WO2017172764A1

WO2017172764A1 - Modified cell line and method of determining tauopathies

Info

Publication number: WO2017172764A1
Application number: PCT/US2017/024536
Authority: WO
Inventors: Amanda L. WOERMAN; Steven Olson; Atsushi Aoyagi; Stanley B. Prusiner; Smita Patel; Sabeen KAZMI
Original assignee: The Regents Of The University Of California; Daiichi Sankyo Company, Limited
Priority date: 2016-04-01
Filing date: 2017-03-28
Publication date: 2017-10-05

Abstract

A cell line comprised of cells stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform. The cell line is used in a variety of methods which make it possible to identify and quantify a range of tauopathies including Alzheimer's disease, chronic traumatic encephalopathy, and progressive super nuclear palsy.

Description

MODIFIED CELL LINE AND METHOD OF DETERMINING TAUOPATHIES

GOVERNMENT RIGHTS

[0001] This invention was made with government support under federal grant nos.

AG002132, AG010770, AG021601, AG031220 awarded by National Institutes of Health. The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

[0002] The invention relates to genetically engineered cell lines transfected with nucleotide sequences which enable the cell lines to be used in the detection, identification, and quantification of a range of different tauopathies including Alzheimer's disease, chronic traumatic encephalopathy, and progressive super nuclear palsy.

BACKGROUND OF THE INVENTION

[0003] In the central nervous system, the protein tau, which is highly soluble and unstructured, binds to microtubules to stabilize and promote their polymerization in neurons (Iqbal K, et al. (1986) Defective brain microtubule assembly in Alzheimer's disease. Lancet 2:421-426). However, when tau misfolds into a conformation rich in β-sheet structure, it becomes a prion, inducing additional tau misfolding and aggregation.

[0004] Tau aggregation ultimately results in the formation of a number of

pathological lesions, including neurofibrillary tangles (NFTs) (Wischik CM, et al. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. USA 85:4506-4510; Iqbal K, et al. (1989) Identification and localization of a τ peptide to paired helical filaments of Alzheimer disease. Proc. Natl. Acad. Sci. USA 86:5646-5650), Pick bodies (Love S, Saitoh T, Quijada S, Cole GM, & Terry RD (1988) Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. /.

Neuropathol. Exp. Neurol. 47:393—4-05; Murayama S, Mori H, Ihara Y, & Tomonaga M (1990) Immunocytochemical and ultrastructural studies of Pick's disease. Ann. Neurol. 27:394-405), and globose tangles (Love S, Saitoh T, Quijada S, Cole GM, & Terry RD (1988) Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. /. Neuropathol. Exp. Neurol. 47:393-405; Flament S, Delacourte A, Verny M, Hauw J-J, & Javoy-Agid F (1991) Abnormal Tau proteins in progressive supranuclear palsy: similarities and differences with the neurofibrillary degeneration of the Alzheimer type. Acta Neuropathol. 81:591-596). This progressive spreading of tau prions is responsible for a class of

neurodegenerative diseases termed tauopathies, which includes Alzheimer's disease (AD), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Pick's disease (PiD), and progressive supranuclear palsy (PSP).

The recognition that a single protein, tau, gives rise to an array of pathologies and diseases was a result of the partial purification of tau from NFTs in AD patient samples (Wischik CM, et al. (1988) Isolation of a fragment of tau derived from the core of the paired helical filament of Alzheimer disease. Proc. Natl. Acad. Sci. USA 85:4506-4510). Antibodies raised against the protein sequence demonstrated that diseases once thought to be unrelated, such as PiD and PSP, were in fact caused by the same protein (Love S, Saitoh T, Quijada S, Cole GM, & Terry RD (1988) Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. /. Neuropathol. Exp. Neurol. 47:393-405; Feany MB & Dickson DW (1996) Neurodegenerative disorders with extensive tau pathology: a comparative study and review. Ann. Neurol. 40: 139-148). This work also led to the cloning and sequencing of tau cDNA, leading to the discovery that tau is expressed as 6 different isoforms (Goedert M, Spillantini MG, Potier MC, Ulrich J, & Crowther RA (1989) Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain. EMBO J. 8:393-399; Goedert M, Spillantini MG, Cairns NJ, & Crowther RA (1992) Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. Neuron 8: 159-168). These isoforms arise from alternative splicing of mRNA transcribed from the tau gene, MAPT, and are comprised of 2 variable regions. In the N-terminal region, 0, 1, or 2 insertions (ON, IN, or 2N) arise from alternative splicing of exons 2 and 3, while in the C-terminal repeat domain (RD), exclusion or inclusion of exon 10 gives rise to either 3 repeats (3R) or 4 repeats (4R), respectively (Andreadis A, Brown WM, & Kosik KS (1992) Structure and novel exons of the human τ gene. Biochemistry 31 : 10626-10633). As such, tau isoforms can be identified by the number of inclusions present in each region (1N3R versus 2N4R, for example).

[0006] This observation provides a method for categorizing tauopathies based upon the isoforms present in the brains of patients suffering from the various diseases. For example, while the Pick bodies seen in PiD patients are typically comprised of 3R tau isoforms (Delacourte A, et al. (1996) Specific pathological Tau protein variants characterize Pick's disease. /. Neuropathol. Exp. Neurol. 55: 159-168), the globose tangles seen in PSP and the astrocytic plaques in CBD typically consist of aggregated 4R tau (Flament S, Delacourte A, Verny M, Hauw J-J, & Javoy-Agid F (1991) Abnormal Tau proteins in progressive supranuclear palsy: similarities and differences with the neurofibrillary degeneration of the Alzheimer type. Acta Neuropathol. 81:591-596; Ksiezak-Reding H, et al. (1994) Ultrastructure and biochemical composition of paired helical filaments in corticobasal degeneration. Am. J. Pathol. 145: 1496-1508; Lee VM-Y & Trojanowski JQ (1999)

Neurodegenerative tauopathies: human disease and transgenic mouse models.

Neuron 24:507-510).

[0007] Interestingly, while tau expression in the brain is usually composed of an approximately equimolar ratio of the 3R and 4R isoforms, mutations first identified in 1998 were found to affect the splicing of tau mRNA, resulting in increased expression of the 4R tau isoforms over the 3R isoforms (Hong M, et al. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282: 1914-1917; D'Souza I, et al. (1999) Missense and silent tau gene mutations cause frontotemporal dementia with parkinsonism-chromosome 17 type, by affecting multiple alternative RNA splicing regulatory elements. Proc. Natl. Acad. Sci. USA 96:5598-5603). These mutations result in dominantly inherited tauopathies, including PSP and CBD. In addition to the "pure" tauopathies comprised of either 3R or 4R tau isoforms, the mixed tauopathies, including AD and CTE, arise from aggregation of both 3R and 4R tau (Goedert M, Spillantini MG, Cairns NJ, & Crowther RA (1992) Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms. Neuron 8: 159-168;Hong M, et al. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282: 1914-1917; Schmidt ML, Zhukareva V, Newell KL, Lee VM, & Trojanowski JQ (2001) Tau isoform profile and phosphorylation state in dementia pugilistica recapitulate Alzheimer's disease. Acta Neuropathol. 101:518-524).

[0008] Research on prion protein (PrP), the first protein discovered to cause disease by protein-induced protein misfolding, shows that the distinct diseases caused by aggregation of the misfolded PrP scrapie (PrPSc; as opposed to the soluble PrPC) are attributable to differences in the prion strain (Peretz D, et al. (2001) Strain-specified relative conformational stability of the scrapie prion protein. Protein Sci.

10:854-863). For example, it is thought that Creutzfeldt- Jakob Disease (CJD) is the result of PrPSc misfolding into one conformation while Fatal Familial Insomnia (FFI) arises from misfolding into a different protein conformation. This hypothesis is supported by the observation that one mutation at codon 178 in PrP, resulting in an asparagine replacing an aspartic acid, will either cause a patient to develop CJD or FFI, depending on which polymorphism a patient carries at codon 129 (Goldfarb LG, et al. (1992) Fatal familial insomnia and familial Creutzfeldt- Jakob disease: disease phenotype determined by a DNA polymorphism. Science 258:806-808). Similarly, it is posited that the unique diseases arising from tau prions also result as differences in tau prion strains (Prusiner SB (2013) Biology and genetics of prions causing neurodegeneration. Annu. Rev. Genet. 47:601-623). However, the factors giving rise to these differences are poorly understood.

SUMMARY OF THE INVENTION

[0009] Neurodegenerative diseases caused by the accumulation of misfolded and aggregated tau are collectively described as tauopathies. In these diseases, tau prions spread throughout a patient's brain via protein-induced protein misfolding.

Historically, tauopathies are classified by the isoforms of tau found in disease. These isoforms arise by the exclusion or inclusion of exon 10, and consist of either 3 repeats (3R) or 4 repeats (4R). Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) contain only the 4R isoforms of tau, whereas Pick's disease (PiD) is characterized by the exclusive presence of 3R tau. Alternatively,

Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE) are described as mixed tauopathies due to aggregation of both 3R and 4R tau. The present invention shows that the isoforms present in each disease extend beyond a categorical description of the different tauopathies, and that they define the prion replication process. The invention includes HEK293T cells expressing different isoforms of tau fused to yellow fluorescent protein (YFP): the repeat domain of 4R tau, 3R tau, or both. After isolating tau prions from the five tauopathy samples and one control sample, we found that only the 4R tauopathies, including PSP and CBD, could infect cells expressing 4R tau alone (Tau4R(LM)-YFP and TauRD(LM)-YFP) (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288), however, only tau prions isolated from the 3R tauopathy PiD could infect the 3R tau cells (Tau3R(VM)-YFP). Infection was visualized by the formation of bright yellow foci in the cells.

Interestingly, tau prions from all diseases, including AD and CTE, infected the Tau3R4R-YFP cells. Together these results indicate that tau prion propagation is dictated by the isoforms of tau comprising each prion strain. Importantly, they also demonstrate that both AD and CTE are propagated by tau prion strains comprised of 3R and 4R tau.

[0010] The progressive nature of neurodegenerative diseases arises from the spread of prions, or misfolded proteins, in the brain. In the case of tauopathies, the protein tau, which is expressed with either 3 repeat domains (3R) or 4 repeat domains (4R), induces the misfolding of additional tau proteins. Traditional sub-categorization of these diseases is based on the isoforms of tau - 3R, 4R, or both - found in neurofibrillary tangles and other aggregates in patient brain samples. The present invention provides cell assays specific to 3R tau and 4R tau to demonstrate that 3R tau prions can only propagate in the presence of 3R tau, and 4R tau prions require 4R tau to propagate. The invention shows that Alzheimer's disease and chronic traumatic encephalopathy are caused by tau prion strains requiring both the 3R and 4R isoforms to propagate. The present invention uses the cell line described here in various methods to provide a mechanistic basis for the categorization of tauopathies.

[0011] While the practice of categorizing tauopathies as 3R, 4R, or mixed

tauopathies has largely been a descriptive exercise for the last 25 years, we conceptualized that tau prions giving rise to each tauopathy are strain specific, and that these strains are dictated by the isoforms of tau involved. Using previously described human embryonic kidney (HEK293) cells expressing the RD of 4R tau with the two familial mutations P301L and V337M (TauRD(LM)-YFP) (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288) to quantify tau prions isolated from PSP patient samples (Woerman AL, et al. (2015) Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A.

112:E4949-E4958).

[0012] To examine the role of the repeat domain in tau prion strains, we developed a panel of cell lines expressing several variations of tau isoform fusion proteins: the RD of 3R tau, the RD of 4R tau, and the combination of both RDs. Specific cell lines included HEK293T cell lines. Using tau prions isolated from several tauopathies, we found that only 4R tau prions, which were isolated from CBD and PSP patient samples, infect the cells expressing 4R tau. We also found that only 3R tau prions isolated from PiD patient samples infect the 3R expressing cells. Importantly, cells expressing both 3R and 4R tau were infected by tau prions isolated from AD, CTE, PSP, CBD, and PiD patients. Together, these findings demonstrate that tau prion strains are isoform specific, providing critical insight into the pathogenesis of all tauopathies.

[0013] Our results support our original conceptualization that the progressive

neurodegeneration in both Alzheimer's disease and chronic traumatic

encephalopathy is attributable to the spreading of tau prions.

[0014] The invention includes a cell line comprised of cells stably transfected with

(a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform.

[0015] Another aspect of the invention is the cell line wherein the cells are

transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

[0016] The cell lines are used in various methods of determining characteristics of neurological diseases and other methodologies as disclosed and described herein.

[0017] The present invention provides

(1) A cell line, comprising cells stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform;

(2) The cell line of (1), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform; (3) The cell line of any of (1) and (2), wherein the cells are human cells;

(4) The cell line of any of (1) to (3), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells, U373 cells, and LUHMES cells;

(5) The cell line of any of (1) to (4), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule;

(6) The cell line of any of (1) to (5), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules;

(7) The cell line of (5), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags;

(8) The cell line of (5), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein;

(9) The cell line of (8), wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase;

(10) The cell line of (5), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody;

(11) The cell line of (10), wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag;

(12) The cell line of (1), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein;

(13) The cell line of (12), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments;

(14) A method of detecting a form of a tau protein in a sample wherein the tau protein form is associated with a neurological disease, the method comprising:

(a) growing a cell line comprising cells stably transfected with (i) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (ii) a nucleotide sequence encoding repeat domains of a 3R tau isoform wherein the sequences (i) and (ii) are fused to a sequence encoding a detectable reporter molecule, or sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein;

(b) adding a sample to the cell line; and

(c) observing the presence of the detectable reporter molecule as an indication of the presence of a tau protein form in the sample;

(15) The method of (14), wherein step (a) comprises growing a plurality of different cell lines in a plurality of different containers wherein different cell lines in the different containers are transfected with different nucleotide sequences which encode different repeat domains of a 4R tau isoform and different nucleotide sequence encoding repeat domains of different 3R tau isoforms;

wherein step (b) comprises adding a biological tissue sample to the different containers; and

wherein step (c) comprises observing the presence of a detectable reporter molecule as an indication of the presence of a particular tau isoform in the biological sample;

(16) The method of (14), wherein the sample is selected from the group consisting of human blood serum and human cerebral spinal fluid.

(17) The method of (14), wherein the tau protein form is associated with a neurological disease selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, Pick's disease, argyrophilic grain disease, corticobasal degeneration, progressive supranuclear palsy, globular glial tauopathy, NFT dementia, tau astrogliopathy in elderly, and post-traumatic stress disorder;

(18) The method of (14), wherein the tau protein form is associated with

Alzheimer's disease;

(19) The method of (14), wherein the sample is a biological sample selected from the group consisting of brain tissue, blood, serum, cerebral spinal fluid, olfactory epithelium, nasal or sinus tissue, saliva, urine, submandibular gland, liver, lymph node, and skin; and wherein the cells are human cells;

(20) The method of any of (14) to (19), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform; (21) The method of any of (14) to (20), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells;

(22) The method of any of (14) to (21), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule;

(23) The method of any of (14) to (22), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent or luminescent protein and epitope tags;

(24) The method of (22), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein;

(25) The method of any of (14) to (24), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules;

(26) The method of any of (14) to (25), wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase;

(27) The method of (22), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody;

(28) The method of (27), wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag;

(29) The method of (27), wherein the epitope tag is a Myc-tag;

(30) The method of any of (14) to (29), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein;.

(31) The method of (30), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments;

(32) A method of testing a compound, comprising:

growing a cell line comprising cells stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform wherein the sequences (a) and (b) are fused to a sequence encoding a detectable reporter molecule, or sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein;

adding a form of tau protein and a compound to the cell line; and observing for the presence of the detectable reporter molecule;

(33) The method of (32), further comprising:

comparing observed presence of the detectable reporter molecule to a known standard;

(34) The method of any of (32) and (33), further comprising:

determining an ability of the compound to hinder formation of a form of tau protein aggregate based on a difference between the known standard and the observed presence of the detectable reporter molecule;

(35) The method of any of (32) to (34), wherein the known standard is a substantially identical cell line to which the compound has not been added;

(36) The method of any of (32) to (35), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform;

(37) The method of any of (32) to (36), wherein the cells are human cells;

(38) The method of any of (32) to (37), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells;

(39) The method of any of (32) to (38), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule;

(40) The method of any of (32) to (39), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules;

(41) The method of any of (32) to (40), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags;

(42) The method of (32), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein; (43) The method of (42), wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase;

(44) The method of (32), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody;

(45) The method of (44), wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag;

(46) The method of (44), wherein the epitope tag is a Myc-tag;

(47) The method of any of (32) to (46), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein;

(48) The method of any of (32) to (47), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments;

(49) A method of determining progress of a neurological disease in an individual, comprising:

(b) adding a first sample obtained from an individual to be tested to the cell line;

(c) observing the presence of the detectable reporter molecule and thereby quantifying the presence of the a form of tau protein in the sample;

(d) repeating steps (a), (b) and (c) with a second sample in step (b) wherein the second sample is obtained from the same individual at a point in time different from the first sample;

(e) quantifying the detectable label in (d); and (f) comparing the quantifying in (c) with the quantifying in (e) to determine progress of neurological disease in the individual;

(50) The method of (49), wherein a plurality of different sequences encoding a plurality of different reporter molecules are fused to the sequences (i) and (ii);

(51) The method of any of (49) to (50), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules;

(52) The method of any of (49) to (51), wherein the expression of the different reporter molecules is related to a different form of a tau protein wherein the tau protein is associated with a neurological disease;

(53) The method of any of (49) to (52), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform;

(54) The method of any of (49) to (53), wherein the cells are human cells;

(55) The method of any of (49) to (54), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells;

(56) The method of any of (49) to (55), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule;

(57) The method of any of (49) to (56), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent or luminescent protein and epitope tags;

(58) The method of any of (49) to (57), wherein the detectable reporter molecule is selected from the group consisting of a luminescent protein and a fluorescent protein;

(59) The method of (58), wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase;

(60) The method of any of (49) to (56), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody;

(61) The method of (60), wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag; (62) The method of any of (49) to (61), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein ;

(63) The method of any of (49) to (62), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments;

(64) A method of determining characteristics of a neurological disease affecting an individual, comprising:

(b) adding a sample obtained from an individual to be tested to the cell line and allowing the cell line to grow;

(c) observing the presence of the detectable reporter molecule in the cell line at plurality of different points in time;

(d) recording the observed presence of the detectable reported molecule at the different points in time;

(e) comparing the recorded presence of the detectable reporter molecule to a plurality of known standards related to different neurological diseases at different points in time; and

(f) making a diagnosis of a type of neurological disease based on the comparing step (e);

(65) The method of (64), wherein a plurality of different sequences encoding a plurality of different reporter molecules are fused to the sequences (i) and (ii);

(66) The method of any of (64) to (65), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules;

(67) The method of any of (64) to (66), wherein the expression of the different reporter molecules is related to a different form of a tau protein wherein the tau protein is associated with a neurological disease;

(68) The method of any of (64) to (67), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform;

(69) The method of any of (64) to (68), wherein the cells are human cells;

(70) The method of any of (64) to (69), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells;

(71) The method of any of (64) to (70), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule;

(72) The method of any of (64) to (71), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags;

(73) The method of any of (64) to (71), wherein the detectable reporter molecule is selected from the group consisting of a luminescent protein and fluorescent protein;

(74) The method of (73), wherein the fluorescent protein or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase;

(75) The method of any of (64) to (71), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody;

(76) The method of (75) wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag;

(77) The method of any of (64) to (76), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein; and

(78) The method of any of (64) to (77), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments. (79) An assay kit, comprising:

a plurality of stably transfected cells wherein the cells are transfected with a sequence encoding a tau protein associated with a neurological disease, which sequence is operatively fused to a sequence encoding a detectable reporter molecule;

a container holding the cells; and

instructions for using the kit in a manner whereby a sample added to the container results in generating a detectable signal when the sample contains a tau protein associated with a neurological disease.

(80) The assay kit of (79), wherein the cells are stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform.

(81) The assay kit of (80), wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

(82) The assay kit of any of (80) and (81), wherein the cells are human cells.

(83) The assay kit of (79), wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells, U373 cells, and LUHMES cells.

(84) The assay kit of any of (80) to (83), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

(85) The assay kit of any of (80) to (84), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

(86) The assay kit of any one of (80) to (85), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags.

(87) The assay kit of any one of (80) to (85), wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein.

(88) The assay kit of (87), wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

(89) The assay kit of any one of (80) to (85), wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

(90) The assay kit of (89), wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

(91) The assay kit of (79), wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

(92) The assay kit of (91), wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

(93) The assay kit of any one of (79( to (92), further comprising:

a plurality of containers wherein each container holds stably transfected cells different from the stably transfected cells in any other container.

(94) The assay kit of any one of (79) to (92), wherein the container holds a plurality of cells stably transfected with a different sequence encoding a different tau protein associated with a different neurological disease, and

further wherein each sequence encoding a different tau protein is operatively connected to a sequence encoding a different detectable reporter molecule,

whereby a different signal is generated for each different neurological disease corresponding to a disease of a patient from which a sample is obtained.

[0018] One aspect of the invention is an assay kit. The kit is comprised of (a) one or more genetically engineered cell lines as described herein; (b) one or more individual containers for each cell line; (c) a reagent solution; and (d) instructions for use. The container(s) of the kit may be labeled with a cell line name and/or a disease which corresponds to an indication of a patient having that disease when a sample from a patient is placed in a container with a given cell line, mixed with a reagent and results in a given signal being generated.

[0019] Multiple different versions of the kit can be generated using different

combinations of cell lines and labels as described herein. For example, rather than using multiple containers labeled with different diseases and/or cell lines, a single container would include multiple cell lines. Each different cell line in the container can include a different label which generates a different color for a positive result. Thus, the container and/or instructions would describe the disease associated with the generation of a particular color/signal.

[0020] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the cell lines and methods as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures:

[0022] Figure 1 includes A, B, C and D which are conceptual figures of tau fusion proteins expressed in human embryonic kidney cells. Human embryonic kidney (HEK) cells were engineered to express the tau RD fused to a fluorescent protein, which facilitated live-cell imaging. In Figure 1(A) TauRD(LM)-YFP cells express the RD of 4R tau (amino acids 243-375), including the familial tauopathy mutations P301L and V337M, fused to yellow fluorescent protein (YFP) (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288). In Figure 1 (B) Tau 3R(VM)-YFP cells express the RD of 3R tau (which excludes amino acids 275-305) with the L266V and V337M mutations. This protein fragment is also fused to YFP with the same 18 amino acid linker used in the TauRD(LM)-YFP cells. In Figure 1 (C) Cells expressing both the 3R and 4R tau isoforms were co-transfected with the constructs shown in A and B, and are identified as Tau3R4R-YFP. In Figure 1 (D) A fourth cell line was developed switching the fluorescent protein fused to the 4R isoform from YFP to mCherry. These cells are denoted by Tau3R4R-YM.

[0023] Figure 2 includes six photos of actual cell cultures showing 4R tau prions only infect TauRD(LM)-YFP cells. Tau prions were isolated from one control and five tauopathy patient samples using phosphotungstic acid (PTA) precipitation. The resulting pellets were resuspended in DPBS and incubated with TauRD(LM)-YFP cells. The 4R tauopathy PSP readily infected the cells. Additionally, tau prions isolated from a second 4R tauopathy, CBD, also infected cells expressing the 4R tau isoform. However, tau prions isolated from the 3R tauopathy, PiD, or the mixed tauopathies, AD and CTE, were unable to infected the TauRD(LM)-YFP cells, and were indistinguishable from the control sample.

[0024] Figure 3 includes six photos of actual cell cultures where Pick's disease prions only infect Tau3R(VM)-YFP cells. Tau prions isolated from the same six samples tested in Fig. 1 were tested on HEK cells expressing the 3R tau isoform (Tau3R(VM)-YFP). PiD prions, which were unable to infect TauRD(LM)-YFP cells, were the only tau prions capable of infecting cells expressing the 3R isoform. PSP, CBD, CTE, and AD prions were unable to infect the cells, yielding similar results as the control patient sample.

[0025] Figure 4 includes six photos of actual cell cultures demonstrating

Alzheimer's disease and chronic traumatic encephalopathy tau prion strains contain both 3R and 4R tau isoforms. Tau prions isolated from all five tauopathies using phosphotungstic acid (PTA) precipitation infected Tau3R4R-YFP cells. The cells, which express the repeat domains of both 3R and 4R tau were susceptible to infection by the 4R tauopathies, PSP and CBD, as well as the 3R tauopathy, PiD. Additionally, tau prions from the two mixed tauopathies, CTE and AD, also infected the cells. The control sample, however, did not induce aggregate formation.

[0026] Figure 5 includes A, B and C and shows that human tau prions can be serially propagated in cells. In Figure 5(A) Cell lines that stably maintain tau aggregates were developed from PSP and CBD patient samples. Isolated tau prions were used to infect the TauRD(LM)-YFP cells. Monoclonal lines of the infected cells were established. Lysate from these cell lines was collected and used to reinfect

TauRD(LM)-YFP cells. In Figure 5 (B) Clones derived from the PSP patient sample are capable of reinfecting naive TauRD(LM)-YFP cells at a concentration of 1 μg total protein per well. In Figure 5 (C) CBD-derived clones continue to propagate tau prions in naive TauRD(LM)-YFP cells using 0.2 μg total protein per well.

[0027] Figure 6 includes specific examples of nucleotide sequences (and the amino acid sequences encoded by them) used in transfecting cells of the present invention.

[0028] Figure 7 includes the graph of Figure 7 A and the seven photos of Figure 7B.

Tau prions were isolated from human brain homogenates by precipitating prion aggregates with sodium PTA. Protein aggregates were then incubated for 4 d with TauRD(LM)-YFP cells, which express the RD of 4R tau containing the mutations P301L and V337M. This protein fragment is fused to YFP and is expressed in HEK293 cells. (A) The graph of Figure 7A provides quantification of cell infection using control (n = 6), PiD (n = 6), AD (n = 7), CTE (n = 5), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patient samples. Total fluorescence in each image was normalized to the cell count. Prions isolated from the 4R tauopathies AGD

(P < 0.05), CBD (P < 0.001), and PSP (P < 0.001) showed a significant increase in infectivity over the control samples, whereas PiD (P = 0.74), AD (P = 0.17), and CTE (P = 0.41) did not. Data shown as mean from five images per well in six wells. *P < 0.05. All values shown in Table S2. (B) Figure 7B shows representative images of TauRD(LM)-YFP cells infected with AGD, CBD, and PSP, but not control, PiD, AD, and CTE patient samples. YFP shown in green. (Scale bar, 50 μιη.)

[0029] Figure 8 includes the graph of Figure 8 A and the seven photos of Figure 8B.

Tau prions were precipitated from control, PiD, AD, CTE, AGD, CBD, and PSP patient samples using sodium PTA. The resulting protein pellets were incubated for 4 d with Tau3R(VM)-YFP cells expressing the repeat domain of 3R tau containing the L266V and V337M mutations and fused to YFP. (A) Quantification of cell infection using control (n = 6), PiD (n = 6), AD (n = 7), CTE (n = 5), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patient samples was determined by summing the total fluorescence in each image and normalizing to the cell count. Prions isolated from PiD alone infected the Tau3R(VM)-YFP cells (P < 0.001) compared to controls. AD (P = 0.94), CTE (P = 0.18), AGD (P = 0.81), CBD (P = 0.67), and PSP (P = 0.14) showed no infectivity. Data shown as mean of five images per well in six wells. *P < 0.001. All values shown in Table S2. (B) Representative images of HEK293T cells infected with PiD, but not control, AD, CTE, AGD, CBD, or PSP patient samples. YFP shown in green. (Scale bar, 50 μιη.)

[0030] Figure 9 includes the graph of 9 A and the seven photos of Figure 9B. Tau prions were isolated using sodium PTA from control, PiD, AD, CTE, AGD, CBD, and PSP patient samples, and were then incubated with Tau3R4R-YFP cells for 4 d. Tau3R4R-YFP cells express both the 3R and 4R repeat domains of tau with mutations L266V and V337M (3R) and P301L and V337M (4R). (A) Quantification of cell infection using control (n = 6), PiD (n = 6), AD (n = 7), CTE (n = 5), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patient samples was determined by standardizing the total fluorescence in each image to the total cell count. Prions from all six tauopathies, including AD and CTE, infected the Tau3R4R-YFP cells, whereas the control samples showed no infection (PiD, P = 0.07; AD, P = 0.05; CTE, P < 0.001; AGD, P < 0.01 ; CBD, P < 0.001; PSP, P < 0.001). *P < 0.01. Data shown as mean from five images per well in six wells. All values shown in Table S2. (B) Representative images of HEK293T cells infected with PiD, AD, CTE, AGD, CBD, and PSP, but not control patient samples. YFP shown in green. (Scale bar, 50 um.)

[0031] Figure 10 includes the gel imagine of Figure 10A, the bar graph of Figure

10B, the graph of Figure IOC and the five photos of Figure 10D. HEK293T cells with a higher expression of the same fusion protein as the TauRD(LM)-YFP cells were created (Tau4R(LM)-YFP cells). (A) Western blot analysis of TauRD(LM)-YFP and Tau4R(LM)-YFP cells in the absence and presence of infection with lysate from Clone 9 cells, which stably propagate synthetic tau prions. The 4R(LM)-YFP construct was probed with anti-GFP (green fluorescent protein) antibody (top blot). The membrane was reprobed for vinculin as a loading control (bottom blot). (B) Quantification of protein expression in the Western blot was performed using ImageJ software. Expression levels were normalized to basal TauRD(LM)-YFP expression in HEK293 cells. (C and D) Crude brain homogenates from control (n =6), PiD (n = 6), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patient samples were diluted in DPBS and incubated with Tau4R(LM)-YFP cells for 4 d. (C) Quantification of cell infection after incubation was determined by dividing the total fluorescence in each image by the total cell count. Tau prions from the 4R tauopathies were all capable of infecting the Tau4R(LM) - YFP cells (AGD, P < 0.001; CBD, P < 0.05; PSP, P < 0.001), whereas the control samples did not. Of the six PiD samples, two infected the Tau4R(LM) - YFP cells (PiD3 and PiD4) and the other four did not (P = 0.41). *P < 0.05. Data shown as mean of five images per well in six wells. All values shown in Table S2 (Fig. 13). (D) Representative images of HEK293T cells infected with AGD, CBD, and PSP, but not control or PiD patient samples. YFP shown in green. (Scale bar, 50 μιη.)

[0032] Figure 11 includes the graph of Figure 11 A, two photos of Figure 1 IB, bar graph of Figure 11C and two photos of Figure 11D . Crude brain homogenate from control, AD, and CTE patient samples were diluted in DPBS and incubated for 4 d with Tau4R(LM)-YFP cells. (A) Quantification of cell infection with control (n = 6; data also shown in Fig. AC), AD (n = 7), and CTE (n = 5) patient samples was performed by standardizing the total fluorescence by the cell count. Both AD (P = 0.1) and CTE (P < 0.05) patient samples infected the Tau4R(LM)-YFP cells. *P < 0.05. Data shown as mean of five images per well in six wells. All values shown in Table S2 (Fig 13). (B) Representative images of HEK293T cells infected with AD and CTE. YFP shown in green. (Scale bar, 50 μιη.) (C) Quantification of cell infection using two brain regions from three CTE patient samples. The frontal pole and temporal pole contain a significantly different concentration of tau prions in two of the CTE patient samples tested. Data shown as mean + SD measured from five images per well in six wells. All values shown in Table S2 (Fig 13). *P < 0.05. (D) Representative images of HEK293T cells infected with samples from the frontal and temporal poles from patient CTE2. YFP shown in green. Scale is the same as shown in (B).

[0033] Figure 12 is Table SI showing information on Tau fusion protein constructs as expressed in specific human cell lines. Figure 12 is referred to throughout as Table SI.

[0034] Figure 13 is Table S2 consisting of Figures 13A and 13B which show

information on infection of tau-expressing mammalian cells with taupathy patient samples. Figure 13 is referred to throughout as Table S2.

[0035] For Figures 14-17, images in green labelled 0N4R Tau-YFP, 3R-YFP, and 4R-YFP were visualized using the FITC filter and show yellow fluorescent protein (YFP). Images in red labelled 0N3R Tau-mCherry, 4R-mCherry and 3R-mCherry were visualized using the dsRed filter and show mCherry. Images in blue labeled 4R-CFP were visualized using the DAPI filter and show the cyan fluorescent protein (CFP).

[0036] Figure 14 includes a conceptual diagram in Figure 14a of a tau fusion proteins expressed in HEK293T cells and six photos from Figure 14b. Tau prions selectively template 0N3R or 0N4R tau fusion proteins in cells expressing full-length tau isoforms. Tau prions isolated from P301S^+/+ mice (which develop spontaneous 4R tau prions; Allen B, et al. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. /. Neurosci. 22:9340-9351), progressive supranuclear palsy (PSP), and Pick's disease (PiD) samples were incubated with cells co-expressing both 0N3R tau fused to mCherry and 0N4R tau fused to YFP. (A) Schematic of isoforms expressed in the HEK293T cells. (B) P301S^+/+ and PSP patient samples, which both contain 4R tau prions, induced aggregation of 0N4R-YFP tau. PiD patient samples, which contain 3R tau prions, induced aggregation of 0N3R-mCherry tau.

[0037] Figure 15 includes a conceptual diagram in Figure 15a of a tau fusion protein

expressed in HEK293T cells and four photos from Figure 15b. Co-expression of the repeat domains of 3R and 4R tau using the P2A construct yields more robust infection than cells expressing full-length tau. Tau prions isolated from the P301S^+/+ mice were incubated with cells expressing a P2A construct containing the repeat domain (RD) of both 3R-YFP and 4R-mCherry tau. (A) Schematic of construct expressed in HEK293T cells. (B) Infection of cells with P301S^+/+ brain homogenate, which contains 4R tau prions, induced aggregation of 4R-mCherry tau, but not 3R-YFP tau. Control mouse samples had no effect on the cells.

[0038] Figure 16 includes a conceptual diagram in Figure 16a of a tau fusion protein

expressed in HEK293T cells and eight photos from Figures 16b and 16c. Tau prions show selective isoform infection. Tau prions isolated from P301S^+/+ mice, PSP, and PiD patients were incubated with cells expressing a P2A construct containing 4R(LM)-YFP tau and 3R(VM)-mCherry tau. (A) Schematic of the P2A construct expressed in HEK293T cells. (B) Infection of cells with P301S^+/+ brain homogenate, which contains 4R tau prions, induced aggregation of 4R-YFP tau, but not

3R-mCherry tau. Control mouse samples had no effect. (C) Tau prions isolated from PSP prions selectively induced aggregation of 4R-YFP tau, while tau prions from PiD selectively induced aggregation of 3R-mCherry tau.

[0039] Figure 17 includes a conceptual diagram in Figure 17a of a tau fusion protein

expressed in HEK293T cells and four photos from Figure 17b. Mouse tau prions show selective infection of 4R-CFP tau. Tau prions isolated from P301S^+/+ mice were incubated with cells expressing a P2A construct containing 3R-tau fused to YFP and 4R-tau fused to CFP. (A) Schematic of the construct expressed in

HEK293T cells. (B) P301S^+/+ 4R tau prions induced aggregation of 4R-CFP tau, but had no effect on 3R-YFP tau. Control mouse samples had no effect on the cells. DETAILED DESCRIPTION OF THE INVENTION

[0040] Before the present cell lines and methods are described, it is to be understood that this invention is not limited to particular cells of method described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0041] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0042] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0043] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell or cell line" includes a plurality of such cells and reference to "the step" includes reference to one or more steps and equivalents thereof known to those skilled in the art, and so forth. [0044] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0045] The present invention relates to a cell line comprised of cells stably

transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform.

[0046] The nucleotide sequence for the repeat domain of the 4R tau isoform is shown as SEQ ID No. 13 in Figure 6 and the nucleotide sequence of the repeat domain of the 3R tau isoform is SEQ ID No. 14 of Figure 6. However, it should be understood that the invention encompasses variations of these sequences which provide the same result in terms of detecting a protein associated with a neurological disease. Thus, the sequences include, for example, variations of SEQ ID No. 13 which includes 1 or more degenerate codons encoding the same amino acid sequence as SEQ ID No. 1. Further, with respect to the repeat domain of the 3R tau isoform, the SEQ ID No. 14 includes one or more degenerate codons which result in the encoding of the amino acid sequence of SEQ ID No. 2. In addition, the term "sequence encoding the repeat domain of the 4R tau isoform" and "sequence encoding the repeat domain of the 3R tau isoform" includes variations of up to 1, 2, 3, 4, or 5 codons different from SEQ IDs No. 13 and 14 respectively. Alternately, the sequences include varied sequences from SEQ ID Nos: 13 and 14 with a variation of 1%, 2%, 3%, 4%, 5%, 10%, or 20% which result in encoding proteins which allow for the operation of the invention as described and disclosed herein.

[0047] The sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform can be fused to a sequence encoding a detectable reporter molecule, or sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein. The cell line, comprising cells stably transfected with sequences mentioned above is useful for (1) detecting a form of a tau protein in a sample wherein the tau protein form is associated with a neurological disease, (2) testing a compound for an ability of the compound in treating neurological diseases associated with tau protein, (3) determining progress of a neurological disease in an individual, and (4) determining characteristics of a neurological disease affecting an individual.

[0048] Examples of detectable reporter molecules includes a fluorescent protein, a luminescent protein, epitope tags or other reporter molecules known to the person skill in the art. Additionally, the use of multiple reporter molecules on the two isoforms can be used to identify not just neurological diseases but the characteristics of a neurological disease such as 3R tauopathy, 4R tauopathy, or both 3R and 4R tauopathy.

[0049] Furthermore, in another embodiment, sequences encoding one or more repeat domains of 4R tau and one or more repeat domains of 3R tau and/or the entire 4R tau isoform and entire 3R tau isoform are operatively fused into a construct with the sequences encoding complimentary fragments which generate a detectable label when the two complementary fragments are brought together. The resulting construct is a cell line, which will generate a detectable reporter molecule when two complementary fragments are brought together such as in a prion or a protein aggregate associated with a neurological disease. Examples of complementary fragments of such detectable labels include split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments. The use of complementary fragments can be used to detect protein-protein interaction.

[0050] Examples of the cells used in the present invention include mammalian cells, preferably human cells, more preferably, embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells.

[0051] Examples of the neurological disease as described herein, include

Alzheimer's disease, chronic traumatic encephalopathy, Pick's disease, argyrophilic grain disease, corticobasal degeneration, progressive supranuclear palsy, globular glial tauopathy, NFT dementia, tau astrogliopathy in elderly, and post-traumatic stress disorder or other tauopathies.

[0052] Examples of samples as described herein, include biological tissue obtained from a subject such as brain tissue, blood, serum, cerebral spinal fluid, olfactory epithelium, nasal or sinus tissue, saliva, urine, submandibular gland, liver, lymph node, and skin. EXAMPLES

[0053] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Results

Developing tau isoform-specific assays

[0054] Initial studies using the HEK293 cells expressing the TauRD(LM)-YFP fusion protein, developed by Marc Diamond's group, demonstrated that in the presence of tau prions (both synthetic and natural), the prions would induce tau aggregation in the cells, which is visualized as bright yellow foci (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288.). Recently, we adapted the TauRD(LM)-YFP cell assay to a 384- well plate format, performing live-cell imaging after incubating the cells for 4 days (Woerman AL, et al. (2015) Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A.

112:E4949-E4958). To improve the robustness of the assay, we used

phosphotungstic acid (PTA), which has been shown to selectively isolate aggregated protein from soluble protein (Safar J, et al. (1998) Eight prion strains have PrP^Sc molecules with different conformations. Nat. Med. 4: 1157-1165), to precipitate the tau prions. With this automated and high-throughput approach, we were able to increase the percentage of cells infected with aggregates from -4% to -61%, greatly increasing the dynamic range of the assay.

[0055] Building on these initial studies in HEK293 cells, we developed a panel of

HEK293T cell lines expressing tau fusion proteins (Fig. 1). In the first line, we expressed the same 4R tau RD from the TauRD(LM)-YFP cells, where amino acids 243-375 of 4R tau containing the familial P301L and V337M mutation were fused to yellow fluorescent protein (YFP) using an 18 amino acid linker sequence

(EFCSRRYRGPGIHRSPTA; SEQ ID NO: 25) This cell line is referred to as Tau4R(LM)-YFP The second we line developed expresses the 3R tau isoform (omitting amino acids 375-305 from the 4R isoform; Fig. IB). Because residue 301 is not expressed in the RD of the 3R tau isoforms, we instead used the familial mutation K266V along with V337M. This construct is also fused to YFP using the same linker sequence, and is identified as Tau3R(VM)-YFP. Finally, we made versions of HEK293T cells that express both the 3R and the 4R tau isoforms. In the first (Fig. 1C), we have combined the two individual constructs, such that both isoforms are fused to YFP, denoted as Tau3R4R-YFP. In the second (Fig. ID), we replaced the YFP tag on the Tau4R(LM) protein with an mCherry tag. These cells are designated as Tau3R4R-YM.

[0056] In addition to cell lines that express the repeat domain of 3R and 4R tau fused to identical reporter molecules, we prepared HEK293T cells that express 0N4R tau with the P301S mutation fused to YFP and 0N3R tau with the K266V and V337M mutations fused to mCherry (Fig 14). We also made cell lines in which the constructs were tandemly connected with the P2A peptide. In one HEK293T cell line, Tau3R(VM)-YFP and Tau4R(LM) -mCherry were tandemly expressed via the P2A construct (Fig. 15). In another cell line, the YFP and mCherry reporter molecules were reversed to give Tau3R(VM)-mCherry and Tau4R(LM)-YFP separated by the P2A construct (Fig. 16). The cyan fluorescent protein (CFP) was also used. Tau 3R(M)-YFP and Tau 4R(LM)-CFP were connected with the P2A construct and expressed in HEK293T cells (Fig. 17)

4R tauopathies are propagated by 4R tau prions

[0057] Using the TauRD(LM)-YFP (Fig. 2) cells, we tested the ability of five

different tauopathies to transmit tau prions to the cells, in comparison to a control sample. The control sample is from an individual void of any neuropathological lesions upon autopsy. For the tauopathy samples, we selected two 4R diseases (progressive supranuclear palsy, PSP; and corticobasal degeneration, CBD), the prototypical 3R disease (Pick's disease, PiD), and two mixed tauopathies (Alzheimer's disease, AD; and chronic traumatic encephalopathy, CTE). A 10% (wt/vol) brain homogenate from each sample was made in Dulbecco's

phosphate -buffered saline (DPBS) without calcium or magnesium before digesting the sample in 2% (vol/vol) sarkosyl and 0.5% (vol/vol) benzonase. Importantly, the addition of benzonase results in the digestion of all nucleic acids in the sample, leaving only protein, which we then incubated with 2% (vol/vol) PTA overnight before pelleting by centrifuging at 16,000 x g for 30 min at room temperature. After removing the supernatant, the precipitation was repeated a second time before the samples were resuspended in DPBS. The samples were then incubated with the TauRD(LM)-YFP cells for 4 days in the presence of Lipofectamine 2000, which increased the efficiency of protein uptake by the cells. The live cells were imaged using the IN Cell 6000, and analyzed for the presence of YFP-positive aggregates.

[0058] As expected, the control patient sample had no effect on the

TauRD(LM)-YFP cells. Tau prions isolated from the two 4R tauopathies, PSP and CBD, however, did infect the cells, inducing the formation of tau aggregates visualized by bright yellow foci. Interestingly, tau prions isolated from the 3R sample, PiD, and the two mixed tauopathy samples, AD and CTE, were unable to propagate in the cells.

Pick's disease prions are 3R tau-specific

[0059] Hypothesizing that the descriptive subclassification of the tauopathies into

3R, 4R, and mixed tauopathy diseases, based upon neuropathological assessment, is a result of the isoform of tau prion giving rise to the disease, we next tested the same set of samples on the Tau3R(VM)-YFP cells (Fig. 3). While again the control sample was unable to induce aggregate formation in the cells, we also found that the PSP and CBD samples, which we showed to contain tau prions, were unable to propagate in the presence of 3R tau, indicating that the prion strains giving rise to these diseases require the 4R tau isoform to propagate. However, tau prions isolated from the 3R tauopathy, Pick's disease, were able to infect the cells and induce aggregate formation. This sample was unable to infect the TauRD(LM)-YFP cells, suggesting that the strain of tau prion giving rise to PiD is 3R tau-specific. Similar to PSP and CBD, we also found that tau prions from AD and CTE were unable to infect the Tau3R(VM)-YFP cells. Alzheimer's disease and chronic traumatic encephalopathy are tau prion diseases

[0060] Our findings that (1) PSP and CBD could only infect the HEK293 or

HEK293T cells as long as the 4R isoform of tau was present, and (2) PiD prions could only propagate in HEK293T cells expressing the 3R isoform of tau, indicated that cells expressing both isoforms of tau should be able to propagate both the 3R and the 4R tauopthies, as both substrates would be available for templating.

[0061] To test this hypothesis, we incubated the same six samples with the

Tau3R4R-YFP cells (Fig. 4). As expected, the PSP and CBD patient samples, as well as the PiD samples, all infected the cells, inducing tau aggregation. Moreover, when the cells were incubated with the mixed tauopathy samples, tau prions isolated from both CTE and AD infected the Tau3R4R-YFP cells. The control patient sample did not induce aggregate formation, indicating that the cell line does not develop spontaneous aggregates, and that the aggregates seen in the cells incubated with either the CTE or the AD patient sample are truly a result of tau prion propagation.

Tau prion propagation is isoform specific

[0062] Summarizing the data collected in our panel of tau HEK293 or HEK293T cell assays, it is evident that propagation of tau prions is dependent upon the isoform(s) of tau involved in each prion strain (Table 1). While we have traditionally used the tau isoform(s) present in each pathology as a method for categorizing the various tauopathies, these data reveal that these subgroupings are also instructive about the prion strains that give rise to each disease. In order to propagate, each tau prion strain requires a specific substrate, be it 3R, 4R, or both isoforms of tau. In the presence of the 4R repeat domain, PSP and CBD can infect both the TauRD(LM)-YFP cells and the Tau3R4R-YFP cells. Similarly, PiD can propagate in both the Tau3R(VM)-YFP cells and the Tau3R4R-YFP cells due to the availability of the 3R isoform as a substrate. Importantly, both CTE and AD, which are composed of 3R and 4R tau aggregates, can only propagate in the Tau3R4R-YFP cell line, suggesting that the strain(s) of tau prions giving rise to these two diseases uses both the 3R and 4R isoforms of the tau protein. [0063] Table 1. Propagation of tau prion strains is isoform dependent

[0064] To further investigate this specificity, we tested the infectivity of cells that express both tau isoforms fused to distinct fluorescent probes. When these cells were exposed to samples from a P301S^+/+ mouse (Allen B, et al. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. /. Neurosci. 22:9340-9351), which overexpresses 0N4R human tau and develops spontaneous tau prions, 4R-specific tau prion propagation is observed (Figures 14 to 17). This specificity is demonstrated in Figs. 15 & 16. When the RD of 4R tau was tagged with mCherry and the RD of 3R tau was fused to YFP, tau prion infection in these cells using the P301S^+/+ mouse samples only induced aggregation of 4R-mCherry tau, and had no effect on 3R-YFP tau (Fig. 15B). Likewise, when the RD of 4R tau expressed in the cells was fused to YFP and the RD of 3R tau was fused to mCherry, incubation with the P301S^+/+ tau prions selectively induced aggregation of 4R-YFP tau and had no effect on 3R-mCherry tau (Fig. 16B). This specificity remains consistent with human samples. In these cells, PSP patient samples induce 4R-YFP tau aggregates only, whereas PiD patient samples induced 3R-mCherry tau aggregates only (Fig. 16C). Similarly, tau prion selectively remained consistent using the P301S^+/+ mouse samples when the mCherry fluorescent tag was replaced with cyan fluorescent protein (CFP), demonstrating the ability of multiple reporter molecules to yield similar findings (Fig. 17). Serial propagation of human tau prions in cultured cells

[0065] A central characteristic of prions is the ability to continue passaging, or propagating, the misfolded protein from one cell to another. To test whether or not we could passage the tau aggregates induced by these diseases in the HEK293 or HEK293T cells, we developed monoclonal populations of the cells that stably express tau aggregates (Fig. 5). Using PSP and CBD patient samples, we used PTA to precipitate the tau prions, and then infected the TauRD(LM)-YFP cells. Monoclonal populations of cells expressing aggregates were isolated and established. Lysate from these clones was collected and used to infect naive TauRD(LM)-YFP cells at a concentration of 1 μg total protein per well for the PSP-derived clone (Fig. 5B) and 0.2 μg total protein per well for the CBD-derived clone (Fig. 5C). Both the PSP and CBD-derived clones were capable of reinfecting the naive cells, demonstrating the aggregates isolated from these tauopathies via PTA precipitation are tau prions.

[0066] Using cells that express 3R and 4R tau fused to different reporter molecules, , provides an unambiguous method for identifying the tauopathies present in a sample. For example, samples from a 4R tauopathy are identified by the reporter molecule fused to 4R tau in the cell, and samples from a 3R tauopathy are identified by the reporter molecule fused to 3R tau in the cell. Mixed tauopathies will result in aggregates that containing both 3R and 4R reporter molecules, facilitating the ability to better stratify and diagnose tauopathy patients.

Discussion

[0067] Increasing evidence supports the hypothesis that the progressive nature of tau-related neurodegenerative diseases (tauopathies) is due to the spread of tau prions, resulting in widespread tau aggregation in later stages of disease. While it is well recognized that distinct isoforms of tau are present in aggregates associated with the various tauopathies, and that each disease is identifiable by unique structural features of these aggregates observed in neuropathological studies, the relationship between these two observations has not been elucidated. Here we demonstrate that the prion strain giving rise to each disease is tau isoform specific (3R in PiD, 4R in PSP and CBD, and both 3R and 4R in AD and CTE), providing important insight into how the phenotypic differences across the tauopathies arise, and how the disease progresses in the brain.

[0068] Previous work with synthetic tau prions provides additional support for these findings. Using the recombinant K18 and K19 constructs, which encode the 4R and 3R repeat domains of tau respectively, Dinkel et al. demonstrated that K19 aggregates could not seed Kl 8 monomer, indicating a seeding barrier exists between incompatible tau prion strains (Dinkel PD, Siddiqua A, Huynh H, Shah M, & Margittai M (2011) Variations in filament conformation dictate seeding barrier between three- and four-repeat tau. Biochemistry 50:4330^4336). Additionally, recombinant 1N3R or 1N4R human tau fibrils were used to test cross-isoform seeding in the neuroblastoma cell line, SH-SY5Y (Nonaka T, Watanabe ST, Iwatsubo T, & Hasegawa M (2010) Seeded aggregation and toxicity of a-synuclein and tau: cellular models of neurodegenerative diseases. /. Biol. Chem.

285:34885-34898). Transient expression of either 1N3R or 1N4R tau was initiated 14 h prior to exposing the cells to the tau fibrils. When the authors attempted to infect the cells with the 1N4R fibrils, only the cells expressing 4R tau developed aggregates. Similarly, the 1N3R fibrils could only propagate in the 3R-expressing cells, demonstrating isoform-specificity in tau prions.

[0069] Several in vivo findings have also demonstrated homotypic seeding of tau prions. In 2009, Michel Goedert and Markus Tolnay demonstrated that brain extract prepared from symptomatic P301S mice (Allen B, et al. (2002) Abundant tau filaments and nonapoptotic neurodegeneration in transgenic mice expressing human P301S tau protein. /. Neurosci. 22:9340-9351) induced tau neuropathology after inoculation into the ALZ17 mouse (Probst A, et al. (2000) Axonopathy and amyotrophy in mice transgenic for human four-repeat tau protein. Acta Neuropathol. 99:469^481 ; Clavaguera F, et al. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11 :909-913). While the P301S (human 0N4R tau with the P301S mutation) and ALZ17 (wildtype human 2N4R tau) models differ slightly, the ability of the diseased P301S mouse to transmit NFTs, neuropil threads, and coiled bodies to the ALZ17 mouse demonstrates homotypic seeding of 4R tau. Using the same P301S model, Ahmed et al. demonstrated that brain extract prepared from aged, symptomatic animals could be inoculated into two-month old pre-symptomatic mice to induce NFT pathology within 2 weeks of inoculation, resulting in rapid propagation of tau prions (Ahmed Z, et al. (2014) A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity. Acta Neuropathol. 127:667-683). In the PS 19 mouse model (human 1N4R tau with the P301S mutation) (Yoshiyama Y, et al. (2007) Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53:337-351), Virginia Lee's group inoculated mice with preformed fibrils to test tau seeding (Iba M, et al. (2013) Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy. /. Neurosci. 33: 1024-1037). Regardless of the fibrils used, either 2N4R tau with the P301S mutation (T40/PS) or K18 with the P301L mutation (K18/PL), inoculations with 4R-expressing fibrils induced tau neuropathology in young PS 19 mice, while inoculation with a-synuclein fibrils had no effect. Additional studies in the PS 19 model from Marc Diamond's group showed tau prions isolated from the Clone 9 and Clone 10 cells, which stably propagate 4R tau prions in the HEK293 TauRD(LM)-YFP cells, induced distinct neuropathologies in the mice (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288). Not only do these studies contribute to the data supporting homotypic seeding of tau prions, but they also demonstrate the ability of distinct 4R tau prion strains to give rise to unique pathologies in vivo. This, in particular, supports the hypothesis that the heterogeneity of human tauopathies arises from distinct prion strains.

Although most reports in the literature support the idea of isoform-specific prion strains, a few studies demonstrate contrasting findings. In 2013, Clavaguera et al. inoculated brain homogenate from a number of tauopathies into the ALZ17 mice and found the 4R tauopathies argyrophilic grain disease (AGD), PSP, and CBD induced tau neuropathology that was similar to the distinct hallmarks associated with the diseases (Clavaguera F, et al. (2013) Brain homogenates from human tauopathies induce tau inclusions in mouse brain. Proc. Natl. Acad. Sci. USA 110:9535-9540). However, they also found that PiD, AD, and tangle-only dementia (TD) were capable of inducing argyrophilic inclusions. While mice predominantly express 4R tau, they do express low levels of the 3R tau isoforms (McMillan P, et al. (2008) Tau isoform regulation is region- and cell-specific in mouse brain. /. Comp. Neurol.

511 :788-803), and it cannot be ruled out that the endogenous mouse tau contributed to this finding. It has also been shown that tau oligomers isolated from PSP patient samples are capable of seeding both 3R and 4R tau in a cell-free system (Gerson JE, et al. (2014) Characterization of tau oligomeric seeds in progressive supranuclear palsy. Acta Neuropathol. Commun. 2:73). Here, fibrilization of 3R or 4R monomer was measured by thioflavin T (ThT) incorporation after seeding with tau oligomers isolated from a PSP patient sample. Additionally, AD and PiD have previously been reported to infect TauRD(LM)-YFP cells expressing 4R tau only (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288). Some of these discordant results may arise from differences in assay conditions. In particular, given the robust and automated read out of our assay, if a low number of cells exposed to a given AD or PiD patient sample developed aggregates, those aggregates would not be detected above the background fluorescence in the assay.

Importantly, the results presented here also provide evidence that the progressive neurodegeneration in Alzheimer's disease and chronic traumatic encephalopathy are propagated by tau prions that require both 3R and 4R tau isoforms. AD, the most common neurodegenerative disease (Association As (2014) 2014 Alzheimer's disease facts and figures, in Alzheimers Dement. , p 80), is referred to as a secondary taupoathy due to the presence of two pathological features:

β-amyloid (Αβ) plaques and tau NFTs. Αβ is produced by cleavage of the amyloid precursor protein (APP), and can become a prion capable of propagating in vivo (Watts JC, et al. (2014) Serial propagation of distinct strains of Αβ prions from Alzheimer's disease patients. Proc. Natl. Acad. Sci. U.S.A. 111: 10323-10328; Watts JC, et al. (2011) Bioluminescence imaging of Abeta deposition in bigenic mouse models of Alzheimer's disease. Proc. Natl. Acad. Sci. USA 108:2528-2533; Stohr J, et al. (2012) Purified and synthetic Alzheimer's amyloid beta (Αβ) prions. Proc. Natl. Acad. Sci. U.S.A. 109: 11025-11030; Meyer-Luehmann M, et al. (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313: 1781-1784; Morales R, Duran-Aniotz C, Castilla J, Estrada LD, & Soto C (2012) De novo induction of amyloid-β deposition in vivo. Mol. Psychiatry

17: 1347-1353; Rosen RF, et al. (2012) Exogenous seeding of cerebral beta-amyloid deposition in betaAPP-transgenic rats. /. Neurochem. 120:660-666). However, tau NFT accumulation is highly associated with cognitive decline (Duyckaerts C, et al. (1997) Modeling the relation between neurofibrillary tangles and intellectual status. Neurobiol. Aging. 18:267-273; Bennett DA, Schneider JA, Wilson RS, Bienias JL, & Arnold SE (2004) Neurofibrillary tangles mediate the association of amyloid load with clinical Alzheimer disease and level of cognitive function. Arch Neurol.

61:378-384; Sabbagh MN, et al. (2010) Functional, global and cognitive decline correlates to accumulation of Alzheimer's pathology in MCI and AD. Curr.

Alzheimer Res. 7:280-286; Nelson PT, et al. (2012) Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. /.

Neuropathol. Exp. Neurol. 71:362-381), suggesting the progressive dementia seen in AD patients arises from the spread of tau prions and not Αβ plaques. Notably, early stages of NFT formation are localized to the locus coeruleus and transentorhinal cortex, but spread by later stages to the hippocampus and neorcortical association areas (Braak H & Braak E (1991) Neuropathological staging of Alzheimer-related changes. Acta Neuropathol. (Berl.) 82:239-259).

CTE, first defined as punch drunk (Martland HS (1928) Punch drunk. /. Am.

Med. Assoc. 91 : 1103-1107), is a progressive neurodegenerative disease caused by repetitive mild traumatic brain injury. Increasingly diagnosed in amateur and professional athletes in contact sports, CTE is now recognized as the signature injury of the recent conflicts in Afghanistan and Iraq as a result of increased exposure to blast waves from improvised explosive devices (Owens BD, et al. (2008) Combat wounds in operation Iraqi Freedom and operation Enduring Freedom. /. Trauma. 64:295-299; Hoge CW, et al. (2008) Mild traumatic brain injury in U.S. Soldiers returning from Iraq. N. Engl. J. Med. 358:453^463; Terrio H, et al. (2009) Traumatic brain injury screening: preliminary findings in a US Army Brigade Combat Team. /. Head Trauma Rehabil. 24: 14-23; Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4). Early stages of the disease present with focal tau pathology near the vasculature within the depths of the cerebral sulci. Tau deposition then becomes widespread throughout the amygdala, hippocampus, and majority of the cerebral cortex as the disease progresses to later stages (Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4).

[0073] Interestingly, tau isolated from AD and CTE patient samples contain all 6 isoforms of tau, with both 3R and 4R tau isoforms isolated from tau aggregates, in particular (Schmidt ML, Zhukareva V, Newell KL, Lee VM, & Trojanowski JQ (2001) Tau isoform profile and phosphorylation state in dementia pugilistica recapitulate Alzheimer's disease. Acta Neuropathol. 101 :518-524). While tau pathology in AD patients is localized to neurons, astrocytic tangles can be prominent in CTE patients (McKee AC, et al. (2013) The spectrum of disease in chronic traumatic encephalopathy. Brain 136:43-64). Additionally, the location of NFTs in the cortical layers also differs between AD and CTE patients. AD patients often develop NFTs in both superficial and deeper cortical layers, with the highest density seen in layers V-VI. CTE patients, on the other hand, typically develop NFTs in the more superficial layers II- III of the neocortex. Despite these differences, the hippocampus is similarly affected by both diseases (Hof PR, et al. (1992) Differential distribution of neurofibrillary tangles in the cerebral cortex of dementia pugilistica and Alzheimer's disease cases. Acta Neuropathol. 85:23-30). That the distinct pathological hallmarks of these two diseases arise from aggregation of the same protein suggests AD and CTE are manifestations of two distinct strains of tau prions.

[0074] The invention shows that Alzheimer's disease and chronic traumatic

encephalopathy are propagated by tau prion strains that require both the 3R and 4R isoforms, and that the propagation of all tau prion strains is isoform-dependent. Drug discovery methodology of the invention uses the inherent differences between tauopathies in order to effectively develop potential therapeutics and diagnostics.

Materials and Methods

Human Tissue Samples

[0075] Frozen brain tissue samples from neuropathologically confirmed cases of

AD, AGD, CBD, PiD, and PSP were received from the UCSF Neurodegenerative Disease Brain Bank, as were two control samples. Frozen brain tissue samples from neuropathologically confirmed cases of CTE were provided by the Alzheimer's Disease Center's Chronic Traumatic Encephalopathy Program at Boston University. Four control samples were provided by Deborah Mash, University of Miami, Coral Gables, Florida.

Human Patient Neuropathology

[0076] Two control samples, as well as the AD, AGD, CBD, PiD, and PSP samples were received from patients enrolled in the UCSF Memory and Aging Center longitudinal clinical research programs. Fresh brains were cut into ~1 cm coronal sections and were alternately fixed in 10% neutral buffered formalin for 72 h or rapidly frozen. Neuropathological diagnoses were made in accordance with consensus diagnostic criteria (Mackenzie IR, et al. (2010) Nomenclature and nosology for neuropathologic subtypes of frontotemporal lobar degeneration: an update. Acta Neuropathol. 119: 1-4) using histological and immunohistochemical methods previously described (Kim E-J, et al. (2012) Selective frontoinsular von Economo neuron and fork cell loss in early behavioral variant frontotemporal dementia. Cereb. Cortex 22:251-259). Patient samples were selected by W.W.S. and L.T.G. The CTE patient samples were provided by the Chronic Traumatic

Encephalopathy Program at Boston University's Alzheimer's Disease Center and the Veterans Affairs Boston Healthcare System. Neuropathological processing was carried out as described previously (Vonsattel JPG, et al. (1995) An improved approach to prepare human brains for research. /. Neuropathol. Exp. Neurol.

54:42-56). Staining and neuropathological assessment of patient samples are described in (McKee AC, et al. (2013) The spectrum of disease in chronic traumatic encephalopathy. Brain 136:43-64). Patient samples were selected by A.C.M.

Cell Line Development

[0077] Constructs encoding the RD of 4R tau (amino acids 243-375 corresponding to 2N4R tau) containing the mutations P301L and V337M, and the RD of 3R tau (amino acids 243-274; 306-375) containing the mutations L266V and V337M were fused with YFP at the C-terminus with an 18-amino-acid flexible linker

(EFCSRRYRGPGIHRSPTA). These constructs were introduced into the pIRESpuro3 vector (Clontech) or the pIRESblaS vector in which the puromycin resistance gene of the pIRESpuro3 was replaced with the blasticidin resistance gene. All tau fusion protein constructs are listed with amino acid residues, mutations, and background cell line in Table SI. HEK293T cells (ATCC) were cultured in

Dulbecco's Modified Eagle's medium (DMEM) supplemented with 50 units/mL penicillin, 50 μg/mL streptomycin, and 10% fetal bovine serum (Thermo Fisher). Cultures were maintained in a humidified atmosphere of 5% CO₂ at 37 °C. Cells plated in DMEM were transfected using Lipofectamine 2000 (Thermo Fisher). Stable cells were selected in DMEM containing 1 μg/mL puromycin or 10 μg/mL blasticidin S (Thermo Fisher). Monoclonal lines were generated by limiting dilution of polyclonal cell populations in 96- or 384- well plates.

[0078] Constructs encoding the RD of 4R tau (amino acids 243-375 corresponding to 2N4R tau) containing the mutations P301L and V337M, and the RD of 3R tau (amino acids 243-274; 306-375) containing the mutations L266V and V337M, were fused with either YFP, mCherry, or CFP at the C-terminus with an 18-amino-acid flexible linker (EFCSRRYRGPGIHRSPTA). These constructs were either separately introduced or tandemly connected with P2A peptide and introduced into the pIRESpuro3 vector (Clontech) or the pIRESblaS vector in which the puromycin resistance gene of the pIRESpuro3 was replaced with the blasticidin resistance gene. All tau fusion protein constructs are listed with amino acid residues, mutations, and background cell line in Table SI. HEK293T cells (ATCC) were cultured in

Dulbecco's Modified Eagle's medium (DMEM) supplemented with 50 units/mL.

Cell Aggregation Assay

[0079] Cells were plated in 384-well plates with black polystyrene walls (Greiner) with 0.012 μg Hoechst 33342 (Thermo Fisher) at a density of 1,000 cells/well

[TauRD(LM)-YFP] , 3,000 cells/well [Tau3R(VM)-YFP and Tau4R(LM)-YFP] , or 4,000 cells/well (Tau3 R4R-YFP) . Cells were incubated at 37 °C for 2-4 h to allow adherence to the plate. Lipofectamine 2000 (1.5% final volume; Thermo Fisher) and OptiMEM (78.5% final volume; Thermo Fisher) were premixed and added to each patient sample before incubating at room temperature for 2 h. Patient samples were plated in 6 replicate wells. Plates were then incubated at 37 °C in a humidified atmosphere of 5% C02 for 4 days before imaging on the IN Cell Analyzer 6000 (GE Healthcare). Images of both the DAPI and FITC channels were collected from 5 different regions in each well. The images were analyzed using the IN Cell Developer software with algorithms developed to identify intracellular aggregates only in live cells.

Cell Assay Sample Preparation

[0080] Patient tissue samples were homogenized to 10% (wt/vol) in calcium- and magnesium- free DPBS using an Omni Tip (Omni International) and PowerGen homogenizer (Fisher Scientific). Samples were aliquoted and stored at -80 °C.

[0081] PTA precipitation was performed as described (Safar J, et al. (1998) Eight prion strains have PrP^Sc molecules with different conformations. Nat. Med.

4: 1157-1165). Briefly, 10% brain homogenate was incubated in 2% sarkosyl (Sigma) and 0.5% benzonase (Sigma) at 37 °C with constant agitation in an orbital shaker for 2 h. Sodium PTA (Sigma) was dissolved in ddH20, and the pH was adjusted to 7.0. PTA was added to the samples to a final concentration of 2%, which was then incubated overnight in the same conditions. The sample was centrifuged at 16,000 x g for 30 min at room temperature, and the supernatant was removed. The resulting pellet was resuspended in 2% sarkosyl in PBS and 2% PTA in ddH20, pH 7.0. The sample was again incubated for at least 1 h prior to a second centrifugation. The supernatant was again removed, and the pellet was resuspended in PBS using 10% of the initial starting volume. This suspension was diluted in DPBS (1:40 for

TauRD(LM)-YFP cells, 1 : 10 for Tau3R(VM)-YFP cells, and 1 :4 for

Tau3R4R-YFP cells) prior to incubating with Lipofectamine 2000 and OptiMEM.

[0082] Crude brain homogenate was diluted 1 :40 in DPBS (for Tau4R(LM)-YFP cells) prior to incubating with Lipofectamine 2000 and OptiMEM.

Clone 9 Infection Assay

[0083] TauRD(LM)-YFP and Tau4R(LM)-YFP cells were plated at a density of 4 x

105 cells/well in a 6-well plate (Costar). Cells were incubated at 37 °C for 2-A h to allow adherence to the plate. Clone 9 lysate was diluted in DPBS (1.5 μg/μL) and incubated in Lipofectamine 2000 (final concentration of 2% for TauRD(LM)-YFP cells and 1.2% for Tau4R(LM)-YFP cells) for 1.5 h. OptiMEM was added to the samples (78% of final concentration for TauRD(LM)-YFP cells and 78.8% of final concentration for Tau4R(LM)-YFP cells). Plates were then incubated at 37 °C in a humidified atmosphere of 5% C02 for 3 d before collecting ly sates.

Production of Cell Lysate

[0084] Cell lysates were prepared by incubating washed, confluent cells in cold radioimmunoprecipitation assay (RIP A) buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% DOC, 0.1% SDS) containing cOmplete EDTA-free protease inhibitor cocktail (Roche) for 10 min on ice. Cells were then collected using a cell scraper (Celltreat). Using a 22G needle and syringe (BD), lysate was produced by drawing and expelling the collected cells through the needle 10 times. The lysate was centrifuged at 3,000 x g for 10 min. The supernatant was collected, and the protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce).

Immunoblotting

[0085] Cell lysates were diluted to a concentration of 1 mg/mL in DPBS and were then combined with 4x NuPAGE loading buffer (final concentration of lx; Thermo Fisher) and lOx reducing agent (final concentration lx; Thermo Fisher), boiled for 10 min, and loaded onto a 4-12% Novex bis-tris gel (Thermo Fisher). SDS/PAGE was performed using MES buffer (Thermo Fisher). Gels were transferred to a PVDF membrane (Thermo Fisher) using a wet transfer system. The membrane was blocked for 30 min in blocking buffer [5% (wt/vol) nonfat milk in lx Tris-buffered saline containing 0.05% (vol/vol) Tween 20 (TBST)] and incubated with primary antibody overnight at 4 °C. GFP conjugated to horseradish peroxidase primary antibody (Santa Cruz) was used at 1:1,000. The membrane was washed three times with lx TBST before developing with enhanced chemiluminescent detection (GE

Healthcare) and exposing to X-ray film. The membrane was stripped with Restore Western Blot Stripping Buffer (Thermo Fisher), blocked for 30 min in blocking buffer, and reprobed with primary antibody overnight at 4°C. Primary antibody for vinculin (Abeam), the protein loading control, was used at 1:10,000). The membrane was washed three times with lx TBST, incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1: 10,000; Bio-Rad) for 1 h, and washed a final time in lx TBST. Western blot analysis was then completed as described above. Quantification of Western blot band intensity was done in ImageJ software (NIH).

Statistical Analysis

[0086] Cell infection data are presented as mean + SD. Values represent averages of five images collected from each well of a 384-well plate. Technical replicates for each patient sample were averaged across six wells. Statistical comparisons between control and diseased patient samples were performed using a mixed effects model on data in their original scale. A mixed effects model was used to incorporate correlation among fluorescence per cell measurements collected from the same patient sample (technical replicates), as opposed to other statistical models that assume technical replicates are independent measures. Data were kept in the original scale because the data are distributed approximately normal for most measures, in contrast to other distributional assumptions that would require a data transformation, such as a log-normal distribution. Statistical comparisons between the two brain regions in the three CTE patient samples were done using a Student's t-test with unequal variance. Statistical significance for all tests was determined with a P value < 0.05.

Propagation of 4R tauopathies in cultured cells requires expression of 4R tau

[0087] An enhanced cellular assay to quantify tau prions in PSP patient samples is described in (Woerman AL, et al. (2015) Propagation of prions causing

synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A.

112:E4949-E4958.) based on initial studies using HEK293 cells expressing the TauRD(LM)-YFP fusion protein developed by Marc Diamond and colleagues (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288.). They demonstrated that tau prions (both synthetic and natural) induced protein aggregation in the cells after 12 d of incubation. The TauRD(LM)-YFP cell assay was modified to a 384-well-plate format and performed live-cell imaging after incubating the cells for 4 d with PSP patient samples (Woerman AL, et al. (2015) Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 112:Ε4949-Ε4958.)· To improve the robustness of the assay, we precipitated tau prions with sodium phosphotungstate (PTA), which selectively isolates aggregated proteins, including PrPSc, tau, and a-synuclein, from soluble proteins (Woerman AL, et al. (2015) Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 112:E4949-E4958. Safar J, et al. (1998) Eight prion strains have PrPSc molecules with different conformations. Nat. Med. 4: 1157-1165.) . Adapting this automated and high-throughput approach, we increased the percentage of cells infected with aggregates from ~4 to -61%, greatly enhancing the dynamic range of the assay.

[0088] Using this experimental design, we tested a number of patient samples, including samples from control (n = 6), PiD (n = 6), AD (n = 7), CTE (n = 5), AGD (n = 2), CBD (n = 5), and PSP (n = 6) patients (Fig. 7). Control samples were from patients devoid of any detectable neuropathological lesions upon autopsy. Tauopathy patient samples were selected from the prototypical 3R tauopathy PiD; the 4R tauopathies AGD, CBD, and PSP; and two mixed tauopathies, AD and CTE. The brain regions sampled for each patient are listed in Table S2.

[0089] A 10% (wt/vol) brain homogenate from each patient sample was prepared in

Dulbecco's phosphate-buffered saline (DPBS) before digesting the sample in 2% (vol/vol) sarkosyl and 0.5% (vol/vol) benzonase. Importantly, the addition of benzonase resulted in the digestion of all nucleic acids in the sample, leaving only protein, which we then incubated with 2% (vol/vol) PTA overnight before pelleting by centrifugation. The resulting pellets were diluted 1:40 in DPBS and incubated with TauRD(LM)-YFP cells for 4 d in the presence of Lipofectamine 2000 to increase the efficiency of protein uptake. The live cells were imaged using the IN Cell Analyzer 6000, collecting DAPI and FITC images from five regions in each of six technical replicate wells per sample. Images were then analyzed for the presence of YFP-positive aggregates. Previous quantification of infection measured the percentage of cells containing aggregates. However, to improve the window size of the assay, infection was measured by normalizing the total fluorescence of aggregates in each FITC image by the cell count (DxA/cell). This measurement was calculated across all five images from each well; the average and standard deviation were then determined for the six replicate wells. The control samples did not infect the TauRD(LM)- YFP cells (average fluorescence per cell measurement of 1.6 + 0.2 x 10³ A.U.), whereas tau prions isolated from PSP patient samples robustly induced aggregate formation (74 + 25 x 10³ A.U.; P < 0.001 ; Fig. 7). Initial publication of the TauRD(LM)-YFP cells found tau prions from PiD, AD, AGD, and CBD patient samples infected the cells (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288). However, while we found that we could propagate AGD (22 + 10 x 10³ A.U.; P < 0.05) and CBD (39 + 11 x 10³ A.U.; P < 0.001) prions, neither PiD (3.6 + 1.5 x 10³ A.U.; P = 0.74) nor AD (9.5 + 2.8 x 10³ A.U.; P = 0.17) patient samples produced a substantive infection. In addition, incubation with CTE patient samples was also unable to yield a robust infection in the TauRD(LM)-YFP cells (6.7 + 2.5 x 10³ A.U.; = 0.41).

[0090] Strikingly, of the samples tested here, only the 4R tauopathies yielded a robust infection in the cells. The TauRD(LM)-YFP cells express the RD of 4R tau, suggesting that propagation of tau prions in these cells is dependent upon the presence of 4R tau prions in the disease. Visual comparison of the cells infected with AGD, CBD, or PSP shows distinct aggregate phenotypes (Fig. 7B), consistent with previous reports (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288.). While AGD-induced aggregates were diffuse, CBD aggregates presented as small punctate throughout the cytoplasm. In contrast, PSP induced large, bright puncta. These phenotypic differences between the diseases are reflected in the quantification of the infection; the larger and brighter the aggregates, the greater the fluorescence measurement (Fig. 7A).

PiD prions are 3R tau specific

[0091] Our finding that only pure 4R tauopathies propagate in cells expressing 4R tau led us to hypothesize that the propagation of 3R tau prions requires the presence of 3R tau. To address this question, we developed HEK293T cells that express the RD of 3R tau with the familial L266V and V337M mutations fused to YFP

(Tau3R(VM)-YFP). We diluted the PTA-precipitated patient samples 1: 10 in DPBS and incubated them with the 3R-expressing cell line for 4 d (Fig. 8). The control patient samples did not infect the Tau3R(VM)-YFP cells (average fluorescence per cell measurement of 3.2 + 1.1 x 10³ A.U.); however, tau prions isolated from the PiD patient samples propagated in the cells (42 + 21 x 10³ A.U.; P < 0.001). Supporting the hypothesis that tau prions are isoform- specific, the AGD (1.9 + 1.0 x 10³ A.U.; P = 0.81), CBD (5.3 + 2.2 x 10³ A.U.; P = 0.67), and PSP (9.4 + 3.4 x 10³ A.U.; P = 0.14) patient samples, which infected the 4R tau cells, did not yield robust infection. Consistent with the TauRD(LM)-YFP results, neither the AD (3.0 + 0.7 x 10³ A.U.; P = 0.94) nor the CTE (9.0 + 2.7 x 10³ A.U.; P = 0.18) patient samples infected the cells. Importantly, these results indicate that the notable aggregates in the

Tau3R(VM)-YFP cells following incubation with PiD samples, but not the other tauopathies (Fig. 8B), arise from the propagation of 3R-tau-specific prions isolated from PiD patient samples.

Identification of AD and CTE prions

[0092] Discovering that the propagation of 3R and 4R tauopathies in mammalian cells is isoform- specific, we posited that cells expressing both 3R and 4R tau isoforms could be infected by PiD samples, as well as by AGD, CBD, and PSP extracts. Testing this hypothesis, we developed HEK293T cells expressing both the Tau4R(LM)-YFP construct expressed in the 4R cells and the Tau3R(VM)-YFP construct expressed in the 3R cells (Tau3R4R-YFP). After isolating tau prions from the patient samples via PTA precipitation, the samples were diluted 1:4 in DPBS and incubated with the Tau3R4R-YFP cells for 4 d (Fig. 9). Incubating the cells with the control patient samples yielded no infection (average fluorescence per cell of 5.3 + 1.0 x 10³ A.U.; Fig. 9A; Table S2). However, because both the 3R and 4R tau isoforms were available as substrates for prion templating, not only did the PiD samples induce aggregates (15 + 3.8 x 10³ A.U.; P = 0.07), but the AGD (25 + 2.2 x 10³ A.U.; P < 0.01), CBD (32 + 9.4 x 10³ A.U.; P < 0.001), and PSP (28 + 7.7 x 10³ A.U.; P < 0.001) patient samples infected the cells as well. Interestingly, the PiD-induced tau aggregates are visibly smaller than the aggregates induced by the 4R tauopathies (Fig. 9B). The smaller aggregates yield a lower fluorescence value, reflected in the quantification of infection (Fig. 9A), and likely the lack of statistical significance. These results are reminiscent of earlier studies in which the replication of distinct PrP^Sc strains in Tg mice expressing both mouse and hamster PrP^c was dependent upon inoculation of either mouse or hamster PrP^Sc, demonstrating homotypic seeding of PrP prions (Prusiner SB, et al. (1990) Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63:673-686.)·

[0093] We also hypothesized that co-expressing both the 3R and 4R isoforms in the cells would support infection by the mixed tauopathies (AD and CTE). When we incubated the Tau3R4R-YFP cells with AD (15 + 4.0 x 10³ A.U.; P = 0.05) and CTE (47 + 24 x 10³ A.U.; P < 0.001) patient samples, we found that the samples induced tau aggregates after 4 d (Fig. 9). Infection with the AD patient samples was highly reproducible with low variability, but the quantification of the infection was not significant. It is important to note that a result of expressing both 3R and 4R fusion proteins is that YFP expression is increased relative to each individual fusion proteins. One limitation that arises from this approach is a reduction in the window size of the assay. The impact of this limitation can be clearly seen by the higher background of the cells incubated with control patient samples, and it may be influencing our ability to statistically separate the PiD and AD patient samples from the control samples, despite the presence of aggregates in the cells (Fig. 9B).

[0094] Remarkably, infection of Tau3R4R-YFP cells across the five CTE patient samples was highly variable compared to the five other groups. Neuropathological assessment of the CTE patients produced a diagnosis of CTE stage IV for all five patients, suggesting that this variability may be attributable to differences in sampling from patients with a disease that is highly focal in nature (Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4-1-4-11.).

Cell assay sensitivity is enhanced by over expression of the 4R tau fusion protein

[0095] Quantification of tau-prion infection in the Tau3R4R-YFP cells overall showed a reduced window size and therefore decreased infectivity of the pure tauopathies. Positing that reduced expression of the individual isoforms, compared to the TauRD(LM)-YFP and Tau3R(VM)-YFP cells, was responsible for decreasing susceptibility to infection, we tested the relationship between protein expression and sensitivity by developing a new HEK293T cell line with increased expression of the 4R RD and again containing the P301L and V337M mutations (Tau4R(LM) - YFP) . Comparing the TauRD(LM)-YFP cells with the new Tau4R(LM)-YFP cells, we examined expression of the fusion protein in the presence and absence of infection (Fig. 10A & B). Lysate from Clone 9 cells, which are TauRD(LM)-YFP cells that stably propagate infection with synthetic tau prions, has previously been shown to induce aggregate formation in TauRD(LM)-YFP cells (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288.)· Here, we found that in both the presence and absence of infection with Clone 9 lysate, expression of the fusion protein is higher in the new Tau4R(LM)-YFP cells compared to the original TauRD(LM)-YFP cells, as visualized by Western blot analysis (Fig. 10A) and quantified in ImageJ software (Fig. 10B).

[0096] We then tested the susceptibility of the Tau4R(LM)-YFP cells to infection with PiD, AGD, CBD, and PSP prions (Fig. IOC and D). Although we previously used PTA to precipitate and concentrate tau prions before infecting the

TauRD(LM)-YFP cells, we instead incubated crude brain homogenate diluted 1 :40 in DPBS with the cells for 4 d. Surprisingly, we found that crude homogenate alone was capable of transmitting AGD (150 + 140 x 10³ A.U.; P < 0.001), CBD (55 + 24 x 10³ A.U.; P < 0.05), and PSP (140 + 74 x 10³ A.U.; P < 0.001) prions to the new cell line, whereas the control samples showed no infection (2.0 + 0.9 x 10³ A.U.; Fig. IOC; Table S2. Visual assessment of the infected cells (Fig. 10D) shows a similar result as seen in the TauRD(LM)-YFP cells (Fig. 7B); infection with AGD, CBD, and PSP samples induced cellular aggregates with distinct phenotypes. AGD-induced aggregates were large and round, whereas PSP prions induced tau aggregation throughout the cytoplasm of the cells but not in the dimmer nuclei. Compared to these larger aggregates, infection with CBD yielded a mixture of phenotypes, but the aggregates were predominantly smaller and dimmer punctate. This phenotypic difference ultimately contributes to variations in fluorescence values measured from the different patient groups (Fig. IOC) and is a likely explanation for a lack of significance following infection with CBD patient samples.

[0097] Intriguingly, when we incubated the Tau4R(LM)-YFP cells with the PiD patient samples, we found one patient sample induced robust aggregate formation, one sample induced weak infection, and the other four showed very low infectivity (20 ± 21 x 10³ A.U.; P = 0.41; Fig. IOC; Table S2). It is important to note that although PiD has traditionally been classified as a 3R tauopathy, based upon the initial discovery of 3R tau only in Pick's bodies (Delacourte A, et al. (1996) Specific pathological Tau protein variants characterize Pick's disease. J. Neuropathol. Exp. Neurol. 55: 159-168.), patients with a mixture of 3R and 4R tau accumulation in cortical gray and white matter have been identified (Zhukareva V, et al. (2002) Sporadic Pick's disease: a tauopathy characterized by a spectrum of pathological τ isoforms in gray and white matter. Ann. Neurol. 51:730-739.). It is possible that the increased sensitivity of the Tau4R(LM)-YFP cells facilitated detection of 4R tau aggregates in Pick's disease, as exemplified by the two patient samples shown here. These results suggest that we may now be better able to assess 4R tau accumulation in PiD using our assay, improving our overall understanding of tau prion propagation in this patient group.

Over expression of 4R tau supports propagation of AD and CTE prions

[0098] Following our observation that the Tau4R(LM)-YFP cells enabled detection of 4R tau in two of the PiD patient samples tested, which were previously unidentifiable, we decided to test whether or not overexpression of 4R tau also facilitates propagation of AD and CTE prions. After incubating crude brain homogenate from AD and CTE patients diluted 1 :40 in DPBS with Tau4R(LM)-YFP cells for 4 d, the cells were imaged and analyzed for infection. Remarkably, the increased sensitivity of the new HEK293T cells enabled infection with both AD (37 ± 20 x 10³ A.U.; P = 0.1) and CTE (49 + 34 x 10³ A.U.; P < 0.005) patient samples, demonstrating the presence of tau prions in AD and CTE patients (Fig. 11A and B). These results, combined with the successful infection of the Tau3R4R-YFP cells, suggest that the progressive nature of AD and CTE is due to the spread of tau prions throughout the CNS.

[0099] To demonstrate the specificity of these findings, we tested samples from two different brain regions on the Tau4R(LM)-YFP cells. As mentioned previously, CTE is characterized by focal deposits of tau tangles, typically originating in the depths of cortical sulci before spreading to additional brain regions (Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of

neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4-1-4-11.). In three of the five CTE patients (CTE1, CTE2, and CTE3), we sampled both the temporal and frontal poles (Fig. 11C and D). Although infectivity of both brain regions for patient CTE1 was poor, the sulcus from the temporal pole tested from CTE patients 2 and 3 contained significantly more tau prions than the sulcus from the frontal pole that was tested (P < 0.05). Post-mortem analysis of these three patients found accumulation of NFTs in both the frontal and temporal poles. Our finding here that tau prions are highly localized within patient samples argues that the standardization of tissue collection, analysis, and sampling is necessary to improve ongoing research into the pathogenesis of CTE.

Findings

[00100] The findings presented here demonstrate that tau prion propagation is

dependent on the tau isoform(s) comprising each prion strain. Traditionally, categorization of tauopathies has been based on the tau isoform(s) identified in specific neuropathologies, but our findings reveal that such classifications are also representative of the prion strains giving rise to each disease. The propagation of each of these strains requires a specific substrate, whether it is 3R, 4R, or both isoforms of tau. AGD, CBD, and PSP infect the 4R-containing cell lines

TauRD(LM)-YFP, Tau3R4R-YFP, and Tau4R(LM)-YFP. Similarly, PiD prions infect both the Tau3R(VM)-YFP and Tau3R4R-YFP cell lines because the 3R isoform is available as substrate. Importantly, both AD and CTE, which are composed of 3R and 4R tau aggregates, were originally found only to propagate in the Tau3R4R-YFP cell line, suggesting that the strain(s) of tau prions giving rise to these two diseases requires both the 3R and 4R isoforms. Unexpectedly, increasing the expression level of the 4R tau isoform in the Tau4R(LM)-YFP cells facilitated propagation not only of AGD, CBD, and PSP, but also of AD and CTE. The biological mechanism by which this occurs remains to be elucidated.

[00101] An expanding wealth of evidence argues that the formation of NFTs is one consequence of tau misfolding. This conformational change self-propagates, resulting in the accumulation of β-sheet-rich tau filaments and the spread of NFTs from neuron to neuron. By quantifying the aggregation of mutant tau fragments fused to YFP, we report that brain extracts from both AD and CTE patients infect mammalian cells, providing evidence that both patient samples contain tau prions. Importantly, this finding implies that tau is not inert but rather is biologically active, facilitating self-propagation throughout the CNS. As such, tau prions are important drug targets in the discovery of neurotherapeutics for these fatal disorders.

[00102] Similar to PrP^Sc and multiple system atrophy (MSA) prions, which coalesce into amyloid plaques and glial cytoplasmic inclusions, respectively, tau also acquires an alternative conformation that is enriched for β-sheet structure in fibrils that form straight and paired helical filaments ( Kidd M (1963) Paired helical filaments in electron microscopy in Alzheimer's disease. Nature 197: 192-193., Prusiner SB, et al. (1983) Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35:349-358., Prusiner SB, et al. (2015) Evidence for a-synuclein prions causing multiple system atrophy in humans with signs of Parkinson's disease. Proc. Natl. Acad. Sci. U.S.A. 112:E5308-E5317.) Measuring the kinetics of PrP^Sc and MSA propagation following intracerebral injection into Tg mice has been important in discerning the underlying biology of these prions. However, while intracerebral injection of tau fibrils into Tg mice expressing tau transgenes has resulted in tau neuropathology, such experiments have been inconclusive with respect to assessing the kinetics and accumulation of tau prions ( Clavaguera F, et al. (2013) Brain homogenates from human tauopathies induce tau inclusions in mouse brain. Proc. Natl. Acad. Sci. USA 110:9535-9540.). As performed here, a more informative bioassay for these measurements employs a fusion protein, which combines a mutated tau fragment with YFP, expressed in cultured mammalian cells (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288., Woerman AL, et al. (2015)

Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A. 112:E4949-E4958., Kfoury N, Holmes BB, Jiang H, Holtzman DM, & Diamond MI (2012) Trans-cellular propagation of Tau aggregation by fibrillar species. /. Biol. Chem. 287: 19440-19451.).

[00103] As described by us and others (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288.), six different neurodegenerative diseases, termed tauopathies, are characterized by the replication and spread of tau aggregates. Our findings argue that groups of tau prion strains responsible for each disease are influenced by the tau isoforms present— i.e., 3R tau in PiD; 4R tau in AGD, CBD, and PSP; and a combination of both 3R and 4R in AD and CTE. Although the isoform specificity in the tauopathies was previously established (Schmidt ML, Zhukareva V, Newell KL, Lee VM, & Trojanowski JQ (2001) Tau isoform profile and phosphorylation state in dementia pugilistica recapitulate Alzheimer's disease. Acta Neuropathol.

101 :518-524., Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, & Hof PR (2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Brain Res. Rev. 33:95-130.), the discovery that the isoforms play an integral role in the formation of tau prion strains adds another layer of complexity to these diseases and provides an explanation for the range of phenotypes seen in patients. New molecular probes based on these differences are needed for the development of clinical diagnostics. Our research here shows that it is possible to discriminate between various tau prion strains, although additional studies are needed to understand how these strains arise and maintain specificity.

[00104] Previous work with synthetic tau prions provides additional support for the isoform-directed specificity of tau prion replication. Using the recombinant K18 and K19 constructs, which encode the 4R and 3R RDs of tau, respectively, Dinkel et al. (Dinkel PD, Siddiqua A, Huynh H, Shah M, & Margittai M (2011) Variations in filament conformation dictate seeding barrier between three- and four-repeat tau. Biochemistry 50:4330-4336.) demonstrated that heparin-induced K19 aggregates could not seed filaments formed from K18 monomers. In a separate experiment, recombinant 1N3R or 1N4R human tau fibrils were used to test the cross-isoform prion replication barrier in the neuroblastoma cell line SH-SY5Y (Nonaka T, Watanabe ST, Iwatsubo T, & Hasegawa M (2010) Seeded aggregation and toxicity of a-synuclein and tau: cellular models of neurodegenerative diseases. /. Biol. Chem. 285:34885-34898.). Transient expression of either 1N3R or 1N4R tau was initiated 14 h prior to exposing the cells to the tau fibrils. When the authors attempted to infect the cells with the 1N4R fibrils, only the cells expressing 4R tau developed aggregates. Similarly, the 1N3R fibrils only propagated in the 3R-expressing cells, demonstrating isoform specificity in synthetic prions.

[00105] In addition to these in vitro findings, several in vivo studies have

demonstrated homotypic seeding of tau. Clavaguera and colleagues used two similar Tg mouse models, P301S (human 0N4R tau with the P301S mutation) and ALZ17 (wild-type human 2N4R tau), to demonstrate that inoculating diseased P301S mouse brain extracts into ALZ17 mice induced NFTs, neuropil threads, and coiled bodies by self-templating the 4R tau transgene (Clavaguera F, et al. (2009) Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11:909-913.)· Using the same P301S model, other groups found that brain extracts prepared from aged, symptomatic animals could be inoculated into 2-month-old pre-symptomatic mice to induce NFT pathology within 2 weeks of inoculation, demonstrating rapid propagation of 4R tau prions (Ahmed Z, et al. (2014) A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity. Acta Neuropathol.

127:667-683.). Similarly, recombinant human 2N4R tau with the P301S mutation (T40/PS) and K18 with the P301Lmutation (K18/PL) were fibrillized in the presence of heparin and inoculated into young PS 19 mice (human 1N4R tau with the P301S mutation) (Yoshiyama Y, et al. (2007) Synapse loss and microglial activation precede tangles in a P301S tauopathy mouse model. Neuron 53:337-351.). Both of these 4R-containing fibrils induced tau neuropathology in the animals, whereas inoculation of a-synuclein fibrils had no effect, highlighting the homotypic propagation of synthetic 4R tau prions in PS19 mice (Iba M, et al. (2013) Synthetic tau fibrils mediate transmission of neurofibrillary tangles in a transgenic mouse model of Alzheimer's-like tauopathy. /. Neurosci. 33: 1024-1037.).

[00106] This body of work is reminiscent of earlier studies with Tg mice expressing Syrian hamster (SHa) PrP (Prusiner SB, et al. (1990) Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63:673-686.). These animals expressed both mouse (MoPrP^c) and SHaPrP^c, and were therefore able to propagate either MoPrP^Sc or SHaPrP^Sc depending on the inoculum chosen; MoPrP^Sc prions stimulated the formation of nascent MoPrP^Sc prions, whereas inoculated SHaPrP^Sc prions initiated the formation of de novo SHaPrP^Sc prions. These results demonstrated that homotypic seeding is compatible with prion replication, but heterotypic templating is inefficient, as seen by the inability of SHaPrP^Sc to template MoPrP^c.

[00107] Importantly, the results presented here demonstrate that the propagation of AD and CTE involves the conversion of both the 3R and 4R tau isoforms into prions. In contrast, AGD, CBD, and PSP tau prions are composed of 4R tau exclusively, as demonstrated by both SDS-PAGE Western blotting of human brain extracts (Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, & Hof PR (2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Brain Res. Rev. 33:95-130) and HEK cell bioassays (Sanders DW, et al. (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82: 1271-1288., Woerman AL, et al. (2015) Propagation of prions causing synucleinopathies in cultured cells. Proc. Natl. Acad. Sci. U.S.A.

112:E4949-E4958.). Tau prions in PiD typically consist of only 3R tau, as shown by SDS-PAGE (Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, & Hof PR (2000) Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Brain Res. Rev. 33:95-130.) and the HEK293T cell bioassays described here. However, astrocytic 4R tau aggregates have been identified in PiD patient samples (Zhukareva V, et al. (2002) Sporadic Pick's disease: a tauopathy characterized by a spectrum of pathological τ isoforms in gray and white matter. Ann. Neurol. 51:730-739.) and were likely detected in the Tau4R(LM)-YFP cells.

The initial finding that both 3R and 4R tau are required to propagate AD and CTE prions in HEK293T cells, and the presence of tau aggregates containing both 3R and 4R isoforms in AD and CTE patient samples (Schmidt ML, Zhukareva V, Newell KL, Lee VM, & Trojanowski JQ (2001) Tau isoform profile and phosphorylation state in dementia pugilistica recapitulate Alzheimer's disease. Acta Neuropathol. 101:518-524.) suggests AD and CTE prion strains are misfolded in a conformation that incorporates both tau isoforms. However, the propagation of AD and CTE prions in the Tau4R(LM)-YFP cells seemingly contradicts our initial conclusion, and suggests that tau misfolding may involve distinct 3R and 4R conformations. The strongest argument against this interpretation is the inability of the samples to infect the Tau3R(VM)-YFP and TauRD(LM)-YFP cells. If AD and CTE samples contained distinct 3R and 4R aggregates, those prions should propagate in the respective cell lines, based upon our findings with PiD, AGD, CBD, and PSP A second explanation could be small amounts of isolated 4R aggregates along with the mixed 3R/4R tau aggregates typically associated with the diseases. Due to its greater sensitivity, only the Tau4R(LM)-YFP cells were able to propagate the minor 4R component of the total aggregated tau, compared to the TauRD(LM)-YFP cells. One possible source of distinct 4R aggregates in these samples is aging-related tau astroglipathy (ARTAG), which is selectively immunostained with 4R tau antibodies, even in PiD patients (Kovacs GG, et al. (2016) Aging-related tau astrogliopathy (ART AG): harmonized evaluation strategy. Acta Neuropathol. 131:87-102.).

Consistent with this finding, two PiD patient samples infected the more sensitive Tau4R(LM)-YFP cells, suggesting the propagation of AD and CTE prions in the same cell line may arise from the presence of ARTAG in the brain regions sampled, as well. However, astrocytic inclusions are more commonly associated with CTE than AD, and the two patient groups overall showed similar infectivity in the Tau4R(LM)-YFP cells.

[00109] A third explanation of our seemingly disparate findings may lie in the kinetic differences between the two RDs. In vitro analysis of the aggregation propensity of the 6 tau isoforms, the 4R isoforms have a higher rate of aggregation than the 3R isoforms; the 4R fibrils showed a decrease in the rate of tau fibril nucleation, and a higher rate of fibril extension (Zhong Q, Congdon EE, Nagaraja HN, & Kuret J (2012) Tau isoform composition influences rate and extent of filament formation. /. Biol. Chem. 287:20711-20719.). This work suggests 4R tau is intrinsically more aggregation-prone than 3R tau. Recognizing this important difference, it is plausible that the high expression of 4R tau in the Tau4R(LM)-YFP cells is seeded by the 4R component of the tau prions in AD and CTE, which then continue to propagate. Certainly the ongoing work to develop the [18FJAV-1451 PET tracer into a diagnostic tool for tauopathy patients is consistent with the idea that AD prions are comprised of both 3R and 4R tau, rather than the presence of distinct 3R and 4R aggregates in patients. Reportedly, [18FJAV-1451 selectively binds to pathologic tau in AD patient samples compared to PSP and PiD patient samples (Gomez F, Lin Y-G, Liang Q, Mintun M, & Attardo G (2016) Quantitative assessment of

[18FJAV-1451 distribution in AD, PSP and PiD post-mortem brain tissue sections relative to that of the anti-tau antibody AT8. /. Nucl. Med. 57:348.), suggesting a conformation difference may exist between tau in AD versus tau in the pure tauopathies. Our conclusion, that AD prions contain both 3R and 4R tau isoforms, whereas PSP and PiD contain either 4R or 3R, respectively, provides one possible explanation for the initial results reported for [18FJAV-1451.

[00110] Multiple lines of evidence argue that Αβ undergoes a conformational change that initiates the pathogenesis of AD, and many investigators argue that Αβ accumulation begins decades before the clinical onset of dementia. Our work and that of others argues that the Αβ peptide can misfold into an Αβ prion that can spread through the CNS (Ridley RM, Baker HF, Windle CP, & Cummings RM (2006) Very long term studies of the seeding of beta-amyloidosis in primates. /. Neural Transm. 113: 1243-1251 ; Meyer- Luehmann M, et al. (2006) Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host. Science 313: 1781-1784; Stohr J, et al. (2012) Purified and synthetic Alzheimer's amyloid beta (Αβ) prions. Proc. Natl. Acad. Sci. U.S.A. 109: 11025-11030; Stohr J, et al. (2014) Distinct synthetic Αβ prion strains producing different amyloid deposits in bigenic mice. Proc. Natl. Acad. Sci. U.S.A. 111: 10329-10334; Watts JC, et al. (2014) Serial propagation of distinct strains of Αβ prions from Alzheimer's disease patients. Proc. Natl. Acad. Sci. U.S.A. 111 : 10323-10328). Αβ prions not only polymerize into fibrils that coalesce into plaques, but also may facilitate the conversion of tau proteins into prions, as suggested by the amyloid cascade hypothesis (Hardy J & Allsop D (1991) Amyloid deposition as the central event in the aetiology of Alzheimer's disease. Trends Pharmacol. Sci. 12:383-388.). Molecular genetics research has shown that the majority of fAD patients harbor mutations in APP or the presenilin genes (Weggen S & Beher D (2012) Molecular consequences of amyloid precursor protein and presenilin mutations causing autosomal-dominant Alzheimer's disease. Alzheimers Res. Ther. 4:9.). These missense mutations demonstrate that fAD cases are caused by mutant Αβ peptides or by increased levels of Αβ. In contrast, mutations in the tau gene, MAPT, do not cause fAD; instead, tau mutations cause familial FTDs, including inherited PiD, AGD, CBD, and PSP (Lee VM, Goedert M, & Trojanowski JQ (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24: 1121-1159.). These genetic studies have been crucial in separating AD, considered a secondary tauopathy, from the primary tauopathies.

We and others have proposed that tau prions polymerize into paired-helical and straight filaments that form NFTs in AD patients. Work by others has demonstrated that NFTs correlate with dementia, and the absence of NFTs is generally accompanied by normal cognition (Jansen WJ, et al. (2015) Prevalence of cerebral amyloid pathology in persons without dementia: a meta-analysis. JAMA 313: 1924-1938., 8. Serrano-Pozo A, et al. (2016) Thai amyloid stages do not significantly impact the correlation between neuropathological change and cognition in the Alzheimer disease continuum. J. Neuropathol. Exp. Neurol. 75:516-526., Hof PR et al. (1992) Differential distribution of neurofibrillary tangles in the cerebral cortex of dementia pugilistica and Alzheimer's disease cases. Acta Neuropathol. 85:23-30.)· Our studies establish that biologically active tau prions are found in AD patient samples, and the spreading of these tau prions may be responsible for the progressive dementia seen in patients.

In contrast to AD, the tau prions identified in CTE patient samples likely arise as a result of repetitive mild traumatic brain injury (TBI). CTE, first defined as "punch drunk" (Martland HS (1928) Punch drunk. /. Am. Med. Assoc.

91: 1103-1107.), has been increasingly diagnosed in amateur and professional athletes in contact sports, garnering wide attention from studies of demented professional football players in the United States. In addition, many soldiers who suffer mild TBIs later develop post-traumatic stress disorders and progressive dementia. Many studies argue that these signature injuries of the recent conflicts in Afghanistan and Iraq, are the result of exposure to blast waves from improvised explosive devices (Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4-1-4-11., Terrio H, et al. (2009) Traumatic brain injury screening: preliminary findings in a US Army Brigade Combat Team. /. Head Trauma Rehabil. 24: 14-23.). In the early stages of CTE, focal tau pathology is present near the vasculature within the depths of the cerebral sulci, but as the disease progresses, tau deposition spreads throughout the amygdala, hippocampus, and majority of the cerebral cortex (Stein TD, Alvarez VE, & McKee AC (2014) Chronic traumatic encephalopathy: a spectrum of

neuropathological changes following repetitive brain trauma in athletes and military personnel. Alzheimers Res. Ther. 6:4-1-4-11.). Similar to AD, biochemical analyses of NFTs isolated from CTE patients have shown that these aggregates consist of both 3R and 4R tau isoforms (Schmidt ML, Zhukareva V, Newell KL, Lee VM, & Trojanowski JQ (2001) Tau isoform profile and phosphorylation state in dementia pugilistica recapitulate Alzheimer's disease. Acta Neuropathol. 101:518-524.). Although tau pathology in AD patients is localized to neurons, astrocytic tangles can be prominent in CTE patients (McKee AC, et al. (2013) The spectrum of disease in chronic traumatic encephalopathy. Brain 136:43-64.). Additionally, the location of NFTs in the cortical layers also differs between AD and CTE patients. AD patients often develop NFTs in both superficial and deeper cortical layers, with the highest density seen in layers V-VI. On the other hand, CTE patients typically develop NFTs in the more superficial layers II— III of the neocortex. Despite these regional differences, the hippocampus is similarly affected by both diseases (Hof PR, et al. (1992) Differential distribution of neurofibrillary tangles in the cerebral cortex of dementia pugilistica and Alzheimer's disease cases. Acta Neuropathol. 85:23-30.), which is likely a major contributor to the progressive dementia seen in these patients.

[00113] In summary, the findings presented here use HEK cells expressing mutant tau fragments fused to YFP to demonstrate that tau prions isolated from patients with tauopathies, including AD and CTE, can infect mammalian cells. Notably, we used a panel of cell lines to show that the tau isoforms identified in the neuropathological lesions associated with each tauopathy play a critical role in the formation and propagation of tau prion strains. While these results provide critical insight into the pathogenesis of each tauopathy, the involvement of 3R and 4R tau isoforms in the propagation of AD and CTE suggests a complex mechanism of prion replication, where heterodimers interact through a process, as yet undefined, to produce different strains of tau prions causing these two diseases. Most importantly, the identification of biologically active tau prions in all of the patient samples tested here suggests that developing successful anti-tau neurotherapeutics will require inhibiting the ability of tau prion strains to propagate.

[00114] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMS That which is claimed is:

1. A cell line, comprising cells stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform.

2. The cell line of claim 1, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

3. The cell line of any of claims 1 and 2, wherein the cells are human cells.

4. The cell line of any of claims 1-3, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells, U373 cells, and LUHMES cells.

5. The cell line of any of claims 1-4, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

6. The cell line of any of claims 1-5, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

7. The cell line of claim 5, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags.

8. The cell line of claim 5, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein.

9. The cell line of claim 8, wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

10. The cell line of claim 5, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

11. The cell line of claim 10, wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

12. The cell line of claim 1 , wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein .

13. The cell line of claim 12, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

14. A method of detecting a form of a tau protein in a sample wherein the tau protein form is associated with a neurological disease, the method comprising:

(b) adding a sample to the cell line; and

(c) observing the presence of the detectable reporter molecule as an indication of the presence of a tau protein form in the sample.

15. The method of claim 14, wherein step (a) comprises growing a plurality of different cell lines in a plurality of different containers wherein different cell lines in the different containers are transfected with different nucleotide sequences which encode different repeat domains of a 4R tau isoform and different nucleotide sequence encoding repeat domains of different 3R tau isoforms;

wherein step (b) comprises adding a sample to the different containers; and wherein step (c) comprises observing the presence of a detectable reporter molecule as an indication of the presence of particular tau isoform(s) in the biological sample.

16. The method of claim 14, wherein the sample is selected from the group consisting of human blood serum and human cerebral spinal fluid.

17. The method of claim 14, wherein the tau protein form is associated with a neurological disease selected from the group consisting of Alzheimer's disease, chronic traumatic encephalopathy, Pick's disease, argyrophilic grain disease, corticobasal degeneration, progressive supranuclear palsy, globular glial tauopathy, NFT dementia, tau astrogliopathy in elderly, and post-traumatic stress disorder.

18. The method of claim 14, wherein the tau protein form is associated with Alzheimer's disease.

19. The method of claim 14, wherein the sample is a biological sample selected from the group consisting of brain tissue, blood, serum, cerebral spinal fluid, olfactory epithelium, nasal or sinus tissue, saliva, urine, submandibular gland, liver, lymph node, and skin; and

20. The method of any of claims 14-18, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

21. The method of any claim 14-19, wherein the cells are human cells.

22. The method of any of claims 14-20, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells.

23. The method of any of claims 14-22, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

24. The method of any of claims 14-23, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent or luminescent protein and epitope tags.

25. The method of any of claims 14-24, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

26. The method of any of claims 14-25, wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

27. The method of claim 22, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

28. The method of claim 27, wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

29. The method of claim 27, wherein the epitope tag is a Myc-tag.

30. The method of any of claims 14-29, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

31. The method of claim 30, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

32. A method of testing a compound, comprising:

adding a form of tau protein and a compound to the cell line; and observing for the presence of the detectable reporter molecule.

33. The method of claim 32, further comprising:

comparing observed presence of the detectable reporter molecule to a known standard.

34. The method of any of claims 32 and 33, further comprising:

determining an ability of the compound to hinder formation of a form of tau protein aggregate based on a difference between the known standard and the observed presence of the detectable reporter molecule.

35. The method of any of claims 32-34, wherein the known standard is a substantially identical cell line to which the compound has not been added.

36. The method of any of claims 32-35, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

37. The method of any of claims 32-36, wherein the cells are human cells.

38. The method of any of claims 32-37, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells.

39. The method of any of claims 32-38, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

40. The method of any of claims 32-39, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

41. The method of any of claims 32-40, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags.

42. The method of claim 32, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein.

43. The method of claim 42, wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

44. The method of claim 32, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

45. The method of claim 44, wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

46. The method of claim 44, wherein the epitope tag is a Myc-tag.

47. The method of any of claims 32-46, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

48. The method of any of claims 32-47, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

49. A method of determining progress of a neurological disease in an individual, comprising:

(e) quantifying the detectable label in (d); and

(f) comparing the quantifying in (c) with the quantifying in (e) to determine progress of neurological disease in the individual.

50. The method of claim 49, wherein a plurality of different sequences encoding a plurality of different reporter molecules are fused to the sequences (i) and (ii).

51. The method of any of claims 49-50, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

52. The method of any of claims 49-51, wherein the expression of the different reporter molecules is related to a different form of a tau protein wherein the tau protein is associated with a neurological disease.

53. The method of any of claims 49-52, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

54. The method of any of claims 49-53, wherein the cells are human cells.

55. The method of any of claims 49-54, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells.

56. The method of any of claims 49-55, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

57. The method of any of claims 49-56, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent or luminescent protein and epitope tags.

58. The method of any of claims 49-57, wherein the detectable reporter molecule is selected from the group consisting of a luminescent protein and a fluorescent protein.

59. The method of claim 58, wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

60. The method of any of claims 49-56, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

61. The method of claim 60, wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

62. The method of any of claims 49-61, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

63. The method of any of claims 49-62, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

64. A method of determining characteristics of a neurological disease affecting an individual, comprising:

(f) making a diagnosis of a type of neurological disease based on the comparing step (e).

65. The method of claim 64, wherein a plurality of different sequences encoding a plurality of different reporter molecules are fused to the sequences (i) and (ii).

66. The method of any of claims 64-65, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

67. The method of any of claims 64-66, wherein the expression of the different reporter molecules is related to a different form of a tau protein wherein the tau protein is associated with a neurological disease.

68. The method of any of claims 64-67, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

69. The method of any of claims 64-68, wherein the cells are human cells.

70. The method of any of claims 64-69, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells U373 cells, and LUHMES cells.

71. The method of any of claims 64-70, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

72. The method of any of claims 64-71, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags.

73. The method of any of claims 64-71, wherein the detectable reporter molecule is selected from the group consisting of a luminescent protein and fluorescent protein.

74. The method of claim 73, wherein the fluorescent protein or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

75. The method of any of claims 64-71, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

76. The method of claim 75 wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

77. The method of any of claims 64-76, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

78. The method of any of claims 64-77, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

79. An assay kit, comprising:

a container holding the cells; and

80. The assay kit of claim 79, wherein the cells are stably transfected with (a) a nucleotide sequence encoding repeat domains of a 4R tau isoform; and (b) a nucleotide sequence encoding repeat domains of a 3R tau isoform.

81. The assay kit of claim 80, wherein the cells are transfected with (a) a nucleotide sequence encoding a 4R tau isoform; and (b) a nucleotide sequence encoding a 3R tau isoform.

82. The assay kit of any of claims 80 and 81, wherein the cells are human cells.

83. The assay kit of claim 79, wherein the cells are selected from the group consisting of embryonic kidney cells (HEK293, HEK293A, HEK293T), H4 cells, SH-SY5Y cells, NT2 cells, IMR32 cells, HeLa cells, U373 cells, and LUHMES cells.

84. The assay kit of any of claims 80-83, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a sequence encoding a detectable reporter molecule.

85. The assay kit of any of claims 80-84, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to a plurality of sequences encoding a plurality of detectable reporter molecules.

86. The assay kit of any one of claims 80-85, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein, luminescent protein and epitope tags.

87. The assay kit of any one of claims 80-85, wherein the detectable reporter molecule is selected from the group consisting of a fluorescent protein and a luminescent protein.

88. The assay kit of claim 87, wherein the fluorescent or luminescent protein is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, red fluorescent protein, mCherry, tdTomato, Far-red fluorescent protein, orange fluorescent protein, and luciferase.

89. The assay kit of any one of claims 80-85, wherein the detectable reporter molecule is an epitope tag which is recognized by a high affinity antibody.

90. The assay kit of claim 89, wherein the epitope tag is selected from the group consisting of Myc-tag, V5-tag, HA-tag, E-tag, AviTag, FLAG -tag, SPB-tag, S-tag, His-tag, polyglutamate tag, calmodulin tag, Softag, Strep-tag, TC tag, VSV-tag, and Xpress tag.

91. The assay kit of claim 79, wherein the sequences encoding the repeat domain of the 4R tau isoform and the repeat domain of the 3R tau isoform are fused to sequences encoding complementary fragments, said fragments generate a detectable reporter molecule when one fragment is brought into contact with its complementary partner protein.

92. The assay kit of claim 91, wherein the complementary fragments are split luciferase fragments, split green fluorescent protein fragments, split dihydrofolate reductase fragments, split beta-galactosidase fragments, or split beta-lactamase fragments.

93. The assay kit of any one of claims 79-92, further comprising:

94. The assay kit of any one of claims 79-92, wherein the container holds a plurality of cells stably transfected with a different sequence encoding a different tau protein associated with a different neurological disease, and