Genome-wide association study and functional validation implicates JADE1 in tauopathy

Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-β (Aβ) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aβ toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.


Introduction
Primary age-related tauopathy (PART) is nearly ubiquitously observed with varying degrees of severity in the brains of aged individuals characterized by the presence of neurofibrillary tangles (NFT) composed of abnormal aggregates of tau protein. These NFTs are regionally, morphologically, biochemically and ultrastructurally identical to those in early to moderate stage Alzheimer disease (AD), yet develops in the absence of Aβ plaques 1 . A neuropathological diagnosis of PART is separate from a clinical diagnosis of cognitive impairment; hence individuals with PART can be normal, mildly cognitively impaired (MCI) or have dementia 2,3 . Most individuals with PART remain cognitively normal, however some develop amnestic cognitive changes [4][5][6][7] . The similarities between PART and AD allows for a unique opportunity to focus specifically on mechanisms of tau-mediated AD-type neurodegeneration. The neuropathological diagnosis of PART is complicated by accompanying age-related comorbid dementing pathologies, therefore discovering unique molecular drivers is challenging antemortem [8][9][10][11][12][13] . Given the similarities between PART and AD NFTs, PART as a diagnostic construct would have greater value if it were shown to arise independently 14,15 . Alternatively, PART might be a component of the AD spectrum, and Aβ pathology might have eventually developed in such individuals had they lived longer 15,16 . Thus, the question remains as to what extent a neuropathological diagnosis of PART diverges from or has similar risk factors to AD and other dementias 17,18 .
Much of the mechanistic knowledge surrounding tauopathy stems from genetic studies 19 . Autosomal dominant mutations in the microtubule-associated protein tau gene (MAPT) in coding regions can interfere with microtubule binding or promote transition to toxic forms. Also, mutations in splice sites disrupt alternative pre-mRNA splicing of the tau mRNA transcript, modifying the ratio of three repeat (3R) and four repeat (4R) tau isoforms leading to downstream pathological sequelae 20 . Additionally, alternative splicing of the N-terminal exons may also play a role in modulating toxic tau 21,22 . However, MAPT mutations are rare, whereas PART occurs sporadically and nearly ubiquitously with advanced age. Thus, PART provides a unique opportunity to understand common genetic drivers of tauopathy and their association with abnormalities in tau proteostasis and isoform expression. Prior research focusing on common genetic variation has identified two distinct haplotypes in the MAPT 17q21.31 locus defined by a large ~900kb inversion region that gives rise to two major haplotypes, H1 and H2. The more common H1 haplotype has was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Outside of PART, one of the largest AD GWAS has identified 29 risk loci implicating 215 potential causative genes 32 . However, it has been suggested that the overwhelmingly strong signal on the APOE locus could mask associations independently related to tauopathy 23 . Another study examining candidate genes in aging cohorts confirmed the lack of the association between APOE ε 4 and PART, as well as a decreased frequency of AD risk alleles in the BIN1, PTK2B, and CR1 loci 33 . Taken together, these data suggest an unexplored genetic risk driving tauopathy that might be revealed by conducting genome-wide association studies in PART.
While PART represents a compelling opportunity to focus on amyloid-independent mechanisms of neurofibrillary degeneration, assembling an autopsy cohort large enough to assesses its genetics on a genome-wide scale has not yet been attempted. In collaboration with twenty-one domestic and international brain biorepositories, we performed the first genome-wide association study in PART and compared our findings to known tauopathy risk loci. We then localized expression of candidate genes in our strongest risk locus (chromosome 4q28.2) using single cell RNA-sequencing and immunohistochemistry. Finally, we validated our findings biochemically in human postmortem brain and functionally using an in vivo Drosophila model. The work presented here not only furthers the investigation of the genetics of PART, but also suggests a novel role for JADE1 in tauopathy.
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Cohort
Fresh-frozen brain tissue was obtained from the contributing centers (Supplementary Table 1). All tissue was used in accordance with the relevant guidelines and regulations of the respective institutions. The neuropathological assessments were performed at the respective centers using standardized criteria outlined by the National Institutes on Aging-Alzheimer's Association (NIA-AA) and National Alzheimer Coordinating Center (NACC) which has a high degree of interrater reliability among centers 34,35 . Inclusion criteria were individuals with normal cognition, mild cognitive impairment (any type) and dementia. Cognitive status was determined either premortem or postmortem by a clinical chart review, mini-mental score, or clinical dementia rating 36,37 . Both sexes were included and ages ranged from 51 to 108 years at death.
Neuropathological inclusion criteria were Braak tangle stage of 0-IV and neuritic amyloid plaque severity CERAD score of 0 38,39 . In addition, tissue sections were obtained and reevaluated by the study investigators to confirm the lack of amyloid and degree of PART tau pathology 40

Genotyping
High-throughput isolation of DNA was performed using the MagMAX DNA Multi-Sample Ultra 2.0 Kit on a KingFisher Flex robotic DNA isolation system (Thermofisher, Waltham, MA). Fresh frozen cortical brain tissue (20-40 mg) was placed into a deep-well plate and treated with 480 µl of Proteinase K mix (Proteinase K, phosphate buffered saline [pH 7.4], Binding Enhancer) and incubated overnight at 65°C at 800 rpm on a shaking plate. Genomic DNA was isolated and purified using magnetic particles. DNA quality control was performed using a nanodrop spectrophotometer (concentration > 50ng/µl, 260/280 ratio 1.7-2.2).
Genotyping was performed using single nucleotide polymorphism (SNP) microarrays (Infinium Global was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Genetic analysis PLINK v1.9 was used to perform quality control 42 . SNP exclusion criteria included minor allele frequency <1%, genotyping call-rate filter less then 95%, and Hardy-Weinberg threshold of 1×10 - 6 43 . Individuals with discordant sex, non-European ancestry, genotyping failure of >5%, or relatedness of >0.1 were excluded. A principal component analysis (PCA) was performed to identify population substructure using EIGENSTRAT and the 1000 genomes reference panel 44,45 . Samples were excluded if they were five standard deviations away from the European population cluster ( Supplementary Fig. 4). PCA was performed again on the genomic data with non-Europeans excluded to generate new PCs, of which the first four were used as covariates in the regression model. All data was imputed on the University of Michigan server using minimac3 and HRC reference panel 46,47 . Imputed variants with MAF <0.01 and a dosage R 2 <0.7 were excluded. Downstream analyses were based on the most likely genotype. A quantitative trait association test was run on 647 PART cases vs. Gaussian-normalized Braak stage using conditional linear regression and age, sex, principal component 1-4 and SNP chip array as covariates. The analysis was run separately on each genotyping array and a meta-analysis was performed using METAL 48 . Regional genome-wide association plots were created with LocusZoom, other plots were created using R 49 .

Single-cell mRNA profiling in tangle-containing neurons
Identification of differentially expressed genes in single neuronal somata with and without NFTs was performed by analyzing a transcriptomic dataset of isolated neurons from post-mortem human brain from individuals with and without AD reported by Otero-Garcia et. al. 2021 50 . This dataset consists of single neuronal soma transcriptomes from Brodmann area 9 subjected to fluorescence-activated cell sorting (FACS) using p-tau (AT8) and MAP2 antisera to differentiate single NFT-positive and NFT-negative cells.
Immunohistochemistry was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
For slides that were doubled-labeled DAB and alkaline phosphatase were used for visualization. Slides were stained with antibodies to JADE1 (1:100, Proteintech, Rosemont, IL) and hyperphosphorylated tau (ptau, AT8, 1:1000, Invitrogen, Waltham, MA). To ensure specificity of the JADE1 antisera, a peptide competition was performed using a blocking peptide. The antisera and paired peptide were pre-incubated for 24 hr. Whole slide images (WSI) were visualized and scanned using an Aperio CS2 digital slide scanner (Leica Biosystems, Wetzlar Germany). In addition to PART cases, neuropathologically confirmed cases of AD, PSP, CTE, CBD, argyrophilic grain disease (AGD) and Pick disease (PiD) (n=3 for each, Supplemental table 6) were examined for convergent or divergent staining patterns.

Biochemical analysis
Western blots were performed using homogenized fresh-frozen brain tissue. Samples were placed in a Horseradish peroxidase-labeled secondary anti-rabbit antisera (1:20,000; Vector Labs, Burlingame, CA) was detected by SuperSignal West Pico PLUS Chemiluminescent Substrate or Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). To quantify and standardize protein levels, GAPDH was detected with GAPDH antisera (6C5, 1:20,000; Abcam, Cambridge, MA) and total protein was detected with Amido Black (Sigma-Aldrich, St. Louis, MO) as previously described 51 . Chemiluminescence was measured in a ChemiDoc Imaging System (Bio-Rad), and relative optical densities were determined by using AlphaEaseFC software, version 4.0.1 (Alpha Innotech, San Jose, CA), normalized to GAPDH and total protein loaded.
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The copyright holder for this preprint (which this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.30.450599 doi: bioRxiv preprint Co-immunoprecipitation (IP) assays were performed using fresh-frozen brain tissue was homogenized in a glass-Teflon homogenizer at 500 rpm in 10 volumes (wt/vol) of ice-cold lysis buffer containing 50 mM Tris, pH 7.8, 0.5 % NP40, 150 mM NaCl, 1 mM EDTA, and Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Samples were incubated on ice for 30 min, centrifuged at 1000 x g for 10 min and the supernatant was collected as an input and used for immunoprecipitation. In a microcentrifuge tube, 70 ul of supernatant, Lysis buffer (930 µl) and 2 µg of either JADE1 antisera or anti-0N tau antisera (EPR21726, Abcam, Cambridge, MA) were combined and incubated overnight at 4 °C. Two controls were also set up, one without the antisera and the other with 2 µg of IgG isotype control antisera, either normal rabbit IgG (PeproTech, Rocky Hill, NJ) or Mouse IgG1 kappa, (clone: P3.6.2.8.1, Thermo Fisher Scientific). Twenty µl of Pierce Protein A/G Agarose beads (Thermo Fisher Scientific) was added to each reaction, and the mixture was incubated for 1 hr at 4 °C. Agarose beads were pelleted at 1000 x g for 5 min at 4 °C, supernatant was removed, 1 ml of ice-cold lysis buffer was added, was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

In vivo Drosophila model
Drosophila stocks, crosses, and aging were performed at 25 °C for the duration of the experiment and an equal number of male and female flies were used for each experiment. The GAL4-UAS expression system and the pan-neuronal elav-GAL4 driver were used to control transgenic human tau and rno expression.
Analyses were run on four fly groups (Bloomington stock rno RNAi line number 57774): elav-GAL4 driver in the heterozygous state (control, elav-GAL4/+), elav-Gal4 positive plus rno RNAi positive group (control + was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. rno RNAi , elav-GAL4/+;UAS-rno RNAi /+), elav-Gal4 positive transgenic human UAS-tau R406W tau group (0N4R Tau, elav-GAL4/+;UAS-Tau R406W ), elav-Gal4 positive human transgenic UAS-tau R406W tau plus rno RNAi positive group (0N4R Tau + rno RNAi , elav-GAL4/+;UAS-Tau R406W /UAS-rno RNAi ). An additional rno RNAi lines were used but was did not produce progeny when crossed to tau transgenic Drosophila, as often occurs with strong enhancers (Bloomington stock rno RNAi line number 62880). Flies were aged to 10 days, at which point terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay was performed in the fly brain (n=6 per genotype) and a blinded assessment of the fly eye phenotype was performed (n=16 total). TUNEL was performed on 4 μ m formalin-fixed paraffin-embedded fly heads. Quantification of TUNELpositive nuclei was performed throughout the entire brain using DAB-based detection and bright field microscopy. Fly eye phenotype scoring was performed blindly using light microscopy (n=16). The blinded rater semi-quantitively evaluated the eye for four distinct qualities including roughness, size, shape and conical shape on a 1 to 5 scale (five being the most severe phenotype) and an average summary score was calculated.

Statistical analysis
For GWAS, statistical analysis was performed in PLINK and our genome wide significance value was < 5 × 10 -8 , which is Bonferroni-corrected for all the independent common SNPs across the human genome.
Genome wide suggestive significant value was set at < 5 × 10 -6 . All other statistical analyses were performed in R. For non-normally distributed data a Wilcox test was used to test for significance, and an ANOVA was used for normally distributed data.
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Results
We assembled the first cohort of individuals with primary age-related tauopathy (PART) in wh individuals were neuropathologically confirmed to be devoid of any neuritic plaques. Autopsy brain samples (n=647) were obtained from 21 brain banks. While each center had performed a compreh neurodegeneration workup, we restained and reassessed these cases for PART pathology as a com of our ongoing histological studies 40 . Neurofibrillary tangles (NFTs) were assessed using the Braak system which ranged from 0-IV with all stages well represented, as is consistent with PART (Table   average age Fig. 1a). Lastly, we investigated the effect of age of de Braak stage with respect to cognitive status and found a positive correlation that does not sign change when cognitive status is accounted for in the model ( Supplementary Fig. 1a,b). In summa cohort consists of primarily older individuals, with a range of clinical cognitive symptoms, as well as a spectrum of regional tau distribution, demonstrating the diversity of both the clinical and neuropatho features of the condition.
Using this cohort, we ran a quantitative trait association analysis across the entire genome to novel genetic risk loci in PART. Using Braak stage as a quantitative trait revealed a genom associated signal on chromosome 4q28.2 (Fig. 1a, Fig. 2a). The genome wide significant variant (rs56405341) has a mino frequency of 0.27. The locusZoom plot indicates our significant SNP is not directly in an intron specific gene, but near C4orf33, SCLT1 and JADE1 (Figure 1b). We also observed in our regional p the lead SNP has a total of 22 other SNPs in Linkage Disequilibrium (r 2 > 0. 8  otomized upporting s ratio (p was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
We also examined candidate SNPs previously found to be associated with AD and prog supranuclear palsy (PSP) in prior GWAS studies to explore convergent and divergent genetic risk (T Supplementary table 5). Of the 52 candidates investigated, we found five associated with PART ( SLC   MS4A6A, HS3ST1, MAPT, and EIF2AK3). rs12590654, which is associated with SLC24A4, had the significance level (p=0.001). rs1582763, rs2081545, rs7935829, were all associated with MS4A6A ( 0.01, 0.02 respectively). The remaining AD SNP, rs7657553, was associated with HS3ST1 (p=0.0 found two variants that overlapped with PSP risk. rs242557 (p=0.02) in the MAPT locus, and rs7 (p=0.03) in the EIF2AK3 locus. In summary, seven of the 52 probed risk AD and PSP SNPs significant associations (p<0.05) in PART.
Next, we refocused on our strongest association at the 4q28.2 locus. Examination o expression quantitative trait loci (eQTL) using the Brain-eMeta eQTL summary data (derived fr datasets; Genotype-Tissue Expression v6, the CommonMind Consortium, Religious Orders Stu Memory and Aging Project, the Brain eQTL Almanac project, the Architecture of Gene Expressio eQTLGen) did not contain significant SNP associated eQTLs for any of the genes in the 4q28.2 lo Because our GWAS quantitative trait was specific to tau pathology, we then examined mRNA exp levels of all the genes contained in the locus using a novel single-cell soma RNA sequencing datase measured transcriptomic changes specifically in tangle-bearing neurons and non-tangle-bearing n ( Fig. 2a-c). Using this dataset, we found that tangle-bearing excitatory neurons significantly differ expressed JADE1 compared to non-tangle-bearing excitatory neurons (p=1.04×10 -61 ). Conversely C and SCLT1 had low levels of expression regardless of cell type and tangle status. Additionally, we ob an upregulation of JADE1 expression in tangle-bearing inhibitory neurons however it was stat ggest the ogressive (  The copyright holder for this preprint (which this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.30.450599 doi: bioRxiv preprint underpowered due to low cell abundance and high variance. Furthermore, we observed two subpopulations of excitatory neurons in which JADE1 was significantly differentially expressed (adjusted p=4.55×10 -15 ,

7.82×10 -8 ). We then examined the relative average expression and percentage of cells expressing JADE1
and observed increases in both metrics in tangle-containing neuronal populations compared to non-tanglecontaining neuronal populations (Fig. 2d-f). SCLT1 and C4orf33 had nominal expression levels in a substantially smaller percentage of neurons. Taken together, these data suggest increased JADE1 expression is unique to specific populations of neurons which also contain NFTs. Because we did not observe significant differences in C4orf33 or SCLT1 expression across the two groups, and the signals were very low, these data suggest that JADE1 as the strongest candidate gene in the locus.
Given our evidence that JADE1 is genetically and transcriptionally associated with NFT pathology, we conducted an immunohistochemical study using specific antisera to JADE1 in our collection of postmortem tauopathy brain tissue (Fig. 3). We assessed tauopathies that are known to involve preferentially tau isoforms with 3 microtubule-binding domain repeats (3R), 4 microtubule-binding domain repeats (4R) or a mixture of the two. We found strong and specific JADE1 immunopositivity in structures morphologically indicative of mature aggregate containing intracellular NFT in not only PART, but also the other mixed 3R/4R tauopathies (i.e., AD and chronic traumatic encephalopathy , Fig 3a-f). NFT pathology in PSP, corticobasal degeneration (CBD), and argyrophilic grain disease (AGD) were also immunopositive for JADE1 ( Fig. 3g-l). Notably, gliofibrillary pathology in these diseases (i.e., aging-related tau astrogliopathy, tufted astrocytes, and astroglial plaques) were also immunopositive. Surprisingly, no signal was detected in NFT in Pick disease (PiD), a predominately 3R tauopathy (Fig. 3m,n). Double labeling experiments showed that early pre-NFT were negative for JADE1 suggesting that this factor begins to coalesce into NFT at the transition to the aggregate stage (Fig. 3o). To ensure the antibody was specifically targeting the JADE1 peptide and not binding non-specifically to NFT pathology, we blocked the JADE1 antibody and did not observe any staining in neurons that had the morphological features of an NFT (Fig. 3p). These findings indicate that JADE1 protein expression is localized specifically to cells with mature NFTs. Furthermore, JADE1 upregulation does not occur in PiD, the only 3R tauopathy examined, suggesting isoform specificity of expression. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. We then biochemically examined protein expression of JADE1 by western blotting using crude lysates derived from the entorhinal cortex and hippocampus proper (cornu ammonis 1-4 and dentate gyrus) of PART and AD individuals. JADE1 exists as 2 isoforms, JADE1 long (JADE1L) and JADE1 short (JADE1S), both of which contain proline, glutamic acid, serine, threonine (PEST) domains and 2 PHD fingers. However, the long form is 333 amino acid sequences greater in length and contains an additional PEST domain as well as a nuclear localization signal (Fig 4a). A Western blot analysis revealed a strong signal in both brain regions and diseases for JADE1S, but no bands were observed in the expected weight for JADE1L, indicating the observed signal is specific to the short isoform of JADE1 (Fig. 4b). This signal is in agreement with the immunohistochemical data given we did not observe a nuclear JADE1 signal which would suggest JADE1L, the form containing the nuclear localization signal, was also expressed.
Furthermore, cytoplasmic colocalization of JADE1 and NFTs immunohistochemically raises a possibility that they form a functional complex in tauopathy brains. To examine this, co-immunoprecipitation (IP) was performed using crude brain lysate from PART individuals as the input. We first IPed using JADE1 antisera and observed a banding pattern that suggests JADE1 pulls down tau near the molecular weight of the 0N4R isoform (Fig. 4 c). To confirm these results, we reverse co-IPed JADE1 with 0N tau antisera and observed a banding pattern indicating the JADE1S isoform was pulled down (Fig. 4d). To investigate the observed JADE1 immunoprecipitated lower banding pattern (Fig. 4c), we treated this form with protein phosphatase over time and observed a shift over time to the expected 58 kDa weight (Fig. 4e), indicating JADE1 antisera was not able to recognize the more abundant native phosphorylated JADE1S and instead a low abundant species of the protein. This data suggests the predominant form of the JADE1S input is likely being biologically modified (i.e., phosphorylation, acetylation, etc.). Staining with C-terminal isoform-specific anti-tau antibodies (3R and 4R tau) revealed that the co-immunoprecipitated tau was predominantly 4R, thus 0N4R tau (Fig. 4f,g). We then ran western blots for the IPed JADE1 using multiple phospho-tau sitespecific antibodies including RZ3 (pThr231), PHF1 (pThr181), CP13 (Ser202), AT8 (Ser202, pThr205), S214 (Ser214) and PG5 (Ser409) of which multiple phosphor tau epitopes showed prominent reactivity (Fig.   4h). Lastly, to confirm protein-protein interactions of Jade1S and 0N tau, proximity-ligation assays (PLA) were performed in fixed hippocampus from PART individuals. The PLA technique utilizes one pair of oligonucleotide-labeled antibodies that detects different epitopes of the two proteins in close proximity was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.30.450599 doi: bioRxiv preprint (maximum 30-40 nm apart). The prominent red fluorescence signal surrounding the nuclei of a neuron indicates the close proximity of these two proteins (Fig. 4i), whereas control samples lacking one of the two antisera did not show any signal ( Supplementary Fig. 7e,f). In summary, the biochemical data strongly indicates a JADE1S, 0N4R tau immunocomplex.
Lastly, we asked whether JADE1 plays a functional role in tau-induced neurotoxicity. We used a Drosophila model that overexpresses human mutant 0N4R tau 55 as well as RNAi-mediated reduction of rhinoceros (rno), the highest matched JADE1 human ortholog in Drosophila. We first blindly evaluated the fly eye phenotype using a semi-quantitative assessment of eye size, roughness, overall shape, and conical shape and saw a significant increase in eye severity between tau transgenic Drosophila and tau transgenic Drosophila with rno knockdown (Fig. 5a-e, p=8.7 ×10 -5 ). We did not observe significant differences between rno rnai and control group in the absence of transgenic tau. To directly quantify neurodegeneration in the Drosophila brain, we quantified TUNEL-positive cells throughout the Drosophila brain. We find that rno knockdown significantly enhances neurotoxicity in tau transgenic Drosophila but is not sufficient to induce neurotoxicity in control flies based on TUNEL staining (p=0.008, Fig. 5f-i). These data provide in vivo evidence that JADE1/rno loss plays a mechanistic role in promoting neurotoxicity in tauopathy, and suggest that proper functioning of JADE1/rno is protective was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Discussion
Genome-wide association studies (GWAS) have enabled advances in our understanding of sporadic tauopathies 19,[56][57][58][59][60][61] . Yet, in the context of the growing numbers of genes associated with Alzheimer disease (AD) 32 , direct links with tau proteinopathy have been challenging to pinpoint as association signals show minimal overlap with factors classically implicated in tauopathy (e.g., proteostasis, tau protein kinases, etc.). This is not surprising given the ubiquity and heterogeneity of tau and other pathological changes in the aging human brain that are variably associated with cognitive impairment, which is the phenotype in most genetic studies despite the fact that it is a non-specific trait. We performed an autopsy-based GWAS, which minimizes classification errors and other issues with clinical studies of dementia, assembling the largest cohort of post-mortem brain tissues from aged individuals devoid of amyloid pathology with a goal of identifying factors independently associated with primary age-related tauopathy (PART). In doing so, we sought to provide genetic evidence that might clarify the controversial relationship between PART and AD, which it closely resembles neuropathologically. We failed to find an association with APOE ε4, the strongest common risk allele for sporadic late-onset AD. We did, however, find associations with other candidate loci in AD and progressive supranuclear palsy (SLC24A4, MS4A6A, HS3ST1, MAPT and EIF2AK3), as well as a novel genome-wide significant association at the chromosome 4q28.2 locus. Our data indicate that among genes in this locus, only the gene for apoptosis and differentiation in epithelia 1 (JADE1), a member of a small protein family that serves as a multifunctional adaptor implicated in renal and other cancers [62][63][64] , is upregulated in tangle-bearing neurons on both the mRNA and protein levels. This accumulation of JADE1 protein in NFT is not specific to PART but occurs in AD and all tauopathies with accumulation of 4R isoforms, but not in Pick disease which is a 3R tauopathy, indicating that this is generally a shared feature.
We also show that JADE1 binds 0N4R tau, an isoform proposed to be a critical driver of tau pathology 65,66 .
Finally, experiments in Drosophila show that reducing expression of the JADE1 homolog rhinoceros (rno) exacerbates tau-induced neurotoxicity in vivo. Together, these findings strongly argue that JADE1 is a factor broadly capable of protecting neurons from neurofibrillary cell death that links PART to the tauopathic component of AD.
We confirm that the genetics of PART has a partial overlap with sporadic late-onset AD and replicated the consistent finding showing the lack of a signal in the APOE locus despite its strong was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.30.450599 doi: bioRxiv preprint association with AD 32,67-70 . It has been shown previously that PART individuals have a higher APOE 2 allele frequency which distinguishes PART neuropathologically and genetically from AD [71][72][73] . We have reported the frequency of the APOE ε 4 allele is lower in PART 28 and other studies have revealed similar findings in independent cohorts 6,33 . It should be noted that these values can fail to reach significance when cross comparing groups with varying degrees of neuritic plaque pathology 33,74 . Recent studies in mice and humans have indicated that the APOE ε 4 allele may exacerbate tau pathology independently of Aβ plaques 75 , however other human studies failed to show an interaction 76,77 . These results add to the strong evidence that PART is entirely independent of APOE ε 4 regardless of amyloid plaque pathology.
17q21.31 MAPT locus is the strongest genetic risk factor for PSP 26 , which we and others had previously reported is also associated with PART 28,31 . The MAPT H1 haplotype has also been associated with AD 78-80 , however this region has a complex haplotype structure and may be more important in specific AD subgroups given the modest signal and variable findings in these association studies 81,82 . Intriguingly, in one AD GWAS using clinically ascertained individuals, removal of APOE ε 4 carriers enhanced signals in the 17q21.31 locus 23 . In the present study, there was only a modest association of MAPT with PART. This result may stem from differences in cohort selection with previous studies focusing on extremes, while we included a range of pathological severity, especially mildly affected individuals. Together, these data highlight that further investigation of the role of 17q21.31 MAPT locus in PART is warranted.
With regards to genes other than APOE and MAPT, we found four additional association signals in PART that overlap with either AD or PSP. Eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) encodes an endoplasmic reticulum (ER) membrane protein critical for the unfolded protein response (UPR) 83,84 . Activation of the UPR has been observed and positively correlated with tau pathology, but not with Aβ plaque burden, in the hippocampus of aged cognitively normal individuals 83 . Solute carrier family 24 member 4 (SLC24A4), a gene in the locus most strongly associated with PART and AD, is a member of the potassium-dependent sodium/calcium exchanger protein family and is involved in neural development, however little is known about its possible function in AD 85,86 . Additionally, we identified an association of PART with the membrane spanning 4-domains A6A (MS4A6A) locus, which contains the binding regions for the transcription factor PU.1 which is selectively expressed in brain microglia and macrophages 87 . The last overlapping genetic locus that contains heparan sulfate-glucosamine 3-sulfotransferase 1 (HS3ST1), has was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Taken together, these data are compatible with the hypothesis that the genes we identified in our GWAS are possible modulators of tau pathology.
Our study highlighted a novel locus on chromosome 4q28.2, that previously gave a suggestive signal in an independent autopsy GWAS in AD that also used Braak NFT stage as an endophenotype and a second GWAS focusing on dichotomized CSF Aβ positivity 53,90 . This prompted us to focus on this locus for further validation and functional studies. Because we failed to identify expression quantitative trait locus in both blood and brain datasets, we hypothesized that given our trait was tangle-specific, modulation of mRNA expression of genes in the locus might also be cell-type specific. This hypothesis was also motivated by the increase in genetic to transcriptomic associations found in cell specific populations in other contexts [91][92][93] . Our results indicate that of the genes in the 4q28.2 locus, only JADE1 mRNA was significantly and differently expressed in tangle-bearing neurons. Our immunohistochemical studies also showed JADE1 protein accumulation in both neuronal and glial tangle containing cells, validating these findings. Thus, JADE1 is most likely responsible for the GWAS signal at this locus.
Our immunohistochemical studies indicate that JADE1 is potentially involved broadly in tauopathies.
We observed immunopositivity not only in PART tangles, but also in tauopathies with aggregates containing 4R tau and mixed tauopathies with aggregates containing both 3R and 4R. The absence of staining in Pick disease, the only tauopathy with 3R tau aggregates examined, was surprising. Our biochemical studies suggest that JADE1 protein specifically interacts with 0N4R tau that is phosphorylated on epitopes known to be hyperphosphorylated in NFT. Our proximity ligation assay confirms the direct interaction between JADE1 protein and tau. Studies using cryo-EM and mass spectrometry have shown the ultrastructure of tau aggregates at unprecedented resolution, and it has been reported that 0N4R has a unique single β -sheet conformation for the fibril core [94][95][96] . Intriguingly, recent mass spectrometry profiling studies of human postmortem brain tissues have suggested that changes in 0N4R tau isoform specifically is an early event in tauopathy 97 . Double labeling experiments indicate that JADE1 increases shortly after the pre-tangle stage, accumulating alongside insoluble tau aggregates during the transition to the intercellular tangle stage perhaps reflecting a reactive/protective compensatory role 98 . was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 1, 2021. ; Our findings provide important clues as to how JADE1 may be functioning in tauopathy. JADE1 has been previously implicated as a renal tumor suppressor involved in apoptosis, well as inhibition of Wnt signaling by ubiquitylating β -catenin, and interactions with cell-cycle regulators 62,[99][100][101][102][103] . Of the two JADE1 isoforms, only the short form which lacks a nuclear localization signal was observed, which was consistent with the cytoplasmic localization of the protein we observed by immunohistochemistry. This prompted us to hypothesize that JADE1S functions in the cytoplasm to mediate tauopathy. Our in vivo studies in which we reduced rno (the closest JADE1 ortholog) levels in Drosophila overexpressing mutant human 0N4R tau significantly enhanced the tau-induced rough eye phenotype as well as TUNEL-positive cells, a marker of apoptotic DNA fragmentation. While JADE1 has been shown to promote apoptosis in some contexts, our RNAi knockdown experiments suggest that proper functioning of JADE1 may be neuroprotective.

Consistent with this, other studies have demonstrated that loss of rno function attenuates apoptosis in
Drosophila 104 . Because previous studies have found JADE1 to be stabilized by the von Hippel-Lindau tumor suppressor which is a component of an E3 ubiquitin-protein ligase activity, JADE1 may be working with 0N4R tau through a similar mechanism to promote ubiquitin-mediated clearance of tau [105][106][107] .
Our study has several notable limitations. Our sample size is small for GWAS standards; however, it should be noted that it is still the largest study of its kind. Given that PART is currently only reliably diagnosed postmortem, the study is limited by the availability of tissue that meet our strict criteria. Our study also relies on neuropathological assessments performed at multiple centers that may cause batch effects. While Braak staging is highly reproducible, with one report showing that across brains and raters the kappa score was greater than 0.90 108 , it is a semiquantitative (ordinal) variable. Further studies using a more quantitative approach to measuring tau burden that we have shown more closely align with functional clinical measures in PART may reveal additional candidates 109 . Additional follow-up studies in experimental models are necessary further to validate our findings.
In conclusion, therapies for AD in clinical trials are moving towards targeting tau due to the lack of clinical efficacy of Aβ modulating therapeutic approaches 110 . Here, by focusing on individuals with PART who lack Aβ plaques, we enriched our cohort for signals related to tau proteinopathy. Our analysis provides additional evidence that PART overlaps with but has considerable differences from AD. This interdisciplinary approach led to the identification of JADE1, which interacts with 0N4R tau, and is protective was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 1, 2021. ; https://doi.org/10.1101/2021.06.30.450599 doi: bioRxiv preprint in vivo. Thus, JADE1 is a potential novel biomarker that differentiates tauopathies. Further understanding the genetics of PART will provide pathways for rationally designed therapeutics for degenerative tauopathies. was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which this version posted July 1, 2021. ; (e,f) A ting status, was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.   was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.