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

Abstract

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.

Fig. 1

Genome-wide association study (GWAS) in primary age-related tauopathy. a Quantitative trait GWAS was performed using normalized Braak neurofibrillary tangle stage with age, sex, principal components (PCs), and genotyping SNP array as covariates (n = 647). The threshold for genome-wide significance (p < 5 × 10⁻⁸) is indicated by the solid grey line; the suggestive line (p < 5 × 10⁻⁶) is indicated by the dotted line. b LocusZoom plot shows a strong signal with multiple SNPs in linkage disequilibrium on chromosome 4q28.2. The x axis is the base pair position, and the y axis is the – log₁₀ of the p value for the association with Braak stage. The blue line represents the recombination rate. c Association between single-nucleotide polymorphism (SNP), rs56405341 and Braak tangle stage (adjusted for age and sex). Pairwise comparisons using Wilcoxon rank sum test, AA–AG p = 0.024, AA–GG p = 3.3 × 10⁻⁵, AG–GG p = 7.2 × 10⁻⁵

Fig. 2

Bulk and single-cell sequencing reveals JADE1 mRNA is modulated by rs56405341 and upregulated in tangle-bearing neurons. a Bulk RNA sequencing data yielded a significant lead SNP (rs56405341) eQTL (p = 0.038) in the dorsolateral prefrontal cortex of 452 postmortem human brain samples. Expression of JADE1 decreases with in subjects carrying the minor allele. Single cell sequencing data of populations of neurons with and without neurofibrillary tangles isolated from human post-mortem brain samples separated using fluorescence-activated cell sorting and transcriptomic profiles from single-cell RNA-sequencing were subsequently analyzed. b In 2 unique excitatory neuronal populations (Ex1 and Ex2) JADE1 mRNA was significantly differentially expressed in the tangle bearing neurons (adjusted p = 7.82 × 10⁻⁸, 4.55 × 10⁻¹⁵); in comparing the overall population of tangle-bearing excitatory neurons (Ex-total) to non-tangle bearing neurons the difference is highly significant (adjusted p = 1.04 × 10⁻⁶¹). c, d Other two genes in the locus, C4orf33 and SCLT1, were overall nominally expressed in both excitatory neuronal groups, as well as subclusters (Supplementary Fig. 5 b, c, online resource). e Dot plot showing average relative expression and percent expression of the candidate genes in the locus. Both JADE1 relative average expression and percentage of cells expressed was higher than C4orf33 and SCLT1. f, g t-Distributed stochastic neighbor embedding (tSNE) plots showing the different populations neurons, tangle bearing status, and relative expression of JADE1 in neuronal subpopulations

Fig. 3

Selective immunolabeling of tau aggregates containing tau with four microtubule-binding domain repeats (4R), but not three (3R), in post-mortem human tauopathy brains with antisera targeting JADE1 protein. Immunohistochemical staining with phospho-tau (p-tau) specific antisera (AT8) and JADE1 specific antisera demonstrates neurofibrillary tangle (NFT) formation marked by the presence of JADE1 in specific populations of neurons and glia. a, b Primary age-related tauopathy (PART, n = 3) NFTs contain JADE1 positive staining in the soma and neurites in the entorhinal cortex. c, d Alzheimer disease (AD, n = 3) individual with Aβ and AT8-positive neuritic plaques and NFTs (subiculum) also display JADE1 immunopositivity in dystrophic neurites and NFTs. e, f Chronic traumatic encephalopathy (CTE, n = 3) contains positive p-tau staining around a blood vessel in the depth of a neocortical sulcus that is immunopositive for JADE1. g, h AT8 positive tufted astrocytes, oligodendroglial coiled bodies, and NFTs are positive in the subthalamic nucleus in an individual with progressive supranuclear palsy (PSP, n = 3) which are also in immunopositive for JADE1. i, j Astrocytic plaques in corticobasal degeneration (CBD, n = 3) and extensive thread-like pathology positive for p-tau and JADE1 in the neocortex. k, l In the cornu ammonis 1 (CA1) sector in an individual with argyrophilic grain disease (AGD, n = 3), abundant grains that are immunopositive for p-tau and JADE1 are evident. m, n Pick disease (PiD), a 3R tauopathy, with Pick bodies in the dentate gyrus that are immunopositive for p-tau but negative for JADE1. o Double staining of a PART entorhinal cortex showing the absence of JADE1 (brown) staining in early pre-tangles, but the presence of p-tau (pink, see inset). p Negative control using a peptide competition despite the presence of a tangle (inset). Scale bar, 100 μm

Fig. 4

JADE1S protein interacts with tau containing four microtubule-binding domain repeats (4R) but not 3R in post-mortem human brain tissue (a) Schematic of the two JADE1 isoforms, JADE1S and JADE1L. b Representative immunoblot using antisera targeting JADE1 in entorhinal cortex and cornu ammonis in individuals with primary age-related tauopathy (PART) and Alzheimer disease (AD) shows JADE1S but not JADE1L at the expected molecular weight. GAPDH was used as a loading standard. c Immunoprecipitation using JADE1 antisera co-immunoprecipitates tau the with a molecular weight near the 0N4R isoform (40 kDa). d Reverse immunoprecipitation using 0N tau antisera co-immunoprecipitates the JADE1S isoform. e Pulled down form of JADE1S molecular weight shifts downward after treatment with lambda protein phosphatase. f, g Co-immunoprecipitated tau with JADE1 stained with C-terminal isoform specific anti-tau antisera are the 0N4R isoform and not the 3R isoform. h Co-immunoprecipitated tau was positively stained with a panel of phospho-tau specific antibodies with the most signal coming from pThr231 (RZ3), pSer396/pSer404 (PHF1), pSer214 (S214), and pSer409 (PG5). i Proximity–ligation assay showing positive fluorescence signal (red) around the nucleus of neurons (blue) indicating the close association between JADE1 and 0N tau detected using the corresponding two primary antibodies in the soma (inset). Scale bar, 20 μm

Fig. 5

RNAi-mediated knockdown of the JADE1 ortholog rhinoceros (rno) enhances tau-induced toxicity in Drosophila. a–e Scanning electron micrograph images showing rno^RNAi significantly enhances the tau-induced rough eye phenotype (size, roughness, shape, and conical shape, p = 8.7 × 10⁻⁵, n = 16 per genotype). Scale bar 100 μm. f–j Rno^RNAi significantly increases the number of terminal deoxynucleotidyl transferase dUTP nick end-labels (TUNEL) in tau transgenic Drosophila compared to tau expressed alone (p = 0.008, n = 6 per genotype). TUNEL was performed at day 10 of adulthood and quantified in the entire fly brain. Representative images are shown from the cortex. An equal number of male and female flies were used for each experiment