BIOMARKERS AND THERAPEUTIC TARGETS FOR ALZHEIMER'S DISEASE

TECHNICAL FIELD

Alzheimer's disease (AD) is the most common age-dependent neurodegenerative disorder associated with progressive decline in cognitive function and hallmark pathological markers. The present invention is directed to a drug screening method for evaluating effectiveness of potential drugs in the treatment of Alzheimer disease and the identification of potential new targets fro drug discovery.

BACKGROUND AND PRIOR ART

Alzheimer's disease (AD) is the most common age-dependent neurodegenerative disorder associated with progressive decline in cognitive function and hallmark pathological markers. Its incidence is rising as the average age of the population increases, since age is the biggest risk factor for AD. In its early stages AD is characterized by the progressive loss of the ability to perform activities of daily living (ADL), such as making a meal and shopping.

So far, the development of transgenic models of AD has been used to provide tools to achieve an understanding of pathogenic mechanisms and develop new therapies. However, the validity of these models is overwhelmingly based on the ability of over-expression of amyloid precursor protein (APP) and tau mutations to cause the pathological plaques and neurofibrillary tangles (NFTs) observed in the AD brain. It is important to find an animal model that develops a comprehensive set of events associated with the AD pathology. The search for a good animal model of AD, preferably rodent, is one of the imperatives in present medical research.

Octodon degus, a South American rodent endemic to Chile, has been recently found to naturally develop histopathological signs of AD with age: accumulation of soluble Αβ oligomers and tau protein phosphorylation, as well as cognitive decline in spatial memory (T-maze) and object recognition memory (ORM) (Inestrosa et al., 2005, "Human-like rodent amyloid-peptide determines Alzheimer pathology in aged wild-type Octodon degus". Neurobiology of Aging 26, 1023-1028.; Ardiles et al., 2012, "Postsynaptic dysfunction is associated with spatial and object recognition memory loss in a natural model of Alzheimer's disease". Proc. Natl. Acad. Sci. USA 109(34), 13835-40). O. degus can live approximately 9-10 years in captivity, and from 3 years of age O. degus has been found to spontaneously develop an AD-like neuropathology. The natural onset and development of neurodegeneration, without the need of genetic manipulation, validate O. degus as a suitable animal model for studying AD.

The present invention is directed to characterize, through gene expression profiling of O. degus at the onset of the AD-like neuropathology, and more particularly to identify specific genes that can be used as biomarkers for use in diagnosing Alzheimer's disease at an early stage, as well as the use of said genes as therapeutic targets for developing drugs and/or treatments directed to treat AD.

In this invention, the global gene expression profile of O. degus by RNA sequencing, at the time of onset of the AD-like neuropathology was used to identify candidate genes for diagnosis and/or as therapeutic targets.

In the present invention, many genes were identified as potential targets that can be used as diagnostic markers for AD, as well as potential targets for therapeutic treatments. In particular, the inventors identified Ataxin; Myosin; Dynein; Serum response factor; Glycogen branching enzyme; Rap guanine nucleotide exchange factor (GEF) 3, among many others, as potential target genes that can be used as markers for AD early diagnostic or as therapeutic targets, Nevertheless, a prior art search found that these genes were already mentioned in relation with AD, for example:

• Ataxin (SCA1 ) is identified in WO2014113843A1 , wherein a method is described for

determining DNA methylation in different diseases, mentioning Alzheimer, indicating that SCA1 is a potential target; WO2010015592A2 describes an immunoassay for diagnostic of different diseases, among which Alzheimer is mentioned and wherein Ataxin is mentioned as a potential target; WO2009128948A1 describes antibodies and compositions for treatment of conditions, among which Alzheimer's disease is mentioned, here Ataxin is described as a potential target.

• Myosin is mentioned in WO2012075361 A2, which describes a treatment method for diseases, among which Alzheimer is mentioned, and that the therapeutic compound binds or is directed to myosin.

• Dynein is described in WO2004032614A2 as part of a non-human animal model for

neurological diseases, among which Alzheimer is mentioned.

• Serum response factor (SRF), mentioned in WO2007059103A2 describes that elevated levels of SRF are associated with AD.

• Glycogen branching enzyme, mentioned in WO2005089462A2, wherein methods are

described for the treatment of central nervous system (CNS) disorders, and wherein Alzheimer is mentioned as one of those CNS disorders.

• Rap guanine nucleotide exchange factor (GEF) 3 or EPAC is described in WO2007094755A2, as a target to improve cognitive functions, indicating that one case wherein the treatment would work is AD.

The identification of these genes in Degus provides strong evidence that the present approach can identify novel markers and targets. This is highlighted by the fact that 20 additional genes were identified, which to the best knowledge of the inventors, have not been previously disclosed as being linked to AD. The invention is directed to these 20 genes, wherein their use as biomarkers for early detection of AD is disclosed, as well as their use as therapeutic targets. SUMMARY OF THE INVENTION

The present invention describes novel gene biomarkers for early detection of AD, as well as their use as therapeutic targets.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes different genes that are upregulated or downregulated in an O. degus that developed an AD-like neuropathology, wherein said gene upregulation or downregulation is determined when comparing to a healthy individual that has not developed an AD-like neuropathology.

A particular aspect of the invention consists of an in vitro method for the diagnosis and/or prognosis of Alzheimer's disease by means of determining the expression level of a gene or set of genes in a biological sample and comparing said level of expression to a reference value.

In the context of the present invention, a reference value is understood as the level of mRNA and/or the protein encoded by a marker gene of the present invention, present in a healthy individual who does not suffer from AD or other diseases affecting levels of the mRNA or of the protein encoded by said marker genes.

In a more specific embodiment of the invention, the marker genes described in Table 1 , are upregulated when compared to a reference value.

Table 1 : Upregulated genes in an individual with AD.

Gene ID Gene Name NCBI ID Description

mitochondrial import inner

101577103 LOC1015771 03 XP_004626537.1 membrane translocase subunit

Tim9-like

eukaryotic translation initiation

101578360 LOC101578360 XP_004646324.1

factor 3 subunit J-A-like

deoxynucleotidyltransferase,

101581436 Dnttip2 XP_004635808.1

terminal, interacting protein 2

structural maintenance of

101576125 LOC101576125 XP_004638150.1

chromosomes protein 2-like

aspartate-tRNA ligase,

101571416 LOC101571416 LOC101571416

cytoplasm ic-like

101586169 LOC1015861 69 LOC101586169 protein SET-like

charged multivesicular body protein

101585432 LOC101585432 XR_192886.1

2B pseudogene

Sjogren syndrome antigen B

101583373 LOC101583373 LOC101583373

(autoantigen La) pseudogene

101570614 rpl35 XP_004642851 .1 mitochondrial ribosomal protein L35 sodium- and chloride-dependent

101567012 LOC101567012 XP_004640732.1

transporter XTRP3-like

serum response factor-binding

101562841 LOC101562841 XP_004631397.1

protein 1 -like

transformer 2 beta homolog

101575010 LOC101575010 LOC101575010

(Drosophila) pseudogene

The gene sequences are incorporated herein as reference based on their accession numbers Gene- ID, Gene-Name and/or NCBI-ID.

In a different embodiment of the invention, the marker genes described in Table 2, are downregulated when compared to a reference value:

Table 2: Downregulated genes in an individual with AD.

Gore ID Gere Na e NCBI ID Description

101590027 Atxnl XP_004628395.1 ataxin 1

101583652 Shisa7 XP_004647733.1 shisa homolog 7 (Xenopus laevis)

pleckstrin homology domain

101577104 Plekhbl XP_004641981 .1 containing, family B (evectins)

member 1 Goro-ID Gci-o-Na iio NCBI-ID Description

ADAM metallopeptidase with

101574476 Adamts9 XP_004644841 .1

thrombospondin type 1 motif, 9

101566459 Rnf175 XP_004638783.1 ring finger protein 175

Rap guanine nucleotide exchange

101559582 Rapgef3 XP_004638650.1

factor (GEF) 3

heterogeneous nuclear

101569339 LOC101569339 LOC101569339

ribonucleoprotein C-like membrane bound 0-

101560338 Mboat7 XP_004648407.1 acyltransferase domain containing

7

101573992 Prr7 XP_004632329.1 proline rich 7 (synaptic)

101580624 111 rl2 XP_004633315.1 interleukin 1 receptor-like 2

immunoglobulin superfamily,

101564503 Igsf9 XP_004640000.1

member 9

single stranded DNA binding

101565617 Ssbp4 XP_004646914.1

protein 4

101579610 LOC101579610 XP_004624373.1 protein PRRC2A-like

101568150 Celf3 XP_004632405.1 CUGBP, Elav-like family member 3

SH3 and multiple ankyrin repeat

101580332 Shankl XP_004646456.1

domains 1

Solute carrier family 1 1 (proton- coupled divalent metal ion

101565926 Slc1 1 a1 XP_004638302.1

transporters),

member 1

phosphatidylinositol transfer

101564208 Pitpnm2 XP_004635938.1

protein, membrane-associated 2 myosin, light chain 6B, alkali,

101592420 Myl6b XP_004647133.1

smooth muscle and non-muscle hairy/enhancer-of-split related with

101588144 Hey1 XP_00463981 1 .1

YRPW motif 1

maestro heat-like repeat family

10157261 1 Mroh7 XP_004642657.1

member 7

101581889 Pld4 XP_004645471 .1 phospholipase D family, member 4

101572335 LOC101572335 LOC101572335 galectin-3-like

family with sequence similarity 189,

101568310 Fam189a1 XP_004623502.1

member A1

101573142 Csmdl XP_004642588.1 CUB and Sushi multiple domains 1 tRNA splicing endonuclease 34

101559693 Tsen34 XP_004648404.1

homolog (S. cerevisiae) heterogeneous nuclear

101560631 LOC101560631 XP_004636713.1

ribonucleoprotein A3-like

TATA box-binding protein- associated factor, RNA polymerase

101593180 LOC101593180 XP_004646623.1

I,

subunit C-like

lipid phosphate phosphatase-

101566956 LOC101566956 XP_004632883.1

related protein type 2-like zinc finger, DHHC-type containing

101565177 Zdhhc8 XP_004628024.1

8

101584049 Gpr6 XP_004637546.1 G protein-coupled receptor 6

discs, large (Drosophila) homolog-

101592746 Dlgap2 XP_004642567.1

associated protein 2

101590084 Gfap XP_004634000.1 glial fibrillary acidic protein

101571439 Egr3 XP_004630715.1 early growth response 3

101568827 Dock6 XP_004632889.1 dedicator of cytokinesis 6 Goro-ID Gci-o-Na iio NCBI-ID Description

potassium intermediate/small conductance calcium-activated

101575671 Kcnn3 XP_004629861 .1

channel,

subfamily N, member 3 cat eye syndrome chromosome

101562751 Cecr6 XP_004638906.1

region, candidate 6

101565477 Drd2 XP_004646431 .1 dopamine receptor D2

101573587 Sh3pxd2a XP_004631565.1 SH3 and PX domains 2A

101588412 Kiaal 522 XP_004641035.1 KIAA1522 ortholog

101565680 Fibcdl XP_004640503.1 fibrinogen C domain containing 1 potassium voltage-gated channel,

101586672 Kcnh3 XP_004637839.1 subfamily H (eag-related), member

3

HIG1 hypoxia inducible domain

101572506 LOC101572506 LOC101572506

family, member 1 A pseudogene

101574590 Otof XP_004627490.1 otoferlin

archaelysin family metallopeptidase

101563222 Amz1 XP_004629750.1

1

101572107 Cdk5 XP_004629047.1 cyclin-dependent kinase 5

101563077 RasM Oa XP_004628100.1 RAS-like, family 10, member A

Rho guanine nucleotide exchange

101567515 Arhgef15 XP_004638512.1

factor (GEF) 15

101562918 Kif21 b XP_004625384.1 kinesin family member 21 B

101578531 Arhgap33 XP_004636799.1 Rho GTPase activating protein 33 cellular repressor of E1 A-stimulated

101579040 Creg2 XP_004633312.1

genes 2

calmodulin regulated spectrin-

101585021 Camsap3 XP_004644954.1 associated protein family, member

3

kelch repeat and BTB (POZ)

101574321 Kbtbdl 1 XP_004642590.1

domain containing 1 1

brain-specific angiogenesis inhibitor

101588433 Bai2 XP_004642991 .1

2

101583950 Ier5l XP_004640630.1 immediate early response 5-like

101592547 LOC101592547 LOC101592547 Krueppel-like factor 9-like

101565887 Capn5 XP_004632583.1 calpain 5

101572524 Neurl XP_004631562.1 neuralized homolog (Drosophila) eukaryotic translation initiation

101564819 LOC101564819 LOC101564819

factor 2 subunit 3, Y-linked-like cytochrome c oxidase assembly

101589777 LOC101589777 LOC101589777

protein COX1 1 , mitochondrial-like

101561216 LOC101561216 LOC101561216 ribosomal protein SA pseudogene

101583771 Insl3 XP_004646954.1 insulin-like 3 (Leydig cell)

hematological and neurological

101570899 Hn1 l XP_004627948.1

expressed 1 -like

101584008 Ptk2b XP_004630745.1 protein tyrosine kinase 2 beta calpain, small subunit 1

101586485 LOC101586485 LOC101586485

pseudogene

latent transforming growth factor

101576491 Ltbp4 XP_004648038.1

beta binding protein 4

WNT1 inducible signaling pathway

101574586 Wispl XP_004642545.1

protein 1

101591493 Pdzd4 XP_004645171 .1 PDZ domain containing 4 Gorc-ID Gcro-Na iio NCBI-ID Description

bromodomain testis-specific

101581650 LOC101581650 } <P_004646548.1

protein-like

ubiquitin-conjugating enzyme E2 C-

101586885 LOC101586885 ) <P_004630066.1

like

chromosome unknown open

101576286 LOC101576286 ) <P_004641228.1

reading frame, human C1 orf95

101573784 Nbeal2 ) <P_004640744.1 neurobeachin-like 2

The gene sequences are incorporated herein as reference based on their accession numbers Gene- ID, Gene-Name, NCBI-ID.

In a preferred embodiment of the present invention, the marker genes upregulated in an individual with AD when compared to a reference value are selected among mitochondrial import inner membrane translocase subunit Tim9-like or TIM9; eukaryotic translation initiation factor 3 subunit J-A- like; structural maintenance of chromosomes protein 2-like; aspartate-tRNA ligase, cytoplasmic-like; charged multivesicular body protein 2B pseudogene; heterogeneous nuclear ribonucleoprotein L-like or HNRNPLL or HNRPLL or SRRF or BLOCK24; Tolloid-like protein 1 or TLL1 ; Kinesin family member 11 ; calretinin-like; serine/threonine kinase 17a.

In a preferred embodiment of the present invention, the marker genes downregulated in an individual with AD when compared to a reference value are selected among Dock6 (Dedicator of cytokinesis 6) or Zir1 ; cyclin-dependent kinase 5 or Cdk5; ADAM metallopeptidase with thrombospondin type 1 motif, 9; galectin-3-like; immunoglobulin superfamily, member 9; G protein-coupled receptor 6; brain- specific angiogenesis inhibitor 2; neurobeachin-like 2; DLGAP2 or Disks large-associated protein 2; or Tsen34 or tRNA splicing endonuclease 34 in a biological sample and comparing said level with a reference value, in which the alteration of said level is indicative of Alzheimer's disease.

According to a preferred embodiment of the present invention, the biological sample is a brain tissue sample or a biological fluid, said biological fluid preferably comprises cerebrospinal fluid, blood or plasma.

In a particular embodiment, determination of the expression level of the gene marker is carried out by means of measuring the amount of mRNA encoded by said gene or fragments thereof. In a more specific embodiment, said measurement of mRNA encoding at least one of the marker genes or fragments thereof is preferably carried out by means of amplification, using oligonucleotides specific for PCR, SDA or any other cDNA amplification method allowing a quantitative estimation of the levels of the marker gene or genes.

In a further specific embodiment, mRNA measurement of marker gene or genes or fragments thereof is preferably carried out by means of DNA biochips made with oligonucleotides or microarrays.

In a second aspect of the invention, the expression level of the marker gene is determined by measuring the amount of protein encoded by said gene or of fragments thereof. Different methods can be used for measuring protein amounts, such as for example Western-blot, immunoassays, immunohistochemical assays, mass spectrometry, or any other mechanism that would allow a quantitative estimation of the levels of protein of the gene marker.

In a further embodiment of the invention are provided metabolites which are produced as part of the biochemical function of the genes listed.

A further embodiment of the invention consists of a kit for the diagnosis and/or prognosis of Alzheimer's disease comprising the necessary reagents for carrying out the determination of the expression level of a gene or genes marker selected among mitochondrial import inner membrane translocase subunit Tim9-like or TIM9; eukaryotic translation initiation factor 3 subunit J-A-like; structural maintenance of chromosomes protein 2-like; aspartate-tRNA ligase, cytoplasmic-like; charged multivesicular body protein 2B pseudogene; heterogeneous nuclear ribonucleoprotein L-like or HNRNPLL or HNRPLL or SRRF or BLOCK24; Tolloid-like protein 1 or TLL1 ; Kinesin family member 11 ; calretinin-like; serine/threonine kinase 17a; Dock6 (Dedicator of cytokinesis 6) or Zir1 ; cyclin- dependent kinase 5 or Cdk5; ADAM metallopeptidase with thrombospondin type 1 motif, 9; galectin-3- like; immunoglobulin superfamily, member 9; G protein-coupled receptor 6; brain-specific angiogenesis inhibitor 2; neurobeachin-like 2; DLGAP2 or Disks large-associated protein 2; or Tsen34 or tRNA splicing endonuclease 34, or combinations thereof.

EXAMPLES

A whole transcriptome analysis using RNA-sequencing on Octodon degus was performed.

Animals were housed in standard metal cages with a layer of wood shaving as bedding and containing a small metallic box (with a single entrance), under natural photoperiod, in an air- conditioned animal room.

They were fed with rabbit pellet and alfalfa and provided water ad libitum during the entire experimental period.

A set of animals was used in this study when they were three-year old.

The burrowing test was used as a behavior test of early living to assess between control and AD-like degus, as previously described in the international patent application PCT/IB2014/059208.

The quantity of soluble Αβ oligomers (Αβ1 -42) was determined using using MALDI-Tof mass spectrometry according to Gelfanova et al., 2007 (Quantitative analysis of amyloid-β peptides in cerebrospinal fluid using immunoprecipitation and MALDI-Tof mass spectrometry. Briefings in functional genomics & proteomics, 6(2), 149-158).

For whole transcriptome analysis, whole brain samples from two groups (n=7 animals) were used according to their brain Αβ1 -42 quantification.

Total RNA was isolated using PureLink RNA Mini kit and mRNA and enrichment was carried out using MicroPoly(A)Purist kit. RNA-sequencing of paired-end libraries was done using ABI SOLID 5500x1 system.

For reads alignment Lifescope genomic analysis pipeline was used. For read counts per gene FLUXcapacitor was used.

For differential gene expression analysis, edgeR package in R was used. biomaRT package in R was used to address gene annotation and Gene Ontology (GO) term IDs.

For gene ontology classification analysis clusterProfiler packages in R and Panther were used. Results

1- STATISTICS OF THE OCTODON DEGUS REFERENCE TRANSCRIPTOME

^■ Size of genome: 2,995,872,505 (~ 3GB) ^■ Number of chromosomes: 58 ^■ Number of scaffolds: 7,134 ^■ Number of contigs: 259,904

^■ Number of predicted transcripts: 26,838 ^■ Number of predicted protein sequences: 26,248

Mapping results (PASA) 24,002/26,838 (89.4%) 1 . min. identity between transcript and genome: 98% 2. min. length of the transcripts aligned to the genome: 98% 3. introns as long as 500kb

2- VOLCANO PLOT TO VISUALIZE DIFFERENTIAL EXPRESSED GENES WITH FDR<0.05

We obtained an average throughput of 150 million reads per sample with 150X estimated depth coverage. Multi-dimensional scaling plot were used to assess the biological coefficient of variation in the samples.

The criteria used to identify differentially expressed genes were: count per million reads (CPM)>1 , LogFC [1 ,-1] and False Discovery Rate (FDR) < 0.05. Differential expressed genes according these criteria were analyzed using hierarchical clustering and Gene-Ontology (GO) enrichment analysis.

Samples replicates cluster according to their biological groups (PB:Poor-Burrowers, GB:Good- Burrowers). Biological processes such us development and immune system response were enriched in GO-terms analysis. INDUSTRIAL APPLICABILITY

The present invention has an important application in the pharmaceutical industry, for AD diagnostic kits, as well as drug development.