Blood-Based Diagnostic Assays for Alzheimer's Disease

Abstract

The present invention relates to a set of biomarkers and methods of use thereof for the diagnosis, staging, treatment, and assessment of treatment response for neurocognitive disorders characterized by tau toxicity, such as Alzheimer's disease. At least three positions of phosphorylated peptides are included that are to be used as biomarkers in screening for neurocognitive disorders.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 225,423, filed July 23, 2021, the entire contents of which are hereby fully incorporated by reference.

[0002] (Reference to electronically submitted sequence listing) This application further incorporates by reference a concurrently filed Sequence Listing submitted electronically via EFS-Web as a file named "SequenceListing.xml" created on July 22, 2022. The Sequence Listing contained in this document is a part of this application and is incorporated herein by reference in its entirety.

[0003] (Technical field) The present invention relates to a set of biomarkers and methods of use thereof for the diagnosis, staging, treatment, and assessment of treatment response in neurocognitive disorders characterized by tau toxicity, such as Alzheimer's disease. [Background technology]

[0004] (background) Alzheimer's disease (AD) is one of the largest healthcare burdens, affecting 35 million people worldwide and projected to increase to 115 million by 2050. [Wimo, Alzheimer's Disease International World Report 2010. The Global Economic Impact of Dementia, Alzheimer's Disease International (2010)]. AD is a devastating dementia that initially manifests as progressive memory loss and can later include neuropsychiatric symptoms such as depression, delusions, agitation, and even aggression. Currently, available AD treatments are limited to cognitive enhancing drugs with limited and short-lived efficacy.

[0005] Until now, the diagnosis of AD could only be confirmed at autopsy by the presence of amyloid deposits and neurofibrillary tangles (NFTs) containing the microtubule-associated protein tau. Current clinical diagnoses of AD meet the criteria for probable AD according to the NINCDS-ADRDA Working Group in the DSM-IV TR and McKhann et al., Neurology 34(7):939-944 (1984). The initial diagnostic criteria presented in McKhann et al., which are based mainly on subjective assessment, require that the presence of cognitive impairment and suspected dementia syndromes be confirmed by neuropsychological testing for a clinical diagnosis of possible or probable AD; however, they require histopathological confirmation (microscopic examination of brain tissue) for a definitive diagnosis.

[0006] The criteria define eight cognitive domains that may be impaired in AD. These cognitive domains are: memory, language, perceptual abilities, attention, constructional abilities, orientation, problem solving, and functional abilities. Motor, sensory, and coordination disorders are not seen early in the disease. These criteria have demonstrated good reliability and validity and are used herein as the basis for claiming a clinical diagnosis of AD.

[0007] This diagnosis has not traditionally been able to be determined by laboratory assays. Such assays are primarily important in identifying other possible causes of dementia that must be ruled out before a diagnosis of Alzheimer's disease can be made with confidence. Neuropsychological testing provides confirmatory evidence of the diagnosis of dementia and helps to evaluate the course and response to treatment. The criteria proposed by McKhann et al. above are intended to serve as a guide for the diagnosis of probable, possible, and definite Alzheimer's disease; these criteria are likely to be revised as more definitive information becomes available.

[0008] Diagnostic criteria have been refined very recently to include a prodromal stage (early symptoms occurring before the full-blown symptoms of the disease appear) called "mild cognitive impairment due to AD (MCI)". This new diagnosis reflects a desire to treat the disease early, since neuropathology is estimated to begin 10 years before symptoms appear. [Trojanowski et al., Alzheimers Dement 6, 230-238 (2010)]. Clinical trials of potential disease-modifying treatments have been very disappointing, in part because even "early stage" patients already have substantial pathology with large amyloid-β (Aβ) burdens and significant synaptic defects and inflammation.

[0009] According to Petersen et al., Arch Neurol 56(3):303-308 (1999), the main difference between control and MCI subjects was in the memory domain, with other cognitive functions being comparable. However, when MCI subjects were compared with patients with the mildest AD, memory performance was similar, but the AD patients were more impaired in other cognitive domains. Longitudinal performance showed that MCI subjects declined at a rate greater than that of controls, but less rapidly than patients with mild AD.

[0010] Patients fulfilling the criteria for MCI can be distinguished from healthy control subjects and from subjects with minimal AD, and appear to constitute a clinical entity that can be characterized for therapeutic intervention.

[0011] Amyloid-β (Aβ) is a peptide of 39-42 amino acid residues in length that is generated in vivo by specific proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretases. 42 comprises residues 677-713 of the APP protein, which is itself a 770-residue transmembrane protein with the designation P05067 in the UniProtKB / Swiss-Prot system. 42Although it is generally believed to be the primary causative agent of AD, its mechanisms underlying AD neuropathology are debated.

[0012] Until recently, only symptom-based assays discussed in McKhann et al. above were available to diagnose the presence of Alzheimer's disease in living patients. Most recently, starting with Eli Lilly's Amyvid® in April 2012, followed by GE Healthcare's Vizamyl® and Piramal Imaging's Neuraceq®, PET scanning technology has been used to assay for AD in living humans. Radiolabeled positron-emitting compounds injected intravenously bind to Aβ in brain plaques.

[0013] Although accurate, PET scan assays are inconvenient for patients in that they must place their head in a relatively confined space within a scintillation detector and remain relatively still. PET scan assays are also expensive, especially when compared to more conventional human blood tests that require drawing a few milliliters of blood to provide as many as 40 different assays (which, unfortunately, do not yet include a commercially available test for AD).

[0014] Discovered 45 years ago as the first non-muscle actin-binding protein [Hartwig et al., J Biol Chem 250:5696-5705 (1975); Wang et al., Proc Natl Acad Sci USA 72:4483-4486 (1975)], filamin [FLN] is a family of cytoskeletal proteins expressed in non-muscle cells - filamins A (FLNA) and B, but not C. Human FLNA has been given the identifier P21333 in the UniProtKB / Swiss-Prot database and contains a sequence of 2647 amino acid residues (approximately 280 kDa). This protein is sometimes referred to in the art as actin-binding protein (ABP-280). [Gorlin et al., J Cell Biol 111:1089-1105 (1990)].

[0015] FLNA protein anchors various transmembrane proteins to the actin cytoskeleton and functions as a scaffold for a wide range of cytoplasmic signaling proteins. Filamin is essential for mammalian cell motility and serves as an interface for protein-protein interactions [van der Flier et al., Biochim Biophys Acta 1538:99-117 (2001)]. In addition to its role in cell motility, FLNA has increasingly been shown to regulate cell signaling by interacting with various receptors and signaling molecules [Stossel et al., Nat Rev Mol Cell Biol 2:138-145 (2001); Feng et al., Nat Cell Biol 6:1034-1038 (2004)].

[0016] The FLNA protein consists of an N-terminal actin-binding domain (ABD) and a rod-shaped domain of 24 immunoglobulin-like repeat domains (IgFLNa), each approximately 96 amino acid residues long and numbered from the N-terminus, interrupted by two 30 amino acid residue flexible loops or hinges. The IgFLNa are numbered from 1 to 24, starting near the N-terminus and ending near the C-terminus. A loop designated H1 is between repeats 15 and 16, and a loop designated H2 is between repeats 23 and 24 [Gorlin et al., J Cell Biol 111:1089-1105 (1990); van der Flier et al., Biochim Biophys Acta 1538:99-117 (2001)].

[0017] H1 and H2 are cleaved by calpains and caspases [Gorlin et al., J Cell Biol 111:1089-1105 (1990); Browne et al., J Biol Chem 275:39262-39266 (2000)]. Cleavage at H1 occurs between amino acid residues 1762 and 1764, generating an approximately 170 kDa fragment consisting of the ABD and repeats 1-15 (IgFLNa-1-15) plus an approximately 110 kDa polypeptide fragment consisting of repeats 16-24 (IgFLNa-16-24).

[0018] It should be noted that the UniProtKB / Swiss-Prot database lists the C-terminus of repeat 15 at position 1740 and the N-terminus of repeat 16 at amino acid residue position 1779. On the other hand, Gorlin et al., supra, places the calpain cleavage site between residues 1762 and 1764, whereas Garcia et al., Arch Biochem Biophys 446:140-150 (2006), places the site between residues 1761 and 1762. Similarly, Gorlin et al., supra, states that the previous authors (Hartwig et al., J Cell Biol 87:841-848 (1980)) reported on page 1089 that the full-length FLNA molecule had a molecular weight of 270 kDa, and then on page 1093 that the protein had a molecular weight of 280 kDa.

[0019] Garcia et al., supra, reported that calpain cleaves full-length FLNA into polypeptide fragments of 180, 100, 90, and 10 kDa, whereas Bedolla et al., Clin Cancer Res 15(3):788-796 (2009), reported proteolytic fragments of 170, 110, and 90 kDa cleaved from the 110 kDa fragment. Browne et al., supra, reported that granzyme B (grB), a protease of cytotoxic T lymphocytes, cleaves filamin in cooperation with the lytic protein perforin, and that filamin is cleaved in a caspase-dependent manner after ligation to the Fas receptor. Western blots of lysates of dying Jurkat cells identified two caspase-cleaved polypeptides derived from the C-terminal region of FLNA with masses of approximately 110 and 95 kDa. Purified grB cleaved filamin into several polypeptides, including those with masses of approximately 205, 200, and 110 kDa. A polyclonal rabbit antibody raised against a fusion protein containing 476 amino acid residues from the C-terminal region of FLNA (positions 2172-2647) was used. Umeda et al., J Biochem 130:535-542 (2001), found somewhat similar results (C-terminal 135, 120, and 110 kDa polypeptide fragments) for proteolysis by caspase-3 in U937 monoblastic leukemia and Jurkat human T-lymphoblastic cells.

[0020] Note that IgFLNa-16-24 is said to have a mass of about 110 kDa in Loy et al., Proc Natl Acad Sci, USA, 100(8):4562-4567 (2003). The 110 kDa polypeptide (IgFLNa-16-24) is further cleaved by calpain with H2, with longer digestion times, to yield a 90 kDa fragment (IgFLNa-16-23) containing repeats 16-23 [Gorlin et al., J Cell Biol 111:1089-1105 (1990); van der Flier et al., Biochim Biophys Acta 1538:99-117 (2001)].

[0021] As discussed above, due to differences in residue positions and some molecular weights of full-length FLNA and its proteolytic fragments reported in the art, the full-length FLNA molecule and the smaller FLNA cleavage products are considered to have molecular weights of "approximately" 280 kDa and "approximately" 90 kDa, respectively.

[0022] FLNA promotes orthogonal branching of actin filaments and links actin filaments to membrane glycoproteins. Filamin A dimerizes through a carboxy-terminal repeat (repeat 24) near the transmembrane region, providing an intracellular V-shaped structure that is important for function.

[0023] Each V-shaped FLNA dimer has two antiparallel self-binding domains24 that form the vertices of the "V" and the remaining domains that extend like beads on a string with each of their N-terminal ABD portions attached to actin molecules. More recently, it has been reported that rod segment 1 (IgFLNa-1-15) at the C-terminus of the ABD forms an extended linear structure without obvious interdomain interactions. Rod segment 2 (IgFLNa-16-23) adopts a compact structure due to multiple interdomain interactions in which domains 16-17, 18-19, and 20-21 form paired structures. [Heikkinen et al., J Biol Chem, 284:25450-25458 (2009); Lad et al., EMBO J, 26:3993-4004 (2007)].

[0024] Proteolysis of FLNA is regulated in part by phosphorylation on Ser 2152 (S2152) in its repeat 20 (IgFLNa-20), which has been reported to render the full-length protein stable and less susceptible to cleavage [Gorlin et al., J Cell Biol 111:1089-1105 (1990); Garcia et al., Arch Biochem Biophys 446:140-150 (2006); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)].

[0025] Loy et al., Proc Natl Acad Sci, USA, 100(8):4562-4567 (2003), report that an H1 cleavage product containing repeats 16-24 and having a molecular weight of approximately 100 kDa colocalizes with the androgen receptor in the nucleus in prostate cancer cells. The researchers noted that FLNA is typically considered a cytoplasmic structural molecule, and characterized their finding that the approximately 100 kDa polypeptide produced by calpain cleavage further functions as a nuclear regulator of the androgen receptor as "completely unexpected" (p. 4565).

[0026] The approximately 100 kDa FLNA fragment found in the cell nucleus is not phosphorylated on S2152. Indeed, phosphorylation on S2152 has been reported to prevent cleavage of full-length FLNA by calpain in prostate cancer lines and platelets [Garcia et al., Arch Biochem Biophys 446:140-150 (2006); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)].

[0027] Wang et al., Oncogene 26:6061-6070 (2007) showed that nuclear localization of FLNA correlates with hormone dependence in prostate cancer. An unphosphorylated ≈90 kDa fragment (IgFLNa-16-23) translocates to the nucleus of hormone-naive cells in androgen-dependent disease. In contrast, in hormone-refractory androgen-independent prostate tumor cells, FLNA is phosphorylated, preventing its cleavage and nuclear translocation. These authors and coworkers subsequently showed not only that prostate cancer metastasis correlates with the cytoplasmic localization of FLNA, but also that metastasis can be prevented by cleavage and subsequent nuclear translocation of the phosphorylated protein [Bedolla et al., Clin Cancer Res. 15(3):788-796 (2009)].

[0028] As a key regulator of the cytoskeletal network, FLNA interacts with many proteins involved in cancer metastasis [Yue et al., Cell & Biosci 3:7 (2013)] and many other diseases. Thus, Nakamura et al., Cell Adh Migr. 5(2):160-169 (2011) discusses the history of research on FLNA and points out that this protein functions as a scaffold for over 90 binding partners, including channels, receptors, intracellular signaling molecules, and transcription factors.

[0029] FLNA has also been implicated in tumor progression. FLNA knockout mice show reduced oncogenic activity of K-Ras, including downstream activation of ERK and Akt. [Nallapalli et al., Mol Cancer 11:50 (2012)]. Many different cancers, including colorectal and pancreatic cancers [Uhlen et al., Mol Cell Proteomics 4:1920-1932 (2005)] and glioblastomas [Sun et al., Cancer Cell 9:287-300 (2006)], show high levels of FLNA expression in contrast to low levels in corresponding normal tissues.

[0030] Inhibition of FLNA expression sensitizes cancer cells to both cisplatin and radiation [Sun et al., Cancer Cell 9:287-300 (2006)], and FLNA deficiency in cancer cells similarly sensitizes cancer cells to chemotherapeutic agents [Yue et al., DNA Repair (Amst) 11:192-200 (2012)] and radiation [Yue et al., Cancer Res 69:7978-7985 (2009); Yuan et al., J Biol Chem 276:48318-48324 (2001)]. On the other hand, Jiang et al., Int.J. Biol.Sci. 9:67-77 (2013) reported that inhibition of filamin A expression reduced metastasis in nude mice implanted with melanoma and breast cancer cells.

[0031] Phosphorylation is increasingly recognized as a global regulator of cellular activity, and aberrant phosphorylation has been implicated in a host of human diseases, particularly cancer. Protein phosphorylation is the enzymatic merging of a hydroxyl group and a phosphate group (-OPO3 -2 ) replacement.

[0032] Phosphorylation and its reverse reaction, dephosphorylation, occur through the action of two important enzyme types. Protein kinases phosphorylate proteins by transferring a phosphate group from a nucleotide triphosphate such as adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to their target proteins. This process is balanced by the action of protein phosphatases, which can subsequently remove the phosphate group.

[0033] Therefore, the amount of phosphate bound to a protein at a particular time is determined by the relative activity of that protein and the particular associated kinase and phosphatase enzymes specific for the particular amino acid residue undergoing phosphorylation / dephosphorylation. If the phosphorylated protein is an enzyme, phosphorylation and dephosphorylation can essentially act as a switch, turning it on and off in a regulated manner to affect its enzymatic activity. Phosphorylation can similarly regulate nonenzymatic protein-protein interactions by promoting binding to partner proteins.

[0034] Protein phosphorylation can play an important role in intracellular signal transduction. Many of the proteins that compose signal transduction pathways, from cell surface tyrosine kinase receptors to downstream effector proteins, are kinases, many of which are serine / threonine kinases.

[0035] FLNA is phosphorylated at many positions in its protein sequence in both normal and diseased cells, such as cancer cells. For example, the enzyme PAK1 (EC 2.7.11.1) is a protein kinase of the STE20 family that regulates cell motility and morphology. FLNA phosphorylation at position 2152 by PAK1 is required for PAK1-mediated actin cytoskeleton rearrangement and PAK1-mediated membrane ruffling. [Vadlamudi et al., Nat. Cell Biol. 4:681-690 (2002); Woo et al., Mol Cell Biol. 24(7):3025-3035 (2004)]. Cyclin B1 / Cdk1 (EC:2.7.11.22; EC:2.7.11.23) phosphorylates serine 1436 in vitro during FLNA-dependent actin remodeling. [Cukier et al., FEBS Letters 581(8):1661-1672(2007)].

[0036] The UniProtKB / Swiss-Prot database entry for human FLNA (No. P21333) lists published reports that the following amino acid residue positions: 11, 1081, 1084, 1089, 1286, 1338, 1459, 1533, 1630, 1734, 2053, 2152, 2158, 2284, 2327, 2336, 2414, and 2510 are phosphorylated under different circumstances. Additionally, polyclonal and monoclonal antibodies that immunoreact with FLNA phosphorylated at serine-1083, tyrosine-1046, serine-1458, serine-2152, and serine-2522 (phosphorylated FLNA) are commercially available from one or more of Abgent (San Diego, CA), Abcam® (Beverly, MA), Bioss (Woburn, MA), and GeneTex (Irvine, CA).

[0037] The 90 kDa FLNA fragment, which can be localized in the nucleus and interact with transcription factors, contains the serine-2152 residue that can be phosphorylated. However, the previously discussed 90 kDa FLNA fragment that is localized in the nucleus does not contain phosphorylation at serine-2152. In fact, it has been reported that phosphorylation of FLNA at serine-2152 ("pS2152" FLNA) protects FLNA from proteolysis to form the 90 kDa fragment [Garcia et al., Arch Biochem Biophys 446:140-150 (2006); Gorlin et al., J Cell Biol 111:1089-1105 (1990); and Chen et al., J Biol Chem 264(24):14282-14289 (1989)].

[0038] Toxic signaling of amyloid-β42 (Aβ42) through the α7-nicotinic acetylcholine receptor (α7nAChR), which leads to tau phosphorylation and the formation of neurofibrillary tangles associated with Alzheimer's disease, has been shown to require recruitment of the scaffolding protein FLNA, which activates TLR4 via CD14. Wang et al., J. Neurosci. 32(29):9773-9784 (18 July 2012). The paper also shows that PTI-125 (simufilam) similarly reduces the association of FLNA with toll-like receptor 4 (TLR4), resulting in anti-inflammatory effects by preventing cytokine release. Summary of the Invention

[0039] BRIEF SUMMARY OF THE PRESENTINVENTION The scaffolding protein FLNA has been recognized to be associated with the development of Alzheimer's disease through its involvement in Aβ42 signaling via α7nAChR, and the abnormally folded form of FLNA, which is believed to be a relevant factor in this process, is the target of PTI-125 (simufilam), a small molecule antagonist of FLNA currently being investigated for use in the treatment of AD. Interestingly, FLNA is also strongly expressed in platelets, where it is thought to regulate normal platelet function, and mutations in FLNA can cause platelet-related disorders. Platelets also express functional α7nAChR (Schedel et al., ArteriosclThrom, Vas, 31:928-934 (2011.)). There are also reports that platelets can shuttle Aβ42 to the brain (Catricala et al., ImmunAgeing 9:20 (2012)). FLNA is also known to be processed in platelets upon activation [Buitrago et al., bioRxiv 307397], which may help generate abnormally folded forms of FLNA capable of binding to Aβ42 and / or α7 nAChR both inside platelets and in the brain following platelet lysis.

[0040] Current research has not been able to create a viable diagnostic assay based on FLNA. In fact, current diagnostic methods for Alzheimer's disease usually require tissue samples (e.g., for postmortem confirmation) or cerebrospinal fluid obtained by invasive surgical procedures. The present disclosure addresses these and other shortcomings associated with current assays by providing new blood-based assays and methods that can be used for the diagnosis of AD and other tauopathies, as well as kits and reagents related to the assays and methods, based on novel biomarkers (e.g., FLNA with a specific phosphorylation profile) or fragments thereof, for use in methods for the diagnosis, staging, treatment, and evaluation of treatment response of neurocognitive disorders such as Alzheimer's disease. Furthermore, the present invention provides a novel target for the development of new therapeutics for tauopathies or for repurposing existing therapeutics that were not originally designed for the treatment of neurocognitive disorders such as tauopathies.

[0041] In a first general aspect, the present disclosure provides a biomarker panel comprising one or more phosphorylated peptides obtained from in vitro digestion of Filamin A (SEQ ID NO: 1). In some embodiments, the FLNA is contained within or obtained from a biological fluid or tissue sample taken from a subject suspected of having a neurological disease (e.g., Alzheimer's disease or other tauopathies). For simplicity, example sequences are provided based on digestion with the proteases trypsin, GluC, and AspN. Those skilled in the art will understand that digestion with other proteases will generate alternative peptide sequences containing the same phosphorylated residues, and all such alternatives are encompassed herein.

[0042] In some embodiments, the phosphopeptide is phosphorylated at residue serine 2152 of SEQ ID NO:1. In some embodiments, the phosphopeptide has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5 (i.e., a fragment of full-length FLNA). In some embodiments, a biomarker panel can include multiple phosphorylated fragments of FLNA, e.g., two or more phosphopeptides each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs:2-5, where at least one of the two or more phosphopeptides is phosphorylated at a position corresponding to serine 2152 of SEQ ID NO:1 (full-length FLNA).

[0043] In some embodiments, the phosphopeptide is phosphorylated at residue serine 2143 of SEQ ID NO: 1. In some embodiments, the phosphopeptide has the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9 (i.e., a fragment of full-length FLNA). In some embodiments, a biomarker panel can include multiple phosphorylated fragments of FLNA, e.g., two or more phosphopeptides each comprising a fragment of FLNA and having the amino acid sequences of SEQ ID NOs: 6-9, where at least one of the two or more phosphopeptides is phosphorylated at a position corresponding to serine 2143 of SEQ ID NO: 1 (full-length FLNA).

[0044] In some embodiments, the phosphopeptide is phosphorylated at residue serine 2180 of SEQ ID NO: 1. In some embodiments, the phosphopeptide has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12 (i.e., a fragment of full-length FLNA). In some embodiments, a biomarker panel can include multiple phosphorylated fragments of FLNA, e.g., two or more phosphopeptides each comprising a fragment of FLNA and having the amino acid sequences of SEQ ID NOs: 10-12, where the two or more phosphopeptides are phosphorylated at a position corresponding to residue serine 2180 of SEQ ID NO: 1 (full-length FLNA).

[0045] In some embodiments, the phosphopeptide is phosphorylated at residue serine 1459 of SEQ ID NO: 1. In some embodiments, the phosphopeptide has the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 (i.e., a fragment of full-length FLNA). In some embodiments, a biomarker panel can include multiple phosphorylated fragments of FLNA, e.g., two or more phosphopeptides each comprising a fragment of FLNA and having the amino acid sequences of SEQ ID NOs: 13-15, where the two or more phosphopeptides are phosphorylated at a position corresponding to residue serine 1459 of SEQ ID NO: 1 (full-length FLNA).

[0046] In some embodiments, the biomarker panel comprises a plurality of phosphopeptides, where each phosphopeptide is a fragment of FLNA (SEQ ID NO: 1) and contains phosphorylation sites at positions corresponding to serine 1459, 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO: 1). For example, a biomarker panel can include a plurality of phosphopeptides, each having a sequence of SEQ ID NOs:2-15, and phosphorylated at positions corresponding to serine 1459, 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO:1). In some embodiments, a biomarker panel can include one or more phosphopeptides that include a fragment of full-length FLNA that is phosphorylated at two or more of the phosphorylation sites identified above (e.g., serine 1459, 2143, 2152, and / or 2180 of full-length FLNA).

[0047] In some embodiments, the biomarker panel includes at least two, three, four, five, six, seven, eight, nine, or ten different phosphopeptides, each phosphorylated at sites corresponding to serine residues 1459, 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO:1).

[0048] In some embodiments, the biomarker panel further comprises one or more peptides listed in Table 4 obtainable from in vitro digestion of Filamin A with a protease or combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO: 1. In some embodiments, the peptide is proteotypic to FLNA.

[0049] In a second general aspect, the disclosure provides a panel of biomarkers comprising: (i) one or more peptides obtained from in vitro digestion of filamin A with a protease or a combination of proteases, each of the one or more peptides comprising an amino acid sequence present in SEQ ID NO:1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, proteins listed in Table 1. In some embodiments, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin. In some embodiments, filamin A and one or more proteins listed in Table 1 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some embodiments, the neurological disease is Alzheimer's disease or another tauopathy. In some embodiments, the one or more peptides obtained from in vitro digestion of filamin A comprise one or more phosphorylation sites. In some embodiments, the one or more phosphorylation sites correspond to serine residues 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO:1).

[0050] In a third general aspect, the disclosure provides a panel of biomarkers comprising: (i) one or more peptides obtained from in vitro digestion of filamin A with a protease or a combination of proteases, each of the one or more peptides comprising an amino acid sequence present in SEQ ID NO:1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, proteins listed in Table 2. In some embodiments, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin. In some embodiments, filamin A and one or more proteins listed in Table 2 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some embodiments, the neurological disease is Alzheimer's disease or another tauopathy. In some embodiments, the one or more peptides obtained from in vitro digestion of filamin A comprise one or more phosphorylation sites. In some embodiments, the one or more phosphorylation sites correspond to serine residues 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO:1).

[0051] In a fourth general aspect, the disclosure provides a panel of biomarkers comprising: (i) one or more peptides obtained from in vitro digestion of filamin A with a protease or a combination of proteases, each of the one or more peptides comprising an amino acid sequence present in SEQ ID NO:1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, proteins listed in Table 3. In some embodiments, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin. In some embodiments, filamin A and one or more proteins listed in Table 3 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some embodiments, the neurological disease is Alzheimer's disease or another tauopathy. In some embodiments, the one or more peptides obtained from in vitro digestion of filamin A comprise one or more phosphorylation sites. In some embodiments, the one or more phosphorylation sites correspond to serine residues 2143, 2152, and / or 2180 of full-length FLNA (SEQ ID NO:1).

[0052] In a fifth general aspect, the disclosure provides a panel of biomarkers comprising: (i) one or more peptides obtained from in vitro digestion of filamin A with a protease or a combination of proteases, each of the one or more peptides comprising an amino acid sequence present in SEQ ID NO:1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, proteins listed in Tables 1, 2, 3, or 4. In some embodiments, the one or more peptides are obtained from in vitro digestion with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin. In some embodiments, filamin A and one or more proteins listed in Table 1 are contained within a biological fluid or tissue sample obtained from a subject suspected of having a neurological disease. In some embodiments, the neurological disease is Alzheimer's disease or another tauopathy.

[0053] In a sixth general aspect, the disclosure provides a biomarker panel comprising one or more synthetic peptides, wherein one or more of the synthetic peptides are enriched with heavy isotopes of H, C, N, O, and / or S. In some embodiments, one or more of the synthetic peptides comprises an amino acid sequence present in Filamin A (SEQ ID NO:1); or present in one of the proteins listed in Tables 1, 2, 3, or 4. In some embodiments, one or more of the synthetic peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of SEQ ID NO:1 or one of the proteins listed in Tables 1, 2, 3, or 4 with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin.

[0054] In a seventh general aspect, the disclosure is a method for the diagnosis / prognosis of a neurological disorder, comprising: obtaining a fluid or tissue sample from a subject (optionally a subject suspected of having a neurological disorder, such as Alzheimer's disease); digesting one or more proteinaceous materials (e.g., proteins and / or polypeptides) in the fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and / or chymotrypsin); and detecting and / or measuring the level of one or more peptides produced from the digestion using mass spectrometry. In some embodiments, each of the one or more peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of filamin A (SEQ ID NO:1) or one of the proteins listed in Tables 1, 2, or 3.

[0055] In an eighth general aspect, the disclosure provides a method of monitoring the progression of a neurological disorder, the method comprising: (a) obtaining a bodily fluid or tissue sample from a subject (optionally a subject suspected of having a neurological disorder, such as Alzheimer's disease) at a first time point; (b) digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and / or chymotrypsin); (c) detecting and / or measuring the level of one or more peptides produced from the digestion using mass spectrometry; and (d) repeating steps (a)-(c) at a second time point. In some embodiments, the method further comprises a step (e) of determining whether a therapeutic treatment administered to the subject is effective to treat the neurological disorder based on the detected and / or measured levels of the one or more peptides in step (c). In some embodiments, each of the one or more peptides comprises an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of filamin A (SEQ ID NO:1) or one of the proteins listed in Table 1, Table 2 or Table 3.

[0056] In a ninth general aspect, the disclosure provides a method of diagnosing or monitoring the progression of a neurological disorder (such as Alzheimer's disease or another tauopathy), comprising: obtaining a bodily fluid or tissue sample from a subject; digesting one or more proteins in the bodily fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and / or chymotrypsin); and measuring the level of any of one or more phosphorylated peptide fragments produced from digestion of filamin A (SEQ ID NO:1). In some embodiments, the one or more fragments comprise phosphorylated serine at positions corresponding to serine 2152 and / or 2143 of filamin A (SEQ ID NO:1).

[0057] In some embodiments, the method further comprises determining a ratio of two or more of the peptide fragments. In some embodiments, the ratio comprises a ratio of fragments phosphorylated at a position corresponding to serine 2143 of filamin A (SEQ ID NO:1) to fragments phosphorylated at a position corresponding to serine 2152 of filamin A (SEQ ID NO:1). In some embodiments, the ratio is determined using two or more peptides, each comprising a sequence of SEQ ID NO:2-9. In some embodiments, the ratio comprises a ratio of fragments phosphorylated at a position corresponding to serine 2143 of filamin A (SEQ ID NO:1) to fragments phosphorylated at a position corresponding to serine 2152 of filamin A (SEQ ID NO:1); where a ratio of <5 indicates that the subject does not have Alzheimer's disease and a ratio of >10 indicates that the subject has Alzheimer's disease.

[0058] In a tenth general aspect, the disclosure provides a method of diagnosing or monitoring the progression of a neurological disorder (such as Alzheimer's disease or another tauopathy), comprising obtaining a body fluid or tissue sample from a subject; digesting one or more proteins in the body fluid or tissue sample with one or more proteases (e.g., trypsin, GluC, ArgC, AspN, and / or chymotrypsin); and measuring the level of any of one or more phosphorylated peptides produced from digestion of filamin A (SEQ ID NO:1) and one or more peptides derived from integrin alpha-IIb, integrin beta-3, and / or linker for activation of T-cell family member 1, where the presence of integrin alpha-IIb, integrin beta-3, and / or linker for activation of T-cell family member 1 is indicative of in vitro or artificial platelet activation. [Brief description of the drawings]

[0059] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows a representation of the TMTcalibrator™ mass spectrometry workflow. After removal of the top 14 abundant proteins, plasma samples were digested and labeled with TMTpro™ reagent. In parallel, brain lysates were also digested and labeled with TMTpro™ reagent. The plasma and brain lysate digests were then mixed and a small aliquot was analyzed for overall protein expression by tandem mass spectrometry (LC-MS / MS). The phosphopeptide fraction was enriched from the remaining mixture and analyzed by LC-MS / MS. Data analysis identified peptide sequences and relative abundances based on TMTpro™ reporter ions, and linear modeling was used to identify features indicative of differential expression in AD and control samples.

[0060] [Diagram 2]Figure 2 shows histograms of the expression profile of FLNA in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. Values ​​are expressed as log2 ratios compared to the reference brain lysate channel.

[0061] [Diagram 3] FIG. 3 shows tryptic peptides phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1) in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] 1 shows histograms of expression profiles of TMTpro™ reporter ion intensities. Values ​​are expressed as isotope-corrected TMTpro™ reporter ion intensities.

[0062] [Figure 4] FIG. 4 shows tryptic peptides phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1) in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] 1 shows histograms of expression profiles of TMTpro™ reporter ion intensities. Values ​​are expressed as isotope-corrected TMTpro™ reporter ion intensities.

[0063] [Diagram 5] FIG. 5 shows tryptic peptides phosphorylated at serine 2143 of FLNA (SEQ ID NO: 1) in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] 1 shows histograms of expression profiles of TMTpro™ reporter ion intensities. Values ​​are expressed as isotope-corrected TMTpro™ reporter ion intensities.

[0064] [Figure 6] FIG. 6 shows tryptic peptides phosphorylated at serine 2180 of FLNA (SEQ ID NO: 1) in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] 1 shows histograms of expression profiles of TMTpro™ reporter ion intensities. Values ​​are expressed as isotope-corrected TMTpro™ reporter ion intensities.

[0065] [Figure 7] FIG. 7 is divided into four parts as FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D, and shows the four FLNA phosphorylated peptides in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] (SEQ ID NO:3; Figure 7A) [ka] (SEQ ID NO: 10; Figure 7B) [ka] (SEQ ID NO: 6; FIG. 7C ) and [ka] (SEQ ID NO: 13; FIG. 7D) are four box plots showing relative expression. Values ​​shown are log2 ratios compared to the reference brain lysate channel.

[0066] [Figure 8]Figure 8 shows heat maps of log2 transformed phosphopeptide expression levels compared to brain lysate channels in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes in three panels, Figure 8A, Figure 8B, and Figure 8C. The original gene names of the regulated phosphopeptides are shown to the right of the figures in each panel. Figure 8A shows regulated phosphopeptides in AD cases and aged controls. Figure 8B shows regulated phosphopeptides in AD cases and young controls. Figure 8C shows regulated phosphopeptides in AD cases and all controls.

[0067] [Figure 9] FIG. 9 shows, in three parts as FIG. 9A, FIG. 9B, and FIG. 9C, the tryptic peptide phosphorylated at serine 2143 of FLNA (SEQ ID NO: 1) in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] (FIG. 9A), phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1). [ka] (FIG. 9B), and phosphorylated at serine 2152 of FLNA (SEQ ID NO: 1). [ka] (SEQ ID NO: 138) (FIG. 9C) provides a histogram showing the expression profile of TMTpro™ reporter ion intensity. Values ​​are expressed as isotope-corrected TMTpro™ reporter ion intensity.

[0068] [Figure 10]FIG. 10 is divided into four parts as FIG. 10A, FIG. 10B, FIG. 10C, and FIG. 10D, and shows the four FLNA phosphorylated peptides in six AD plasma samples prepared with Histopaque®-1077 and six plasma samples collected in EDTA tubes. [ka] (SEQ ID NO: 1; Figure 10A) [ka] (SEQ ID NO:3; FIG. 10B) [ka] (SEQ ID NO: 186; FIG. 10C ) and [ka] (SEQ ID NO: 13; FIG. 10D) show four box plots depicting the relative expression of (SEQ ID NO: 13; FIG. 10D). Values ​​shown are the log2 ratios compared to the reference brain lysate channel. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0069] Detailed Description of the Invention For the purposes of interpreting this specification, the following definitions and abbreviations shall apply, unless otherwise stated and whenever appropriate, and terms used in the singular shall include the plural and vice versa.

[0070] The term "biomarker" includes all biologically relevant forms of the identified protein, including post-translational modifications. For example, biomarkers can exist in glycosylated, phosphorylated, multimeric, fragmented, or precursor forms. Biomarker fragments can be naturally occurring or, for example, enzymatically produced and still retain the biologically active function of the intact protein. Fragments are typically at least about 10 amino acids in length, usually at least about 50 amino acids in length, and can be as long as 300 amino acids or longer.

[0071] The term "canonical sequence" is used herein to refer to the most common and / or most similar sequence among orthologous species. Unless otherwise specified, canonical sequence herein refers to a human sequence.

[0072] The peptide sequences disclosed herein are represented using the IUPAC one-letter code. The use of lower case letters indicates modifications such as: "c"-carbamidomethylated cystine; "m"-oxidized methionine; "n"-deamidated asparagine; "q"-deamidated glutamine; and "k"-TMT-labeled lysine. A lower case letter at the N-terminus represents a TMT-modified amino acid. Additionally, the symbol "[p]" is used to indicate phosphorylation on the following amino acid, e.g., "[p]s" represents phosphorylated serine; "[p]t" represents phosphorylated threonine; and "[p]y" represents phosphorylated tyrosine.

[0073] The term "KEGG pathway" refers to a collection of hand-drawn pathway maps that represent molecular interaction and reaction networks related to metabolism, genetic information processing, environmental information processing, cellular processes, biological systems, human diseases, and drug discovery. "KEGG pathway mapping" is the process of mapping molecular datasets, especially large datasets in genomics, transcriptomics, proteomics, and metabolomics, onto the KEGG pathway map for biological interpretation of higher-level systemic functions (http: / / www.genome.jp / kegg / pathway.html).

[0074] The term "concentration or amount" refers to the relative concentration or amount of a biomarker in a sample, as determined by LC-MS / MS label-free quantification approaches, such as area under the curve and spectral counting.

[0075] The terms "comparing" or "comparing" or their grammatical equivalents refer to determining the relative concentration or amount of a biomarker in a sample compared to other samples (e.g., protein concentrations or amounts stored in a proprietary or public database).

[0076] The term "reference concentration or amount" refers to, but is not limited to, a protein concentration or amount stored in a proprietary or public database. A "reference concentration or amount" can be obtained from large-scale screening of patients or by reference to a known or previously determined correlation between such determinations and clinical information in control patients. For example, a reference value can be determined by comparison with the concentration or amount of a biomarker in a control subject, e.g., a healthy individual (i.e., an individual without dementia) of similar age and sex as the subject. Alternatively, the reference value can be a value that can be found in the literature, such as the presence of the ApoE 24 allele, where the presence or absence of mutations at positions 112 and 158 represent the standard to be compared, or levels of total tau (T-tau) in the CSF of >350 ng / L, phosphorylated tau (P-tau) of >80 ng / L, and A-42 of <530 ng / L (Hansson et al., Lancet Neural. 5(3):228-234 (2006)). Furthermore, the reference value can be obtained from the same subject at one or more time points that temporally precede the test time point. Such a prior sample can be taken one week or more, one month or more, three months or more, and most preferably six months or more prior to the date of the test time point. In some embodiments, multiple prior samples are compared longitudinally, and the slope of the change in biomarker expression, if any, can be calculated as a correlate of cognitive change, such as decline, as typically noted.

[0077] The term "control" or, as used herein, "non-AD control" or "non-AD subject" refers to a tissue or fluid sample taken from a human or non-human subject who is cognitively normal or who has been diagnosed with or exhibits symptoms of a cognitive abnormality, but who is defined as a non-AD subject with respect to existing biochemical tests.

[0078] The terms "selected reaction monitoring", "SRM" and "MRM" refer to mass spectrometry assays in which precursor ions of known mass to charge ratios representing known biomarkers are preferentially targeted for analysis by tandem mass spectrometry in an ion trap or triple quadrupole mass spectrometer. During mass spectrometric analysis, the parent ions are fragmented and the number of selected daughter ions of a second predefined mass to charge ratio are counted. Typically, the method includes an equivalent precursor ion that has a predefined number of stable isotope substitutions but is otherwise chemically identical to the target ion to serve as a quantitative internal standard.

[0079] The terms "parallel reaction monitoring" and "PRM" refer to a mass spectrometry assay in which precursor ions of known mass-to-charge ratios representing known biomarkers are preferentially targeted for analysis by tandem mass spectrometry on an Orbitrap™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA). During analysis, the parent ions are fragmented and the number of each daughter ion is counted. Typically, the method includes an equivalent precursor ion that has a predefined number of stable isotope substitutions but is otherwise chemically identical to the target ion to serve as a quantitative internal standard.

[0080] The term "proteotypic" means a peptide that uniquely represents the protein from which it is derived such that the sequence of amino acid residues in the peptide is not found in any other protein from the same species, apart from other expressed isoforms or splice variants from the same gene.

[0081] The term "phosphorylated peptide" means a peptide that contains at least one amino acid modified by the addition of a phosphate group.

[0082] The term "isolated" or its grammatical equivalents is used throughout this specification to mean that a protein, peptide, antibody, polynucleotide, or, in some cases, a chemical molecule, is present in a physical environment that is different from that in which it may be found in nature.

[0083] As used herein, the term "subject" includes any human or non-human animal.

[0084] The term "non-human animals" includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, rodents, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0085] The terms "treat," "treating," "treatment," "prevent," "preventing," or "prevention," or grammatical equivalents thereof, include therapeutic treatments, prophylactic treatments, and applications in which a subject reduces the risk of developing a disorder or other risk factors. Treatment does not require a complete cure of a disorder, but encompasses the alleviation of symptoms or underlying risk factors.

[0086] The term "diagnosis" or its grammatical equivalents as used herein includes any information provided regarding the presence or absence or absence or possibility of a disorder in a patient. This further includes providing information regarding the type or classification of the disorder or symptoms experienced or that may be experienced in association with the disorder. This can include, for example, diagnosing the severity of the disorder. The term "diagnosis" encompasses the prognosis of the medical course of the disorder, for example, its duration, severity, and the course of progression from mild cognitive impairment (MCI) to AD or other dementia.

[0087] The term "staging" or its grammatical equivalents as used herein means to identify the stage of neurocognitive disorder, particularly AD, in a subject.For example, AD is characterized by three or seven stages, depending on the diagnostic framework used.Global Dementia Scale is one such measure of global function.It is measured by the assessment of severity, including cognition and function, against a set of standardized severity criteria.

[0088] The term "efficacy" refers to the capacity of a given intervention (e.g., a drug, a medical device, a surgical procedure, etc.) for beneficial change. If efficacy is demonstrated, the intervention is likely to be at least as good as other available interventions to which it is being compared. The terms "efficacy" and "effectiveness" are used interchangeably herein.

[0089] The term "comprising" indicates that subject matter includes all the elements listed, but may optionally include additional unnamed elements.

[0090] The term "and / or" as used herein should be construed as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0091] Unless the context indicates otherwise, the definitions of features / terms above are not intended to be limited to any particular aspect or embodiment of the present invention, but rather apply equally to all aspects and embodiments described herein.

[0092] The following abbreviations shall be understood in the context of the present specification as follows: CSF (cerebrospinal fluid); LBD (dementia with Lewy bodies); FTD (frontotemporal dementia); VaD (vascular dementia); ALS (amyotrophic lateral sclerosis); CJD (Creutzhert-Jakob disease); CNS (central nervous system); TMT® (Tandem Mass Tag®); TEAB (tetraethylammonium bicarbonate); TFA (trifluoroacetic acid); SDS (sodium dodecyl sulfate); TCEP (tris(2-carboxyethyl)phosphine); ACN (acetonitrile); Da (Daltons); HPLC (high performance liquid chromatography); FA (formic acid); LC-MS / MS (liquid chromatography with tandem mass spectrometry detection); MS (mass spectrometry); MS / MS or MS2 (tandem MS); MS / MS / MS or MS3 (triple MS); PAGE (polyacrylamide gel electrophoresis); SCX (strong cation exchange); ppm (parts per million); TiO2 (titanium dioxide); IMAC (iron metal affinity chromatography).

[0093] In AD pathogenesis, the accumulation of amyloid-β peptide (Aβ) interacts with signaling pathways that regulate the phosphorylation of tau. Hyperphosphorylation of tau disrupts its normal function in regulating axonal transport, leading to the accumulation of neurofibrillary tangles and toxic species of soluble tau. Currently, there is no cure for AD.

[0094] For AD, there are few approved treatments and limited effectiveness, most of which serve to slow or delay progression. The identification and development of new therapies for the treatment of AD and other tauopathies is heavily affected by the lack of effective diagnostic, prognostic, and predictive biomarkers, as well as the lack of new targets for the design of new therapies. Currently, AD can only be definitively diagnosed by brain biopsy or at autopsy after the patient has died. Obviously, in clinical settings, brain biopsies are rarely performed, and diagnosis is still mainly based on the history of symptoms and relies on neurological, psychometric, and biochemical tests.

[0095] These latter tests include evaluation of ApoE e4 allele status and evaluation of amyloid beta, tau, and phosphorylated tau in cerebrospinal fluid. Nevertheless, these current methods are still not satisfactory not only for early diagnosis of AD and other tauopathies, but also for predicting the progression of neurological diseases, which is important for recruiting patients into clinical trials, designing new treatments, and predicting the effectiveness of current and new treatments. Thus, the present disclosure provides new blood-based assays and methods that can be used to diagnose AD and other tauopathies, as well as to monitor or predict the progression of these diseases (e.g., to guide treatment decisions).

[0096] An ideal diagnostic biomarker should have high specificity for disease versus non-disease and high sensitivity for distinguishing between types and stages of disease. Prognostic biomarkers should reflect the intensity and severity of pathological changes and predict their future course from the very early stages of disease before degeneration is observed to the advanced stages of disease. Pharmacodynamic biomarkers should provide a reliable indication of whether an administered treatment is effective based on changes in the levels of disease-related proteins in easily accessible body fluids such as blood, blood products including platelets, serum, and most preferably, plasma and CSF. It is also desirable that such pharmacodynamic biomarkers can provide guidance to clinicians when to stop treatment or switch to a different treatment.

[0097] New targets must be effective, safe, meet clinical and commercial needs, and above all be "druggable". A "druggable" target is accessible to a putative drug molecule, whether a small molecule or a larger biological molecule, and when bound, elicits a biological response that can be measured both in vitro and in vivo, i.e., its inhibition or activation provides a therapeutic effect in a pathological condition. Thus, there remains a need for proteins and / or peptides that can function with good sensitivity and / or specificity as biomarkers in the diagnosis, staging, prognostic monitoring, and evaluation of treatment efficacy in patients with Alzheimer's disease and other tauopathies, and serve as new targets for the development of new therapeutics.

[0098] Filamin A (FLNA) is a ubiquitously expressed actin-binding protein that regulates cell morphology and is particularly expressed in structured cells such as neurons, although expression levels are usually higher in other organs such as lung, kidney, and muscle (Human Protein Atlas). FLNA can be phosphorylated at many serine, threonine, and tyrosine residues in vivo, and the parent protein of about 280 kDa is processed to form fragments of about 110 kDa and 90 kDa. It has previously been shown that all three major isoforms of FLNA can be phosphorylated at serine 2152, and that the relative intensity of staining in Western blots using a pS2152-specific antibody can distinguish Alzheimer's disease (AD) patients from cognitively normal controls.

[0099] Modification of FLNA by proteolytic cleavage and phosphorylation is believed to promote structural and / or conformational changes of the protein in the brain. Other disease-specific factors yet to be identified may also alter the conformation of FLNA. FLNA with altered conformation is believed to play a role in the toxic signaling mechanism of Aβ oligomers. In Alzheimer's disease patients, altered FLNA interacts independently with both α7-nicotinic acetylcholine receptor (α7nAChR) and toll-like receptor 4 (TLR4). Signaling through α7nAChR has been reported to hyperphosphorylate tau, and signaling through TLR4 has been reported to induce neuroinflammation.

[0100] Furthermore, FLNA is a regulator of the actin cytoskeleton that is important for synaptic function, suggesting another mechanism by which FLNA may contribute to cognitive dysfunction. Altered FLNA is therefore a promising target for new AD therapeutics and is a target of simufilam (PTI-125), a small molecule structural modulator that reduces or prevents the pathological effects of altered FLNA.

[0101] The use of pS2152-specific antibodies for the diagnosis of AD has not yet been validated. In part, this may be due to the antibody's potential to bind to unphosphorylated protein, or it may reflect a lack of consistency in phosphorylation in the human population. In addition to the use of pS2152 antibodies in Western blots, where individual isoforms can be easily distinguished based on their molecular weight, the ability to distinguish FLNA isoforms (where the pS2152 epitope is present in each isoform) in liquid-phase assays such as ELISA remains a challenge. Furthermore, the widespread expression of FLNA, particularly that found in platelets, renders measurements in peripheral fluids prone to potential false positives. To date, it has not been shown that measurements of specific FLNA isoforms or phosphorylation can be reliably used to diagnose AD.

[0102] Considering the limitations of antibody-based FLNA profiling, the present disclosure provides a more specific bottom-up mass spectrometry method for profiling the distribution of FLNA-derived tryptic peptides in patient plasma, particularly focusing on the measurement of specific phosphorylation events.Specifically, in some embodiments, the present disclosure provides an assay using the TMTcalibrator™ method (US Pat. No. 10,976,321; [Russel et al., Rapid Commun. Mass Spectrom. 31:153-159 (2017)]), in which an isobaric labeled digest of AD brain tissue lysate is mixed with a similarly labeled plasma sample, and the brain lysate digest is present at various concentrations, the sum of which is in excess compared to the total concentration of digest from the plasma channel.By this arrangement, the high concentration of disease-related proteins in brain lysate acts as a booster for the same proteins present in plasma, even though the concentration is usually below that required for bottom-up mass spectrometry. Using this approach, the ability to measure FLNA-derived peptides is greatly improved and allows the analysis of a much broader set of proteins commonly expressed in AD brain and plasma.

[0103] Table 1. Biomarkers in Group A (significantly regulated proteins) [Table 1] TIFF2024529417000018.tif245170

[0104] Table 2. Biomarkers in Group B (significantly regulated peptides) [Table 2] TIFF2024529417000020.tif249170TIFF2024529417000021.tif248170

[0105] Table 3. Biomarkers for Group C (additional phosphopeptides) [Table 3] TIFF2024529417000023.tif248170 Table 4A. Filamin A peptides detected by TMTcalibrator™ mass spectrometry [Table 4] TIFF2024529417000025.tif247170TIFF2024529417000026.tif248170TIFF2024529417000027.tif245170

[0106] Table 4B. Filamin A peptides detected by TMTcalibrator™ mass spectrometry [Table 5] TIFF2024529417000029.tif245170TIFF2024529417000030.tif247170TIFF2024529417000031.tif247170 EXAMPLES

[0107] (Example) It should be understood that the above embodiments and the following examples are given by way of illustration and not by way of limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from this description.

[0108] Example 1: TMTcalibrator™ Analysis of Alzheimer's Disease Plasma Prepared with Histopaque®-1077 and EDTA Control Plasma

[0109] (sample)

[0110] Human plasma samples from six controls (three elderly controls, three cognitively unimpaired young subjects) were collected in Vacutainer® tubes (Becton, Dickinson and Company Franklin Lakes, NJ) containing K2EDTA. [Plasma from controls was negative (-) for the 90kDa FLNA biomarker as shown by Western blot using a phosphospecific rabbit polyclonal antibody specific for pS2152 (Origene TA313881, Origene Technologies Rockville, MD).] Within 30 minutes of blood collection, blood was centrifuged at approximately 1000×G for 15 minutes, preferably at 4-5°C. Plasma was collected within 30 minutes of centrifugation. Control plasma was then combined with 5% volume / volume 20X protease and phosphatase inhibitor cocktail, added, mixed thoroughly by vortexing for 1 minute, and dispensed. Protease-phosphate inhibitor cocktail was prepared by dissolving 1X Roche PhosStop EASYpak with 1X Roche Complete Tablets Mini EDTA-free EASYpack (Thomas Scientific, Swedesboro, NJ) in 500 ml of distilled water. Human plasma samples from six AD patients were collected in Vacutainer® tubes and transported to the laboratory for processing. Human plasma from AD patients was positive (+) for the 90 kDa FLNA biomarker as shown by Western blot using a phospho-specific rabbit polyclonal antibody specific for pS2152 (Origene TA313881, Origene Technologies Rockville, MD). In the laboratory, 4 ml of whole blood from the Vacutainer® tube was layered onto 4 ml of Histopaque®-1077 (Sigma-Aldrich) in a 14 ml disposable tube.The disposable tubes were centrifuged at 400g for 30 min at room temperature, after which the plasma was transferred to 1.5 ml Eppendorf tubes for storage, 5% vol / vol 20X protease and phosphatase inhibitor cocktail was added, mixed thoroughly by vortexing for 1 min, and aliquots were stored for use in biomarker assays. Aliquots were stored at -80°C. Aliquots of three postmortem AD brain (Braak stages IV-VI) lysates were provided by Proteome Sciences and used as trigger samples for brain-derived proteins.

[0111] (Analysis method)

[0112] For this experiment, TMTcalibrator™ phosphoproteome analysis (Figure 1) was performed using 12 AD plasma samples with AD brain tissue used as trigger sample. Plasma samples were depleted of high abundance proteins using Top14 high abundance protein removal spin columns. Proteins were digested with trypsin and peptides were labeled with TMTpro™ and mixed to generate one TMTpro™ 16-plex sample (reagents available from Thermo Fisher Scientific, Waltham, MA, USA). Four TMTpro™ channels were used for the AD brain tissue trigger.

[0113] The TMTpro™ 16-plex samples were divided into aliquots for whole proteome analysis (non-enriched) and aliquots for phosphoproteome analysis (phosphopeptide enriched). After phosphopeptide enrichment, six phosphopeptide enriched and six non-enriched fractions were generated. Each fraction was subjected to LC-MS2 analysis using a high-performance Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA.) using a data-dependent acquisition method (using an inclusion list of FLNA peptides for the non-enriched fractions). The raw data were searched using Proteome Discoverer™ v2.5 (Thermo Fisher Scientific). The data were further processed using a proprietary bioinformatics pipeline including filtering, normalization, biostatistics, annotation, and functional analysis. Box plots of proteins of interest were also generated.

[0114] (Sample Management)

[0115] Upon receipt, all samples were visually inspected to assess for unthawed, correct labelling and general integrity. Samples were stored at -80°C. Details were entered into the Laboratory Information Management System (LIMS) under a unique reference number.

[0116] (Brain Melt)

[0117] Available protein from pooled AD brain tissue lysates was used as a calibrator / trigger brain sample.

[0118] (Protein concentration measurement and SDS-PAGE)

[0119] Protein concentrations of plasma, depleted plasma, and brain lysate samples were determined by Bradford protein assay, and each sample was visualized by Coomassie-stained (Imperial Stain, Pierce, Thermo Fisher Scientific) SDS-PAGE 4-20% gradient gels (Criterion, Biorad).

[0120] (protein removal)

[0121] Thirty-five microliters of plasma were removed using HighSelect Top14 High Abundance Protein Depletion (Pierce, Thermo Fisher Scientific) according to the manufacturer's protocol. For some samples, multiple aliquots of 35 μl were removed to obtain sufficient volumes (Table S2).

[0122] (Digestion and TMTpro labeling)

[0123] Calibrator / trigger brain sample: 6.6 mg of pooled brain lysate was reduced (dithiothreitol), alkylated (iodoacetamide), digested (trypsin) to generate peptides, desalted (SepPak® tC18 cartridge, Waters, Milford, MA, USA), aliquoted into four portions reflecting ratios of 1:4:6:10, respectively, and lyophilized.

[0124] Cleared plasma samples for analysis: 180 μg was used per individual cleared plasma sample. Cleared plasma samples were made to equal volumes. Samples were reduced, alkylated, and digested with trypsin to generate peptides, and after desalting (SepPak® tC18 cartridges), samples were lyophilized to dryness.

[0125] Dried peptides (from cleared plasma and brain samples) were dissolved in TEAB / ACN buffer. Peptides were mixed with their respective TMTpro™ reagents (labeling scheme shown in Table 2). TMTpro™ labeled samples were treated with hydroxylamine and the labeled digests were pooled to generate a TMTpro™ 16-plex sample containing 12 cleared plasma samples (12 x 180 μg = 2160 μg) + pooled brain digest mixture (approximately 3780 μg in four channels at a ratio of 1:4:6:10) at a protein mass ratio of 1:1.75. 50 μg of the mixture was purified by solid phase extraction and used to evaluate labeling efficiency and reporter ion distribution (to confirm equimolarity). Two portions of 100 μg each were taken as samples for basic reverse fractionation of the non-enriched population (including a back-up sample) and an approximately 5,690 μg portion was taken for phosphopeptide enrichment.

[0126] (phosphopeptide enrichment)

[0127] Approximately 5,690 μg of the TMTpro™ 16-plex sample was enriched for phosphopeptides using two columns of the High Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific Pierce, Cat. No. A32992) according to the manufacturer's instructions and combined to obtain one pooled phosphopeptide sample.

[0128] (Basic reversed phase fraction (bRP))

[0129] bRP fractionation was performed on A) 100 μg of unenriched sample and B) TMTpro™ 16plex requiring enriched phosphopeptides using the Thermo Scientific™ Pierce™ High pH Reverse Phase Peptide Fractionation Kit (Cat. No. 84868) according to the manufacturer's instructions.

[0130] (Liquid Chromatography Mass Spectrometry (LC-MS / MS))

[0131] Each of the individual fractions (six phosphopeptide-enriched and six non-enriched fractions per 16plex) was analyzed by LC-MS / MS using an EASYnLC-1000 system (both from Thermo Scientific) connected to an Orbitrap Fusion™ Tribrid™ mass spectrometer. Peptides were resuspended in 2% acetonitrile (ACN) containing 0.1% formic acid (FA). From a thermostatically controlled autosampler, samples (approximately 5% of the non-enriched fraction and approximately 25% of the phosphopeptide-enriched fraction) were loaded onto a 2 cm, 75 μm internal diameter (ID) C18 Acclaim PepMap 100 capture column (Thermo Scientific, PN 164946) and separated using an increasing gradient of ACN in 0.1% FA through a 50 cm, 75 μm ID EasySpray analytical column (Thermo Scientific, PN ES803A) at a flow rate of 200 nL / min. The gradient starting conditions were 8% ACN for the non-enriched fraction and 10% ACN for the phosphorylation-enriched fraction. The percentage of organic solvent was successively increased up to 30% ACN.

[0132] Peptide mass spectra were acquired throughout the entire chromatographic run (180 min). The mass spectrometer was operated in data-dependent mode with a full scan at a resolution of 120.000 and MS2 fragment scans acquired at a resolution of 50.000. The duty cycle was set to 3 s, i.e. a full scan was acquired at least every 3 s, followed by an MS2 scan of a fragmented precursor selected from the most abundant target. After being fragmented once, the precursor was excluded from further fragmentation for 30 s.

[0133] (Computer-assisted mass spectrometry)

[0134] A total of 12 individual mass spectrometry raw data files (12 fractions - 6 enriched and 6 non-enriched) were searched in Proteome Discoverer (PD) v2.5 (Thermo Scientific) using the SEQUEST HT search algorithm. Raw spectra were searched against the uniprot reviewed human database (January 2021 version) with full tryptic specificity and up to two missed cleavages. All spectra that did not match the peptide with high confidence were searched again with semi-tryptic specificity to take into account potential cleavage products. Spectra that did not match after the semi-tryptic search were searched again without cleavage specificity. TMTpro™ modifications of N-terminus and lysine residues, as well as carbamidomethylation of cysteine ​​were set as static modifications; oxidation of methionine was considered as a variable modification. Phosphorylation on serine, threonine, and tyrosine residues was set as a variable modification in the search of phosphopeptide enriched fractions only.

[0135] The precursor mass tolerance was set to 20 ppm and the fragment mass tolerance was set to 0.02 Da. The false discovery rate was controlled at 1% at the PSM level by the Percolator node built into Proteome Discoverer. A reporter ion limiter node was set to extract raw intensity values ​​of TMTpro™ 16plex monoisotopic ions (126, 127N, 127C, 128N, 128C, 129N, 129C, 130N, 130C, 131N, 131C, 132N, 132C, 133N, 133C, 134N). All raw reporter ion intensity values ​​were exported to a tab-delimited text file for further processing and bioinformatics analysis.

[0136] (Bioinformatics Data Analysis)

[0137] Statistical analyses were performed using in-house developed software written in the R statistical programming language (R Core Team 2017). All data integration tools were developed to work with TMT®-labeled MS data and included functionality to address isolation interference [Savitski, M. et al., Measuring and managing ratio compression for accurate iTRAQ / TMT quantification. J. Proteome Research 12.8(2013):3586-3598], isotopic crosstalk [Rauniyar, N. et al., Isobaric labeling-based relative quantification in shotgun proteomics. J. Proteome Research 13.12(2014):5293-5309], PSM normalization, and summarization to peptides.

[0138] (statistical analysis)

[0139] Statistical analyses were performed using in-house software and were based on linear modeling [T., Tibshirani et al., The Elements of Statistical Learning: Data Mining, Inference, and Prediction, Vol. 2, pp. 1-758. New York: Springer (2009)]. For feature selection, we choose a relaxed t-statistic (Ritchie, M. et al., "Limma Powers Differential Expression Analyses for RNA-sequencing and Microarray Studies." Nucleic Acids Research 43.7(2015): e47-e47; Phipson, B. et al., "Robust Hyperparameter Estimation Protects Against Hypervariable Genes and Improves Power to Detect Differential Expression." Annals of Applied Statistics 10.2(2016): 946.) which effectively borrows information from the ensemble of features to help infer about each individual feature, and is useful for small datasets.

[0140] Analyses were performed for three different contrasts: Contrast 1 = AD vs. controls (older); Contrast 2 = AD vs. controls (young); Contrast 3 = AD vs. controls (all). For each contrast, fold changes, p-values, and adjusted p-values ​​(calculated using Benjamini-Hochberg FDR) are provided. LIMMA p-values ​​are based on a moderated t-statistic (Ritchie, Phipson) and are the result of borrowing information across all features to overcome the problem of small sample sizes. Multiple testing correction was applied using the Benjamini-Hochberg method.

[0141] (Sample Evaluation)

[0142] Protein determination by modified Bradford assay showed that plasma samples had protein concentrations ranging from 41.9 to 73.6 μg / μl (mean: approximately 59 μg / μl). AD samples showed a lower mean protein concentration compared to controls (48.7 vs. 68.5 μg / μl). SDS-PAGE of all plasma samples showed a very homogenous banding pattern. Plasma samples showed protein concentrations in the expected range, raising no concerns regarding sample quality. Lysis of brain tissue pieces provided sufficient protein for the study. SDS-PAGE of the three brain lysates showed acceptable banding patterns.

[0143] (plasma removal)

[0144] After depletion of the top 14 of 12 plasma samples, the total available protein ranged from 189 to 354 μg. SDS-PAGE of the top 14-depleted plasma samples showed a homogenous banding pattern. Visual evaluation confirmed the typical post-depletion pattern, e.g., albumin bands were clearly reduced. Approximately 95% depletion was achieved.

[0145] (Quality Control for TMTpro™ Analysis)

[0146] Analysis of the TMTpro™ labeling reaction efficiency of the TMTpro™ 16plex experiment showed that ∼98.2% of the N-terminal amino groups were labeled, indicating that labeling was essentially complete. All mass spectrometry runs of fractionated samples passed an internal quality assessment based on the maximum intensity of the total ion chromatogram (TIC), the number of MS scans, and the number of peptide spectral matches (PSMs).

[0147] (Method: Quantified peptides and proteins)

[0148] We report 39,686 peptide sequences with expression values ​​across all 12 patient-derived samples (Table 3). Of these, 9,337 are phosphorylated and associated with 6,047 distinct and well-localized phosphorylation site combinations. The 39,686 peptides are associated with 4,192 distinct protein groups quantified across all 12 samples. Overall, application of the TMTcalibrator™ phosphoproteome workflow to human plasma provided excellent coverage of the plasma proteome and phosphoproteome.

[0149] Table 5. Number of features detected and quantified [Table 6]

[0150] In Table 5, peptide rows represent both non-phosphorylated and phosphorylated peptides, the latter of which are listed in the following row (italics). Note that quantified features allow for a certain number of missing values ​​that are imputed during data preprocessing.

[0151] (Feature Selection: Phosphopeptide)

[0152] We found a strong upregulation of phosphopeptides in the AD group compared to controls, with many phosphopeptides with expression changes of more than six-fold.No significantly downregulated phosphopeptides were detected at the applied thresholds.

[0153] The most highly regulated phosphorylated peptide was a peptide derived from junctional adhesion molecule A (F11R, also known as JAM-1), containing a modification on serine 284. [ka] The next most highly regulated phosphopeptide was from caveolae-associated protein 2 (CAVIN2, also known as SDPR), modified on serine 241 (logFC: 6.6, contrast #1). [ka] (logFC: 6.5, contrast #1). The third most highly regulated phosphopeptide was [ka] (logFC: 6.4, contrast #1)--a sequence shared between filamin A (FLNA) and filamin B (FLNB) and modified at serine 2143 (FLNA) and serine 2098 (FLNB).

[0154] Looking at the profile of the four quantified FLNA phosphopeptides (Figure 7), phosphorylated FLNA appears to be more abundant in AD compared to the overall control population. However, there are some expression differences between young and aged controls. The "RAPSVAN" peptide, which contains pS2152, is found at similar levels in AD and young controls, but is significantly lower in cognitively healthy aged controls. Conversely, the "CSGPG" peptide, which contains pS1459, is higher in AD and aged controls, and significantly lower in young controls.

[0155] We also performed post-hoc analyses to determine whether the selected phosphopeptides could explain the differences between the experimental classes by the significance of their expression changes (Figure 8). For all comparisons, the selected phosphopeptides showed good discrimination between the AD and control groups, with the expression differences between the groups accounting for approximately 95% (PC1) of the total variance in the data set. Based on these results, this peptide set was not associated with significant differences between the two control groups (PC2<2%). These results are reflected in the heatmap, in which the control groups were intermingled on the sample clustering dendrogram (Figure 4), while the AD group formed a distinct subcluster with significantly higher expression values.

[0156] (Feature Selection: Protein)

[0157] All contrasts revealed strong upregulation of proteins in disease compared to controls, with many proteins exhibiting expression changes of more than four-fold. At the applied threshold, only two significantly downregulated proteins were detected.

[0158] Across all three contrasts, we observed a significant degree of consistency between proteins, both in their identity and the magnitude of fold change. The most highly regulated protein was bridging integrator 2 (BIN2) (logFC: 4.5, contrast #1). The second most highly regulated protein was integrin alpha-IIb (ITGA2B) (logFC: 4.5, contrast #1). The third most highly regulated protein was protein S100-A12 (S100A12) (logFC: 3.6, contrast #1). Of the three previously described highly regulated phosphopeptides, the corresponding total protein expression (FLNA and CAVIN2) was associated with significantly increased levels in disease samples (FLNA logFC: 1.8, contrast #1; CAVIN2 logFC: 1.9, contrast #1).

[0159] Taking a closer look at the observed expression profile between the three sample groups for the protein of interest, FLNA (Figure 2), the same trend was revealed at the protein level as previously observed at the phosphopeptide level: FLNA expression was higher in the AD group compared to controls. Comparing the two control groups by themselves showed similar expression, although the mean value of the young group without cognitive impairment was slightly higher compared to the elderly control group.

[0160] (FLNA Coverage Summary)

[0161] Filamin A (FLNA) was successfully detected at the protein and phosphopeptide levels and was found to be significantly regulated in AD patients compared to old and young controls. The protein itself was quantified by a standard search of tryptic peptides with 85 PSMs from 46 peptides excluding phosphopeptides (Table 6). Of the five phosphopeptides detected, four could be quantified in all individuals (see, for example, Table 7). Phosphopeptides [ka] did not provide a sufficient number of data points for the control sample because the signal was below the detectable range. However, plotting the reporter ion intensity values ​​of each peptide spectral match shows that this peptide was successfully detected in the AD sample, in contrast to the control, and that the peptide covering the same phosphorylation site of the protein (S2152) was also detected. [ka] It becomes clear that this is consistent with the data generated for (Figure 9).

[0162] Table 6. Quantified peptides of FLNA (non-phosphorylated) [Table 7]

[0163] Table 7. Phosphopeptides of target protein FLNA [Table 8]

[0164] Overview of phosphopeptides where phosphorylation sites of the target protein FLNA were determined. The positions of ambiguously located sites with confidence scores below a set threshold (75%) are indicated with the letter "?". Although the strong regulation of FLNA peptides is optimistic, there is a risk that signals may be caused by post-collection artifacts, e.g., platelet activation, since AD ​​and control plasma samples are prepared in different ways. To investigate this and provide an expanded panel of biomarkers to control artifact signals, we performed further experiments as shown in Examples 2 and 3.

[0165] Example 2: Analysis of changes in FLNA, phosphorylated FLNA, and other proteins in Histopaque® 1077 plasma samples

[0166] In this example, we used fresh plasma samples from AD and control cohorts prepared by the same process believed to cause platelet activation. Again, six samples from controls (three elderly controls, three young people without cognitive impairment) (FLNA band negative or "-") and six AD patients (FLNA band positive or "+") were analyzed using TMTcalibrator™ with the same AD brain triggers as described in Example 1. Whole blood from AD and control cohorts was collected and processed using the Histopaque® 1077 process described above.

[0167] (Sample Processing)

[0168] After thawing the plasma samples, all processing steps were performed as described in Example 1. Mass spectrometry was performed using an inclusive list of all FLNA peptides detected in Example 1.

[0169] (result)

[0170] Using a dedicated filamin A inclusion scheme during MS acquisition, we obtained good coverage (~40%) of the filamin A sequence. No significant changes in abundance were observed for the protein as a whole, but specific parts of the sequence showed various changes in abundance across different groups. We also observed only subtle differences in the expression levels of two of the quantified phosphorylation sites.

[0171] In addition to FLNA, we quantified a total of 29,786 peptides and 3,297 phosphorylation sites from 3,493 proteins, providing a useful resource for identifying plasma markers dependent on Alzheimer's disease pathology. Statistical analysis demonstrated the presence of a panel of phosphopeptide and protein features that facilitated separation of experimental classes and were potential biomarkers independent of platelet activation status. Functional enrichment indicated changes in cytoskeleton organization, complement cascade regulation, and lipoprotein metabolism.

[0172] (FLNA Coverage)

[0173] FLNA was identified in 220 PSMs for 77 (phosphorylated) peptides and quantified in a standard search for tryptic and semi-tryptic peptides by 158 PSMs from 54 peptides excluding phosphopeptides (Table 4). All four detected phosphopeptides could be quantified across all individuals (Table 5 contains statistical data). Two peptides were identified only in the control sample: [ka] (aa 8-24, SEQ ID NO: xx) was identified in 4 of 6 controls (3 young and 1 elderly); and [ka] (SEQ ID NO: vv) was identified in only one young control. [ka] (aa 1636-1644; sequence number CC) was identified only in Alzheimer's disease samples, with values ​​obtained for 5 of 6 patients.

[0174] Surprisingly, the level of platelet activation is likely to be similar between AD and control samples by using Histopaque® 1077 preparation, but the increase previously observed in AD is lost.In fact, the level of free plasma FLNA is lower in AD samples, suggesting that AD platelets are less activated than platelets from cognitively healthy controls.The inventors believe that this loss of regulation is due to the significant release of FLNA in the control plasma samples of this study.

[0175] Table 8. Quantification of FLNA in Histopaque® 1077 plasma samples [Table 9]

[0176] We also identified four phosphorylated FLNA peptides, three of which were seen in Example 1 and one unique to the present analysis. Unlike the previous study, the levels of the two peptides containing pS2152 did not show any modulation between the AD and control groups, but there was a trend towards a higher expression of the "RAPSVAN" peptide in AD and aged controls, suggesting that this, at least peripherally, may be related to the disease rather than to the processing of the samples. Conversely, the peptide "CSGPG" containing pS1459 showed a strong upregulation in healthy elderly subjects, which may indicate a protective post-translational modification. Interestingly, it was also elevated in the AD group, which was seen in the previous study but not in this one, which may suggest that this site is rather produced artifactually and requires further evaluation. The threonine phosphorylation site at residue T2336 showed a similar behavior to the previously described serine phosphorylation: the levels detected in aged controls were higher compared to both Alzheimer's disease patients and young healthy controls.

[0177] Table 9. Quantified phosphopeptides derived from FLNA in Histopaque® 1077 plasma [Table 10]

[0178] Example 3: TMTcalibrator™ Analysis of AD and Control EDTA Plasma Samples

[0179] The results of Example 2 provide evidence that some modulation of protein expression can be promoted by sample preparation methods. Therefore, we completed the study by analyzing AD fresh samples and control plasma samples prepared from EDTA blood collection tubes under all identical conditions. All other conditions are as described in Examples 1 and 2. We expected that the level of platelet activation would be lowest in this series of samples, which would better reflect standard clinical practice.

[0180] (result)

[0181] FLNA was successfully detected at the protein and phosphopeptide levels. We detected 60 unmodified peptides associated with FLNA, 54 of which were unique to FLNA, and 42 could be quantified in all 12 samples. The protein was found to be significantly upregulated (approximately 2-fold) in AD samples compared to healthy controls. Three of the four detected phosphopeptides associated with FLNA were used for quantification. These peptides carry modifications at phosphorylation sites pS1459, pS2143, and pS2152. All three phosphopeptide signatures were more abundant in the AD group, as seen in Example 1, but the degree of regulation was less intense here. We hypothesize that this reflects a true biological difference in FLNA abundance in Alzheimer's disease patients. When platelet activation is prominent, the amount of FLNA released is sufficient to mimic that seen in the disease and even reverse the relative abundance signature between healthy and AD patients.

[0182] Table 10. Relative expression of phosphorylated FLNA peptides in EDTA plasma [Table 11]

[0183] Table 11. Relative expression of non-phosphorylated FLNA in EDTA plasma. [Table 12]

[0184] In addition to FLNA, we quantified a total of 31,382 peptides, 7,677 phosphopeptides, and 3,478 proteins. Statistical analysis revealed multiple significantly regulated phosphopeptide and protein signatures that facilitated separation of experimental classes. Functional enrichment indicated changes in platelet activation, cell-extracellular matrix interactions, and cell junction organization.

[0185] We obtained a good characterization of filamin A in AD plasma and control K2EDTA plasma. In contrast to the results obtained with plasma prepared by the Histopaque® 1077 method, quantitative differences in the abundance of filamin A and several other protein features were detected in the present study. These results were comparable to the original study (40-224), although the observed differences were less pronounced.

[0186] The present inventors have found various tryptic peptides associated with FLNA that are well regulated between AD and control groups.The present inventors believe that these results can serve as a good basis for developing targeted mass spectrometry of FLNA with diagnostic and prognostic utility in the context of selecting and monitoring patients for treatment with simufilam.

[0187] Example 4: Comparison of protein expression in AD and cognitively normal individuals across three different sample preparation methods

[0188] We compared the relative expression of various proteins, including FLNA and phosphorylated peptides of FLNA, in the various plasma groups: AD Histopaque® 1077 vs. control EDTA, AD Histopaque® 1077 vs. control Histopaque® 1077, and AD EDTA vs. control EDTA. This confirmed the suitability of FLNA, and in particular the phosphorylated epitope of FLNA, to serve as a peripheral biomarker for Alzheimer's disease. However, it is clear that potential activation of platelets using non-standard plasma preparations could potentially mimic the elevated FLNA levels seen in AD, and therefore we searched for peptides and proteins whose levels are elevated in Histopaque® 1077 plasma but not detected in EDTA plasma. We also focused on a specific subset of such proteins that have been reported to be associated with platelet biology.

[0189] The relative expression of FLNA is clearly affected by the use of Histopaque® 1077 for plasma preparation. In general, we find that the relative level of phosphorylated FLNA is particularly increased in cognitively healthy individuals compared to AD patients (Table 12). In Example 1, Histopaque® 1077 is used to prepare AD plasma, whereas the control is prepared with EDTA. All three phosphorylated FLNA peptides detected in Example 1 are found to be increased in the AD group, but two of them show reduced levels in the AD group compared to the control group, both when prepared by Histopaque® 1077. For FLNA pS1459, the degree of regulation in the AD group compared to the control group is found to be further expanded in the EDTA-prepared plasma samples tested in Example 3.

[0190] Table 12. Relative expression of FLNA phosphopeptides in plasma from AD patients compared to controls for Examples 1-3 [Table 13]

[0191] The inventors also identified three peptides that can be used to assess the level of artifactual platelet activation during preparation of plasma or serum. These peptides are derived from proteins with annotations to a role in platelet biology, and were differentially expressed in AD and control groups when Histopaque® 1077 was used only in AD samples, were not modulated when Histopaque® 1077 was used for both cohorts, and were not detected in either group when EDTA was used to prepare plasma. In this regard, inclusion of these peptides or their respective proteins in the method of the invention can be used to indicate samples in which platelet activation has occurred, and fresh samples should be tested where the absence of these peptides / proteins indicates a good sample.

[0192] Table 13. Peptides that indicate excessive platelet activation [Table 14]

[0193] Throughout this specification, various patents, patent applications, and / or other types of publications (e.g., journal articles and books) are referenced. The disclosures of all patents, patent applications, and publications cited herein are incorporated by reference in their entirety for all purposes.

Claims

1. A biomarker panel comprising one or more phosphorylated peptides obtained from in vitro digestion of filamin A having the polypeptide sequence of SEQ ID NO:

1.

2. 2. The biomarker panel of claim 1, wherein the filamin A is contained in or obtained from a biological fluid or tissue sample taken from a subject suspected of having a neurological disease.

3. 3. The biomarker panel of claim 2, wherein the neurological disease is Alzheimer's disease.

4. 2. The biomarker panel of claim 1, wherein said phosphopeptide is phosphorylated at a residue corresponding to serine 2152 of SEQ ID NO:

1.

5. 2. The biomarker panel of claim 1, wherein the phosphorylated peptide has the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

5.

6. The biomarker panel of claim 1, wherein the biomarker panel comprises multiple phosphorylated fragments of FLNA, optionally two or more fragments or three or more fragments.

7. The biomarker panel of claim 1, wherein the biomarker panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different phosphorylated fragments of FLNA.

8. The biomarker panel of claim 1, wherein the biomarker panel comprises two or more phosphopeptides, each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs: 2 to 5, wherein at least one of the two or more phosphopeptides is phosphorylated at a position corresponding to serine 2152 of SEQ ID NO:

1.

9. 2. The biomarker panel of claim 1, wherein said phosphopeptide is phosphorylated at a position corresponding to serine 2143 of SEQ ID NO:

1.

10. 2. The biomarker panel of claim 1, wherein the phosphorylated peptide has the amino acid sequence of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

11. The biomarker panel of claim 1, wherein the biomarker panel comprises two or more phosphopeptides, each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs: 6 to 9, wherein at least one of the two or more phosphopeptides is phosphorylated at a position corresponding to serine 2143 of SEQ ID NO:

1.

12. 2. The biomarker panel of claim 1, wherein said phosphopeptide is phosphorylated at a position corresponding to serine 2180 of SEQ ID NO:

1.

13. 2. The biomarker panel of claim 1, wherein the phosphorylated peptide has the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

14. 2. The biomarker panel of claim 1, wherein the biomarker panel comprises two or more phosphopeptides, each comprising a fragment of FLNA and having the amino acid sequence of any one of SEQ ID NOs: 10-12, wherein at least one of the two or more phosphopeptides is phosphorylated at a position corresponding to serine 2180 in SEQ ID NO:

1.

15. A biomarker panel comprising a plurality of phosphopeptides, wherein each phosphopeptide is a fragment of SEQ ID NO:1 and comprises a phosphorylation site at a position corresponding to serine 2143, 2152, and / or 2180 of SEQ ID NO:

1.

16. A biomarker panel comprising a plurality of phosphopeptides, each having the sequence of SEQ ID NOs: 2-12, and being phosphorylated at positions corresponding to serine 2143, 2152, and / or 2180 of SEQ ID NO:

1.

17. 16. The biomarker panel of claim 15, wherein the one or more phosphopeptides are phosphorylated at two or more of serine 2143, serine 2152, and / or serine 2180 of SEQ ID NO:

1.

18. 17. The biomarker panel of claim 16, wherein the one or more phosphopeptides are phosphorylated at two or more of serine 2143, serine 2152, and / or serine 2180 of SEQ ID NO:

1.

19. A biomarker panel comprising at least two, three, four, five, six, seven, eight, nine, or ten different phosphopeptides, each phosphorylated at a site corresponding to serine residues 2143, 2152, and / or 2180 of SEQ ID NO:

1.

20. 20. The biomarker panel of any one of claims 1-19, further comprising one or more peptides listed in Table 4.

21. 21. The biomarker panel of claim 20, wherein the one or more peptides are obtained from in vitro digestion of filamin A using a protease or a combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO:

1.

22. The biomarker panel of any one of claims 1 to 19, wherein said one or more phosphorylated peptides are proteotypic for FLNA.

23. A biomarker panel comprising: (i) one or more peptides obtained from in vitro digestion of filamin A with a protease or a combination of proteases, wherein each of the one or more peptides comprises an amino acid sequence present in SEQ ID NO: 1; and (ii) one or more peptides obtained from in vitro digestion of one or more, optionally two or more, proteins listed in Tables 1-4.

24. 20. The biomarker panel of any one of claims 1-19, wherein said one or more peptides or said one or more phosphorylated peptides are obtained from in vitro digestion using one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin.

25. 20. The biomarker panel of any one of claims 1-19, comprising one or more synthetic peptides, wherein one or more of said synthetic peptides are enriched with heavy isotopes of H, C, N, O, and / or S.

26. 26. The biomarker panel of Claim 25, wherein one or more of said synthetic peptides comprise an amino acid sequence corresponding to the sequence of a peptide obtained from in vitro digestion of SEQ ID NO: 1 or one of the proteins listed in Tables 1, 2, 3, or 4 with one or a combination of proteases selected from trypsin, GluC, ArgC, AspN, and / or chymotrypsin.

27. 1. A method for obtaining data for identifying / prognosing a neurological disorder, comprising: Removing a bodily fluid or tissue sample from the subject; digesting one or more proteins in the body fluid or tissue sample with one or more proteases; and detecting and / or measuring the level of one or more peptides produced from said digestion using mass spectrometry.

28. 28. The method of claim 27, wherein the subject is suspected of having Alzheimer's disease.

29. 28. The method of claim 27, wherein the digesting step is carried out with trypsin, GluC, ArgC, AspN, chymotrypsin, or any combination thereof.

30. 28. The method of claim 27, further comprising determining a ratio of two or more of the peptides produced from said digestion.

31. 31. The method of claim 30, wherein the ratio comprises the ratio of fragments phosphorylated at a position corresponding to serine 2143 of SEQ ID NO:1 to fragments phosphorylated at a position corresponding to serine 2152 of SEQ ID NO:

1.

32. 32. The method of claim 31, further comprising determining that the subject has Alzheimer's disease if the ratio of fragments phosphorylated at a position corresponding to serine 2143 of SEQ ID NO: 1 to fragments phosphorylated at a position corresponding to serine 2152 of SEQ ID NO: 1 is greater than 10.

0.

33. 32. The method of claim 31, further comprising determining that the subject does not have Alzheimer's disease if the ratio of fragments phosphorylated at a position corresponding to serine 2143 of SEQ ID NO: 1 to fragments phosphorylated at a position corresponding to serine 2152 of SEQ ID NO: 1 is less than 5.

0.

34. 1. A method of obtaining data for monitoring the progression of a neurological disorder, comprising: (a) obtaining a body fluid or tissue sample from the subject at a first time point; (b) digesting one or more proteins in the body fluid or tissue sample with one or more proteases; (c) detecting and / or measuring the level of one or more peptides produced from the digestion using mass spectrometry; and (d) removing a bodily fluid or tissue sample from the subject at a second time point; and repeating steps (b)-(c).

35. 35. The method of claim 34, wherein the subject is suspected of having Alzheimer's disease.

36. 35. The method of claim 34, wherein said digesting step is carried out with trypsin, GluC, ArgC, AspN, chymotrypsin, or any combination thereof.

37. 35. The method of claim 34, further comprising the step (f) of determining whether a therapeutic treatment administered to the subject is effective in treating the neurological disorder based on the detected and / or measured levels of the one or more peptides.

38. 35. The method of claim 34, further comprising determining a ratio of two or more of the peptides produced from said digestion.

39. 39. The method of claim 38, wherein the ratio comprises the ratio of fragments phosphorylated at a position corresponding to serine 2143 of SEQ ID NO:1 to fragments phosphorylated at a position corresponding to serine 2152 of SEQ ID NO:

1.

40. 40. The method of claim 39, further comprising determining the subject as progressing towards Alzheimer's disease if the ratio is higher at the second time point compared to the first time point.

41. 40. The method of claim 39, further comprising determining that the treatment administered to the subject is ineffective in slowing, preventing, or treating the neurological disorder in the subject if the ratio is higher at the second time point compared to the first time point.

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