia.

The term“diagnosis” is known to the person skilled in the art. As used herein“diagnosis” is understood as becoming aware of a particular medical condition, complication; the determination of the nature of the condition; or the distinguishing of the condition from another. It refers both to the process of attempting to determine or identify the possible condition, and to the opinion reached by this process. A diagnosis, in the sense of diagnostic procedure, can be regarded as an attempt at classification of an individual's condition into separate and distinct categories that allow medical decisions about treatment and prognosis to be made. Subsequently, a diagnostic opinion is often described in terms of a condition.

The diagnostic in vitro method of the second aspect of the invention can be performed with a sample of: (a) an asymptomatic individual which has already been identified as being suspicious of suffering from a neurodegenerative or a neuropsychiatric disorder, (b) an individual already diagnosed of a neurodegenerative or a neuropsychiatric disorder, as a complementary confirmatory diagnostic test or (c) an individual with high risk of suffering a neurodegenerative or a neuropsychiatric disorder.

“Prognosis” as used herein refers to the prediction of the probable progression and outcome of the disease as well as the monitoring of the disease progression. As an illustrative example, in case of dementia prognosis includes: stages of a

neurodegenerative or neuropsychiatric disorder without cognitive decline, with mild cognitive decline, with moderate cognitive decline, with moderately severe cognitive decline, severe cognitive decline, moderate dementia and severe dementia.

In a particular embodiment of the methods of the invention, optionally in combination with any embodiment provided above or below, the test sample is selected from the group consisting of serum, plasma, saliva, pleural or cerebral spinal fluid (CSF), blood, urine, feces, mucus, cell extracts, tissue extracts and pus.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the test sample is a biological fluid selected from CSF, blood, plasma, serum, saliva and urine. Advantageously, the obtaining of these test samples is typically much less invasive and traumatizing than obtaining a solid tissue biopsy sample. In a particular embodiment, optionally in combination with any of the embodiments provided above or below, the test sample is CSF. The CSF is in immediate contact with the nervous system and is readily obtainable using routine means for the skilled person in the art.

Neurexin-2A, also known asNRXN2, NRX2A, Neurexin-2 or KIAA0921 has the Uniprot database accession number Q9P2S2, October 1 , 2000 -v1. This protein is a neuronal cell surface protein that may be involved in cell recognition and cell adhesion, as well as intracellular signalling. Neurexin-3A, also known as NRXN3, NRX3A, Neurexin-3, 14orf60 or KIAA0743 has the Uniprot database accession number Q9Y4C0, March 3, 2009 - v4. It is a neuronal cell surface protein, and as other members of its family, may be involved in cell recognition, cell adhesion and intracellular signalling.

Thy-1 membrane glycoprotein, also known as THY1 , CDw90, Thy-1 antigen or

CD_antigen: CD90, has the Uniprot database accession number P04216, May 2, 2002 - v2. This protein may play a role in cell-cell or cell-ligand interactions during

synaptogenesis and other events in the brain.

VAMP-2, also known as Vesicle-associated membrane protein2, VAMP2, synaptobrevin-2 or SYB2 has the Uniprot database accession number P63027, February 9, 2010-v3. It is involved in the targeting and/or fusion of transport vesicles to their target membrane. Moreover, this protein modulates the gating characteristics of the delayed rectifier voltage- dependent potassium channel KCNB1.

Tenascin-R, also known as TN-R, TNR, Janusin or Restrictin has the Uniprot database accession number Q92752, November 2, 2010 -v3. It is a neural extracellular matrix (ECM) protein involved in interactions with different cells and matrix components. These interactions can influence cellular behaviour by either evoking a stable adhesion and differentiation, or repulsion and inhibition of neurite growth. Binding to cell surface gangliosides inhibits RGD-dependent integrin-mediated cell adhesion and results in an inhibition of PTK2/FAK1 (FAK) phosphorylation and cell detachment. Binding to membrane surface sulfatides results in an oligodendrocyte adhesion and differentiation.

Its interaction with CNTN1 induces a repulsion of neurons and an inhibition of neurite outgrowth. Additionally it may play a crucial role in clustering and regulation of activity of sodium channels at nodes of Ranvier by its interaction with SCN2B. GluR4, also known as Glutamate receptor 4, GRIA4, AMPA-selective glutamate receptor 4, GluR-D or GluA4 has the Uniprot database accession number P48058, December 16, 2008 - v2. It is a receptor for glutamate that functions as ligand-gated ion channel in the central nervous system and plays an important role in excitatory synaptic transmission. L- glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system. Binding of the excitatory neurotransmitter L-glutamate induces a conformation change, leading to the opening of the cation channel, and thereby converts the chemical signal to an electrical impulse. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4 or CACNG7 or CACNG8, shows resensitization, which is characterized by a delayed accumulation of current flux upon continued application of glutamate.

Neuroligin-2, also known as NLGN2 or KIAA1366, has the Uniprot database accession number Q8NFZ4, October 1 , 2002 -v1. This protein is a transmembrane scaffolding protein involved in cell-cell interactions via its interactions with neurexin family members. Mediates cell-cell interactions both in neurons and in other types of cells, such as

Langerhans beta cells. Plays a role in synapse function and synaptic signal transmission, especially via gamma-aminobutyric acid receptors (GABA (A) receptors). It functions recruiting and clustering synaptic proteins. Promotes clustering of postsynaptic GABRG2 and GPHN. Modulates signalling by inhibitory synapses, and thereby plays a role in controlling the ratio of signalling by excitatory and inhibitory synapses and information processing. Required for normal signal amplitude from inhibitory synapses, but is not essential for normal signal frequency. May promote the initial formation of synapses, but is not essential for this. In vitro, triggers the de novo formation of presynaptic structures. Mediates cell-cell interactions between Langerhans beta cells and modulates insulin secretion.

GluR2, also known as Glutamate receptor-2, GRIA2, AMPA-selective glutamate receptor 2, GluR-B, GluR-K2 or GluA2 has the Uniprot database accession number P42262, October 10, 2002 -v3. It is a receptor for glutamate that functions as ligand-gated ion channel in the central nervous system and plays an important role in excitatory synaptic transmission. L-glutamate acts as an excitatory neurotransmitter at many synapses in the central nervous system. Binding of the excitatory neurotransmitter L-glutamate induces a conformation change, leading to the opening of the cation channel, and thereby converts the chemical signal to an electrical impulse. The receptor then desensitizes rapidly and enters a transient inactive state, characterized by the presence of bound agonist. In the presence of CACNG4 or CACNG7 or CACNG8, shows resensitization, which is characterized by a delayed accumulation of current flux upon continued application of glutamate.

Syntaxin-1 , also known as Syntaxin-1 B1 , or Syntaxin-1 B2 has the Uniprot database accession number P61266, May 10, 2004 -v1. This protein is potentially involved in docking of synaptic vesicles at presynaptic active zones. In addition, it may mediate Ca2+- regulation of exocytosis acrosomal reaction in sperm.

Calsyntenin-1 , also known as CLSTN1 , Alcadein-alpha, Alzheimer-related cadherin-like protein, Non-classical cadherin XB31 alpha or SAIc-alpha has the Uniprot database accession number 094985, May 1 , 1999 - v1. This protein induces KLC1 association with vesicles and functions as a cargo in axonal anterograde transport. Complex formation with APBA2 and APP, stabilizes APP metabolism and enhances APBA2-mediated

suppression of beta-APP40 secretion, due to the retardation of intracellular APP maturation. In complex with APBA2 and C99, a C-terminal APP fragment, abolishes C99 interaction with PSEN1 and thus APP C99 cleavage by gamma-secretase, most probably through stabilization of the direct interaction between APBA2 and APP. The intracellular fragment AlcICD suppresses APBB1 -dependent transactivation stimulated by APP C- terminal intracellular fragment (AICD), most probably by competing with AICD for APBB1- binding. May modulate calcium-mediated postsynaptic signals.

Uniprot database accession numbers and information provided above for the biomarkers object of the invention, correspond to versions accessible on December 2017.

In the present invention, the terms“level” and“amount” are interchangeably used and have the same meaning.

In the present invention, the term“expression product” of a marker is to be understood as encompassing the mRNA product, full-length protein product or a proteotypic fragment thereof, depending on the detection tecnique to be used. Thus, when it is determined the “level of the expression product”, it can refer to the level of mRNA, or to the level of the full-length protein or to the level of a proteotypic fragment.

In one embodiment of any of the methods of the present invention, optionally in combination with any of the embodiments provided above or below, the“expression product” is a protein fragment. In another embodiment, this protein fragment has a length from 5 to 35 amino acids. These protein fragments can be obtained by, for instance, proteolysis, using commercial reagents.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment provided above or below, the method comprises determining the level of expression of one or more Neurexin-2A protein fragments (same or different), said fragments comprising the amino acid sequence SEQ ID NO: 1

(corresponds to the amino acid residues 161-170 of the whole sequence provided in Uniprot accession number: Q9P2S2), SEQ ID NO: 2 (corresponds to the amino acid residues 184-198 of the whole sequence provided in Uniprot accession number:

Q9P2S2), and/or SEQ ID NO: 3 ((correspond to the amino acid residues 478-485 of the whole sequence provided in Uniprot accession number: Q9P2S2). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Neurexin-2A protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 1 , SEQ ID NO: 2, and/or SEQ ID NO: 3, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Neurexin-2A protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO: 2, and/or SEQ ID NO: 3, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Neurexin-2A protein fragments of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of Neurexin-3A protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 4 (corresponds to the amino acid residues 49-56 of the whole sequence provided in Uniprot accession number: Q9Y4C0), SEQ ID NO: 5

(corresponds to the amino acid residues 293-301 of the whole sequence provided in Uniprot accession number: Q9Y4C0), and/or SEQ ID NO: 6 (corresponds to the amino acid residues 537-549 of the whole sequence provided in Uniprot accession number: Q9Y4C0). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Neurexin-3A protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of one or more Neurexin-3A protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 6, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Neurexin-3A protein fragments of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of Thy-1 protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 7 (corresponds to the amino acid residues 22-35 of the whole sequence provided in Uniprot accession number: P04216), SEQ ID NO: 8 (correspond to the amino acid residues 61-75 of the whole sequence provided in Uniprot accession number: P04216), and/or SEQ ID NO: 9 (correspond to the amino acid residues 88-97 of the whole sequence provided in Uniprot accession number: P04216). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Thy-1 protein fragments (same or different) having a length up to 50 amino acids and comprising the amino acid sequence SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of one or more Thy-1 protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8, and/or SEQ ID NO: 9, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Thy-1 protein fragments of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of VAMP-2 protein fragments comprising the amino acid sequence SEQ ID NO: 10 (correspond to the amino acid residues 32-47 of the whole sequence provided in Uniprot accession number: P63027). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more VAMP-2 protein fragments having a length up to 50 amino acids and comprising the amino acid sequence SEQ ID NO: 10, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of a VAMP- 2 protein fragment consisting of the amino acid sequence of SEQ ID NO: 10, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of GluR4 protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 1 1 (correspond to the amino acid residues 218-230 of the whole sequence provided in Uniprot accession number: P48058), SEQ ID NO: 12

(correspond to the amino acid residues 234-245 of the whole sequence provided in Uniprot accession number: P48058), and/or SEQ ID NO: 13 (correspond to the amino acid residues 354-368 of the whole sequence provided in Uniprot accession number: P48058). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more GluR protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 11 , SEQ ID NO: 12, and/or SEQ ID NO: 13, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of one or more GluR4 protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 1 1 , SEQ ID NO: 12, and/or SEQ ID NO: 13, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of GluR4 protein fragments of SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of Tenascin-R protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 14 (correspond to the amino acid residues 906-922 of the whole sequence provided in Uniprot accession number: Q92752) and/or SEQ ID NO: 15 (correspond to the amino acid residues 923-937 of the whole sequence provided in Uniprot accession number: Q92752). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Tenascin-R protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 14 and/or SEQ ID NO: 15, is determined. In another embodiment, optionally in combination with any of the

embodiments provided above or below, the levels of one or more Tenascin-R protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 14 and/or SEQ ID NO: 15, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Tenascin-R protein fragments of SEQ ID NO: 14 and SEQ ID NO: 15, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of Neuroligin-2 protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 16 (correspond to the amino acid residues 336-346 of the whole sequence provided in Uniprot accession number: Q8NFZ4) and/or SEQ ID NO: 17 (correspond to the amino acid residues 450-469 of the whole sequence provided in Uniprot accession number: Q8NFZ4). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Neuroligin-2 protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 16 and/or SEQ ID NO: 17, is determined. In another embodiment, optionally in combination with any of the

embodiments provided above or below, the levels of one or more Neuroligin-2 protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 16 and/or SEQ ID NO: 17, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Neuroligin-2 protein fragments of SEQ ID NO: 16 and SEQ ID NO: 17, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of GluR2 protein fragments comprising the amino acid sequence SEQ ID NO: 18 (correspond to the amino acid residues 295-312 of the whole sequence provided in Uniprot accession number: P42262). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more GRIA2 protein fragments having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 18, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of a GluR2 protein fragment consisting of the amino acid sequence of SEQ ID NO: 18, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment above or below, the method comprises determining the level of expression of Syntaxin-1 B protein fragments comprising the amino acid sequence SEQ ID NO: 19 (correspond to the amino acid residues 94-107 of the whole sequence provided in Uniprot accession number: P61266). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more GRIA2 protein fragments having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 19, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the levels of a Syntaxin-1 protein fragment consisting of the amino acid sequence of SEQ ID NO: 19, is determined.

In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment provided above or below, the method comprises determining the level of expression of one or more Calsyntenin-1 protein fragments (same or different) comprising the amino acid sequence SEQ ID NO: 20 (correspond to the amino acid residues 235-244 of the whole sequence provided in Uniprot accession number: 094985), SEQ ID NO: 21 (correspond to the amino acid residues 537-545 of the whole sequence provided in Uniprot accession number: 094985), and/or SEQ ID NO: 22 ((correspond to the amino acid residues 684-690 of the whole sequence provided in Uniprot accession number: 094985). In an additional embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more CLSTN1 protein fragments (same or different) having a length up to 35 amino acids and comprising the amino acid sequence SEQ ID NO: 20, SEQ ID NO: 21 , and/or SEQ ID NO: 22, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of one or more Calsyntenin-1 protein fragments (same or different) consisting of the amino acid sequence of SEQ ID NO: 20, SEQ ID NO: 21 , and/or SEQ ID NO: 22, is determined. In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of Calsyntenin-1 protein fragments of SEQ ID NO: 20, SEQ ID NO: 21 , and SEQ ID NO: 22, is determined.

In a particular embodiment, optionally in combination with any embodiment above or below, the method of any of the aspects of the invention comprises determining the level of expression of at least two, three, or four proteins from Neurexin-2A, Neurexin-3A, Thy-

1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in a test sample.

As depicted in Example 1 , among the 10 proteins evaluated, Neurexin-2A, Neurexin-3A, Thy-1 and VAMP-2 levels showed the greatest consistency regarding protein level variations between cohorts.

In a further embodiment of any of the methods of the invention, optionally in combination with any embodiment provided above or below, it is determined the level of expression of one or more of the following proteins: Neurexin-2A, Neurexin-3A, Thy-1 and VAMP-2.

In a particular embodiment of any of the methods provided by the present invention, optionally in combination with any embodiment provided above or below, the method comprises determining the level of expression of NRX2A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated test sample of the individual. In another embodiment of any of the methods provided by the present invention, optionally in combination with any of the embodiments provided above or below, the method comprises determining the level of expression of Neurexin-3A in combination with one or more of the markers: Thy-1 , VAMP-

2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 in an isolated test sample of the individual.

In another embodiment of the invention, optionally in combination with any embodiment above or below, the method or use of the invention comprises determining the level of expression of at least one set of proteins selected from the group consisting of Tenascin- R-VAMP-2, Tenascin-R-Neurexin-3A, Tenascin-R-GluR4, Tenascin-R-Thy-1 , Tenascin-R- Neurexin-2A, Tenascin-R-Neuroligin-2, Tenascin-R-GluR2, Tenascin-R-Syntaxin-1 , Tenascin-R-Calsyntenin-1 , VAMP-2-Neurexin-3A, VAMP-2-GluR4, VAMP-2-Thy-1 , VAMP-2-Neurexin-2A, VAMP-2-Neuroligin-2, VAMP-2-GluR2, VAMP-2-Syntaxin-1 , VAMP-2-Calsyntenin-1 , Neurexin-3A-GluR4, Neurexin-3A-Thy-1 , Neurexin-3A-Neurexin- 2A, Neurexin-3A-Neuroligin-2, Neurexin-3A-GluR2, Neurexin-3A-Syntaxin-1 , Neurexin- 3A-Calsyntenin-1 , GluR4-Thy-1 , GluR4-Neurexin-2A, GluR4-Neuroligin-2, GluR4-GluR2, GluR4-Syntaxin-1 , GluR4-Calsyntenin-1 , Thy-1 -Neurexin-2A, Thy-1 -Neuroligin-2, Thy-1 - GluR2, Thy-1 -Syntaxin-1 , Syntaxin-1 -Thy-1 , Thy-1-Calsyntenin-1 , Neurexin-2A-Syntaxin- 1 , Syntaxin-1 -Neuroligin-2, Neurexin-2A-Neuroligin-2, Neurexin-2A-GluR2, Neurexin-2A- Calsyntenin-1 , Neuroligin-2-GluR2, Neuroligin-2-Calsyntenin-1 , and GluR2-Syntaxin-1 , Syntaxin-1 -Calsyntenin-1 , GluR2-Calsyntenin-1. In another embodiment, all 10 protein markers’ expression product level is determined (i.e., Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 ).

As depicted in Example 1 and 2, the inventors also provide a method to dissect synaptopathy from neurodegeneration. As shown in Tables 2 and 4, the inventors have found that the normalization of the protein levels of the markers of the invention with respect to Neurofilament light chain (Nf-L) levels, a neurodegeneration marker, is informative of the underlying synaptopathy in a neurodegenerative. In all the diseases stages with synaptopathy (PC1 , PC2, PROD, DAT of AD and FTD), the obtained values remained below the normalization levels of the healthy controls (Table 2 and 4), indicating that having these levels below the control is indicative of suffering from synaptopathy. In addition, the inventors found that the values obtained by the normalization, exhibited even more robust statistical power in terms of synaptopathy detection than the markers used alone both in AD and FTD (see Examples 1 and 2). In this regard, the values obtained by the normalization of the markers of the invention vs Nf-L are of high interest for synaptopathy diagnosis, prognosis and monitoring of therapeutic interventions applied in neurodegenerative disorders.

In an embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment provided above or below, the neurodegenerative marker is selected from the group consisting of Nf-L, total Tau, and phosphorylated Tau.

Tau is a microtubule stabilizing protein predominantly expressed in the neurons of the central nervous system. Genetic, transcript and protein alterations in Tau lead to intraneuronal Tau inclusions that are observed in numerous neurodegenerative diseases, termed tauopathies. Tau inclusions associated with neuronal damage, which leads to the leakage of abnormal forms of Tau into the CSF (Schraen-Maschke et al.,“Tau as a biomarker of neurodegenerative diseases”, Biomark Med, 2008, vol. 2, pp 363).

Nf-L, also known as Neurofilament-L or Neurofilament light chain, has the Uniprot database accession number P07196, January 23, 2007 -v3. Nf-L is a scaffolding protein of the neuronal cytoskeleton. CSF expression of Nf-L is increased following axonal injury and in several neurodegenerative diseases such as Alzheimer’s disease, frontotemporal dementia and amyotrophic lateral sclerosis (Bacioglu et al.,“Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in

Neurodegenerative Diseases”, Neuron, 2016, vol.91 , pp56). The increased CSF expression of Tau and Nf-L proteins as a result of neuronal damage are well-accepted biomarkers of neurodegeneration. In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment provided above or below, the neurodegenerative marker is Nf-L. In another embodiment of the method of any of the aspects of the invention, optionally in combination with any embodiment provided above or below, the method comprises determining the level of expression of Nf-L protein fragments (same or different) comprised in the amino acid sequence corresponding to the amino acid residues 1-543 provided in Uniprot accession number: P07196. In an additional embodiment, optionally in combination with any embodiment above or below, the level of one or more Nf-L protein fragments having a length up to 50 amino acids, is determined. Alternatively, in another embodiment, optionally in combination with any embodiment provided above or below, the method comprises determining the level of expression of the whole Nf-L protein. In another embodiment, optionally in combination with any of the embodiments provided above or below, the method comprises determining the level of expression of the whole Nf-L protein by ELISA. In another embodiment of the invention, optionally in combination with any embodiment provided above or below, the method or use of the invention comprises determining the level of expression of at least one set of proteins selected from the group consisting of Neurexin-2A-Nf-L, Neurexin-3A-Nf-L, Thy-1-Nf-L, VAMP-2-Nf-L, Tenascin-R-Nf-L, GluR4- Nf-L, Neuroligin-2-Nf-L, GluR2-Nf-L, Syntaxin-1-Nf-L, and/or Calsyntenin-1-Nf-L.

In an embodiment of any of the methods provided by the invention, optionally in combination with any of the embodiments provided above or below, the method further comprises comparing the level of expression product with a“reference value”.

In the present invention, the term“reference value”, when referred to in any of the methods provided by the present invention, is to be understood as a predefined value of a given marker or a combination of the given molecular markers, which is derived from the levels of said molecular marker or markers in a well-defined sample or group of control samples without any signs of synaptic dysfunction. If the level of expression is determined at the protein level, then the "reference value" is a predefined value of protein amount, whereas if the level of expression is determined at the mRNA level, then the "reference value" is a predefined value of mRNA amount, whereas if the protein levels of a marker(s) of the invention are normalized with respect to Nf-L levels, then the "reference value" is a predefined value of Nf-L normalization. The samples are taken from an individual or group of individuals wherein the presence, absence, stage, or course of the disease has been properly performed previously. This value is used as a threshold to discriminate subjects wherein the condition to be analysed is present from those wherein such condition is absent (for instance, individuals with synaptopathy from individuals without synaptopathy), to determine the stage of the disease, the risk of developing or of being suffering from a synaptopathy and/or neurodegeneration, among others. This reference value is also useful for determining whether the subject has to initiate a therapeutic intervention and how effective the regimen is. The skilled person in the art, making use of the general knowledge, is able to choose the appropriate subject or group of subjects for obtaining the reference control level for each of the methods of the present invention. Methods for obtaining the reference value from the group of subjects selected are well known in the state of the art (Burtis C. A. et al., 2008, Chapter 14, section“Statistical Treatment of Reference Values”). In a particular case“reference value” is a cut-off value defined by means of a conventional ROC analysis (Receiver Operating Characteristic analysis). As the skilled person will appreciate, the optimal cut-off value will be defined according to the particular applications of the diagnostic or prognostic method: purpose, target population for the diagnosis or prognosis, balance between specificity and sensibility, etc.

Each of the markers of the invention is informative by itself. However, in the case that the patient is diagnosed as suffering synaptopathy based in the determination of some of the markers of the invention, the physician can further determine the other markers of the invention to further dissect whether there is neurodegeneration in the patient. When more than one marker of the invention is determined, the physician or person skilled in the art would be able to dissect the correct diagnostic regarding the differential levels of the markers analysed. For example, in the case that 4 markers are determined, and 1 is detected below the levels of the reference value and the other 3 above, it is indicative that the patient suffers synaptopathy and neurodegeneration. In this direction, in one embodiment of the invention, optionally in combination with any embodiment provided above or below, having one or more of the markers determined above the reference value, is indicative that the patient is suffering from both events synaptopathy and neurodegeneration. Otherwise, in another embodiment of the invention, optionally in combination with any embodiment provided above or below, having all the altered markers with levels below the reference value, is indicative that the patient suffers synaptopathy without neurodegeneration.

On the other hand, the present inventors further found that the level of expression products were increased in individuals at later stages of the disease (the prodromal or dementia stages), in whom synaptopathy is progressing in conjunction with

neurodegeneration (as defined by CSF levels of tau protein), which is indicative that the markers not only reflect synaptopathy at the early stages but also in late stages, once neurodegeneration has started (see Table 1 ). In addition, the markers of the invention were also found informative of neurodegeneration in frontotemporal dementia patients, since these proteins were detected in a higher amount in these patients than in the control group (Table 3, below). Hence, in an embodiment of any of the second, third, fourth or sixth methods provided by the present invention, the individual is suffering from or is suspected of suffering a neurodegenerative or neuropsychiatric disorder, and the level of the expression product(s) is higher than the reference value, being indicative that the disease is in the neurodegenerative stage. In one embodiment, optionally in combination with any of the embodiments provided above or below, the individual suffers Alzheimer, and the level of the expression product(s) is higher than the reference value, being indicative that the disease is in the neurodegenerative stage Prodromal AD stage or AD with dementia. In another particular embodiment, optionally in combination with any of the embodiments provided above or below, the individual suffers frontotemporal dementia, and the level of the expression product(s) is higher than the reference value, being indicative that the disease is in the neurodegenerative stage. In another embodiment, optionally in combination with any of the embodiments provided above or below, the individual suffers Alzheimer and the level of the expression product(s) is lower than the reference value, being indicative that the individual suffers a synaptopathy in the absence of widespread neurodegeneration, corresponding to pre-clinical stage 1 AD (PC1 ).

The inventors characterized the levels of the markers of the invention across all stages of the AD continuum, and, as shown below, the marker levels were differentially altered at different clinical stages compared to the control group (Example 1 ). Considering the results obtained by the inventors, the markers of the invention can also be useful to monitor the progression from synaptopathy to stages with neurodegeneration. As shown in FIG.1 , the levels of the markers of the invention were decreased at the earliest preclinical stage of AD and increased in the CSF of individuals at advanced stages of AD.

In an embodiment of second aspect of the invention, optionally in combination with any embodiment provided above or below, wherein the individual is identified to be suffering from AD or FTD or a preclinical stage thereof, the increase in the levels of a marker or marker(s) of the invention with respect to reference value, it is indicative that the neurodegeneration has progressed, wherein neurodegeneration progression is understood as aggravation in the severity of the symptoms or a change in the clinical stratification of the patient to a worse stage. In another embodiment, optionally in combination with any embodiment provided above or below, if the level of the marker(s) does not significantly change, it is indicative that the individual is stabilized at a specific stage of the disease’s continuum. In a further embodiment, optionally in combination with any embodiment provided above or below, if the level of the marker(s) is decreased with respect to reference value or the value previously obtained from said individual, but remains above the normal control levels, this can indicate a positive evolution of the disease, wherein a positive evolution is understood as an improvement in the severity of the symptoms or a change in the clinical stratification of the patient to a better stage. In a further embodiment, optionally in combination with any embodiment provided above or below, when the patient had been identified at a Pre-clinical stage 1 of AD, an increase in the CSF levels of a marker or marker(s) of the invention remaining below the normal control levels, can indicate that there is a positive evolution as defined in the previous embodiments; an increase in the CSF levels of a marker or marker(s) of the invention above the normal control levels, can indicate that there is a negative evolution, that is, a progression of the disease, the underlying synaptopathy, or neurodegeneration; and if the level of the marker(s) does not significantly change or remain significantly below the normal control levels, this can indicate that the patient is in a stable state.

Furthermore, the markers of the invention can be used to monitor not only the disease’s evolution but also to recommend whether to initiate a therapeutic intervention and monitor the efficacy of a therapeutic intervention in a patient already diagnosed with a

neurodegenerative or neuropsychiatric disorder.

In an embodiment of the third aspect of the invention, optionally in combination with any embodiment provided above or below, if the individual is diagnosed of suffering from synaptopathy as defined in the first aspect, or diagnosed of suffering neurodegeneration as defined in the second aspect, then the initiation of the therapeutic intervention is recommended. In another embodiment, if the patient is diagnosed of not suffering synaptopathy or neurodegeneration as defined in the first and second aspects of the invention, the follow-up is performed optionally in consideration of the result of an examination of the patient by a physician.

In the present invention,“to initiate a therapeutic intervention” and the term "treat" are interchangeable. Thus, both are used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in an individual. For example, in relation to Alzheimer's disease and frontotemporal dementia, these terms include relieving, reducing, or alleviating cognitive impairment (such as impairment of memory and/or orientation) or impairment of global functioning (overall functioning, including activities of daily living) and/or slowing down or reversing the progressive deterioration in global or cognitive impairment. Accordingly, these terms also encompass delaying or preventing onset prior to clinical manifestation of a disease or symptom of a disease and/or reducing the risk of developing or worsening of a symptom of a disease.

In the fourth and fifth aspects of the invention, method for determining the efficacy of a therapeutic intervention in a patient already identified of suffering synaptopathy or neurodegeneration are provided. In an embodiment, optionally in combination with any embodiment provided above or below, when the individual suffering from neurodegenerative or neuropsychiatric disorder is identified as suffering synaptopathy, a return to normal levels of these marker(s) or an increase of these marker(s), remaining below reference value (i.e., to the level of a control subject), can indicate that the individual has reacted favourably to the therapeutic intervention and, therefore, said intervention is effective; if the level of the marker(s) does not significantly change, this can indicate that the therapeutic intervention is not effective.

In another embodiment, optionally in combination with any embodiment provided above or below, when the individual suffering from AD is identified as suffering synaptopathy, a return to normal levels of these marker(s) or an increase of these marker(s), remaining below normal levels (i.e., to the level of a control subject), can indicate that the individual has reacted favourably to the therapeutic intervention and, therefore, said intervention is effective; if the level of the marker(s) does not significantly change, this can indicate that the therapeutic intervention is not effective.

In a further embodiment, optionally in combination with any embodiment provided above or below, when the individual suffering neurodegenerative or neuropsychiatric disorder is identified as suffering from neurodegeneration, a decrease or return to a normal levels (i.e., to the level of a control subject) can indicate that the individual has reacted favourably to the therapeutic intervention and, therefore, said therapeutic intervention is effective; if the level of the marker(s) does not significantly change, this can indicate that the therapeutic intervention is not effective.

In a further embodiment, optionally in combination with any embodiment provided above or below, when the individual suffering from symptomatic stages of AD (Prodromal AD stage (PROD), or dementia Alzheimer’s-type (DAT)) or frontotemporal dementia is identified as suffering neurodegeneration, a decrease or return to a normal levels (i.e., to the level of a control subject) can indicate that the individual has reacted favourably to the therapeutic intervention and, therefore, said therapeutic intervention is effective; if the level of the marker(s) does not significantly change, this can indicate that the therapeutic intervention is not effective.

In another particular embodiment of the methods provided by the present invention, optionally in combination with any embodiment above or below, wherein it is determined the level of the protein markers or fragments thereof by a quantitative test selected from the group consisting of an immunological test, bioluminescence, fluorescence,

chemiluminescence, electrochemistry and mass spectrometry In one embodiment of the methods of the invention, optionally in combination with any of the embodiments provided above or below, the level of the marker is determined by mass spectrometry. In this embodiment, the level of proteotypic peptides of the marker

(peptides with an amino acid sequence uniquely associated with the studied protein in a given proteome) is detected by mass spectrometry. Particularly, the level of proteotypic peptides of the marker is detected by Shotgun Liquid Chromatography Mass

Spectrometry (LC-MS/MS) analysis. Briefly, the samples are collected and precipitated with acetone. The protein content is quantified by routine techniques (such as Bradford assays), reduced with a reducing agent (such as DTT), digested with trypsin and LysC overnight and desalted. Samples, then, are analysed using Orbitrap mass analysers, running specific protocols/methods adapted to the nature of the sample.

In another embodiment, optionally in combination with any of the embodiments provided above or below, the level of proteotypic peptides of the marker is detected by Multiple reaction monitoring (MRM) mass spectrometry. Briefly, heavy peptides corresponding to the proteolytic peptide of the marker are synthesised with 613C 415N (Arg) or 613C 215N (Lys) isotopes in orther to generate a library of MS/MS spectra that allowed selection of interference-free transitions for the simultaneous monitoring of the endogenous peptides of interest. Individual samples are precipitated with acetone, redissolved in 6M urea and digested in-solution with LysC and Trypsin overnight. After addition of the corresponding heavy peptide standards. Then samples are analysed using a mass spectrometer coupled to a nano-LC chromatography column, and transitions corresponding to the monitored peptides are visualised and analysed using protocols/methods adapted to sample’s nature.

In an alternative embodiment of the methods of the invention, optionally in combination with any of the embodiments provided above or below, the level of expression is determined by immunochemistry.

The term "immunochemistry" as used herein refers to a variety of techniques for detecting antigens (usually proteins and peptides, and in the present case any of the proteins listed above alone or in combination) in a sample by exploiting the principle of antibodies binding specifically to said antigens. Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine. The immunochemistry technique can be direct or indirect. The direct method is a one-step staining method and involves a labelled antibody (e.g. FITC- conjugated antiserum) reacting directly with the antigen. While this technique utilizes only one antibody and therefore is simple and rapid, the sensitivity is lower due to little signal amplification, such as with indirect methods, and is less commonly used than indirect methods. The indirect method involves an unlabelled primary antibody (first layer) that binds to the target antigen in the sample and a labelled secondary antibody (second layer) that reacts with the primary antibody. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.

Further amplification can be achieved if the secondary antibody is conjugated to several biotin molecules, which can recruit complexes of avidin-, streptavidin or Neutravidin- enzyme. The indirect method, aside from its greater sensitivity, also has the advantage that only a relatively small number of standard conjugated (labelled) secondary antibodies needs to be generated. With the direct method, it would be necessary to label each primary antibody for every antigen of interest. It must be borne in mind that

immunochemistry techniques can also be used to detect certain nucleic acid sequences if a tagged nucleic acid probe (designed to specifically bind to a certain target nucleic acid sequence) can later on be detected with a labelled antibody. Thus, the detection of the protein could be performed by using a tagged nucleic acid designed to bind a specific sequence of the target protein RNA, and then detecting said tagged nucleic acid with a labelled antibody which selectively binds to the tag.

Suitable immunoassay procedures include enzyme-linked immunosorbent assays (ELISA, such as multiplex ELISA), enzyme immunodot assay, agglutination assay, antibody- antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay,

immunocromatography, or other immunoassay formats well-known to the ordinarily skilled artisan, such as radioimmunoassay, as well as protein microarray formats.

In one embodiment, in combination with any of the embodiments provided above or below, the level of the protein is determined by an immunoassay.

In another embodiment, in combination with any of the embodiments provided above or below, the level of expression of protein is determined by ELISA; more in particular multiplex ELISA.

The term "antibody or a fragment thereof able to bind to the target protein(s)" is to be understood as any immunoglobulin or fragment thereof able to selectively bind the target protein(s) referred in the aspects and embodiments of the present invention. It includes monoclonal and polyclonal antibodies. The term "fragment thereof” encompasses any part of an antibody having the size and conformation suitable to bind an epitope of the target protein. Suitable fragments include F(ab), F(ab') and Fv. An "epitope" is the part of the antigen being recognized by the immune system (B-cells, T-cells or antibodies).

There are well known means in the state of the art for preparing and characterizing antibodies. Methods for generating polyclonal antibodies are well-known in the prior art. Briefly, one prepares polyclonal antibodies by immunizing an animal with the protein; then, serum from the immunized animal is collected and the antibodies isolated. A wide range of animal species can be used for the production of the antiserum. Typically the animal used for production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat.

Moreover, monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the protein associated with the disease. The immunizing composition can be administered in an amount effective to stimulate antibody-producing cells. Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation. The immunogen is injected into animals as antigen. The antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks,

approximately, the immunization is repeated with the same antigen.

In another particular embodiment of any of the methods of the invention, the means to carry out the invention form part of a kit. The antibody or fragment thereof for detecting the target protein(s) can be included in a kit. The kit may additionally comprise means

(additives, solvents) to visualize the antibody-protein interactions.

These antibodies can be used as“means” for determining the expression of the target proteins in the seventh aspect of the invention.

In an embodiment of the present invention, optionally in combination with any of the embodiments provided above or below, the means for detecting the level of expression of the proteins are antibodies or fragments thereof that specifically bind to the target protein(s).

In another particular embodiment of the invention, the kit is an ELISA kit. In another embodiment, the kit comprises a solid support and antibodies or fragments thereof, which specifically bind to the target proteins to be detected, these antibodies being conjugated with a reporter molecule capable of producing a signal.

In the present invention, the term“solid support” includes a nitrocellulose membrane, glass or a polymer. The most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of strips, tubes, beads, discs or microplates, or any other surface suitable for conducting an immunoassay.

In an alternative embodiment of the methods provided by the present invention, it is determined the level of expression of mRNA marker(s).

In one embodiment, the level of mRNA is determined via polymerase chain reaction using, for example, oligonucleotide primers that hybridize to one or more polynucleotide of the markers of the invention or complements of such polynucleotides. Within other

embodiments, the level of mRNA is detected using a hybridization technique, employing oligonucleotide probes that hybridize to one or more polynucleotide of the markers of the invention or complements of such polynucleotides.

When using mRNA determination, the method may be carried out by combining isolated mRNA with reagents to convert to cDNA according to standard methods well-known in the art, treating the converted cDNA with amplification reaction reagents (such as cDNA PCR reaction reagents) in a container along with an appropriate mixture of nucleic acid primers; reacting the contents of the container to produce amplification products; and analysing the amplification products to detect the presence of one or more of the polynucleotide of the markers of the invention in the sample. For mRNA, the analysing step may be

accomplished using Northern Blot analysis to detect the presence of polynucleotide of the markers of the invention in the sample. The analysis step may be further accomplished by quantitatively detecting the presence of polynucleotide of the markers of the invention in the amplification product, and comparing the quantity of marker detected against a panel of expected values for the known presence or absence of such markers in normal and diseased tissue derived using similar primers.

In another embodiment, the invention provides a method wherein mRNA is detected by:

(a) isolating mRNA from a sample and combining the mRNA with reagents to convert it to cDNA; (b) treating the converted cDNA with amplification reaction reagents and nucleic acid primers that hybridize to one or more of the polynucleotide of the markers of the invention to produce amplification products; (c) analysing the amplification products for determining the amount of mRNA present encoding the protein of the markers of the invention; and (d) comparing the determined amount of mRNA to an amount detected against a panel of expected values for normal and diseased tissue derived using similar methods. In other embodiments, the invention comprises the use quantitative RT-PCR to quantitatively determine amount of mRNA for protein of the markers of the invention. Further embodiments of the invention use real time RT-PCR for quantification and analysis. In another embodiment, optionally in combination with any of the embodiments provided above or below, the expression product is mRNA and the kit is a microarray.

Furthermore, the present invention further provides the use of kits comprising means for determining one or more of the proteins defined above for performing any of the methods provided herein. In an embodiment of this aspect, optionally in combination with any of the embodiments provided above or below, the kit comprises a solid support and means for detecting the level of expression of one or more of the following proteins Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1.

In another embodiment of this aspect of the invention, optionally in combination with any of the embodiments provided above or below, the kit further comprises means for detecting the level of expression of Nf-L. In another embodiment, optionally in combination with any of the embodiments provided above or below, the kit comprises a solid support and means for detecting the level of expression of at least one set of proteins selected from the group consisting of Tenascin-R-Neurexin-3A, Tenascin-R-Neurexin-3A- Neurexin-2A, Tenascin-R-Neurexin-2A, VAMP-2-Neurexin-3A, VAMP-2-Neurexin-3A- Neurexin-2A, VAMP-2-Neurexin-2A, Neurexin-3A-GluR4, Neurexin-3A-GluR4-Neurexin- 2A, GluR4-Neurexin-2A, Neurexin-3A-Thy-1 , Neurexin-3A-Thy-1-Neurexin-2A, Thy-1- Neurexin-2A, Neurexin-3A-Neurexin-2A, Neurexin-3A-Neuroligin-2, Neurexin-3A- Neuroligin-2-Neurexin-2A, Neurexin-2A-Neuroligin-2, Neurexin-3A-GluR2, Neurexin-3A- GluR2-Neurexin-2A, Neurexin-2A-GluR2, Neurexin-3A-Syntaxin-1 , Neurexin-3A-Syntaxin- 1-Neurexin-2A, Neurexin-2A-Syntaxin-1 , Neurexin-3A-Calsyntenin-1 , Neurexin-3A- Calsyntenin-1-Neurexin-2A, Neurexin-2A-Calsyntenin-1.

In a particular embodiment of the use of the kits, optionally in combination with any of the embodiments provided above or below, the kits are those comprising a solid support and means for detecting the level of expression of two, three, four, five, six, seven, eight, nine, or all ten markers selected from the group consisting of Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, GluR4, Tenascin-R, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1. More particularly, the kits comprise a solid support and means for detecting the level of expression of four of proteins selected.

In an embodiment of the use of the kits, optionally in combination with any of the embodiments provided above or below, the kits are those comprising a solid support and means for detecting the level of expression of Neurexin-2A, Neurexin-3A, Thy-1 , and VAMP-2. In another embodiment of the use of the kits, the kits are those comprising a solid support and means for detecting the level of expression of Neurexin-2A, Neurexin- 3A, Thy-1 , VAMP-2 and Nf-L.

The in vitro methods of the invention provide diagnostic prognostic and/or monitoring information. In one embodiment, the methods of the invention further comprise the steps of (i) collecting the diagnostic, prognostic, and/or monitoring information, and (ii) saving the information in a data carrier.

In the sense of the invention a“data carrier” is to be understood as any means that contain meaningful information data for the diagnosis and-/or prognosis of synaptopathy and or neurodegeneration in an individual suffering from a neurodegenerative or neuropsychiatric disorder, such as paper. The carrier may also be any entity or device capable of carrying the prognosis data. For example, the carrier may comprise a storage medium, such as a ROM, for example a CD ROM or a semiconductor ROM, or a magnetic recording medium, for example a floppy disc or hard disk. Further, the carrier may be a transmissible carrier such as an electrical or optical signal, which may be conveyed via electrical or optical cable or by radio or other means. When the

diagnosis/prognosis/monitoring data are embodied in a signal that may be conveyed directly by a cable or other device or means, the carrier may be constituted by such cable or other device or means. Other carriers relate to USB devices and computer archives. Examples of suitable data carrier are paper, CDs, USB, computer archives in PCs, or sound registration with the same information.

Finally, is another aspect of present invention to provide an algorithm for carrying out any of the methods of diagnosis and/or prognosis as defined in the above aspects. In the sense of the invention, the term“algorithm” is also synonymous of pannel or decision diagrams, predictors and combinatory of data to correctly categorize an individual sample.

According to aspects and embodiments of the invention, diagnosis and/or prognosis of synaptopathy and or neurodegeneration, in an individual suffering from a

neurodegenerative or neuropsychiatric disorder, can be performed using a mathematical algorithm that assesses a detectable level of biomolecules, proteins, antibodies, and/or mRNA, comprising one or more of the biomarkers of diagnosis and prognosis monitoring of synaptopathy and neurodegeneration described above, either in conjunction with or independent of other clinical parameters, to correctly categorize an individual sample as originating from a healthy patient, a patient with stages of a neurodegenerative or neuropsychiatric disorder without cognitive decline, with mild cognitive decline, with moderate cognitive decline, with moderately severe cognitive decline, severe cognitive decline, middle dementia and dementia.

The classification algorithm may be as simple as determining whether or not the amount of a specific biomarker or subset of biomarkers measured are above or below a particular cut-off number. When multiple biomarkers are used, the classification algorithm may be a linear regression formula. Alternatively, the classification algorithm may be the product of any of a number of learning algorithms. In the case of complex classification algorithms, it may be necessary to perform the algorithm on the data, thereby determining the classification, using a computer, e.g., a programmable digital computer. In either case, one can then record the status on tangible medium, for example, in computer-readable format such as a memory drive or disk or simply printed on paper. The result also could be reported on a computer screen. This algorithm is used as diagnostic and/or prognostic method, and is in particular part of the kits for carrying out the methods disclosed in former aspects.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps.

Furthermore, the word“comprise” encompasses the case of“consisting of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Example 1. Biomarkers for the diagnosis and prognosis of synaptopathy and neurodegeneration in Alzheimer’s Disease.

1. Samples, reagents and methods.

1.1.Subjects included in the study and sample description.

Two AD cohorts were analized in order to identify and validate the panel of synaptic proteins of the invention. Subjects included in the exploratory cohort were recruited as part of the Sant Pau Initiative in Neurodegeneration (SPIN) cohort at Hospital Sant Pau (HSP; Barcelona) and by the CITA Foundation (Donostia). Subjects included in the validation cohort were recruited at Hospital Clinic (Barcelona). All participants gave their written consent, and the study was approved by the local ethics committee following the ethical standards recommended by the Helsinki Declaration. All subjects were evaluated by neurologists with expertise in neurodegenerative diseases. Clinical AD stages were classified in accordance with 201 1 NIH-NAA guidelines based on clinical diagnoses, formal cognitive evaluation using a previously published

neuropsychological battery and assessment for amyloidosis markers (PET-amyloid positive and CSF Ab1-42 levels) and neurodegeneration markers (CSF total and/or phosphorylated levels of Tau). Commercially available ELISA kits were used to determine levels of CSF Ab1-42 (InnotestTM b-Amyloid 1-42, Fujirebio-Europe), total Tau

(InnotestTM hTAU Ag, Fujirebio-Europe) and Tau phosphorylated at threonine residue 181 (InnotestTM Phospho-Tau 181 P, Fujirebio-Europe). Neurofilament light chain (Nf-L) was measured in all CSF samples using a commercially available assay (Uman

Diagnostics).

CSF samples were assessed for amyloidosis markers and neurodegeneration markers based on local cut-offs, which have been shown to have high specificity and sensitivity to classify patients with DAT and controls . For the exploratory cohort the cut-offs were as follows: amyloidosis = PET-amyloid positive and/or CSF Ab1-42 < 550 ng/ml,

neurodegeneration = CSF total tau > 350 ng/ml or CSF phosphorylated tau > 61 ng/ml.

For the validation cohort the cut-offs were: amyloidosis = PET-amyloid positive and/or CSF Ab1-42 < 500 ng/ml, neurodegeneration = CSF total tau > 300 ng/ml (age <50) or >450 (age50-70) or >500(age>70) or CSF phosphorylated tau > 75 ng/ml.

The Control group included samples from cognitively normal subjects who were negative for tau and amyloidosis markers and display within the normal range following

neuropsychological evaluation, when accounting for age and education (mostly recruited among patients’ caregivers), were classified as cognitively normal.

Preclinical stage 1 (PC1 ) included cognitively normal subjects, positive for amyloidosis and negative for neurodegeneration markers. Preclinical stage 2 (PC2) included cognitively normal subjects, positive for both markers. Subjects with mild cognitive impairment (aMCI) due to AD and positive for both amyloidosis and neurodegeneration markers were recruited for the prodromal AD group. Patients with dementia due to AD and positive for both amyloidosis and neurodegeneration markers were recruited for the dementia, Alzheimer-type group.

Where possible, subjects included in each group were age and sex-matched. CSF samples were collected following international consensus recommendations (C. E. Teunissen, et al.,“Consensus Guidelines for CSF and Blood Biobanking for CNS

Biomarker Studies” Mult Scler Int, 201 1 , 246412). CSF samples were collected in polypropylene tubes and immediately centrifuged (2000 g for 10 min) to avoid any possible cellular contamination. All samples were processed within 2 hours of the extraction and stored in 1.5 ml polypropylene tubes at -80°C until analysis.

CSF samples were run on the triple quadrupole-Qtrap mass spectrometer (5500 QTrap, ABSciex) in a randomised order with respect to diagnostic group and the researchers performing the mass RMN were blinded to the diagnosis. No outlying samples were identified.

1.2. Multiple reaction monitoring (MRM) mass spectrometry analysis.

Exploratory and validation cohorts samples were evaluated by MRM. Therefore, corresponding heavy peptides of the markers of the invention, were synthesised with 613C 415N (Arg) or 613C 215N (Lys) isotopes (Thermo Fisher). Heavy peptides were used to generate a library of MS/MS spectra that allowed selection of interference-free transitions for the simultaneous monitoring of endogenous peptides in CSF samples.

Individual CSF samples were precipitated with acetone, redissolved in 6M urea and digested in-solution with LysC and Trypsin overnight. After addition of the corresponding heavy peptide standards, 5% volume of each sample was analysed using a triple quadrupole-Qtrap mass spectrometer (5500 QTrap, ABSciex) coupled to a nano-LC chromatography column (2-hour gradient). Measurements were taken in acquisition mode with a 5 minute retention time.

Proteins isolated from Escherichia Coli were digested in parallel with the CSF samples using the same protocol and b-galactosidase was monitored in the E. Coli samples between each CSF sample to avoid carryover and assess the instrument performance.

Transitions corresponding to the monitored peptides were visualised and analysed using Skyline 3.5 (MacLean et al.,“Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics”, 2010, vol 26, pp. 966-968).) based on co-elution of the heavy and endogenous peptides, identical peak shapes and the same order of peak heights for heavy and endogenous transitions. Transitions were first summarised by peptide using Tukey’s Median Polish. Peptides from the same protein showed strong correlation in the Exploratory (p=0.74 to 0.99) and Validation (p=0.46 to 0.98) cohorts. Consequently, the transitions were summarised by protein for all analyses. Transitions affected by between run interference were identified and removed using the “betweenRunlnterferenceScore” (cut-off=<0.7) combined with visual assessment. .

As differences in peptide levels measured in samples from the same diagnostic group across the two sample collections (HSP and CITA) did not reach significance (a=0.05), the data from both collections were pooled and analyzed together.

For statistical data analysis, protein levels comparisons in CSF across dementia stages were made, and the log-2 fold-change between control and each diagnostic group were compared using a mixed effect linear regression model (GroupComparison function in MSstats). To account for multiple testing, the Benjamini-Hochberg (B-H) procedure was applied to the p-values (false discovery rate; FDR = 0.25). p values that were less than the B-H criteria (rank/#tests^*FDR) were considered significant.

2. Results

The inventors had identified 22 peptides which corresponded to 10 proteins, according to searches performed against SwissProt (Human) database, using an internal version of the search algorithm using MASCOT software (http://www.matrixscience.com/), and a further analysis using the software Proteome Discoverer v1.4.

In order to evaluate the individual potential of each one of said protein markers to diagnose synaptopathy and differentiate between the different AD clinical stages, each protein was evaluated by using MRM in two different cohorts comprising CSF samples for control individuals and all stages of the AD continuum (preclinical AD stage 1 , preclinical AD stage 2, prodromal AD, and dementia, Alzherim’s-type; DAT) which were previously stratified following the criteria as defined above under section 1.1.

As shown in FIG 1 and Table 1 below, the inventors found that all 10 biomarkers, Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , were differentially altered in AD patients vs the control (non-AD) group. All ten markers displayed a biphasic pattern, being informative of synaptopathy and neurodegeneration. It is even more remarkable, their potential use in detecting synaptoapthy at early preclinical stages of AD. In this connection, the inventors found that all markers were clearly reduced in the preclinical stage 1 of AD compared with cognitively normal controls (Table 1 ). As mentioned above, in this preclinical stage of the disease the patient is cognitively normal but has brain amyloidosis in the absence of neurodegeneration. Having markers that detect changes in the synapse (the target of amyloid protein) at stages so early in the AD continuum, is of great interest since it’s precisely at early stages of the disease, when the patient remains assymptomatic, where medication has a higher probability to succeed. That is, the markers of the invention will contribute to widen the terapeutic window to treat AD, since they will help in early identification of these patients.

Furthermore, the alteration in the marker levels was biphasic across the different stages of the disease (decreased at preclinical AD stage 1 and increased from preclinical stage 2 onwards). This makes the markers even more informative and, thus, interesting for clinical development assays, since they can be used not only for diagnosis of synaptopathy, but also for prognosis of synaptopathy and neurodegeneration of patients suffering from neurodegenerative and neuropsychiatric disorders, and for testing medical responsivness in these patients.

Preclinical AD 1 control Preclinical AD2:control Prodromal AD:control Dementia (AD):control

Protein N FC log2FC SE pyalue N FC log2FC SE pyalue N FC log2FC SE pyalue N FC log2FC SE pyalue

Calsyntenin-1 57 0,8 -0,3 0,2 0,04 56 1,2 0,3 0,2 1,00 68 1,2 0,3 0,2 0,007 73 1,2 0,3 0,2 0,04

GluR2 57 1,0 0,0 0,3 1,00 56 1,1 0,2 0,3 0,56 68 1,3 0,4 0,2 0,04 73 1,2 0,3 0,2 0,22

GluR4 57 0,8 -0,4 0,2 0,10 56 1,0 0,0 0,3 1,00 68 1,3 0,4 0,2 0,01 73 1,2 0,3 0,2 0,06

Neurexin-2A 57 0,9 -0,2 0,2 0,13 56 1,1 0,1 0,2 0,65 68 1,2 0,3 0,2 0,01 73 1,2 0,2 0,2 0,11

Neurexin-3A 57 0,8 -0,3 0,2 0,03 56 1,2 0,2 0,2 0,381 68 1,3 0,4 0,2 0,009 73 1,2 0,2 0,2 0,15

Neuroligin-2 57 0,8 -0,3 0,2 0,09 56 1,0 0,1 0,2 1,00 68 1,2 0,3 0,2 0,05 73 1,2 0,2 0,2 0,11

Syntaxin-1 B 30 0,8 -0,3 0,2 0,15 30 1,0 -0,1 0,2 0,74 40 1,2 0,3 0,2 0,04 40 1,1 0,2 0,2 0,32

Tenascin-R 57 0,8 -0,3 0,2 0,09 56 1,0 -0,1 0,2 1,00 68 1,3 0,4 0,2 0,02 73 1,2 0,2 0,2 0,20

Thy-1 57 0,8 -0,4 0,2 0,01 56 1,1 0,1 0,2 0,659 68 1,4 0,5 0,2 0,0004 73 1,1 0,2 0,2 0,30

Vamp-2 57 0,9 -0,1 0,2 0,65 56 1,2 0,3 0,2 0,10 68 1,4 0,4 0,2 0,002 73 1,2 0,2 0,2 0,15

Os

Among the 10 proteins evaluated, the inventors found that Neurexin-2A, Neurexin-3A, Thy-1 and VAMP-2 levels were the most consistent between cohorts since they demonstrated a fold-change in the same direction in both cohorts at all stages of the AD continuum.

The inventors further assessed the predictive potential of the markers of the invention normalized to the axonal marker Neurofilament light chain (Nf-L) levels, across all clinical and preclinical stages of AD. The rationale with this normalization was to dissect synaptoapthy from neurodegeneration (a feature seen in many neurodegenerative conditions).

As shown in Table 2 and FIG 2, the normalization of the expression of these markers with respect to the expression of Nf-L in CSF samples, showed even higher power in terms of synaptopathy diagnosis potential.

CD

o

CO

w o

Preclinical AD 1 control Preclinical AD 2:control Prodromal AD:control Dementia (AD):control o cn cn

Protein N FC log2FC SE pvalue N FC log2FC SE pvalue N FC log2FC SE pvalue N FC log2FC SE pvalue CD

Calsyntenin-1:NFL 35 0,6 -0,7 0,3 0,0008 35 0,6 -0,7 0,3 0,004 35 0,5 -0,9 0,2 2,4E-07 35 0,6 -0,8 0,2 2.0E-06

GluR2:Nf-L 35 0,8 -0,4 0,3 0,06 35 0,6 -0,6 0,3 0,01 35 0,5 -0,9 0,3 2,2E-05 35 0,6 -0,8 0,3 9,1 E-06

GluR4:Nf-L 35 0,6 -0,7 0,3 0,006 35 0,6 -0,8 0,3 0,001 35 0,5 -0,9 0,3 4,9E-05 35 0,6 -0,8 0,3 1.2E-04

Neurexin-2A :Nf-L 35 0,7 -0,6 0,3 0,02 35 0,6 -0,7 0,3 0,008 35 0,5 -1,0 0,2 1.6E-06 35 0,6 -0,9 0,2 1.9E-06 3

Neurexin-3A:Nf-L 35 0,6 -0,7 0,3 0,004 35 0,7 -0,6 0,3 0,02 35 0,6 -0,9 0,3 5,0E-06 35 0,6 -0,8 0,2 4.2E-06

Neuroligin-2:Nf-L 35 0,6 -0,6 0,3 0,003 35 0,6 -0,8 0,3 0,0004 35 0,5 -1,0 0,2 1.3E-08 35 0,6 -0,9 0,2 2.4E-07

Syntaxin-1 :Nf-L 35 0,6 -0,7 0,3 0,02 35 0,6 -0,7 0,3 0,0060 35 0,6 -0,7 0,2 9.7E-04 35 0,6 -0,7 0,2 2,1 E-03

Tenasin-R:Nf-L 35 0,6 -0,7 0,3 0,002 35 0,6 -0,8 0,3 0,0007 35 0,5 -0,9 0,2 8.9E-07 35 0,6 -0,9 0,2 6.8E-07

Thy-1:Nf-L 35 0,6 -0,8 0,3 0,002 35 0,6 -0,7 0,3 0,008 35 0,6 -0,8 0,2 1.0E-05 35 0,5 -0,9 0,2 1.2E-06

Vamp-2:Nf-L 35 0,7 -0,5 0,3 0,04 35 0,7 -0,5 0,3 0,03 35 0,6 -0,8 0,2 5.2E-06 35 0,5 -0,9 0,2 1.5E-06

Example 2. Biomarkers of diagnosis and prognosis of Frontotemporal Dementia in CSF.

1. Samples, reagents and methods.

1.1.Subjects included in the study and sample description.

To confirm that the markers of the invention could also found used in other synaptopathy conditions, samples of patients suffering from frontotemporal dementia, another well- defined synaptopathy different from Alzeihmer disease, were analysed.

Subjects included in frontotemporal dementia cohorts were recruited as part of the Sant Pau Initiative in Neurodegeneration (SPIN) cohort at Hospital Sant Pau (HSP; Barcelona). Patients clinically diagnosed with frontotemporal dementia fulfilled criteria for either the behavioural variant (Rascovsky et al.,“Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia”, 2011 , Brain, vol. 134, pp. 2456-2477) or semantic dementia (Gorno-Tempini et al.,“Classification of primary progressive aphasia and its variants”, 2011 , Neurology, vol. 76, pp 1006-1014) and were excluded if positive for both amyloidosis and neurodegeneration markers.

Where possible, subjects included in each group were age and sex-matched. CSF samples were run on the mass spectrometer in a randomised order with respect to diagnostic group and the researchers performing the MRM were blinded to the diagnosis. No outlying samples were identified.

CSF samples were collected following international consensus recommendations

(Teunissen et. al,“Consensus Guidelines for CSF and Blood Biobanking for CNS

Biomarker Studies" Mult Scler Int, 2011, 246412). CSF samples were collected in polypropylene tubes and immediately centrifuged (2000g for 10 min) to avoid any possible cellular contamination. All samples were processed within 2 hours of the extraction and stored in 1.5 ml polypropylene tubes at -80°C until analysis.

1.2. Protein preparation and Multiple reaction monitoring (MRM) mass spectrometry analysis.

Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 proteotypic peptides were evaluated by MRM in samples of frontotemporal dementia patients and their matching controls using the same experimental protocol and data analysis described in previous section 1 .2 from Example 1 .

2. Results

In Table 3 and 4, the comparison of these markers in CSF samples from frontotemporal dementia (FTD) patients and cognitively normal indiviudals, are shown. All ten markers of the invention tested were found significantly increasead in CSF samples from FTD patients (Table 3), confirming their value for diagnosing synaptopathy and

neurodegeneration with a different etiology to AD.

Table 3. Synaptic panel protein levels in the CSF of FTD patients.

Frontotem poral dementia:control

Protein N FC log2FC SE pvalue

Calsyntenin-1 35 1.2 0,3 0,2 0,09

GluR2 35 1 ,5 0,6 0,2 0,02

GluR4 35 1.3 0,4 0,2 0,07

Neurexin-2A 35 1.3 0,4 0,2 0,02

Neurexin-3A 35 1.4 0,4 0,2 0,04

Neuroligin-2 35 1.3 0,4 0,2 0,02

Syntaxin-1 B 35 1 ,2 0,2 0,2 0,13

Tenascin-R 35 1 ,2 0,3 0,2 0,10

Thy-1 35 1 ,2 0,3 0,2 0,1 1

Vamp-2 35 1.4 0,5 0,2 0,03

As above, the inventors assessed the ratio between the markers of the invention levels and levels of Neurofilament-L. As shown below in Table 4, the obtained ratios were also different comparing FTD patients and control subjects. Moreover, they exhibited even more robust statistical power in terms of synaptpathy diagnosis compared with analyszing the proteins alone.

Table 4. Fold-change in Neurexin-2A, Neurexin-3A, Thy-1 and VAMP-2 normalized for Nf-L in the CSF of FTD patients.

Frontotem poral dementia:control

Protein N FC log2FC SE pvalue

Calsyntenin-1 :NFL 35 0,3 -1 ,5 0,3 5,7E-08

GluR2:Nf-L 35 0,4 -1 ,3 0,3 4,5E-05

GluR4:Nf-L 35 0,4 -1 ,5 0,3 5,5E-07

Neurexin-2A :Nf-L 35 0,4 -1 ,4 0,2 4,6E-08

Neurexin-3A:Nf-L 35 0,4 -1 ,4 0,3 3,8E-06

Neuroligin-2:Nf-L 35 0,4 -1 ,4 0,3 3,9E-07

Syntaxin-1 :Nf-L 35 0,3 -1 ,6 0,2 3,2E-09

Tenasin-R:Nf-L 35 0,3 -1 ,5 0,3 1 ,5E-07

Thy-1 :Nf-L 35 0,3 -1 ,5 0,3 5,4E-08

Vamp-2:Nf-L 35 0,4 -1 ,4 0,3 2,1 E-06 Taking all together, the inventors have identified a panel of proteins informative of the underlying synapse integrity loss and neurodegeneration in different neurodegenerative disorders such as AD and FTD.

Example 3. Synaptic expression of the biomarkers of the invention.

1 . Samples, reagents and methods.

Post-mortem tissue from the frontal cortex of human brain donors was collected by the Neurological Tissue Bank at Hospital Clinic (IDIBAPS, Barcelona). 200 mg chunks were cut from frozen frontal cortex tissue blocks of 6 brains and homogenized in cold Buffer A (0.32 M sucrose, 1 mM NaHC0₃, 1 mM MgCI₂, 0.5 mM CaCI₂, 1 :2500

phenylmethylsulfonyl fluoride, 1 pg/ml aprotinin, 1 pg/ml leupeptin). Homogenates were centrifuged at 1400 g for 10 minutes at 4 °C and the supernatant transferred to a new tube. The pellet was resuspended in cold Buffer A and the previous step repeated with centrifugation at 710 g. The two supernatants were combined and centrifuged at 710 g. The supernatant was subjected to a final centrifugation at 30,000 x g for 15 minutes at 4 °C. The pellet was resuspended in Buffer B (0.32 M sucrose, 1 mM NaHC0₃), layered over a sucrose gradient (0.85 M, 1 M, 1.2 M) and centrifuged at 82,500 g for 2 hours. The synaptosomal fraction (a thick white band at the 1 -1 .2 M interface) was collected, diluted in 4x volume of Buffer B and divided into 2 equal aliquots. Both aliquots were centrifuged at 48,200 g for 20 minutes. The first pellet (synaptosome-enriched fraction) was resuspended in Buffer C (50 mM Tris pH 7.4, 1 % SDS) and stored at -80°C. The second pellet was diluted in equal volume Buffer D (50 mM Tris pH 7.4). 1 volume of 2% Triton-X was added and the sample was incubated on ice for 10 minutes and centrifuged at maximum velocity for 30 minutes. The pellet (PSD-enriched fraction) was resuspended in Buffer C and stored at -80°C. The total protein content of homogenate, synaptosome and PSD enriched fractions was quantified by BCA assay. Aliquots containing 20 pg total protein were boiled, diluted in loading buffer (100 mM Tris-HCL, 4% SDS, 20% glycerol, 200 mM DTT, and 200 mM b-mercaptoethanol) and loaded onto a 12% Tris-Tricine gel. The gel was electrophoresed for 2 hours and proteins were transferred to a 0.2 pm nitrocellulaose membrane. The membranes were immunostained for our panel of 10 synaptic proteins; anti-CLSTN1 , anti-Synaptophysin, anti-Thy1 , (Abeam; ab134130, ab8049, ab133350), anti-GluR2, anti-GluR4, anti-Vamp2, anti-PSD95 (Cell Signaling; 13607, 8070, 13508, 3450), Anti-neuroligin-2, anti-Syntaxin-1 B (Synaptic Systems;

129203, 110403), anti-Neurexin3, anti-Tenascin R (Thermo Fisher; PA5-47714, PAS- 47546) and fluorescent dye-conjugated secondary antibodies (LI-COR Biosciences). To determine the enrichment of the markers in synaptosome and PSD fractions relative to the homogenate, the intensity of the corresponding bands were quantified using Odyssey software (Li-COR, Biosciences, Nebraska) and the ratio of each fraction relative to that of the homogenate was calculated for each brain.

For Array Tomography microscopy, a 1 cm³ section was taken from the superior frontal cortex was fixed, dehydrated and polymerised in 100% LRwhite resin. The embedded samples were sectioned using a diamond knife (Diatome, UK) creating a ribbon of at least 20 serial 70 nm thick sections, which were mounted onto coverslips. The ultrathin ribbons were washed with Tris buffer and blocked (0.05% Tween, 0.1 % BSA in Tris) for 5 minutes. Primary antibodies used were as mentioned above for the Western blotting with the exception of the following; anti-Gephyrin, anti-Tenascin-R (Abeam, UK; ab136343, ab121916) anti-GluR2, anti-Neuroligin-2, anti-Neurexin2 (Merck Millipore,

Massachussetts; MAB397, AB15510, ABN97), anti-synaptophysin (Osenses, Australia; oss00029w) and anti-PSD95 (Synaptic Systems, Germany; 124014). Secondary antibodies were alexa-tagged (Thermo Fisher Scientific). Coverslips were mounted onto the slides using Slowfade Gold with DAPI (Thermo Fisher Scientific). Images from the same ROI of each ribbon section were captured using a fully automated epifluorescence upright microscope (custom adapted BX51 , Olympus, Pensylvania) with a 64x 1.2 NA Plan Apochromat objective. Image analysis was performed using Matlab (Mathworks) and lmageJ(87). Images from serial sections were stacked, aligned, thresholded and the non- specific staining (not present in at least 2 consecutive sections) removed using a local threshold based algorithm. Reconstructions containing a representative synapse were generated using the Volume Viewer plug-in of ImageJ with tricubic smooth interpolation. 2. Results

The expression of the 9 of the markers of the invention at the human synapse was evaluated. It was found that in autopsy tissue taken from the frontal cortex of a human donor, immunostaining for all of the markers co-localised with the immunostaining of established markers of the pre-synapse (Synaptophysin-38) and the post-synapse (PSD- 95) tissue using array tomography (AT) microscopy. AT is particularly suited to the study of synapses as it provides improved spatial resolution in the axial plane compared to other light microscopy techniques (i.e., confocal microscopy). Specificity of synaptic expression was further evaluated by quantifying the expression of the markers in fractions enriched for the whole synapse (synaptosome) or the post-synaptic density (PSD). The fractions were extracted from frontal cortex autopsy tissue from 6 human donors. NRX2A could not be analyzed due to the lack of a commercially-available specific antibody suitable for Western blotting. FIG. 3 shows that all 9 markers tested showed greater expression (enriched) in the synaptosome and/or PSD enriched fractions compared with the homogenate. Specifically, Calsyntenin-1 , Neurexin-3A, Syntaxin-1 B and Vamp-2 were enriched in the synaptosome fraction with Vamp-2 in particular, showing greater enrichment than the pre-synaptic marker, synaptophysin. Calsyntenin-1 , GluR2, GluR4, Neuroligin-2, Tenascin-R and Thy-1 were enriched in the PSD fraction.

Citation List

Spires-Jones & Hyman,“The intersection of amyloid beta and tau at synapses in

Alzheimer's disease”, Neuron, 2014, vol. 82, pp. 756-771.

McKhann et al., “The diagnosis of dementia due to Alzheimer's disease:

recommendations from the National Institute on Aging-Alzheimer's Association

workgroups on diagnostic guidelines for Alzheimer's disease” Alzheimers Dement, 201 1 , vol 7, pp. 263-269

Bellucci et al.,“Review: Parkinson’s disease: from synaptic loss to connectome dysfunction”, 2016, Neuropathology and Applied Neurobiology, vol. 42, pp. 77-94

Calabrese et al.,” Synaptic alterations associated with psychiatric disorders: potential as therapeutic target”, 2016 Expert Opinion on Therapeutic Targets, vol. 20(10), pp.1 195- 207.

Thorsell et al.,“Neurogranin in cerebrospinal fluid as a marker of synaptic degeneration in Alzheimer's disease”, Brain Research, 2010, vol. 1362, pp. 13-22)

Brinkmalm et al.,“SNAP-25 is a promising novel cerebrospinal fluid biomarker for synapse degeneration in Alzheimer's disease”, Mol Neurodegener, 2014, vol. 9, pp 53

Schraen-Maschke et al.,“Tau as a biomarker of neurodegenerative diseases”, Biomark Med, 2008, vol. 2, pp 363

Bacioglu et al.,“Neurofilament Light Chain in Blood and CSF as Marker of Disease Progression in Mouse Models and in Neurodegenerative Diseases”, Neuron, 2016, vol.91 , pp56.

Teunissen et al.,“Consensus Guidelines for CSF and Blood Biobanking for CNS

Biomarker Studies”. Mult Scler Int, 201 1 , 246412

MacLean et al.,“Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics”, 2010, vol 26, pp. 966-968

Burtis C. A. et al., 2008, Chapter 14, section“Statistical Treatment of Reference Values”

Rascovsky et al.,“Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia”, 2011 , Brain vol 134, pp. 2456-2477

Gorno-Tempini et al.,“Classification of primary progressive aphasia and its variants”,

201 1 , Neurology, vol 76, pp. 1006-1014

For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

Clauses:

1. An in vitro method of diagnosis and/or prognosis of synaptopathy in an individual, the method comprising:

(i) determining the level of the expression product of:

(ii) comparing it with a reference value, or alternatively

(ibis) determining the level of the expression product of one or more of Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and/or Calsyntenin-1 in an isolated test sample from the individual;

(ivbis) comparing the value obtained in (iiibis) with a reference value, wherein if the level determined in step (i) or (iiibis) is lower than the reference value, this is indicative that the individual suffers synaptopathy.

2. An in vitro method of diagnosis and/or prognosis of neurodegeneration in an individual, the method comprising:

(i) determining the level of expression product of:

(b) one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2,

GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated CSF sample from the individual; or alternatively,

(ii) comparing it with a reference value, wherein if the level in the test sample is higher than the reference value, this is indicative that the individual suffers neurodegeneration.

3. An in vitro method of deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or

neurodegeneration, wherein the method comprises the steps of: (1 ) determining the level of the expression product:

(2) comparing it with a reference value, or alternatively

(4bis) comparing the value obtained in (3bis) with a reference value,

Wherein, if the level in the test sample is different from the reference value, this is indicative that the individual has to start a therapeutic intervention.

4. An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy, the method comprising performing step (i), or alternatively steps (ibis) to (iiibis), as defined in clause 1 before and after starting the therapeutic intervention, wherein: if the level measured once started the intervention is equal or higher than the level measured before starting the intervention, and lower than the reference value it is indicative that the therapeutic intervention is effective in the treatment of the

synaptopathy.

5. An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering neurodegeneration, the method comprising performing step (i) as defined in clause 2 before and after starting the therapeutic intervention, wherein: if the level measured once started the intervention is lower than the level measured before starting the intervention, it is indicative that the therapeutic intervention is effective in the treatment of the neurodegeneration.

6. The method according to any of the preceding clauses, wherein the test sample is selected from the group consisting of serum, plasma, saliva, pleural, cerebral spinal fluid (CSF), blood, amniotic fluid, urine, feces, mucus, cell extracts and pus.

7. The method according to any of the preceding clauses, wherein the individual is a human that suffers or is suspected to be suffering a neurodegenerative or

neuropsychiatric disease.

8. The method according to clause 7, wherein the individual suffers or is suspected of suffering a neurodegenerative disease selected from: Alzheimer disease, a preclinical stage thereof selected from the group consisting of Preclinical stage 1 , Preclinical stage 2, and prodromal stage, or, alternatively, frontotemporal dementia.

9. The method according to any of the preceding clauses, wherein it is determined the level of expression product of:

- NRX2A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 , in an isolated test sample of the individual; or, alternatively,

- Neurexin-3A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin- R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 in an isolated test sample of the individual; or alternatively,

- one set of proteins selected from the group consisting of Neurexin-2A-Nf-L, Neurexin- 3A-Nf-L, Thy-1 -Nf-L, VAMP-2-Nf-L, Tenascin-R-Nf-L, GluR4-Nf-L, Neuroligin-2-Nf-L, GluR2-Nf-L, Syntaxin-1 -Nf-L, and/or Calsyntenin-1 -Nf-L

10. The method according to any of the preceding clauses, wherein it is determined the level of expression of one or more of the following proteins: Neurexin-2A, Neurexin-3A, Thy-1 and VAMP-2.

11. The method according to any of the preceding clauses, wherein the level of expression product corresponds to the level of the protein markers. 12. The method according to clause 11 , wherein the determination of the level of the expression product is performed by a quantitative test selected from the group consisting of an immunological or spectroscopic technique.

13. Use of:

- Neurexin-2A and/or Neurexin-3A as: diagnostic or prognostic marker(s) of synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or

neurodegeneration; or alternatively,

- Neurexin-2A and/or Neurexin-3A in combination with one or more of the markers: Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, and Syntaxin-1 , Calsyntenin-1 , as: diagnostic or prognostic marker(s) of synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or neurodegeneration.

14. Use of a kit for the diagnosis and/or prognosis of synaptopathy and/or

neurodegeneration in an individual, or for deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, the kit comprising a solid support and means for detecting the level of expression of one or more of the following proteins Neurexin-2A, Neurexin-3A, Thy-1 , VAMP-2, Tenascin-R, GluR4, Neuroligin-2, GluR2, Syntaxin-1 , and Calsyntenin-1 and optionally means for detecting the level of expression of Nf-L.

15. The use according to clause 14, wherein the means for detecting the level of expression of the proteins are antibodies or fragments thereof.

Claims

1. An in vitro method of diagnosis and/or prognosis of synaptopathy in an individual, the method comprising: (i) determining the level of the expression product of: Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood; and

(ii) comparing it with a reference value, or alternatively

(iibis) determining the level of expression product of a neurodegenerative protein marker other than the markers referred in step ibis, such as Nf-L;

2. The in vitro method according to claim 1 , wherein it is determined in step (i) the level of expression product of Syntaxin-1.

3. An in vitro method of diagnosis and/or prognosis of neurodegeneration in an individual, the method comprising:

(i) determining the level of expression product of: VAMP-2, Syntaxin-1 , or both in an isolated test sample selected from CSF, serum, plasma and blood; and

4. The in vitro method according to claim 3, wherein it is determined in step (i) the level of expression product of VAMP-2.

5. An in vitro method of deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or

neurodegeneration, wherein the method comprises the steps of: (1 ) determining the level of the expression product: Syntaxin-1 , VAMP-2 or both in an isolated test sample selected from CSF, serum, plasma and blood; and

(2) comparing it with a reference value, or alternatively

(2bis) determining the level of expression product of a neurodegenerative protein marker other than the markers referred in step I bis, such as Nf-L;

(4bis) comparing the value obtained in (3bis) with a reference value, wherein, if the level in the test sample is different from the reference value, this is indicative that the individual has to start a therapeutic intervention.

6. An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy, the method comprising performing step (i), or alternatively steps (ibis) to (iiibis), as defined in claim 1 before and after starting the therapeutic intervention, wherein: if the level measured once started the intervention is equal or higher than the level measured before starting the intervention, and lower than the reference value it is indicative that the therapeutic intervention is effective in the treatment of the

synaptopathy.

7. An in vitro method for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering neurodegeneration, the method comprising performing step (i) as defined in claim 3 before and after starting the therapeutic intervention, wherein: if the level measured once started the intervention is lower than the level measured before starting the intervention, it is indicative that the therapeutic intervention is effective in the treatment of the neurodegeneration.

8. The method according to any of the preceding claims, wherein the test sample referred in step (ibis) or (I bis) is selected from the group consisting of serum, plasma, saliva, pleural, cerebral spinal fluid (CSF), blood, amniotic fluid, urine, feces, mucus, cell extracts and pus; preferably from the group consisting of CSF, serum, plasma, and blood.

9. The method according to any of the preceding claims, wherein the individual is a human that suffers or is suspected to be suffering a neurodegenerative or

neuropsychiatric disease.

10. The method according to claim 9, wherein the individual suffers or is suspected of suffering a neurodegenerative disease selected from: Alzheimer disease, a preclinical stage thereof selected from the group consisting of Preclinical stage 1 , Preclinical stage 2, and prodromal stage, or, alternatively, frontotemporal dementia.

1 1. The method according to any of the preceding claims, wherein it is further determined in step (i) or (1 ) the level of expression product of one or more of: NRX2A, Thy-1 ,

Tenascin-R, GluR4, Neuroligin-2, GluR2, Calsyntenin-1 , and Neurexin-3A,

12. The method according to any of the preceding claims, wherein the level of expression product corresponds to the level of the protein markers.

13. The method according to any of the preceding claims, wherein it is determined the level of expression product of one set of proteins selected from the group consisting of Neurexin-2A:Nf-L, Neurexin-3A:Nf-L, Thy-1 :Nf-L, VAMP-2:Nf-L, Tenascin-R:Nf-L,

GluR4:Nf-L, Neuroligin-2:Nf-L, GluR2:Nf-L, Syntaxin-1 :Nf-L, and/or Calsyntenin-1 :Nf-L.

14. The method according to any of the claims 12-13, wherein the determination of the level of the expression product is performed by a quantitative test selected from the group consisting of an immunological or spectroscopic technique.

15. Use ofSyntaxin-1 and/or VAMP-2 as: diagnostic or prognostic marker(s) of

synaptopathy and/or neurodegeneration; or as marker(s) for deciding or recommending whether to initiate a therapeutic intervention of individual suspicious of suffering a synaptopathy and/or neurodegeneration; or as marker(s) for determining the efficacy of a therapeutic intervention in a patient already diagnosed of suffering synaptopathy and/or neurodegeneration.

16. Use of a kit for the diagnosis and/or prognosis of synaptopathy and/or

neurodegeneration in an individual, or for deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, the kit comprising a solid support and means for detecting the level of expression of one or more of VAMP-2 Syntaxin-1 , and optionally means for detecting the level of expression of Nf-L.

17. The use further comprising means for detecting the level of expression of one or more of Neurexin-2A, Neurexin-3A, Thy-1 , Tenascin-R, GluR4, Neuroligin-2, GluR2, and Calsyntenin-1.

18. Use of a kit for the diagnosis and/or prognosis of synaptopathy and/or

neurodegeneration in an individual, or for deciding or recommending whether to initiate a therapeutic intervention of an individual suspicious of suffering a synaptopathy and/or neurodegeneration, the kit comprising a solid support and means for detecting the level of expression of one or more of VAMP-2 Syntaxin-1 , and means for detecting the level of expression of Nf-L.

19. The use according to any of the claims 16-18, wherein the means for detecting the level of expression of the proteins are antibodies or fragments thereof.