WO2024161163A2 - Diagnosis of vascular dementia - Google Patents

Abstract

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. Altered levels of protein biomarkers are discloses to be useful in the diagnosis of vascular dementia.

Description

Diagnosis of Vascular Dementia

FIELD OF THE INVENTION

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes.

The present inventors have found significantly altered proteins in the mitochondria, MAM, and CSF samples. Most of the changed proteins were involved in protein turnover and import in all three sample types.

Thus, the present inventors have shown by proteomic analysis that chronic cerebral hypoperfusion-induced disturbed proteostasis of mitochondria and MAM is reflected in the CSF.

BACKGROUND ART

Chronic cerebral hypoperfusion (CCH) is a pathological state that is characterized by declining cerebral blood flow (CBF). One of the potential risk factors for vascular dementia (VD) and sporadic Alzheimer's disease (AD) is the decreased blood flow of the brain (Feng et al., 2018; Montaldi et al, 1990). The present inventors performed stepwise bilateral common carotid artery occlusion (BCCAO) which induces oxidative stress, inflammatory response, and disturbed lipid metabolism leading to cognitive impairment (Farkas et al., 2007). In the stepwise BCCAO model, rats have one week of regeneration time between the bilateral common carotid artery occlusions, leading to a gradual decrease in cerebral blood flow. The present inventors previously studied the long-term effects in this model when the CBF is almost completely recovered (Otori et al., 2003); however, cognitive deficits and oxidative stress are apparent (Yadav et al., 2018).

Brain plasticity is the ability of the brain to modify its structure and function in response to the alterations of its environment. Many subcellular organelles can adapt to changing environments of neurons such as synapses and mitochondria. In our previous studies, Volgyi et al. investigated mitochondrial (Volgyi et al., 2017) and mitochondrial- associated membrane (MAM) proteome (Volgyi et al., 2018) alterations in Alzheimer's disease model animals, revealing the relevance of these organelles in neurodegeneration.

Since the brain is one of the most energy-demanding organs and its ATP source relies mostly on oxidative phosphorylation, mitochondrial proteome changes can give insight into its molecular adaptation to CCH. The mitochondria are vital, dynamic, and plastic organelles that are essential for maintaining membrane ion gradients, neurotransmission, and synaptic plasticity, requiring a large amount of energy. Moreover, mitochondria participate in other neuronal processes such as calcium buffering and intracellular signaling. Dysfunctions of mitochondria were implicated in VD and its animal models (Du et al., 2013; Yadav et al., 2018; Li et al., 2016) and several other neurodegenerative disorders (Islam, 2017; Burte et al., 2015; Bhat et al., 2015; Mancuso et al., 2007). In the BCCAO model, increased mitochondrial DNA deletion and structural damage were observed (He et al., 2019). Despite the known role of mitochondria in CCH, our study is the first proteomic analysis of this organelle in the BCCAO model.

Furthermore, mitochondrial dynamics are disturbed in several neurodegenerative disorders (Calkins et al., 2011; Burte et al., 2015). MAM also participates in the protein supply of mitochondria and regulates mitochondrial dynamics (Area- Gomez and Schon, 2017). Besides mitochondrial dynamics, MAM also regulates essential cellular processes, such as fatty acid metabolism, and calcium homeostasis (Krols et al., 2016). Also, it has a main role in the processing of amyloid precursor protein which is dysregulated in AD (Area-Gomez et al., 2012). In addition, MAM dysfunction is involved in other neurodegenerative diseases (Krols et al., 2016); however, its role in CCH is poorly studied.

Cerebrospinal fluid (CSF) has a vital role in the clearance of brain interstitial fluid (Iliff et al., 2012). Due to its direct contact with the brain, its content can reflect biochemical changes in the brain. CSF has detectable protein content; therefore, proteomic alterations in the CSF might provide potential biomarkers that can indicate early, pre-symptomatic pathological alterations, and CSF sampling is feasible in human translational studies.

Additionally, decreased brain metabolism is one of the earliest clinical symptoms of AD which induces mitochondrial dysfunction in the brain (Cardoso et al., 2016). Detecting molecular shifts of mitochondria or MAM in the CSF can provide early and detectable pathological signs of neurodegeneration. In the current study, the present inventors, by monitoring changes induced by BCCAO in the MAM and mitochondria of the frontal cortex, which is one of the brain areas responsible for higher-order cognitive functions and affected in VD (Tatemichi et al., 1995; Thomas et al., 2015), have surprisingly identified a system of protein markers for vascular dementia. The proteome of the CSF was also analysed to reveal whether organellar changes could be detected in body fluids. Mass spectrometry-based (MS-based) and gel-based proteomic methods were used to study the proteomic alterations - induced by stepwise BCCAO - of CSF and the organelles of the frontal cortex, respectively. As an unexpected result, protein function networks have been revealed which organize protein biomarkers useful in diagnosis of vascular dementia.

BRIEF DESCRIPTION OF THE INVENTION la. The invention relates to a diagnostic method for indicating vascular dementia in mammalian patient (alternatively a method for diagnosing a condition related to vascular dementia in a mammalian patient), said method comprising the steps of a) providing a sample of biological fluid being in contact with the nervous system of said patient, preferably selected from the group consisting of a sample of cerebrospinal fluid or blood origin, a sample comprising proteins of mitochondrial origin and a sample comprising mitochondrial-associated membrane, b) measuring level of one or more protein biomarkers of one or more biomarker panels in the sample, preferably a set of protein biomarkers of one or more, preferably two or more biomarker panels in the sample, each of said protein biomarkers being expressed from a respective biomarker coding gene in said patient, preferably in the nervous system of said patient, wherein each of the one or more biomarker panels consist(s) of biomarkers belonging to a protein function network (/common protein function class), preferably a protein network of biomarkers classified according to the cellular function of the expressed proteins, (preferably a common pathway), c) wherein if the level of the protein biomarkers (at least one protein biomarker from each biomarker panels, preferably at least one protein biomarker from each of two or more biomarker panels, preferably at least 2 protein biomarkers from each of two or more biomarker panels, preferably 5 to 10 protein biomarkers from each of two or more biomarker panels, preferably from each 3 biomarker panels) is significantly altered (increased or decreased) in comparison with a reference level, the patient is considered as having vascular dementia or being susceptive of vascular dementia or being at risk of vascular dementia, in particular wherein said protein function network (in particular of the respective protein once expressed from the biomarker coding gene) being selected from the group consisting of energy and carbohydrate metabolism (in particular in mitochondria-associated samples and/or in CSF and blood samples, preferably in MAM sample and/or mitochondrial sample), protein turnover and import (in particular in mitochondria-associated samples and/or in CSF and blood sample, preferably in MAM sample and/or mitochondrial sample), cytoskeletal proteins (in particular in mitochondria-associated samples and/or blood and/or CSF, preferably in MAM sample and/or mitochondrial sample), mitochondrial processes (including blood and/or CSF sample, preferably in MAM sample and/or mitochondrial sample), vesicle transport and/or fusion (including blood and/or CSF sample, preferably in MAM sample and/or mitochondrial sample), fatty acid metabolism (including blood and/or CSF sample, preferably in MAM sample), signaling pathways (including blood and/or CSF sample, preferably in MAM sample and/or mitochondrial sample), ion transport (including blood and/or CSF sample, preferably in MAM sample), redox-state regulation (in particular in blood and/or CSF sample), complement cascade regulation (in particular in blood and/or CSF sample), protein folding (including protein binding involved in protein folding) (GO terms); or preferably: energy and carbohydrate metabolism (in particular in mitochondria-associated samples and/or in blood and/or CSF sample, preferably in MAM sample and/or mitochondrial sample), protein turnover and import (in particular in mitochondria-associated samples and/or blood and/or CSF, preferably in MAM sample and/or mitochondrial sample), cytoskeletal proteins (in particular in mitochondria-associated samples and/or blood and/or CSF, preferably in

MAM sample and/or mitochondrial sample), redox-state regulation (in particular in CSF sample or in blood sample), complement cascade regulation (in particular in blood and/or CSF sample); or preferably (in particular in blood and/or CSF sample): protein turnover and import, redox-state regulation or preferably: energy and carbohydrate metabolism, protein turnover and import, and cytoskeletal proteins. or in particular wherein said function class (GO terms and KEGG pathways) being selected from the group consisting of protein folding (GO term), protein binding involved in protein folding (GO term),

ATP-metabolic processes (GO term), glycolysis/gluconeogenesis (KEGG pathway) biosynthesis of amino acids (KEGG pathway). lb. The invention also relates to a diagnostic method for indicating vascular dementia in mammalian patient (or alternatively to a method for diagnosing a condition related to vascular dementia in a mammalian patient), said method comprising the steps of a) providing a biological sample from said patient, in particular a sample selected from the group consisting of a sample of cerebrospinal fluid and blood origin, a sample comprising proteins of mitochondrial origin and a sample comprising mitochondrial-associated membrane, b) measuring level of one or more protein biomarkers of one or more biomarker panels in the sample, preferably a set of protein biomarkers of one or more, preferably two or more biomarker panels in the sample, each of said protein biomarkers being expressed from a respective biomarker coding gene in said patient, preferably in the nervous system of said patient, wherein each of the one or more biomarker panels consist(s) of biomarkers belonging to a protein function network (/common protein function class), preferably a protein network of biomarkers classified according to the cellular function of the expressed proteins, (preferably a common pathway), c) wherein if the level of the biomarkers (at least one protein biomarker from each biomarker panels, preferably at least one protein biomarker from each of two or more biomarker panels, preferably at least 2 protein biomarkers from each of two or more biomarker panels, preferably 5 to 10 protein biomarkers from each of two or more biomarker panels, preferably from each 3 biomarker panels) is significantly altered (increased or decreased) in comparison with a reference level, the patient is considered as having vascular dementia or being susceptive of vascular dementia or being at risk of vascular dementia, said protein function network ("function class") (/of the respective proteins once expressed from the biomarker coding gene) being selected from the group consisting of energy and carbohydrate metabolism, protein turnover and import, and cytoskeletal proteins protein folding (including protein binding involved in protein folding) (GO terms). lc. The invention also relates to a diagnostic method for indicating vascular dementia in mammalian patient (or alternatively a method for diagnosing a condition related to vascular dementia in a mammalian patient), said method comprising the steps of a) providing a biological fluid sample from said patient, in particular a nervous system related fluid sample from said patient, preferably a sample of blood or cerebrospinal fluid origin, b) measuring level of one or more expression protein biomarkers or protein biomarkers, preferably a set of protein biomarkers of one or more, preferably two or more biomarker panels in the sample, each of said protein biomarkers being expressed from a specific biomarker coding gene in said patient, preferably in the nervous system of said patient, wherein each of the one or more biomarker panels consist(s) of biomarkers belonging to a protein function network (/common protein function class), preferably a protein network of biomarkers classified according to the cellular function of the expressed proteins, (preferably a common pathway), c) wherein if the level of the protein (at least one protein biomarker from each biomarker panels, preferably at least one protein biomarker from each of two or more biomarker panels, preferably at least 2 protein biomarkers from each of two or more biomarker panels, preferably 5 to 10 protein biomarkers from each of two or more biomarker panels, preferably from each 3 biomarker panels) is significantly altered (increased or decreased) in comparison with a reference level, the patient is considered as having vascular dementia or being susceptive of vascular dementia or being at risk of vascular dementia, said protein function network (/function class) (in particular of the respective protein once expressed from the biomarker coding gene) being selected from the group consisting of energy and carbohydrate metabolism (in particular in mitochondria-associated samples and/or in blood and/or CSF sample, preferably in MAM sample and/or mitochondrial sample), protein turnover and import (in particular in mitochondria-associated samples and/or blood and/or CSF, preferably in MAM sample and/or mitochondrial sample), cytoskeletal proteins (in particular in mitochondria-associated samples and/or blood and/or CSF, preferably in MAM sample and/or mitochondrial sample), redox-state regulation (in particular in blood and/or CSF sample), complement cascade regulation (in particular in blood and/or CSF sample); protein folding (including protein binding involved in protein folding) (GO terms); or preferably (in particular in blood and/or CSF sample): protein turnover and import, redox-state regulation or preferably (general): energy and carbohydrate metabolism, protein turnover and import, and cytoskeletal proteins.

OR in particular wherein said function class (GO terms and KEGG pathways) being selected from the group consisting of protein folding (GO term), protein binding involved in protein folding (GO term), ATP-metabolic processes (GO term), glycolysis/gluconeogenesis (KEGG pathway) biosynthesis of amino acids (KEGG pathway).

Id. The invention also relates to a method for assessing in a subject the risk of developing a neurocognitive disorder of vascular origin, in particular wherein said neurocognitive disorder is selected from the group of mild cognitive impairment, Alzheimer's disease of vascular origin, vascular dementia or combinations thereof, said method comprising the steps as defined in paragraph la or lb or lc.

2. The method according to any of paragraphs la to Id wherein step b) of measuring comprises: a) analyzing the sample from said patient to obtain data, preferably quantitative data, for the level of one or more than one biomarker(s); b) comparing the data for said one or more biomarker(s) to corresponding data obtained for one or more than one reference sample or to data for respective pre-determined reference level(s) to identify alteration (an increase or decrease) in the level of said one or more than one biomarker in said sample.

3. The invention relates to method according to any of paragraphs la to 2, wherein protein levels are measured by western blot.

4. The method according to any of paragraphs la to 2, wherein protein levels are measured by a proteomic method, preferably by two-dimensional differential gel electrophoresis (2-D DIGE).

5. The method according to any of paragraphs la to 2, wherein protein levels are measured by a proteomic method, preferably by mass spectrometry.

6. The method according to any of paragraphs la to 5, wherein the level of at least one protein biomarker from a set of biomarkers represented by a group of biomarkers as defined in any of the paragraphs below, preferably a group being biomarkers in a paragraph, or biomarkers in a line or in a preferred option; preferably the level of at least two protein biomarker, preferably at least 3, 4 or 5 (if the case allows) biomarkers represented by a group of biomarkers as defined in any of the paragraphs below, preferably a group being biomarkers in a paragraph, or biomarkers in a line or in a preferred option; the level of at least one, preferably at least two protein biomarker, preferably at least 3, 4 or 5 (if the case allows) protein biomarker from each of two or more biomarker panels in any of the paragraphs below, preferably at least three biomarker panels in any of the paragraphs below.

7. The method according to any of paragraphs la to 6, wherein the biomarker is selected from the group consisting of

ATP5B (AtpSflb) - (energy and carbohydrate metabolism)

ALDOC (Aldoc) - (energy and carbohydrate metabolism)

ENO2 (Eno2) - (energy and carbohydrate metabolism)

PSMA6 (Psma6) - (protein turnover and import, in particular amino acid synthesis or metabolism) WARS (wars) - (protein turnover and import, in particular amino acid synthesis or metabolism).

8. The method according to any of paragraphs la to 6, wherein the level of any or more of the following protein biomarkers is altered in the sample:

Hspa5 has decreased level,

Pdia3 and/or P4hb is/are upregulated, and/or the level of any or more of the following biomarkers is altered in the sample:

T-complex protein subunits 2, 5 (Cct2, Cct5) have decreased level,

P-tubulin subunits (Tubb2a, Tubb4a), and Trapl have decreased level, proteasome subunit alpha type-6 (Psma6) and/or proteasome subunit beta (Psmb4) have a decreased level.

9. The method according to any of paragraphs la to 6, wherein the level of any or more of the following oxidative phosphorylation protein biomarkers is altered in the sample:

ATP synthase subunit beta (Atp5flb),

- Ndufsl and/or the level of any or more of the following heat-shock protein biomarkers is altered in the sample:

Hspdl and/or

Hspa9. and/or the level of any or more of the following branch-chained amino acids (BCAAs) biomarkers is altered in the sample: methylcrotonoyl-CoA carboxylase subunit alpha (Mcccl) has increased level, and/or 3-hydroxyisobutyrate dehydrogenase (Hibadh) has decreased level.

10. The method according to any of paragraphs la to 6, wherein the level of any or more of the following protein turnover and redox state regulation biomarkers is altered in the sample: Eif3j, Uchl3, Psmb4 in the CSF, Psma6 (MAM), and glycine— tRNA ligase (Gars) (M) Glrx and Gstpl (decrease in CSF).

11. The method according to any of paragraphs la to 6 or 10, wherein the biomarker panel consisting of biomarkers belonging to energy and carbohydrate metabolism comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Ndufsl, Pdhal, Aldoc, Gapdh, Nt5cla, Atp5flb, Pdhx, Eno2, Tpil, preferably Ndufsl, Pdhal, Aldoc, Gapdh, Nt5cla, (MAM)

Atp5flb, Ak5, Pcca, (M) and/or

Lgals5, (CSF),

OR (according to localization):

Ndufsl, Atp5flb, Pdhal, Pdhx, Pcca, (mitochondrial localization)

Eno2, Aldoc, Gapdh, Nt5cla, Ak5, Lgals5 (cytoplasmatic localization)

12. The method according to any of paragraphs la 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to protein turnover and import, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2, Wars, Rpsl2, Psma6, Mcccl, Glul, (MAM)

Trapl, Hspa5, P4hb, Gars, Ctsd, Hibadh (M)

Psmb4, Uchl3, Eif3j, Kpnbl, Wfdcl (CSF), preferably: Hspa8, Hspa9, Pdia3, Hspdl, Psma6, Cct5, Mcccl, Glul, P4hb, more preferably Hspa8, Hspa9,

OR (according to localization):

Hspa9, Pdia3, Hspdl, Mcccl, Glul, Trapl, Hspa5, Gars, Hibadh (mitochondrial localization), preferably Hspa9, Pdia3, Hspdl, Mcccl, Glul, more preferably Hspa9,

Hspa8, Cct5, Cct2, Wars, Rpsl2, Psma6, Psmb4, Uchl3, Eif3j, (cytoplasmatic localization); preferably Hspa8, Cct5, Psma6, more preferably Hspa8, preferably of protein folding class:

Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2 (MAM),

Trapl, Hspa5, P4hb; (M), preferably of protein or amino acid synthesis or metabolism, including amino acid synthesis or metabolism class:

Wars, Rpsl2, Psma6, Mcccl, Glul,

Psmb4, Uchl3, Eif3j.

13. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to cytoskeletal proteins, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of

Tubalb, Tuba4a, Tubala, Dpysl2, Dpysl3, Septinll (MAM),

Dpysl2, Tubb2a, Tubb4a, Tubalb, Tmod2 (M)

Capl (CSF), preferably Tubalb, Tuba4a, Tubala, or preferably Dpysl2.

14. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to redox-state regulation, wherein the sample is in particular a blood and/or CSF sample, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Gstpl and Glrx.

15. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to complement cascade regulation, wherein the sample is in particular a blood and/or CSF sample, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Cd59.

16. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to protein folding (including protein binding involved in protein folding), comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2

Trapl, Hspa5, P4hb;

OR (based on GO terms)

HSPA9, HSPA8, HSPA5, HSPD1 (protein refolding), or

FKBP1A, HSPA8, CCT2, CCT5 (chaperone-mediated protein folding) or

HSPA9, PDIA3, HSPA8, CCT2, TRAP1, P4HB, CCT5,

HSPD1 (protein folding) or

17. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to ATP metabolic process, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of HSPA8, ATP5F1B, ATP6V1B2, NDUFS1, AK5.

18. The method according to any of paragraphs la to 6 or to 10, wherein the biomarker panel consisting of biomarkers belonging to any of the following protein function classes, mitochondrial processes (preferably in blood, CSF, MAM sample and/or mitochondrial sample), vesicle transport and/or fusion (preferably in blood, CSF, MAM sample and/or mitochondrial sample), fatty acid metabolism (preferably in blood, CSF and/or MAM sample), signaling pathways (preferably in blood, CSF and/or MAM sample and/or mitochondrial sample), and/or ion transport (preferably in blood, CSF and/or MAM sample).

ATP synthesis or metabolism (preferably in blood, CSF and/or MAM sample).

19. The method according to paragraph 18 wherein the level of any or more of the following protein biomarker(s) is/are altered: mitochondrial processes biomarker Timm50 and/or Slc25al2, vesicular transport biomarker Nsf, vesicular fusion biomarker Stxbpl, fatty acid metabolism biomarker Fabp5, signaling pathway biomarkers Ppp2rla, Fkbpla and/or Prkar2b, preferably TGFbeta signaling pathway biomarker Fkbpla, PKA signaling biomarker Prkar2b, proton transport biomarker Atp6vlb2, and/or

ATP synthesis or metabolism biomarker(s) Atp5flb, Ak5, further mitochondria-related biomarkers Septin3, Mpp3, Mpp6 and/or Emc8 and/or further CSF biomarkers Ppal and/or Anxa2.

20. The method according to any of paragraphs la to 19, wherein the direction of the alteration of level of a protein biomarker (increase or decrease) is as indicated in Table 1 or 2. ABBREVIATIONS

CCH: Chronic cerebral hypoperfusion

CBF: Cerebral blood flow

VD: Vascular dementia

AD: Alzheimer's disease

BCCAO: Bilateral common carotid artery occlusion

MAM: Mitochondrial-associated membrane

2-D DIGE: 2-dimensional differential gel electrophoresis

HPLC-MS/MS: High performance liquid chromatography-tandem mass spectroscopy

MRA: Magnetic resonance angiography

IB-1: Isolation buffer-1

IB-2: Isolation buffer-2

IB-3: Isolation buffer-3

MRB: Mitochondria resuspending buffer

IEF: Isoelectric focusing

FC: Fold change

GO: Gene Ontology

KEGG Kyoto Encyclopedia of Genes and Genomes

ERAD: ER-associated degradation

DEFINITIONS

A "patient" is a mammalian subject who is or intended to be under medical or veterinarian observation, supervision, diagnosis or treatment. Highly preferably the patient is a primate, a hominid or a human.

An "assay method" or shortly "assay" as used herein is an analytical laboratory procedure for detecting, preferably qualitatively or quantitatively assessing or quantitatively or semi-quantitatively measuring the presence, level or amount, or functional activity of a target entity (the herein a protein biomarker). In the present invention the assay method may be for example a research method or a diagnostic method.

"Diagnosis" is understood herein as a process of identifying a condition of a patient, by observation of signs, body parameters of, including measurement or detection of features of a sample obtained from said patient. The condition may be a disease, consequences of disease or post-disease status or health status or reaction of the patient's body to a treatment.

"Vascular dementia" is a common type of dementia caused by reduced blood flow to the brain. The general term describes problems with thought processes including reasoning, planning, judgment, memory, caused by brain damage from impaired blood flow to your brain. In a particular sense vascular dementia is a dementia due to cerebrovascular disease having code 6D81 in ICD-11 (eleventh revision of the International Classification of Diseases, ICD,, World Health Organization, see e.g. Version : 02/2022). A condition related to vascular dementia is a condition which results from vascular dementia or a consequence of dementia or a condition which is considered as a cause of vascular dementia, including vascular dementia itself. Specifically, symptoms of vascular dementia, like changes to memory, thinking, and behavior result from vascular dementia related conditions, like impaired or declining cerebral blood flow, that affect the blood vessels in the brain or interrupt the flow of blood and oxygen supply to the brain.

"Indicating vascular dementia" is understood herein as an object or result wherein the patient is considered as having vascular dementia or having a condition which suggest that the patient will or probably will or may develop vascular dementia (e.g. having a condition related to vascular dementia) or having a condition resulted or caused by vascular dementia being present for a given time period.

The term "biomarker(s)" is generally defined as a measurable substance that can be used as an indicator for ongoing physiological and pathological processes. As used herein the term "biomarker" is particularly understood as a protein expressed from a gene coding therefor wherein the presence or level of said protein is relevant to a condition related to vascular dementia. The more specific terms "expression biomarker" and "protein biomarker" refer to these facts. A protein biomarker includes all biologically relevant forms of the protein identified, including post-translational modifications. For example, the biomarker can be present in a glycosylated, phosphorylated, multimeric, fragmented or precursor form. A biomarker fragment may be naturally occurring or, for example, enzymatically generated and still retaining the biologically active function of the full protein. Fragments will typically be at least about 10 amino acids, usually at least about 50 amino acids in length, and can be as long as 300 amino acids in length or longer.

A protein biomarker in its form expressed is encoded by a biomarker gene. In case of protein biomarkers their biomarker genes are, as examples, listed in Tables 1 to 6 of the specification.

It is to be understood that the sequence of the genes as well as their expression product can be found in e.g. the UNIPROT database [See e.g. UniProt: the Universal Protein Knowledgebase in 2023, Nucleic Acids Res. 51:D523-D531 (2023)].

The skilled person will understand that protein function networks are largely the same across mammals and proteins, based on their functions are largely homologous across species. Therefore, in case of a protein in a given mammalian species and the specific gene encoding it, a corresponding gene of another species can be found. In particular, exemplary studies shown herein were based on rate experiments and protein biomarkers as well as respective protein function networks related to vascular dementia have been identified based on these experiments. Human counterparts of the rat genes and encoded proteins can be easily identified and used in human diagnosis. In particular the skilled person will understand that an analogous condition of the other species, like human, will affect the same protein function networks or preferably the corresponding proteins and therefore the present invention can be adapted across mammalian species by a person skilled in the art.

A "protein function network" (or protein function class) is understood herein as a network (or class) of several proteins having similar and/or (inter)related function; preferably a protein network of biomarkers classified into the same class or network according to the cellular function of the expressed proteins, (preferably a common pathway). As an example, "protein function network" comprises a class of proteins belonging to a defined biological domain. For example, the classes have same, similar or related molecular function, or they may form the same, similar or related cellular component or may take part in a same, similar or related biological process, e.g. pathway.

An example for "protein function networks" are the Gene Ontology (GO) terms [see Ashburner et al. Gene ontology: tool for the unification of biology. Nat Genet. May 2000;25(l):25-9. and The Gene Ontology resource: enriching a GOId mine.

Nucleic Acids Res. Jan 2021;49(Dl):D325-D334.]

An ontology is a formal representation of a body of knowledge within a given domain. Ontologies usually consist of a set of classes (or terms or concepts) with relations that operate between them. The Gene Ontology (GO) describes our knowledge of the biological domain with respect to three aspects: molecular function, cellular component and biological process, each of which comprising terms defining protein function networks.

The term "KEGG pathway" refers to the terms defining pathways in the Kyoto Encyclopedia of Genes and Genomes [Kanehisa, M et al. KEGG for taxonomy-based analysis of pathways and genomes, Nucleic Acids Research, Volume 51, Issue DI, 6 January 2023, Pages D587-D592], a collection of manually drawn pathway maps representing molecular interactions and reaction networks for metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, human diseases and drug development.

The term "level" is understood broadly as a data to characterize the presence, concentration or amount of a protein in an environment like in a sample; preferably the level is a value or set of values indicative of a protein biomarker, e.g. the level of each of the biomarkers herein is a quantified value which is suitable for comparison with another "level" i.e. quantified value obtained similarly, e.g. obtained for the same protein by the same method in or from a different sample, e.g. in or from a control sample. Thus, a level can be increased or decreased in comparison with a control level. The control level may also be a value which is derived from multiple measurement and/or calculations and/or estimations. The term "concentration or amount" may refer to absolute or the relative concentration or amount of biomarker in the sample, for example as determined by any physical method.

"Comparing" two levels is understood herein to include a comparison of quantities expressed in numerical values characterizing said levels to establish which is higher or lower, optionally completed with mathematical procedures (calculation) as the measurement method requires. In an embodiment comparing comprises subtracting two levels, e.g. subtracting two normalized or baseline-corrected level. In an embodiment comparing comprises a mathematical procedure, e.g. transformation carried out on both levels, e.g. calculating logarithm, or other function. In an embodiment comparing comprises making statistics and calculating errors and/or means or averages which is/are considered, and/or assessment of statistics assessment of statistical significance. In an embodiment comparing includes establishing a difference or establishing a ratio of the levels, or values derived from the levels. Comparing also means measuring the relative concentration or amount of a biomarker in a sample relative to other samples (for example protein concentrations or amounts stored in proprietary or public database).

The meaning of "in particular" or "particularly" is to indicate an optional feature which may be cancelled if the definition of the invention or embodiment is complete without the "particular" feature; moreover, said feature may be made obligatory by omitting the term "in particular" or "particularly", while these terms do not state that the optional feature is preferred, they suggest that from a specific point of view it is special or specific and therefore it is to be specifically mentioned ("particularized"). However, if a context or experiment shows that feature so stated to be special is also advantageous for some reason this fact may also be indicated by using the term "preferred" or "preferably".

The term "comprises" or "comprising" or "including" are to be construed here as having a non-exhaustive meaning and allow the addition or involvement of further features or method steps or components to anything which comprises the listed features or method steps or components.

The expression "consisting essentially of" or "comprising substantially" is to be understood as consisting of mandatory features or method steps or components listed in a list e.g. in a claim or paragraph whereas allowing to contain additionally other features or method steps or components which do not materially affect the essential characteristics of the use, method, composition or other subject matter. It is to be understood that "comprises" or "comprising" or "including" can be replaced herein by "consisting essentially of" or "comprising substantially" if so required without addition of new matter.

The singular forms "a", "an" and "the", or at least "a", "an", include plural reference unless the context clearly dictates otherwise.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - MRA recordings of animals 5 weeks after the operations. Representative images of sham-operated (left) and BCCAO (right) rats are shown (a). The common carotid arteries (CC) are occluded, and the basilar artery (B) is thickened in the BCCAO animals. Image analysis of MRA recordings (b), statistical evaluation was performed with non-parametric, Mann-Whitney test (*: P-value < 0.01)

Figure 2 - PCA (a) and hierarchical clustering (b) of MAM could distinguish sham-operated and CCH animals. However, PCA (c) and hierarchical clustering (d) of mitochondria identified one sham-operated animal that differs from the rest. Volcano plots of MAM (e) and mitochondria (f) proteomic results are shown. Pie charts of significantly altered proteins in MAM (g) and mitochondria (h) represent functional annotation with legend (i)

Figure 3 - PCA (a) and hierarchical clustering (b) of CSF could separate sham-operated and CCH animals. Volcano plots of CSF proteomic results (c) and pie chart (d) of functional annotation with legend (e) are shown.

Figure 4 - Enrichment map of top five terms of GO biological process (a), GO molecular function (b), and KEGG pathway (c) are connected to their related proteins labeled with gene names. Fold enrichment and -log(adjusted P-value) of terms are represented with node size and grayscale, respectively. Altered proteins of MAM, mitochondria, and CSF are depicted with circle, hexagon, and rhombus, respectively.

Figure 5 - Altered levels of P4hb (a), Hibadh (b), Trapl (c), and Hspa5 (d) in the mitochondria analyzed by western blot. Specific protein bands and total protein staining (e) are shown. Independent two-tailed Student's t-test was applied (*: P-value < 0.05).

DETAILED DESCRIPTION OF THE INVENTION

Declining cerebral blood flow leads to chronic cerebral hypoperfusion which can induce neurodegenerative disorders, such as vascular dementia. The reduced energy supply of the brain impairs mitochondrial functions that could trigger further damaging cellular processes. The present inventors have carried out stepwise bilateral common carotid occlusions on rats and investigated long-term mitochondrial, mitochondria-associated membrane (MAM), and cerebrospinal fluid (CSF) proteome changes. Samples were studied by gel-based and mass spectrometry-based proteomic analyses. The present inventors have unexpectedly found that several specific protein function networks provide protein biomarkers of altered expression levels indicative of vascular dementia.

The present inventors have carried out gel-based proteomics study on the MAM and mitochondria containing subcellular fractions and found that among 725 and 708 protein spots on the gels of MAM and mitochondria samples, respectively, 17 and 6 spots showed significant change in the MAM and mitochondria sample (Fig. 2a and 2b; Table 3 and Table 4). Altered proteins showed fold changes (FC) between -1.69 to 2.38 in the MAM samples and smaller fold changes (FC) in the mitochondrial samples. From altered spots, HPLC-MS/MS identified 19 proteins from the mitochondria sample, and 35 from MAM (Table 1). Taken together, a modest alteration was observed in the levels of mitochondrial proteins and a more complex proteome modification in the MAM-containing sample.

From the expression changes several observations could be made.

For example, in the MAM sample the highest increase was observed in case of NADH dehydrogenase (Ubiquinone) Fe-S protein 1 (Ndufsl) (the subunit of complex I of the electron transport chain), and heat shock proteins, namely stress-70 protein, mitochondrial (Hspa9 or mortalin), and heat shock cognate 71 kDa protein (Hspa8). Further increased spots with fold changes of 1.67, are comprised of methylcrotonoyl-CoA carboxylase subunit alpha (Mcccl) and vesicle-fusing ATPase (Nsf) which are involved in branch-chained amino acid catabolism and vesicle transport, respectively.

Among the decreased level markers protein disulfide isomerase A3 (Pdia3) chaperone (FC = -1.69), as well as T-complex protein 1 subunit epsilon (Cct5), proteasome subunit alpha type-6 (Psma6), and neuronal-specific septin-3 (Septin 3) with fold changes of -1.62, -1.55, and -1.41 were observed.

In the mitochondrial fraction decreasing protein marker levels (FC = -1.31) involved protein disulfide isomerase (P4hb or Pdial) chaperon, as well as, with a lower negative fold change, endoplasmic reticulum chaperone BiP (Hspa5), ATP synthase subunit beta (Atp5flb), heat shock protein 75 kDa (Trapl), and cytoskeletal protein dihydropyrimidinase- related protein 2 (Dpysl2). The spot most increasing level (FC = - 1.27) was observed in the case of cytoskeletal protein tropomodulin-2 (Tmod2)

Upon analysis of the spots several protein function networks were involved, like branch-chained amino acid catabolism and vesicle transport as well as protein turnover and import and energy and carbohydrate metabolism.

Importantly, several cytoskeletal proteins were involved.

As to the CSF sample CSF proteome analysis was performed using LC-MS/MS. Protein abundance mean ratios of BCCAO and control samples were calculated. By this method 12 proteins with significantly altered abundance with mean ratios between 0.32 and 2.06, using MS-based proteomics were identified. As examples, CD59 glycoprotein (Cd59) and WAP four-disulfide core domain protein 1 (Wfdcl), increased significantly whereas Annexin A2 (Anxa2) showed the lowest mean ratio. Glutaredoxin-1 (Glrx) and glutathione S-transferase P (Gstpl) of the redox state regulation protein function network and further proteins (decreasing proteasome subunit beta (Psmb4), ubiquitin carboxyl-terminal hydrolase isozyme L3 (Uchl3), eukaryotic translation initiation factor 3 subunit J (Eif3j) and importin subunit beta-1 (Kpnbl)); and increasing Wfdcl, an endopeptidase inhibitor) of the protein turnover and import function network have been identified. With functional annotation further biomarkers of the following protein function networks have been identified: ATP metabolic processes, protein folding protein binding involved in protein folding as GO terms as well as glycolysis/gluconeogenesis, biosynthesis of amino acids and neurodegeneration - multiple diseases KEGG pathways.

Briefly, 19, 35, and 12 significantly altered proteins were found in the mitochondria, MAM, and CSF samples, respectively. Most of the changed proteins were involved in protein turnover and import in all three sample types. Decreased levels of proteins involved in protein folding and amino acid catabolism. Reduced levels of several components of protein synthesis and degradation in the CSF as well as in the subcellular fractions have been detected, implying that hypoperfusion-induced altered protein turnover of brain tissue can be detected in the CSF by proteomic analysis.

The skilled person will understand that the protein function networks identified, including those identified by GO terms and KEGG pathways, as well as those listed in Tables 1 and 2 are useful in diagnosis in other species, in particular in humans.

Also the finding is not limited to the exemplary samples shown herein as it is expectable that the protein biomarker changes, and in particular the changes in the functional network level can be identified from blood as well.

The markers of the invention can also be used together with other markers to obtain a more complex diagnostic analysis. While the protein markers providing the higher increase or decrease have a diagnostic value on their own, applying markers sets of multiple (at least two) markers from various marker panels have a significant advantage as defined herein.

Chaperons of the ER protein quality control

MAM is a specialized area of the ER, communicating directly with mitochondria. ER is essential in protein synthesis, folding, and trafficking and several ER chaperones localize to the MAM (Simmen et al., 2010). Upon acute energy restriction, misfolded and unfolded proteins accumulate in the ER lumen (Wang and Kaufman, 2016). In response to ER stress, the unfolded protein response (UPR) is triggered to restore proteostasis by reducing protein synthesis, promoting proper protein folding, and the degradation of misfolded proteins. ER-associated degradation (ERAD) induces misfolded protein clearance by proteasomal degradation. Thus, ERAD and UPR serve to restore normal proteostasis upon stress.

A widely used marker of ER stress is Hspa5 chaperone; we detected its decrease in the mitochondria. Although Hspa5 level showed a declining trend analyzed by western blot, its decrease was not significant. Overexpressing Hspa5 in primary cultured astrocytes protected mitochondria against ischemic stress (Ouyang et al., 2011). Additional ER chaperones implicated in UPR, Pdia3 and P4hb, also changed in our study; and the latter was validated by western blot. Pdia3 reduced ER stress upon ischemia (Yoo et al., 2019) and its mRNA level was upregulated upon glucose-depletion (Zhou et al., 2011). P4hb is upregulated in short-term hypoxic astrocytes and protects neurons from apoptotic cell death in vitro (Tanaka et al., 2000). Chaperone-containing T-complex (TRiC) protein subunits are chaperones that compose the CCT- complex upon interacting with -tubulin. Disruption of CCT- complex reduced ER stress response through altering mitochondrial membrane potential and inducing heat shock protein 75 kDa (Trapl) dependent protein degradation and ERAD (Lin et al., 2012). In our study, we detected decreased levels of T-complex protein subunits 2, 5 (Cct2, Cct5), several P-tubulin subunits (Tubb2a, Tubb4a), and Trapl. It was found that a component of ERAD localizes proteasome subunits to the ER (Tcherpakov et al., 2008) and we detected decreased proteasome subunit alpha type-6 (Psma6) levels in the MAM and proteasome subunit beta (Psmb4) in CSF.

In a CCH model, EPTA staining of hippocampal slices revealed abnormal protein aggregation of newly synthesized proteins in the cytosol, 3 months after the operation (Hai et al., 2013). In addition, elevated levels of ubiquitinated proteins were shown in the chronic BCCAO model (Hai et al, 2011), implying that disturbed proteostasis is a long-term effect of CCH. In conclusion, most of the proteins of the quality control mechanism decreased, implying that ER stress response is hindered in response to chronic hypoperfusion, and its protective role in maintaining normal proteostasis might be diminished.

We detected dihydropyrimidinase-related proteins 2 and 3 (Dpysl 2 and 3) which were also identified in the MAM fraction previously (Poston et al., 2013; Fold i et al., 2013). However, their role in the regulation of MAM is poorly studied.

Altered proteins of mitochondrial oxidative phosphorylation and quality control

The occlusion of the common carotid arteries reduces the cerebral blood flow for several weeks in BCCAO rats. Although the blood flow recovers to the control level (Farkas et al., 2007), subsequently, mitochondrial damage develops (He et al., 2008). Additionally, BCCAO augmented the level of reactive oxygen species and reduced the activity of Mn- superoxide dismutase (Sod2), even 8 weeks after the surgery (Yadav et al., 2018). Two components of the oxidative phosphorylation (OXPHOS) system were altered in our study; one of them is the ATP synthase subunit beta (Atp5flb), and the other is Ndufsl. Altered levels of subunits of the electron transport chain can hinder the correct assembly of the OXPHOS system which can lead to oxidative (Miwa et al., 2014) and possibly proteotoxic stress (Callegari and Dennerlein, 2018).

Mitochondrial quality control is crucial in maintaining normal cellular metabolism. Several proteins and molecular pathways serve to maintain or restore mitochondrial functions such as chaperones, proteases, and import proteins involved in mitochondrial UPR (mtUPR). In the mitochondria, misfolded or damaged proteins are digested and the peptides are transported to the cytosol where they induce mtUPR (Haynes et al., 2013). In our proteomic study, we found 60 kDa heat shock protein (Hspdl also known as Hsp60) and Hspa9 that are involved in mtUPR. Hspa9 is a matrix chaperone and the only component of the mitochondrial import complex that has an ATPase function (Chacinska et al., 2009). Since most of the mitochondrial proteins are encoded in the nucleus, the import and refolding of proteins are essential to maintain mitochondrial proteostasis. Loss of Hspa9 induced proteotoxic stress and autophagic clearance of damaged mitochondria in vitro (Burbulla et al., 2014).

Trapl is a mitochondrial chaperone, involved in oxidative stress response. It reduces mitochondrial respiration, promotes glycolysis, and reduces reactive oxygen species (Yoshida et al., 2013). We have detected decreased level of Trapl in mitochondria in response to long-term hypoperfusion. Furthermore, we confirmed this result by western blot (Fig. 5c).

Cathepsin D (Ctsd) is a lysosomal endopeptidase and its release from lysosomes triggers bax activation (Bidere et al., 2003), mitochondrial cytochrome C release, and transmembrane potential loss during apoptosis upon oxidative stress (Roberg et al., 1999). We detected Ctsd elevation in the mitochondrial fraction. Surprisingly, several cytoskeletal proteins were detected in the mitochondrial samples. However, a- and [3-tubulin were shown to associate with mitochondrial membranes (Carre et al., 2002). Furthermore, tubulins decreased mitochondrial respiration by reducing the permeability of voltage-dependent anion channel for ADP (Rostovtseva et al., 2008; Monge et al., 2008). In our study, levels of several tubulin subunits dropped in mitochondria and MAM samples.

Metabolism of branch-chained amino acids (BCAAs) in dementia

BCAAs (Leu, lie, Vai) are essential amino acids necessary for protein synthesis and also have roles in the regulation of metabolic functions. BCAAs serve as nitrogen donors for amino acids (e.g. glutamine) and neurotransmitter synthesis (e.g. glutamate, gamma-aminobutyric acid).

Decreased levels of BCAAs were detected in the serum levels of patients with Alzheimer's disease and dementia (Tynkkynen et al., 2018). Further, decreased level of leucine (Leu) was also detected in the saliva of Alzheimer's disease and vascular dementia patients (Figueira et al., 2016). We have detected an increased level of methylcrotonoyl-CoA carboxylase subunit alpha (Mcccl) which is responsible for the catabolism of Leu. However, we also measured a decreased level of 3-hydroxyisobutyrate dehydrogenase (Hibadh), an enzyme of valine catabolism, and its decrease was validated by western blot. Thus, our results suggest unbalanced BCAA catabolism in CCH.

Disturbed protein turnover and redox state regulation are reflected in the CSF

While we could not detect significant changes of the same proteins in the CSF and the subcellular fractions, several proteins of protein turnover decreased in the CSF and subcellular fraction proteomes upon stepwise BCCAO, such as Eif3j, Uchl3, Psmb4 in the CSF, Psma6 in the MAM, and glycine--tRNA ligase (Gars) in the mitochondria.

Glrx and Gstpl have main roles in the detoxification of reactive oxygen species which levels' are elevated upon BCCAO (Yadav et al., 2018). Although we did not detect significant alterations of Glrx and Gstpl in the subcellular fraction of the frontal cortex, both proteins decreased in the CSF in our study, and they were shown to localize to mitochondria (Goto et al., 2019; Pai et al., 2007). Furthermore, we previously showed altered Gstpl level in the synaptosomal fraction of the occipital cortex in CCH rats compared to sham-operated controls (Tukacs et al., 2020).

There were several proteins that we could only detect in sham-operated animals (see Additional file 1). For instance, proteasome subunit beta type-7, exportin-1, and electron transfer flavoprotein subunit beta have roles in protein degradation, protein transport from the nucleus, and mitochondrial electron transport, respectively. Lack of their detection can indicate that their levels are below the detection limit or missing from the samples of BCCAO animals. Thus, it further suggests that protein turnover impairment and mitochondrial damage of the brain can be detected in the CSF.

In conclusion, the present invenors have detected reduced levels of several components of protein turnover in the mitochondria, MAM, and CSF of the stepwise BCCAO model, implying that altered cellular processes of brain tissue can be detected in the cerebrospinal fluid by proteomic analysis. EXAMPLES

Materials and methods

Animals

Adult male Wistar rats (aged 3 months, weighing ~220 g) were obtained from Toxi-Coop Ltd. (Budapest, Hungary). Rats were housed under standard laboratory conditions (lights on at 9:00 AM, lights off at 9:00 PM) in temperature- and humidity-controlled rooms with ad libitum access to food and water. All animal care and experimental procedures were following the Council Directive 86/609/EEC, the Hungarian Act of Animal Care and Experimentation (1998, XXVIII). All the procedures conformed to the National Institutes of Health guidelines were in accordance with the guidelines of the local Animal Care and Use Committee and were approved by the local Ethical Committee of Gedeon Richter Pic. (PE/EA/2885- 6/2016). All efforts were carried out to minimize the animals' pain and suffering and to reduce the number of animals used. A total of 16 rats were used in the experiments; they were assigned randomly to operated and sham-operated groups. The blindness of the experimenters whenever was guaranteed it was possible.

Stepwise bilateral occlusion of common carotid arteries

Stepwise bilateral common carotid artery occlusion was performed on rats as previously described (Volgyi et al. 2018, Nyitrai et al. 2018, Tukacs et al., 2020). Briefly, rats were anesthetized with isoflurane (1.5-2% in air) and a ventral midline incision was placed on the neck. First, the left common carotid artery was exposed, gently separated from the vagus nerve, and occluded by three ligatures (2-0). The animals were allowed to regenerate for a week in their home cages. Then, the same surgical procedure was performed on the right common carotid artery too. Rats in the control group underwent a sham operation as they received the same surgical procedures in both steps, but only a thread was placed around the vessels without ligation of the arteries.

Magnetic resonance angiography

The efficacy of the occlusions and the changes in cerebral blood flow were monitored with MRA, on the second and fifth weeks after the second occlusion, based on our previous studies (Nyitrai et al. 2018; Tukacs et al., 2020). Anesthetized animals were scanned in a 9.4 T MRI system (Varian, Medical Systems Inc., Palo Alto, CA, USA) with a free bore of 210 mm, containing a 120 mm inner size gradient coil. Three-dimensional time-of-flight angiography (3D gradient echo) was performed at TR/TE = 30/2.8 msec, resolution = 0.42 x 0.42 x 0.46 mm. The MRA studies were performed to evaluate cervical and intracranial arteries and parenchymal injury. For proteomic experiments, the animals had to meet the criteria as follows: i. lack of gross anatomical abnormalities (i.e., pathologically large or asymmetrical anatomical structures) ii. lack of extensive lesions or any sign of extended tissue impairment. The MRA revealed ischemic lesions in none of the animals. Maximum intensity projection was applied on the volumetric MRA recordings to create 2D images. The images were analyzed by lmageJ2; to discard background noise, the low threshold of 110 was applied on the intensity values. Then, the images were inverted, and mean gray values were obtained. A nonparametric, Mann-Whitney test was applied to the mean gray values of the MRA images with the significance level of 0.05.

Collection of CSF and preparation of mitochondria and MAM samples

CSF was collected and mitochondria and the subcellular fraction containing MAM were prepared from the frontal cortices of sham-operated and operated rats based on the protocol of Wieckowski et al. (2009). Animals were anesthetized with isoflurane (1.5-2% in air), and CSF was collected from cisterna magna, then rats were sacrificed, and their brains were quickly removed from the skull. Brain samples were washed in ice-cold isolation buffer-1 (IB-1) (225 mM mannitol, 75 mM saccharose, 0.5% BSA, 0.5 mM EGTA, and 30 mM Tris-HCI pH 7.4), then three times in ice cold isolation buffer-3 (IB-3) (225 mM mannitol, 75 mM saccharose, and 30 mM Tris-HCI pH 7.4). Brain samples were cut into smaller pieces and washed in IB-1. Brain samples were homogenized in IB-1 supplemented with protease and phosphatase inhibitor cocktail with Dounce Tissue Grinder (Sigma-Aldrich) manually (15 strokes per sample). Homogenized samples were centrifuged at 740 x g for 7 min at 4 °C. Supernatants were centrifuged at 9,000 x g for 10 min at 4 °C and pellets were suspended in isolation buffer-2 (IB-2) (225 mM mannitol, 75 mM saccharose, 0.5% BSA, and 30 mM Tris-HCI pH 7.4). Samples were centrifuged at 10,000 x g for 10 min at 4 °C and pellets were suspended in IB-3. Samples were centrifuged again at 10,000 x g for 10 min at 4 °C and pellets were suspended in mitochondria resuspending buffer (MRB) (250 mM mannitol, 5 mM HEPES (pH 7.4), and 0.5 mM EGTA); this is the crude mitochondrial fraction that contains MAM. The one fourth of each sample was centrifuged at 10,000 x g for 10 min at 4 °C and pellets were resuspended in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris, 5 mM magnesium-acetate). The other three fourth of the fractions were used for pure mitochondria preparation. Samples were layered onto Percoll medium and centrifuged at 95,000 x g for 35 min at 4 °C with SW-40 Ti rotor. The pure mitochondria fraction of the sample was collected from the interface and centrifuged at 6,300 x g for 10 min at 4 °C. Pellets were resuspended and centrifuged again at 6,300x g for 10 min at 4 °C with 70.1 Ti rotor and pellets were resuspended in lysis buffer. All the samples were stored at -80 °C until use.

Proteomic investigation of subcellular fractions

The proteome alterations of MAM-containing and mitochondria samples from the frontal cortex of 6 sham-operated and 6 BCCAO rats were investigated, using two-dimensional differential gel electrophoresis (2-D DIGE). The samples were adjusted to pH 8 and pH 8.5, respectively. Then, their protein concentration was measured by the 2D-Quant kit (GE Healthcare, Chicago, IL, USA). The fluorescent labeling of the mitochondrial proteins was conducted with a CyDye DIGE Fluor Minimal dye labeling kit (Cytiva). From BCCAO and control rats, 50 pg of protein of each sample were randomly labeled with either Cy5 or Cy3 dyes, while the internal sample (containing equal protein amounts (25 pg)) was labeled with Cy2. MAM-containing samples were labeled with CyDye DIGE Fluor labeling kit for Scarce sample (Cytiva). Five pg of protein from each sample and the internal standard were labeled with Cy3 and Cy5, respectively. The differently labeled samples were mixed and rehydrated passively onto Immobiline DryStrip gel strips (24 cm, pH 3-10 NL, GE Healthcare) overnight. Isoelectric focusing (IEF) was performed in an EttanIPGphor 3 IEF unit for 24 h to attain a total of 100 kVh (GE Healthcare). Following IEF, the mitochondrial proteins were reduced and carbamidomethylated using an equilibration buffer containing 1% mercaptoethanol and 2.5% iodoacetamide, respectively. In the case of MAM, proteins were only reduced as previously mentioned. SDS-PAGE separation was performed on 24 x 20 cm, 10% polyacrylamide gels in an EttanDALTsix Electrophoresis System (GE Healthcare). Then, the gels were scanned with a TyphoonTRIO+ scanner (GE Healthcare) using appropriate lasers and filters with the photomultiplier tube biased at 600 V. Differential protein analysis was performed using the DeCyder v7.0 software package (GE Healthcare), employing its Differential Analysis and Biological Variance Analysis modules. The fluorescence intensities of the Cy3 and Cy5 dyes on a particular gel were normalized to the intensity of the Cy2 dye or Cy3 intensities were normalized to the Cy5 dye. Quantitation of the fluorescence intensities of the protein spots and statistical analyses were carried out using the software. Independent two-tailed Student's t-test was performed on spots that are present on at least 80% of the gels and have fold changes of more than ± 1.2. The Benjamini-Hochberg procedure was applied with a false discovery rate of 0.25. In the results, the inventors show the original P-values of only those spots that remained significant after the procedure. Statistically significantly altered protein spots (P< 0.05) were picked for further protein identification. For the identification of proteins in spots of interest, preparative 2-D gel electrophoresis was performed separately using a total of 800 pg of synaptic proteins per gel. Resolved protein spots were visualized by the Colloidal Coomassie Blue G-250 stain (Merck Millipore, Billerica, MA, USA). One preparative gel for each brain region was run, and the selected spots were manually excised from the gels with pipette tips for protein identification. Excised spots were placed in a 1% acetic acid solution.

Mass spectrometry-based protein identification from 2-D gel spots

Proteins in the selected 2-D gel spots were in-gel digested as described in the protocol available online (http://msf.ucsf.edu/proto cols.html). Briefly, gel spots were cut into smaller cubes, washed with 25 mM ammonium- bicarbonate/50% acetonitrile, reduced using 10 mM TCEP, and alkylated with 55 mM MMTS. After dehydration, the gel pieces were rehydrated with 100 ng trypsin (sequencing grade, side chain protected porcine trypsin, Promega) in 20 pl of 25 mM ammonium-bicarbonate. Samples were digested for 4 hours at 37°C. Tryptic peptides were extracted and dried in a vacuum centrifuge.

Samples were reconstructed in 20 pl of 0.1% formic acid before mass spectrometric analysis. Five pl of the digest was injected for LC-MS/MS analyses onto an LTQ-Orbitrap Elite (Thermo Fisher Scientific) mass spectrometer on-line coupled with a nanoAcquity UPLC (Waters) system. In order to shorten the injection time, the sample was injected with a high flow rate to a trap column (Symmetry C18, nanoACQUITY UPLC 2D, V/M 0.180mm x 20mm, 5pm, 100A, Waters,) and a nano column (BEH130, C18 Acquityuplc column, 0.100mm x 100mm, 1.7 pm, 130 A, Waters) was used for the analysis. Gradient elution was applied from 3 to 40 % of eluent B (0.1% formic acid in acetonitrile) in 37 min. Mass spectrometry data were collected in data-dependent manner, a high-resolution survey scan was followed by a maximum of 20 dependent CID spectra analyzed in the ion-trap. Only multiple charged precursor ions were selected for fragmentation and after that, they were excluded for 30 seconds from the repeated selection. A PAVA script (UCSF, MSF, San Francisco, CA) was used for peak picking and our /n-c/oudProteinProspector (version: 5.22.0) server (https://cloud.mta.hu/) was used for database search, with the following parameters: UniProtKB.2019.6.12. random. concat database was filtered for the rat sequences concatenated with the most frequent contaminant proteins (36319 sequences; with 209 additional contaminant protein sequences were searched). Only tryptic peptides were considered, with a maximum of one missed cleavage site. Several variable modifications were set as acetyl (protein N-term), acetyl+oxidation (protein N-term M), Gln->pyro-Glu (N-term Q), Met-loss (protein N-term M), Met-loss+acetyl (protein N-term M), oxidation (M), and carbamidomethyl- and methyltio-cysteine. Mass accuracy was set to 10 ppm for parent and 0.6 Da for fragment ions. Proteins and peptides were accepted with a maximum of 1% FDR. Proteins were rejected with less than 10 unique peptides, 30 peptide counts, or 40% of sequence coverage; accepted proteins are shown in Table 1. CSF proteome analysis using LC-MS/MS

The protein content of the collected rat cerebrospinal fluid samples were treated with trypsin using the S-Trap micro spin columns according to the vendor's protocol (https://protifi.com/pages/protocols) and the resulting peptide mixture was analyzed using a Waters MClass nUPLC-Thermo Orbitrap Fusion Lumos Tribrid LC-MS system in a data-dependent fashion. Proteins were identified using the Protein Prospector BatchTag Web software applying score-based acceptance criteria. In more detail, the tryptic digest was injected onto a trapping column (MClass Symmetry Waters Acquity UPLC Trap Col 2G V/M C18 column, 0.180 mm ID * 20 mm L, 5 Elm particle size, 100 A pore size; loading time: 3 min with 1% B at 5 Hl/min) and after desalting was separated using a nonlinear gradient of 10-50% B in 80 min (solvent A: 0.1% formic acid/water, solvent B: 0.1% formic acid/acetonitrile, flow rate: 400 nl/min) on a Waters separating nanocolumn (nanoAcquity UPLC BEH130 C18 column, 0.075 mm ID*250 mm L, 1.7 Em particle size, 130 A pore size; column temperature: 40 °C). Peptides eluting from the column were analyzed in 2-sec cycles selecting the most abundant multiply charged ions (z=2-6, m/z range: 380-1580) for HCD fragmentation (normalized collision energy: 35%) following each MSI scan. Both MS and MS/MS spectra were collected in the Orbitrap analyzer with a resolution of 120,000 or 15,000, respectively. Raw data were converted into peaklists using the Proteome Discoverer (v2.4 SP1) software. Proteins were identified using the BatchTag Web software of Protein Prospector (v6.3.1.) with the following parameters: database: Uniprot Rattus norvegicus sequences (2020.10.07. version, 36457 sequences) concatenated with a randomized version for each entry and also supplemented with 172 additional sequences representing the most common contaminant proteins (such as trypsin and human keratins); enzyme: trypsin allowing maximum one missed cleavage site; modifications: static: methylthio on Cys, variable: cleavage of Met and/or acetylation of protein N-termini, oxidation of Met, pyroglutamic acid formation from peptide N-terminal Gin, deamidation of Gin or Asn, allowing up to two variable modifications/peptide; mass accuracy: 5 and 20 ppm for precursor and fragment ions, respectively defined as monoisotopic values. Acceptance parameters: minimum score: 22 and 15, maximum E-value: 0.01 and 0.05 on the protein and peptide level, respectively; minimum protein best discriminant score: 0. Peptide level false discovery rate was below 1% for all samples as estimated by the incidence of randomized sequence identifications.

Quantification of the proteins across the sample groups was performed using spectral counting. Three sham-operated control and nine BCCAO-operated rats were compared. Peptide counts of proteins were normalized to the total peptide count found in each sample. Proteins were removed from analysis with more than 1 or 6 missing values among the control or operated rats, respectively. Protein abundance mean ratios of BCCAO and control samples were calculated. Independent two-tailed Student's t-test was performed on proteins of more than 1.5 or less than 0.5 mean ratios. The Benjamini-Hochberg procedure was applied with a false discovery rate of 0.25. In Additional file 2, the inventors show the original P-values of only those proteins that remained significant after the procedure.

Enrichment analyses of altered proteins

Enrichment analysis was performed on altered proteins of MAM, mitochondria, and CSF, using the DAVID Bioinformatics Resources v2022ql (Huang et al., 2009; Sherman et al., 2022) with the background of the rat genome. The functional annotation tool of DAVID was used to evaluate the enrichment of the KEGG pathway, GO biological process, cellular component, and molecular function terms with the EASE score and count threshold of 0.01 and 2, respectively. Benjamini-Hochberg correction was applied for controlling the false discovery rate. Validations of protein changes by western blot

Western blot experiments were carried out on mitochondrial (n = 8) samples to confirm our proteomic results. Samples (30 pg) were diluted with sample loading buffer (1 M Tris-HCI pH = 6.8, 8 \N/ I% SDS, 24 v/v% glycerol, 200 mM DTT, 0.2 v/v% bromophenol blue) and incubated at 96 °C for 5 min. Protein samples were separated on 10% acrylamide gel by SDS-PAGE. Then, the gel was transferred to a PVDF membrane. The membrane was washed with distilled water and stained with Ponceau S stain (0.5 (w/v)% in 1 (v/v)% acetic acid), after washing with distilled water; it was photographed when it air dried. The membrane was destained in 200 pM NaOH and 20% acetonitrile and washed again in distilled water, then blocked with 3% BSA solution (in TBS-T for lh at RT). The membrane was stained with primary antibodies: anti-Hibadh (at 1:1000 dilution) (Sigma-Aldrich, Cat# HPA021002), anti-P4hb (at 1:1000 dilution) (Sigma-Aldrich, Cat# HPA018884), anti-Hspa5 (at 1:1000 dilution) (Thermo Fisher Scientific, Cat# PA1-014A), and anti-Trapl (at 1:2000 dilution) (Thermo Fisher Scientific, Cat# MAI-010) at 4 °C overnight and washed in TBS-T. Secondary antibody staining with A647-conjugated anti-rabbit (Jackson ImmunoResearch, Cat# 711-605-152) and A594-conjugated anti-mouse (Jackson ImmunoResearch, Cat# 715-585-151) was applied in 1:800 dilution for 2 h at RT and washed in TBS-T then TBS. The antibody-labeled membrane was scanned with Typhoon Trio+ scanner (Amersham) with appropriate filter settings and 50 micron resolution. Images were analyzed with ImageJ (version 1.53c); densitometric values of protein bands were determined and normalized to total protein content. Independent two-tailed Student's t-test (P < 0.05) was applied to confirm altered levels of proteins.

Results

Magnetic resonance angiography (MRA) confirmed the efficacy of the occlusions (Fig. lb)

MRA confirmed that the experiment did not affect sham-operated animals, while the blood flow was bilaterally blocked in the common carotid artery in each CCH animal. As a result of the occlusions, the basilar artery thickened in the operated rats (Fig. la), implying it partially overtook the functions of the occluded arteries. Based on image analysis, the mean pixel density of blood vessels significantly (P-value = 0.0026) decreased in CCH animals five weeks after the surgery Mitochondrial and MAM proteome alterations induced by CCH

The current gel-based proteomics study was performed on the MAM and mitochondria containing subcellular fractions of frontal cortices from stepwise BCCAO rats. The inventors have separated 725 and 708 protein spots on the gels of MAM and mitochondria samples, respectively. Seventeen and 6 spots showed significant change in the MAM and mitochondria sample of the CCH rats compared to the sham-operated group, respectively (Fig. 2a and 2b; Table 3 and Table 4). In the MAM samples, altered proteins showed fold changes between -1.69 to 2.38. Three spots showed elevation while 14 decreased compared to the control group. Strikingly, most of the altered proteins in the mitochondria decreased with fold changes (FC) varying between -1.31 and -1.2, and one spot increased with the fold change of 1.27. From altered spots, HPLC-MS/MS identified 19 proteins from the mitochondria sample, and 35 from MAM (Table 1). It is known, that several proteins can be present in one spot and one protein can be found in several spots, the number of significantly altered spots is not equal to the number of identified proteins. Taken together, we observed a modest alteration in the levels of mitochondrial proteins and a more complex proteome modification in the MAM-containing sample. Table 1

List of altered proteins in subcellular fractions of MAM and mitochondria.

Since the MAM comprises the interacting surfaces of the endoplasmic reticulum (ER) and mitochondrial membrane, the subcellular fraction of MAM will inevitably contain some mitochondrial proteins. Here, the spot with the highest fold change (FC = 2.38) consisted of NADH dehydrogenase (Ubiquinone) Fe-S protein 1 (Ndufsl) (the subunit of complex I of the electron transport chain), and heat shock proteins, namely stress-70 protein, mitochondrial (Hspa9 or mortalin), and heat shock cognate 71 kDa protein (Hspa8). While, in the most decreasing spot (FC = -1.69), the inventors detected protein disulfide isomerase A3 (Pdia3) chaperone among others with lower peptide count. The other two increased spots, with fold changes of 1.67, are comprised of methylcrotonoyl-CoA carboxylase subunit alpha (Mcccl) and vesiclefusing ATPase (Nsf) which are involved in branch-chained amino acid catabolism and vesicle transport, respectively. All the other protein spots decreased; T-complex protein 1 subunit epsilon (Cct5), proteasome subunit alpha type-6 (Psma6), and neuronal-specific septin-3 (Septin3) had fold changes of -1.62, -1.55, and -1.41, respectively. In the mitochondrial fraction, the spot decreasing and increasing the most (FC = -1.31 and 1.27) comprised protein disulfide isomerase (P4hb or Pdial) chaperon and cytoskeletal protein tropomodulin-2 (Tmod2), respectively. Other altered spots changed between -1.20 and -1.29; these contained endoplasmic reticulum chaperone BiP (Hspa5), ATP synthase subunit beta (Atp5flb), heat shock protein 75 kDa (Trapl), and cytoskeletal protein dihydropyrimidinase-related protein 2 (Dpysl2).

The annotation of significantly altered proteins can reveal the disturbed cellular processes in the MAM and mitochondria induced by the stepwise BCCAO (Fig. 2c and 2d, respectively). Protein turnover and import showed the highest ratio of altered proteins in the MAM (n = 11; 32%) and mitochondria samples (n = 6; 32%). Energy and carbohydrate metabolism-associated proteins were highly represented in the MAM (n = 10; 29%) and less robustly in the mitochondria (n = 3; 16%) of the frontal cortex. Additionally, the inventors identified cytoskeletal proteins in both mitochondria (n = 5; 26%) and MAM (n = 6; 18%) subcellular fractions. Proteins of fatty acid metabolism (n = 1; 3%) and ion transport (n = 1; 3%) were present only in MAM. Proteins involved in membrane dynamics and mitochondrial processes were also present among the altered proteins of MAM (n = 2; 6%) and mitochondria (n = 2; 10%). In conclusion, our data show a wide disturbance of the protein turnover process and mitochondrial energy metabolism in response to stepwise BCCAO.

Functional proteomic alterations in the CSF reflect cellular organelle changes in the frontal cortex

In the CSF, the inventors have found 12 proteins with significantly altered abundance with mean ratios between 0.32 and 2.06, using MS-based proteomics (Fig. 3a and Table 2; Table 5 (CSF ful az Additional file 1-ben). Two of them, CD59 glycoprotein (Cd59) and WAP four-disulfide core domain protein 1 (Wfdcl), increased by 1.65 and 2.06 fold, respectively. Annexin A2 (Anxa2) showed the lowest mean ratio of 0.32. Functional annotations (Fig. 3b) showed that 17% of altered proteins were involved in redox state regulation; namely, glutaredoxin-1 (Glrx) and glutathione S-transferase P (Gstpl) decreased by 0.53 and 0.52. Protein turnover and import had the highest ratio of altered proteins in the CSF (n = 5; 42 %) similar to the MAM and mitochondria. Four of them were less than halved compared to control namely, proteasome subunit beta (Psmb4), ubiquitin carboxyl-terminal hydrolase isozyme L3 (Uchl3), eukaryotic translation initiation factor 3 subunit J (Eif3j), and importin subunit beta-1 (Kpnbl)); and Wfdcl, an endopeptidase inhibitor, increased. Table 2

List of altered proteins in the CSF and their primary cellular functions and localization

Uniprot accession numbers, gene names, primary function, subcellular localization, and mean ratios of significantly altered proteins in the CSF are shown.

Enrichment analysis of altered proteins upon stepwise BCCAO is in accordance with functional annotation

The enrichment of Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways can give further insight into the disturbed cellular process and related genes affected by stepwise BCCAO (Table 6: Additional file 2). Enrichment maps of the top five terms of GO biological process, molecular function, and KEGG pathways are shown in figure 4. Regarding biological process (GOTERM BP DIRECT) (Fig. 4a), ATP metabolic process was enriched with five related genes Hspa8, Atp5flb, v-type proton ATPase subunit B (Atp6vlb2), Ndufsl, adenylate kinase 5 (Ak5) (32.2 fold, adj. P-value = 4.2E-03). Also, protein folding was significantly overrepresented (20.8 fold, adj. P-value = 5.44E-05). In accordance, protein binding involved in protein folding molecular function (GOTERM MF DIRECT) term was highly enriched (32.2 fold, adj. P-value = 9.02E-04) with five related genes (Hspa9, Hspa8, Cct2, Hspa5, Cct5) (Fig. 4b). Among KEGG pathways (Fig. 4c), the most enriched terms were glycolysis/gluconeogenesis (14.3 fold, adj. P-value = 4.83E-03) and biosynthesis of amino acids (12.5 fold, adj. P-value = 7.25E-03). Also, pathways of neurodegeneration - multiple diseases were enriched (4.5 fold, adj. P-value = 2.24E-03) with 11 related genes (such as Trapl, Psma6, Psmb4, Atp5flb, Hspa5, Ndufsla, and tubulins) (Additional file 2).

Altered proteins of organellar quality control validated by western blot

The inventors analyzed the levels of four proteins involved in mitochondrial and ER quality control and protein folding by western blot to confirm our proteomic results (Fig. 5). P4hb (P-value = 0.026), Hibadh (P-value = 0.031), and Trapl (P- value = 0.046) levels decreased significantly in the mitochondria (Fig. 5a, 5b, and 5c). While the decline of Hspa5 level in the mitochondria was not significant (P-value = 0.11) (Fig. 5d). Table 3

Table 4

Table 5

Table 6

INDUSTRIAL APPLICABILITY

High-throughput methods are powerful tools to track molecular changes upon different treatments in animals, tissues, or cell lines. Two-D DIGE (2-dimensional differential gel electrophoresis) proteomics enables the separation of two or three protein samples labeled by different fluorescent dyes simultaneously on a single gel. This technique makes spot comparison and protein quantification more reliable and reproducible (Marouga et al., 2005).

At the same time, 2-D DIGE has some limitations that must be taken into account. Hydrophobic membrane proteins solubilize poorly in polar detergent-free solvents applied in isoelectric focusing (IEF). This effect aggravates their migration into the gel; thus their detection is limited. Higher molecular weight proteins and the strongly acidic or alkaline ones have difficulties since applied IEF and PAGE allows protein separation in the range of ~3 to ~10 isoelectric points (pl) and 10-100 kDa, respectively. In addition, highly abundant proteins can mask lower-abundant protein alterations if more than one protein runs into the same spot. This can be improved by subcellular fractionation, such as mitochondria and MAM isolation in this study. However, contaminations from other cellular organelles are inevitable and not negligible (Poston et al., 2013). At the same time, the applied preparations are highly reproducible and validated (Wieckowski et al., 2009).

The present protein function network panels and protein biomarkers are nevertheless highly useful to diagnose vascular dementia even at an early stage when cerebral blood flow is impaired and the symptoms are still weak; or even in later stages.

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Claims

1. A diagnostic method for indicating vascular dementia in mammalian patient, said method comprising the steps of a) providing a sample of biological fluid being in contact with the nervous system of said patient, preferably selected from the group consisting of a sample of cerebrospinal fluid or blood origin, b) measuring level of one or more protein biomarkers of one or more biomarker panels in the sample, preferably a set of protein biomarkers of one or more, preferably two or more biomarker panels in the sample, each of said protein biomarkers being expressed from a respective biomarker coding gene in said patient, preferably in the nervous system of said patient, wherein each of the one or more biomarker panels consist(s) of biomarkers belonging to a protein function network, preferably a protein network of biomarkers classified according to the cellular function of the expressed proteins, c) wherein if the level of at least one protein biomarker from each of two or more biomarker panels is significantly altered in comparison with a reference level, the patient is considered as having vascular dementia or being susceptive of vascular dementia or being at risk of vascular dementia, in particular, wherein said protein function network being selected from the group consisting of energy and carbohydrate metabolism, protein turnover and import, cytoskeletal proteins, mitochondrial processes, vesicle transport and/or fusion, fatty acid metabolism, signaling pathways, ion transport, redox-state regulation, complement cascade regulation, protein folding. diagnosing a condition related to vascular dementia in a mammalian patient, protein function network, said method comprising the steps of a) providing a sample selected from the group consisting of mitochondria-associated samples, mitochondrial- associated membrane (MAM) samples, samples of cerebrospinal fluid (CSF) or of blood origin, b) measuring level of one or more protein biomarkers of one or more biomarker panels in the sample, preferably a set of protein biomarkers of one or more, preferably two or more biomarker panels in the sample, each of said protein biomarkers being expressed from a respective biomarker coding gene in the nervous system of said patient, wherein each of the one or more biomarker panels consist(s) of biomarkers belonging to a common protein function class, classified according to the cellular function of the expressed proteins, preferably a common pathway, c) wherein if the level of at least one protein biomarker from each of two or more biomarker panels is significantly increased or decreased in comparison with a reference level, the patient is considered as having vascular dementia or being susceptive of vascular dementia or being at risk of vascular dementia, wherein said protein function class of the respective protein, once expressed from the biomarker coding gene, being selected from the group consisting of energy and carbohydrate metabolism in mitochondria-associated samples, mitochondrial-associated membrane (MAM) samples, CSF samples and/or blood samples,, protein turnover and import in mitochondria-associated samples and/or in CSF and blood samples, preferably in MAM sample and/or mitochondrial sample, cytoskeletal proteins in mitochondria-associated samples and/or blood and/or CSF, preferably in MAM sample and/or mitochondrial sample, mitochondrial processes in MAM sample and/or mitochondrial sample, vesicle transport and/or fusion in MAM sample and/or mitochondrial sample, fatty acid metabolism in MAM sample, signaling pathways in MAM sample and/or mitochondrial sample, ion transport in MAM sample, redox-state regulation in blood and/or CSF sample, complement cascade regulation in blood and/or CSF sample, protein folding protein binding involved in protein folding) (GO terms).

3. The method according to any of claims 1 or 2, wherein said protein function network being selected from the group consisting of energy and carbohydrate metabolism, protein turnover and import, cytoskeletal proteins, redox-state regulation, complement cascade regulation; or preferably: protein turnover and import in blood and/or CSF sample, redox-state regulation in blood and/or CSF sample, or preferably: energy and carbohydrate metabolism, protein turnover and import, and cytoskeletal proteins.

4. The method according to any of claims 1 to 3, wherein said protein function network is a function class given in

GO terms and/or KEGG pathways, being selected from the group consisting of protein folding, protein binding involved in protein folding, ATP-metabolic processes, glycolysis/gluconeogenesis biosynthesis of amino acids.

5. A method for assessing in a subject the risk of developing a neurocognitive disorder of vascular origin, in particular wherein said neurocognitive disorder is selected from the group of mild cognitive impairment, Alzheimer's disease of vascular origin, vascular dementia or combinations thereof said method comprising the steps as defined in any of claims 1 to 4.

6. The method according to any of claims 1 to 5 wherein step b) of measuring comprises: bl) analyzing the sample from said patient to obtain data, preferably quantitative data, for the level of one or more than one biomarker(s); b2) comparing the data for said one or more biomarker(s) to corresponding data obtained for one or more than one reference sample or to data for respective pre-determined reference level(s) to identify alteration (in particular an increase or decrease) in the level of said one or more than one biomarker in said sample.

7. The method according to any of claims 1 to 6, wherein protein levels are measured by a method selected from the group consisting of western blot, two-dimensional differential gel electrophoresis (2-D DIGE), mass spectrometry

8. The method according to any of claims 1 to 7, wherein the alteration of at least 2 protein biomarkers is measured from each of two or more biomarker panels, preferably 5 to 10 protein biomarkers from each of two or more biomarker panels, preferably from each 3 biomarker panels.

9. The method according to any of claims 1 to 8, wherein the biomarker is selected from the group consisting of ATP5B (AtpSflb) - (energy and carbohydrate metabolism) ALDOC (Aldoc) - (energy and carbohydrate metabolism) ENO2 (Eno2) - (energy and carbohydrate metabolism)

PSMA6 (Psma6) - (protein turnover and import, in particular amino acid synthesis or metabolism) WARS (wars) - (protein turnover and import, in particular amino acid synthesis or metabolism).

10. The method according to any of claims 1 to 9, wherein the level of any or more of the following protein biomarkers is altered in the sample:

Hspa5 has decreased level,

Pdia3 and/or P4hb is/are upregulated, and/or the level of any or more of the following biomarkers is altered in the sample: T-complex protein subunits 2, 5 (Cct2, Cct5) have decreased level, P-tubulin subunits (Tubb2a, Tubb4a), and Trapl have decreased level, proteasome subunit alpha type-6 (Psma6) and/or proteasome subunit beta (Psmb4) have a decreased level.

11. The method according to any of claims 1 to 9, wherein the level of any or more of the following oxidative phosphorylation protein biomarkers is altered in the sample: ATP synthase subunit beta (Atp5flb),

Ndufsl and/or the level of any or more of the following heat-shock protein biomarkers is altered in the sample:

Hspdl and/or

Hspa9. and/or the level of any or more of the following branch-chained amino acids (BCAAs) biomarkers is altered in the sample: methylcrotonoyl-CoA carboxylase subunit alpha (Mcccl) has increased level, and/or

3-hydroxyisobutyrate dehydrogenase (Hibadh) has decreased level.

12. The method according to any of claims 1 to 9, wherein the level of any or more of the following protein turnover and redox state regulation biomarkers is altered in the sample:

Eif3j, Uchl3, Psmb4 in the CSF, Psma6, and glycine--tRNA ligase (Gars)

Glrx and Gstpl.

13. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to energy and carbohydrate metabolism comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of

Ndufsl, Pdhal, Aldoc, Gapdh, Nt5cla, Atp5flb, Pdhx, Eno2, Tpil, preferably Ndufsl, Pdhal, Aldoc, Gapdh, Nt5cla, Atp5flb, Ak5, Pcca, Lgals5, OR:

Ndufsl, Atp5flb, Pdhal, Pdhx, Pcca,

Eno2, Aldoc, Gapdh, Nt5cla, Ak5, Lgals5

14. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to protein turnover and import, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of

Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2, Wars, Rpsl2, Psma6, Mcccl, Glu I,

Trapl, Hspa5, P4hb, Gars, Ctsd, Hibadh

Psmb4, Uchl3, Eif3j, Kpnbl, Wfdcl, preferably: Hspa8, Hspa9, Pdia3, Hspdl, Psma6, Cct5, Mcccl, Glul, P4hb, more preferably Hspa8, Hspa9,

OR:

Hspa9, Pdia3, Hspdl, Mcccl, Glul, Trapl, Hspa5, Gars, Hibadh (mitochondrial localization), preferably Hspa9, Pdia3, Hspdl, Mcccl, Glul, more preferably Hspa9,

Hspa8, Cct5, Cct2, Wars, Rpsl2, Psma6, Psmb4, Uchl3, Eif3j, (cytoplasmatic localization); preferably Hspa8, Cct5, Psma6, more preferably Hspa8, preferably of protein folding class: Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2,

Trapl, Hspa5, P4hb; preferably of protein or amino acid synthesis or metabolism, including amino acid synthesis or metabolism class:

Wars, Rpsl2, Psma6, Mcccl, Glul,

Psmb4, Uchl3, Eif3j.

15. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to cytoskeletal proteins, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of

Tubalb, Tuba4a, Tubala, Dpysl2, Dpysl3, Septinll 6,

Dpysl2, Tubb2a, Tubb4a, Tubalb, Tmod2,

Capl, preferably Tubalb, Tuba4a, Tubala, or preferably Dpysl2.

16. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to redox-state regulation, wherein the sample is in particular a blood and/or CSF sample, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Gstpl and Glrx.

17. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to complement cascade regulation, wherein the sample is in particular a blood and/or CSF sample, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of Cd59.

18. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to protein folding (including protein binding involved in protein folding), comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of

Hspa8, Hspa9, Pdia3, Hspdl, Cct5, Cct2

Trapl, Hspa5, P4hb;

OR (based on GO terms)

HSPA9, HSPA8, HSPA5, HSPD1 (protein refolding), or

FKBP1A, HSPA8, CCT2, CCT5 (chaperone-mediated protein folding) or

HSPA9, PDIA3, HSPA8, CCT2, TRAP1, P4HB, CCT5,

HSPD1 (protein folding) or

19. The method according to any of claims 1 to 9, wherein the biomarker panel consisting of biomarkers belonging to ATP metabolic process, comprises one or more, preferably two or more biomarkers encoded by a gene selected from the group consisting of HSPA8, ATP5F1B, ATP6V1B2, NDUFS1, AK5.

20. The method according to any of claims 10 to 19, wherein the biomarker panel is selected from the group consisting of one or more, preferably two or more biomarker panels as defined in claims 10 to 19.

21. The method according to any of claims 1 to 20, wherein the biomarker panel consisting of biomarkers belonging to any of the following protein function classes, mitochondrial processes (preferably in MAM sample and/or mitochondrial sample), vesicle transport and/or fusion (preferably in MAM sample and/or mitochondrial sample), fatty acid metabolism (preferably in MAM sample), signaling pathways (preferably in MAM sample and/or mitochondrial sample), and/or ion transport (preferably in MAM sample).

22. The method according to claim 21 wherein the level of any or more of the following protein biomarker(s) is/are altered: mitochondrial processes biomarker Timm50 and/or Slc25al2, vesicular transport biomarker Nsf, vesicular fusion biomarker Stxbpl, fatty acid metabolism biomarker Fabp5, signaling pathway biomarkers Ppp2rla, Fkbpla and/or Prkar2b, preferably TGFbeta signaling pathway biomarker Fkbpla, PKA signaling biomarker Prkar2b, proton transport biomarker Atp6vlb2, and/or further mitochondria-related biomarkers Septin3, Mpp3, Mpp6 and/or Emc8 and/or further CSF biomarkers Ppal and/or Anxa2.

23. The method according to any of claims 1 to 22, wherein the direction of the alteration of level of a protein biomarker (increase or decrease) is as indicated in Table 1 or 2, if present therein.