Reddy, Joseph S.

AbstractBackgroundProgressive supranuclear palsy (PSP) is a devastating primary tauopathy with rapid progression to death. Although several therapies currently in the development pipeline show promising safety profiles and robust target engagement, few demonstrated significant efficacy in patients, underscoring the need to interrogate additional targets with novel therapeutic modalities to expand the potential therapeutic arsenal. To diversify the therapeutic avenues for PSP and related tauopathies (e.g. Alzheimer’s disease), we systematically integrated multi‐omics data from human brains of PSP and control donors with cross‐species validation to nominate high‐confidence therapeutic targets. We plan to translate our findings into safe and effective treatments for PSP using antisense oligonucleotides (ASO).MethodWe analyzed brain gene expression profiles in PSP and control individuals at bulk tissue (N = 408) and single‐cell levels (snRNAseq, N = 36). Cell‐type‐specific expression perturbations were systematically prioritized using a cross‐species validation paradigm, including rTG4510 tau mice and tau‐overexpressing Drosophila models. We conducted in vitro screening of ASO candidates against our prioritized target genes. ASO efficacy and toxicity were measured. RNAseq experiments will be performed to interrogate off‐target effects. The safety and efficacy of the lead ASOs will be assessed in PSP‐patient‐derived iPSC models.ResultWe previously reported discovery of novel genes with significant differential expression in PSP brains, characterized their brain cell‐specificity and validated them using snRNAseq data. We prioritized 21 genes using gene expression data from a mouse tauopathy model. Validation of these 21 high‐priority genes using a Drosophila tau model nominated astrocytic STOM, KANK2, and DDR2 as potential therapeutic targets for PSP. Knocking down their expression in Drosophila significantly rescues tau‐mediated neurodegenerative pathology. In vitro screening identified ASO leads that reduced the target expressions at mRNA and protein levels with low cellular toxicity.ConclusionWe developed a systems biology pipeline and nominated STOM, KANK2, and DDR2 as candidate gene targets for PSP. We identified ASOs that modulate these targets’ expression without cellular toxicity, suggesting they may be suitable as potential therapeutic candidates. Importantly, the shared pathophysiology and molecular aberrations between PSP and other tauopathies, such as Alzheimer’s disease, suggests that such therapeutic candidates may be repurposed for multiple neurodegenerative diseases, further accelerating, and streamlining the therapeutic pipeline.

AbstractBackgroundGenetic variations have emerged as crucial players in the etiology of Alzheimer’s disease (AD), and they serve for a better understanding of the disease mechanisms; yet the specific roles of these genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Therefore, we generated zebrafish models for AD variants to analyze the in depth molecular and biological functions of these variants.MethodUsing CRISPR/Cas9, we generated a knockout model for abca7, orthologous to human ABCA7. We performed single cell transcriptomics and analyzed the altered genes and molecular pathways in zebrafish. We leveraged data from multiethnic AD cohorts at Mayo Clinic and Columbia University, to perform genetic association studies, co‐expression analyses, in silico interaction mapping, family based variant segregation analyses and epigenetic association studies, and the functional and histological studies in zebrafish.ResultThe abca7± zebrafish reduced astroglial proliferation, synaptic integrity, and microglial response after Aβ42 toxicity. We found that the abca7 loss‐of‐function (LOF) reduced neuropeptide Y (npy) expression as well as Brain‐derived neurotrophic factor (bdnf) and Nerve growth factor receptor (ngfr). Human brain analysis showed reduced NPY in AD, regulatory interaction between NPY and BDNF, genetic variants in NPY associated with AD, and segregation of variants in ABCA7, BDNF and NGFR in families. ABCA7 variants altered the epigenetic codes in NPY, BDNF, and NGFR promoter regions. Human results paralleled with zebrafish findings to indicate an evolutionarily conserved disease mechanism through ABCA7‐NPY signalling axis. NPY administration to zebrafish rescued the phenotypes in abca7 knockout, suggesting a true biological relevance.ConclusionOur results demonstrate a previously unknown link between ABCA7 and NPY in regulation of synaptic integrity and neurogenesis in AD. We propose that ABCA7‐dependent NPY is a resilience factor in vertebrate brains, and this reserve mechanism is impaired in AD.

AbstractBackgroundAlzheimer’s disease (AD) is neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often quantified by Thal phase and Braak stage, respectively. Aβ also frequently deposits in the cerebrovasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. These and related measures often show high variability within AD suggesting distinct underlying mechanisms. We hypothesize that, within the AD brain, neuropathology and levels of core AD‐related proteins are influenced by variations in DNA methylation (DNAm). To test this, we performed epigenome‐wide association studies (EWAS) using DNAm measures from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathologic measures (Braak, Thal, CAA) and brain biochemical levels of five proteins (apoE, Aβ40, Aβ42, tau, p‐tau).MethodsDNAm from neuropathologically‐confirmed AD cases was measured by reduced representation bisulfite sequencing (RRBS) from 471 TCX samples, 200 of which also had CER RRBS. TCX levels of five AD‐related proteins from three tissue fractions (buffer‐, detergent‐, and in‐soluble) were measured previously by ELISA (Liu 2020). CpG methylation (CpGm) was binned by a 15‐state chromatin model (Kundaje 2015) and averaged into CpGm clusters (rCpGm). rCpGms were tested for association with each AD endophenotype and expression levels of nearby genes through multi‐variable linear regression. Replication of significant rCpGms was performed in two independent datasets with AD‐related endophenotypes (Shireby 2022, De Jager 2014).ResultsOur innovative binning method demonstrated biologically relevant CpGm patterns. We found epigenome‐wide significant associations primarily with tau‐related endophenotypes including 93 that had significant and concordant associations in the replication datasets. These rCpGms also significantly associated with expression of nearby genes showing enrichment in oligodendrocyte marker genes including those related to myelination. In vitro validation of tau effects on oligodendrocyte gene expression through DNAm is ongoing.ConclusionsAlthough all endophenotypes tested are core to AD pathophysiology, our results suggest each has a distinct epigenetic architecture underlying their variability in the AD brain. In particular, we found evidence of DNAm variability associating with brain oligodendrocyte gene expression and TCX tau levels. By discovering AD brain endophenotype‐specific DNAm changes, we can identify core components of complex mechanisms revealing important biological insights into AD pathophysiology.

Abstract INTRODUCTION Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, yet our comprehension predominantly relies on studies within non‐Hispanic White (NHW) populations. Here we provide an extensive survey of the proteomic landscape of AD across diverse racial/ethnic groups. METHODS Two cortical regions, from multiple centers, were harmonized by uniform neuropathological diagnosis. Among 998 unique donors, 273 donors self‐identified as African American, 229 as Latino American, and 434 as NHW. RESULTS While amyloid precursor protein and the microtubule‐associated protein tau demonstrated higher abundance in AD brains, no significant race‐related differences were observed. Further proteome‐wide and focused analyses (specific amyloid beta [Aβ] species and the tau domains) supported the absence of racial differences in these AD pathologies within the brain proteome. DISCUSSION Our findings indicate that the racial differences in AD risk and clinical presentation are not underpinned by dramatically divergent patterns in the brain proteome, suggesting that other determinants account for these clinical disparities. Highlights We present a large‐scale proteome (∼10,000 proteins) of DLPFC (998) and STG (244) across AD cases. About 50% of samples were from racially and ethnically diverse brain donors. Key AD proteins (amyloid and tau) correlated with CERAD and Braak stages. No significant race‐related differences in amyloid and tau protein levels were observed in AD brains. AD‐associated protein changes showed a strong correlation between the brain proteomes of African American and White individuals. This dataset advances understanding of ethnoracial‐specific AD pathways and potential therapies.

Abstract INTRODUCTION Multi‐omics studies in Alzheimer's disease (AD) revealed many potential disease pathways and therapeutic targets. Despite their promise of precision medicine, these studies lacked Black Americans (BA) and Latin Americans (LA), who are disproportionately affected by AD. METHODS To bridge this gap, Accelerating Medicines Partnership in Alzheimer's Disease (AMP‐AD) expanded brain multi‐omics profiling to multi‐ethnic donors. RESULTS We generated multi‐omics data and curated and harmonized phenotypic data from BA ( n = 306), LA ( n = 326), or BA and LA ( n = 4) brain donors plus non‐Hispanic White ( n = 252) and other ( n = 20) ethnic groups, to establish a foundational dataset enriched for BA and LA participants. This study describes the data available to the research community, including transcriptome from three brain regions, whole genome sequence, and proteome measures. DISCUSSION The inclusion of traditionally underrepresented groups in multi‐omics studies is essential to discovering the full spectrum of precision medicine targets that will be pertinent to all populations affected with AD. Highlights Accelerating Medicines Partnership in Alzheimer's Disease Diversity Initiative led brain tissue profiling in multi‐ethnic populations. Brain multi‐omics data is generated from Black American, Latin American, and non‐Hispanic White donors. RNA, whole genome sequencing and tandem mass tag proteomicsis completed and shared. Multiple brain regions including caudate, temporal and dorsolateral prefrontal cortex were profiled.

Abstract Background: Breast cancer (BC) patients are at significant risk of therapy-related cardiotoxicity, but prediction of which patients will experience cardiotoxicity and the severity of the outcome are lacking. We hypothesized that germline genetic variants are potential risk factors for cancer treatment-related cardiomyopathy (CTRC) and in this study, we focused on rare non-synonymous variants within 57 genes deemed causative of familial and sporadic forms of idiopathic cardiomyopathies. Methods: We developed a clinical database and biorepository for patients receiving any potentially cardiotoxic cancer therapy at Mayo Clinic Florida. Enrollment is ongoing and includes consent to access external records. Patient data is extracted from records at baseline, completion of treatment and then once per year. The clinical database includes the following datasets: demographics, pathology, chemotherapy, radiation, echocardiography, cardiac MRI, PET/CT scan, blood pressure, BMI, labs, treatment complications. Treatment complications includes pre-existing conditions (smoking, alcohol and drug use history, hypertension, diabetes, coronary artery disease, hyperlipidemia, prior stroke or myocardial infarction, peripheral vascular disease, renal failure); cardiac medications at baseline; cardiac side-effects; time to cardiotoxicity; cardiotoxicity interventions and outcomes. Exome sequencing was performed by the Mayo Clinic NGS core on the first 86 patients in the registry, average of ~120x read depth following Illumina’s standard protocol for the NovaSeq 6000 and S4 flow cell. Genomic variants were extracted for 57 genes known to be causative of dilated, hypertrophic and arrhythmogenic right ventricular cardiomyopathies and channelopathies. Putative risk variants in known cardiomyopathy genes were defined as non-synonymous variants with a frequency of < 0.0004 in the GnomAD database. We compared the number of patients with putative risk variants in those groups with and without CTRC. Results: 84/86 patients had BC diagnosis. Key characteristics are described in the table below. 60/86 (70%) patients had at least one putative risk variant in one of 31 genes (there were no risk variants identified in 27/57 genes). The average number of putative risk variants per patient within the CTRC group was higher than that observed in patients without CTRC (2.06 vs 1.62). Post-hoc analysis suggested this difference is largely from patients with >2 risk variants in the group who experienced CTRC (N=7/17, 41%) compared to those who did not (N=14/69, 20%), p=0.023. Conclusions: Rare missense variants in the causative genes of idiopathic cardiomyopathies are potential risk factors for CTRC and risk may be increased in patients with a higher burden of variants. Analysis of the genetic data from patients that continue to be accrued in this study, including effects of additional clinical risk factors will be undertaken to improve prediction of cardiotoxicity. Table 1 Distribution of putative cardiomyopathy genetic variants in 86 cancer patients with and without treatment-related cardiomyopathy, separated by treatment regimen Citation Format: Nadine Norton, Thien Nguyen, Joseph Reddy, Jinky Harvey, Morgan Weidner, Amanda Arnold, Lily Evans, Laura Vallow, Pooja Advani. Increased burden of genetic variants in idiopathic cardiomyopathy genes is associated with cancer therapy-related cardiomyopathy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-12-08.

AbstractBackgroundA hallmark of Alzheimer’s disease is amyloid‐β (Aβ) deposition in the brain parenchyma as senile plaques which Aß frequently accompanies in the cerebral vasculature as cerebral amyloid angiopathy (CAA). Previous studies demonstrated that the cerebrovascular system is essential for Aβ clearance and interactions exist between cerebrovascular disease and AD. Neuroimaging data, including measures on small vessel disease, amyloid deposits, and microhemorrhages (MCH), can reflect both cerebrovascular disease and AD pathology, thereby serving as intermediate phenotypes of both conditions. We aim to identify genes and co‐expression networks in blood associated with these neuroimaging endophenotypes and the biological pathways enriched in these perturbed transcriptional networks.MethodWe conducted differential gene expression and Weighted Gene Co‐Expression Network analyses (WGCNA) on blood RNA sequencing data of 407 participants from the longitudinally followed Mayo Clinic Study of Aging cohort. The neuroimaging phenotypes include MCH, infarction, and amyloid deposits measured by Pittsburgh compound B positron emission tomography (PiB PET).ResultWGCNA analysis showed a nominally significant association (p<0.05) between several module eigengenes (MEs) and MCH and PiB PET. One module, M42, remained significantly associated with PiB PET after Bonferroni correction for all 43 modules tested. Pathway enrichment analysis of M42 identified two significant pathways (FDR‐adjusted p<0.05) including phosphodiesterase I activity and IgE binding. These pathways contain genes such as MS4A2 encoding the high‐affinity IgE receptor involved in the immune response. Similarly, ENPP2 in the phosphodiesterase I activity pathway represents an additional candidate gene previously implicated in Alzheimer’s disease.ConclusionWe generated co‐expression networks and identified one network module with a statistically significant association between brain amyloid deposits and blood transcriptome data. Further investigations of this network and its enriched pathways are necessary to understand their roles in amyloidosis, CAA, and neurodegeneration. Future studies will include seeking replication of these results in other datasets. Such studies can help elucidate the biological mechanisms of pathogenesis of cerebrovascular damage and amyloid deposits. In addition, in a clinical setting, blood levels of transcripts discovered in this study may serve as predictive or diagnostic biomarkers with mechanistic implications.

AbstractBackgroundGiven the overlap of symptoms between Alzheimer’s disease (AD) and other dementias, it is essential to develop widely accessible biomarkers that will ensure an accurate diagnosis of AD to enable its proper treatment. African Americans (AA) are twice more likely to develop AD than non‐Hispanic whites (NHW) and yet they remain underrepresented in AD research. The few studies conducted in AA suggest that there are different factors that influence AD in this population; therefore, it is possible that different biomarkers and/or therapies may be needed to effectively treat AD in AA. In this study we are testing the hypothesis that genetic variants that show association with AD‐risk in AA and with plasma levels of transcript and proteins may serve as AD biomarkers which are more accessible and less costly than cerebrospinal fluid or neuroimaging biomarkers.MethodsIn 189 AD cases and 183 cognitively unimpaired (CU) AA participants of the Florida Consortium of African American Alzheimer’s Disease Studies (FCA3DS), we performed targeted DNA sequencing of 10 AD‐associated loci. Genetic variants were tested for association with plasma transcripts and plasma total tau protein levels previously measured in this cohort using a nanoString® custom panel (Reddy J, et al. 2022) and Simoa assays (Deniz K, et al. 2021), respectively. In receiver operating characteristic (ROC) analyses, the most significant expression/protein quantitative trait loci (eQTL/pQTL) were added sequentially to a base model that included only age and sex to identify eQTL/pQTL that improved the accuracy to correctly classify AD cases vs. CU.ResultsOf the 5112 variants identified in the targeted sequencing, 71 were nominally associated (p<0.05) with plasma transcript/protein levels and with AD‐risk. ROC analysis of age, sex, eQTL/pQTL in APOE, ABCA7, AKAP9, EPHA1, SORL1, and TLR4, and plasma levels of APP, ABCA7, AKAP9, CD14, CLU, and APOE‐ɛ4 dosage achieved 77.6% area under the curve (AUC) to discriminate AD vs. CU, a 19.9% improvement over a model that only included age and sex.ConclusionsPlasma transcript levels and eQTL/pQTL are promising diagnostic biomarkers for AD that may improve accessibility and reduce costs for a more accurate diagnosis of AD.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, and maintenance of the blood‐brain‐barrier (BBB) properties. The breakdown of BBB in Alzheimer’s disease (AD) is well‐established, but precise underlying molecular changes remain unclear. Additionally, whether GVU molecular alterations observed in AD brains are also detected in blood from living patients is unknown. Further, these GVU molecular perturbations require further investigation in different model systems to identify both human brain‐specific and cross‐species conserved alterations. In this study, we investigated prioritized GVU molecules altered in AD brains for their conservation in blood and cross‐species model systems.MethodsWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in AD and control brains. We analyzed this data to detect cell‐specific GVU molecular perturbations and their interactions. We investigated molecular interactions between vascular and astrocyte clusters, the major cell types of the GVU of the BBB. To determine whether GVU transcriptional alterations detected in the brain are preserved in the blood, existing blood expression, genetic, and neuroimaging data from two longitudinal antemortem cohorts were analyzed. Using model systems, including mouse, drosophila, and zebrafish, we evaluated the cross‐species conservation of the top GVU alterations detected in AD brains.ResultsBrain snRNAseq revealed transcriptional profiles of 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as pericytes, endothelia and perivascular fibroblasts. We identified differentially expressed genes and their enriched pathways within these clusters and observed the highest levels of transcriptional changes within pericytes. Vascular targets that interact with astrocytic ligands have biological functions in cell signaling, angiogenesis, amyloid ß metabolism, and cytoskeletal architecture. We discovered that genetic variants influencing blood expression levels of some of the prioritized GVU genes were associated with neuroimaging burden of cerebrovascular disease in living human cohorts. Our ongoing studies in model systems revealed conservation of some of the top prioritized molecular perturbations across species.ConclusionOur findings prioritized by multiscale, cross‐tissue human data revealed GVU perturbations within interacting pericyte and astrocyte molecules, which are conserved across multiple cross‐species models. These results nominate new molecular targets and mechanistic insights for BBB disruptions in AD.

AbstractBackgroundNeurodegenerative disorders affect different brain regions at different time points and to varying degrees, although the underlying reasons for this variability in regional susceptibility is unknown. Brain regions have different proportions of cell types. This cell proportion variability may be further influenced by presence and degree of brain disorders, including neurodegenerative diseases. We postulate that genetic and other factors that influence brain cell type proportions may also provide insights into brain regional susceptibility to neurodegenerative diseases.MethodsUtilizing bulk transcriptomics data from 7 brain regions obtained from 3 cohorts and corresponding whole genome sequencing (WGS) data, genetic variants were evaluated for association with cell type proportions across brain regions. Utilizing cell type specific marker genes for deconvolution provided by McKenzie et.al, we estimated surrogate variables for proportion (SVP) of various cell types using BRETIGEA. We then evaluated relationship between SVP and AD as well as Age, Sex, APOE ε4 and neuropathology measures. Genetic variants from WGS were then tested for association with SVP to identify distinct and conserved patterns across 6 cell types and 7 brain regions.ResultsAnalysis of SVP by diagnosis, sex, age, APOE ε4 and neuropathology measures identified significant differences in cell proportions in various cell types and brain regions. GWAS of SVP identified 134 loci that reached genome‐wide significance (5e‐8). After conservative adjustment for multiple testing (6 cell types and 7 brain regions; 42 tests), variants proximal to SYNPR and VENTXP7 remained significant (p‐value<1.19E‐09). Variants proximal to SYNPR were associated with increased proportion of oligodendrocytes in cerebellum whereas variants near VENTXP7 were associated with reduced proportion of astrocytes in DLPFC. Variants in SYNPR have previously been associated with neurofibrillary tangles and diffuse plaque measurements while variants in VENTXP7 have been associated with measurement of neurofibrillary tangles, cognitive decline, unipolar depression, and bipolar disorder.ConclusionsIt is likely that there are genetic variants associated with cell proportion in brain, may vary by cell type and brain region, while others may act more broadly. Identification of genetic variants associated with cell proportions in different brain regions may provide insights into affected cells and regional vulnerabilities.