Joshua M Shulman - US grants

Analyses of high-throughput human data, including gene expression profiles of human brains, constitute a powerful strategy for nominating biological networks associated with Alzheimer?s disease (AD) pathophysiology as potential therapeutic targets. The Accelerating Medicines Partnership-Alzheimer?s Disease (AMP-AD) Target Discovery Project has defined consensus, AD-associated molecular networks based on joint analyses of transcriptomic profiles in ~2,000 human brain autopsy samples. The critical next step is to rigorously test computational predictions in experimental animal models to (i) unambiguously link promising networks to specific AD triggers (i.e. Amyloid-ß, Tau, and/or aging), (ii) confirm the network architecture, (iii) validate implicated molecular pathways in the nervous system context, and (iv) discover which molecular changes are truly causal, including distinguishing between amplifiers of pathogenesis versus protective, compensatory responses. We have developed a cross-species strategy for functional dissection of emerging AD molecular networks using high-throughput assays in the nervous system of transgenic Drosophila expressing human wild-type human Tau or the Amyloid-ß peptide. When applied to candidates from AD-associated networks, these rapid and complementary in vivo assays enable identification of those genes and pathways that are likely causal modifiers of Tau- and/or Aß-induced neuronal dysfunction, including both drivers of pathogenesis and compensatory responses. The overall goal of this proposal is to deploy our cross-species strategy to accelerate the discovery, refinement, and functional dissection of AD molecular networks derived from human brain transcriptomes and proteomes. First (AIM 1), transcriptomic and proteomic profiling in Drosophila AD models will be coupled with comprehensive screening of conserved network candidates in order to identify those genes causally linked to neurodegeneration and differentiate in vivo interactions with Tau-, Aß-, or other age-dependent mechanisms. The proposed screen will deploy robotic instrumentation for high-throughput, quantitative analyses of Drosophila motor impairment due to neuronal dysfunction. Second (AIM 2), using systems biology approaches, gene expression and functional data from Drosophila will be integrated with the human AD networks. The resulting multi-scale atlas of AD molecular systems will pinpoint causal networks and key gene/protein drivers with the greatest potential to alter neurodegeneration upon perturbation. Next (AIM 3), we will experimentally confirm the network architecture for the most promising modules highlighting key drivers with roles as AD amplifying/protective factors. Lastly (AIM 4), all project results will be made publicly available via the AMP-AD Knowledge Portal. IMPACT: Our integrative, cross-species discovery strategy will comprehensively probe AD molecular networks with powerful, in vivo functional assays to reveal age- dependent drivers, including amplifiers and protectors, for Tau- and Aß-induced neurotoxicity, and elucidate the complex network architecture underlying AD pathogenesis.

Synucleinopathies, including Lewy body dementia (LBD) and Parkinson?s disease (PD), are common and incurable neurodegenerative disorders with strong evidence for heritability. Loss-of-function variants in Glucocerebrosidase (GBA) cause Gaucher?s disease, a recessive lysosomal storage disorder (LSD). It is estimated that 85% or greater loss of Glucocerebrosidase activity is required to trigger Gaucher?s. Paradoxically however, heterozygous carriers of GBA variants?causing modest reductions in overall enzyme function?have a significantly increased risk of PD and LBD, and GBA alleles also dominantly modify risk of dementia among subjects with PD. Emerging evidence suggests that GBA loss of function may enhance the neurotoxicity of ?-synuclein (?Syn), the pathological protein that aggregates to form brain Lewy bodies in PD and LBD. However, the mechanism by which partial reduction in GBA activity contributes to pathogenesis of synucleinopathy remains elusive. Since most GBA variant carriers do not develop disease in their lifetimes, other factors likely contribute to disease penetrance. In an exome-wide study, we discovered an aggregate genetic variant burden among 54 LSD genes associated with PD risk. In fact, over half of subjects carried at least one variant, and 21% carried 2 or more variants. These results suggest that (i) other LSD genes likely contribute to synucleinopathy, and (ii) LSD gene variants may interact with one another to modify risk and progression of neurodegeneration. In this proposal we test the hypothesis that partial, haploinsufficient loss of function in LSD genes disrupts sphingolipid metabolism, leading to enhanced lysosomal stress and increased vulnerability to ?Syn-induced, age-dependent neurodegeneration. In compelling preliminary studies, we have performed comprehensive genetic manipulations of 94 conserved homologs of human LSD genes in a Drosophila transgenic model of ?Syn-mediated neurodegeneration, identifying GBA and 17 other candidate enhancers. A preponderance of modifiers are implicated in lysosomal metabolism of ceramide and sphingolipids. Here, we will employ the powerful and rapid genetics available in Drosophila to systematically confirm interactions between LSD genes and ?Syn-mediated neurodegeneration and assess impact on ?Syn protein dynamics . To establish clinical relevance, LSD gene modifiers of ?Syn will be examined for associations with PD/LBD pathology in human brain autopsy cohorts . In parallel, the most promising LSD gene modifiers of ?Syn will be interrogated for impact on lysosomal structure and function , and we will perform mass-spectrometry to profile sphingolipid perturbations in a GBA allelic series with graduated reduction in Glucocerebrosidase activity . In sum, this exploratory project will establish a causal chain between partial loss-of-function in GBA and other LSD genes leading to subclinical derangements in lysosomal metabolism, ?Syn neuropathology in the aging brain, neuronal dysfunction and death, and ultimately, the clinical manifestations of PD/LBD.

SUMMARY Alzheimer?s Disease (AD) is projected to affect 13 million people in the US by 2050 and remains neither curable nor preventable. Following remarkable recent progress, the genomic architecture of AD and related dementias (ADRD) is coming into focus. Similar to other common and genetically complex disorders, AD is characterized by substantial locus heterogeneity and polygenic susceptibility: risk or protective alleles are being identified in many distinct genes, and in most individuals, a subset of common and rare variants likely interact to trigger neurodegeneration. The critical next steps include confirmation of the responsible genes, understanding the functional impact of disease-associated variants, elaboration of the relevant cell types and pathways, and determining how polygenic interactions mediate disease risk. We propose an integrated computational and tiered experimental validation strategy to accelerate AD functional genomics, building on advances from the AD Sequencing Project (ADSP) and leveraging powerful technologies available in the fruit fly, Drosophila melanogaster. First (AIM 1), leveraging infrastructure developed for the Clinical Genome Resource and ENCODE projects, we will integrate ADSP results with other human data, including brain transcriptome and epigenome profiles, prioritizing genes and variants for experimental follow-up. Next (AIM 2), using high-throughput Drosophila screening, we will systematically manipulate 2,000 conserved, candidate AD genes in vivo to pinpoint causal modulators of age-dependent neurodegeneration, including interactions with Tau, Aß, and other pathologic triggers. Third (AIM 3), for a subset of 200 prioritized gene candidates, we will generate customized Drosophila strains and characterize cell-type expression and loss-of-function phenotypes. Lastly (AIM 4), for 50 high-priority targets, we will experimentally probe mechanisms in-depth, including testing of cell-type specific requirements (neurons vs. glia) and examining gene-gene interactions that define relevant pathways. We will broadly share all project data and resources with the research community (AIM 5). Our integrative, tiered, cross-species strategy promises rapid functional annotation of ADSP targets using powerful, in vivo assays in the aging nervous system of Drosophila, and is ideally suited for reciprocal cross-validation in complementary mammalian preclinical models. On a scale and timeframe not currently possible in other model systems, our innovative experimental strategy will transcend barriers to translation of human genetic discoveries and catalyze breakthroughs in our understanding AD pathobiology.