Publications

Alzheimer's disease (AD) is a complex neurodegenerative disorder. The relative contribution of the numerous underlying functional mechanisms is poorly understood. To comprehensively understand the context and distribution of pathways that contribute to AD, we performed text-mining to generate an exhaustive, systematic assessment of the breadth and diversity of biological pathways within a corpus of 206,324 dementia publication abstracts. A total of 91% (325/335) of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways have publications containing an association via at least 5 studies, while 63% of pathway terms have at least 50 studies providing a clear association with AD. Despite major technological advances, the same set of top-ranked pathways have been consistently related to AD for 30 years, including AD, immune system, metabolic pathways, cholinergic synapse, long-term depression, proteasome, diabetes, cancer, and chemokine signaling. AD pathways studied appear biased: animal model and human subject studies prioritize different AD pathways. Surprisingly, human genetic discoveries and drug targeting are not enriched in the most frequently studied pathways. Our findings suggest that not only is this disorder incredibly complex, but that its functional reach is also nearly global. As a consequence of our study, research results can now be assessed in the context of the wider AD literature, supporting the design of drug therapies that target a broader range of mechanisms. The results of this study can be explored at www.adpathways.org.

The molecular underpinnings of organ dysfunction in acute COVID-19 and its potential long-term sequelae are under intense investigation. To shed light on these in the context of liver function, we performed single-nucleus RNA-seq and spatial transcriptomic profiling of livers from 17 COVID-19 decedents. We identified hepatocytes positive for SARS-CoV-2 RNA with an expression phenotype resembling infected lung epithelial cells. Integrated analysis and comparisons with healthy controls revealed extensive changes in the cellular composition and expression states in COVID-19 liver, reflecting hepatocellular injury, ductular reaction, pathologic vascular expansion, and fibrogenesis. We also observed Kupffer cell proliferation and erythrocyte progenitors for the first time in a human liver single-cell atlas, resembling similar responses in liver injury in mice and in sepsis, respectively. Despite the absence of a clinical acute liver injury phenotype, endothelial cell composition was dramatically impacted in COVID-19, concomitantly with extensive alterations and profibrogenic activation of reactive cholangiocytes and mesenchymal cells. Our atlas provides novel insights into liver physiology and pathology in COVID-19 and forms a foundational resource for its investigation and understanding.

The SARS-CoV-2 pandemic has caused over 1 million deaths globally, mostly due to acute lung injury and acute respiratory distress syndrome, or direct complications resulting in multiple-organ failures. Little is known about the host tissue immune and cellular responses associated with COVID-19 infection, symptoms, and lethality. To address this, we collected tissues from 11 organs during the clinical autopsy of 17 individuals who succumbed to COVID-19, resulting in a tissue bank of approximately 420 specimens. We generated comprehensive cellular maps capturing COVID-19 biology related to patients' demise through single-cell and single-nucleus RNA-Seq of lung, kidney, liver and heart tissues, and further contextualized our findings through spatial RNA profiling of distinct lung regions. We developed a computational framework that incorporates removal of ambient RNA and automated cell type annotation to facilitate comparison with other healthy and diseased tissue atlases. In the lung, we uncovered significantly altered transcriptional programs within the epithelial, immune, and stromal compartments and cell intrinsic changes in multiple cell types relative to lung tissue from healthy controls. We observed evidence of: alveolar type 2 (AT2) differentiation replacing depleted alveolar type 1 (AT1) lung epithelial cells, as previously seen in fibrosis; a concomitant increase in myofibroblasts reflective of defective tissue repair; and, putative TP63+ intrapulmonary basal-like progenitor (IPBLP) cells, similar to cells identified in H1N1 influenza, that may serve as an emergency cellular reserve for severely damaged alveoli. Together, these findings suggest the activation and failure of multiple avenues for regeneration of the epithelium in these terminal lungs. SARS-CoV-2 RNA reads were enriched in lung mononuclear phagocytic cells and endothelial cells, and these cells expressed distinct host response transcriptional programs. We corroborated the compositional and transcriptional changes in lung tissue through spatial analysis of RNA profiles in situ and distinguished unique tissue host responses between regions with and without viral RNA, and in COVID-19 donor tissues relative to healthy lung. Finally, we analyzed genetic regions implicated in COVID-19 GWAS with transcriptomic data to implicate specific cell types and genes associated with disease severity. Overall, our COVID-19 cell atlas is a foundational dataset to better understand the biological impact of SARS-CoV-2 infection across the human body and empowers the identification of new therapeutic interventions and prevention strategies.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.

COVID-19, which is caused by SARS-CoV-2, can result in acute respiratory distress syndrome and multiple organ failure1-4, but little is known about its pathophysiology. Here we generated single-cell atlases of 24 lung, 16 kidney, 16 liver and 19 heart autopsy tissue samples and spatial atlases of 14 lung samples from donors who died of COVID-19. Integrated computational analysis uncovered substantial remodelling in the lung epithelial, immune and stromal compartments, with evidence of multiple paths of failed tissue regeneration, including defective alveolar type 2 differentiation and expansion of fibroblasts and putative TP63+ intrapulmonary basal-like progenitor cells. Viral RNAs were enriched in mononuclear phagocytic and endothelial lung cells, which induced specific host programs. Spatial analysis in lung distinguished inflammatory host responses in lung regions with and without viral RNA. Analysis of the other tissue atlases showed transcriptional alterations in multiple cell types in heart tissue from donors with COVID-19, and mapped cell types and genes implicated with disease severity based on COVID-19 genome-wide association studies. Our foundational dataset elucidates the biological effect of severe SARS-CoV-2 infection across the body, a key step towards new treatments.

INTRODUCTION: Genome-wide association studies have led to numerous genetic loci associated with Alzheimer's disease (AD). Whole-genome sequencing (WGS) now permits genome-wide analyses to identify rare variants contributing to AD risk.

METHODS: We performed single-variant and spatial clustering-based testing on rare variants (minor allele frequency [MAF] ≤1%) in a family-based WGS-based association study of 2247 subjects from 605 multiplex AD families, followed by replication in 1669 unrelated individuals.

RESULTS: We identified 13 new AD candidate loci that yielded consistent rare-variant signals in discovery and replication cohorts (4 from single-variant, 9 from spatial-clustering), implicating these genes: FNBP1L, SEL1L, LINC00298, PRKCH, C15ORF41, C2CD3, KIF2A, APC, LHX9, NALCN, CTNNA2, SYTL3, and CLSTN2.

DISCUSSION: Downstream analyses of these novel loci highlight synaptic function, in contrast to common AD-associated variants, which implicate innate immunity and amyloid processing. These loci have not been associated previously with AD, emphasizing the ability of WGS to identify AD-associated rare variants, particularly outside of the exome.

With the advent of whole genome-sequencing (WGS) studies, family-based designs enable sex-specific analysis approaches that can be applied to only affected individuals; tests using family-based designs are attractive because they are completely robust against the effects of population substructure. These advantages make family-based association tests (FBATs) that use siblings as well as parents especially suited for the analysis of late-onset diseases such as Alzheimer's Disease (AD). However, the application of FBATs to assess sex-specific effects can require additional filtering steps, as sensitivity to sequencing errors is amplified in this type of analysis. Here, we illustrate the implementation of robust analysis approaches and additional filtering steps that can minimize the chances of false positive-findings due to sex-specific sequencing errors. We apply this approach to two family-based AD datasets and identify four novel loci (GRID1, RIOK3, MCPH1, ZBTB7C) showing sex-specific association with AD risk. Following stringent quality control filtering, the strongest candidate is ZBTB7C (Pinter = 1.83 × 10-7), in which the minor allele of rs1944572 confers increased risk for AD in females and protection in males. ZBTB7C encodes the Zinc Finger and BTB Domain Containing 7C, a transcriptional repressor of membrane metalloproteases (MMP). Members of this MMP family were implicated in AD neuropathology.

Mechanistic disease stratification will be crucial to develop a precision medicine approach for future disease modifying therapy in sporadic Parkinson's disease (sPD). Mitochondrial and lysosomal dysfunction are key mechanisms in the pathogenesis of sPD and therefore promising targets for therapeutic intervention. We investigated mitochondrial and lysosomal function in skin fibroblasts of 100 sPD patients and 50 age-matched controls. A combination of cellular assays, RNA-seq based pathway analysis and genotyping was applied. Distinct subgroups with mitochondrial (mito-sPD) or lysosomal (lyso-sPD) dysfunction were identified. Mitochondrial dysfunction correlated with reduction in complex I and IV protein levels. RNA-seq based pathway analysis revealed marked activation of the lysosomal pathway with enrichment for lysosomal disease gene variants in lyso-sPD. Conversion of fibroblasts to induced neuronal progenitor cells and subsequent differentiation into tyrosine hydroxylase positive neurons confirmed and further enhanced both mitochondrial and lysosomal abnormalities. Treatment with ursodeoxycholic acid improved mitochondrial membrane potential and intracellular ATP levels even in sPD patient fibroblast lines with comparatively mild mitochondrial dysfunction. The results of our study suggest that in-depth phenotyping and focussed assessment of putative neuroprotective compounds in peripheral tissue are a promising approach towards disease stratification and precision medicine in sPD.

We present a consensus atlas of the human brain transcriptome in Alzheimer's disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington's disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies.

Recent genome-wide association studies identified the angiotensin-converting enzyme gene (ACE) as an Alzheimer's disease (AD) risk locus. However, the pathogenic mechanism by which ACE causes AD is unknown. Using whole-genome sequencing, we identified rare ACE coding variants in AD families and investigated one, ACE1 R1279Q, in knockin (KI) mice. Similar to AD, ACE1 was increased in neurons, but not microglia or astrocytes, of KI brains, which became elevated further with age. Angiotensin II (angII) and angII receptor AT1R signaling were also increased in KI brains. Autosomal dominant neurodegeneration and neuroinflammation occurred with aging in KI hippocampus, which were absent in the cortex and cerebellum. Female KI mice exhibited greater hippocampal electroencephalograph disruption and memory impairment compared to males. ACE variant effects were more pronounced in female KI mice, suggesting a mechanism for higher AD risk in women. Hippocampal neurodegeneration was completely rescued by treatment with brain-penetrant drugs that inhibit ACE1 and AT1R. Although ACE variant-induced neurodegeneration did not depend on β-amyloid (Aβ) pathology, amyloidosis in 5XFAD mice crossed to KI mice accelerated neurodegeneration and neuroinflammation, whereas Aβ deposition was unchanged. KI mice had normal blood pressure and cerebrovascular functions. Our findings strongly suggest that increased ACE1/angII signaling causes aging-dependent, Aβ-accelerated selective hippocampal neuron vulnerability and female susceptibility, hallmarks of AD that have hitherto been enigmatic. We conclude that repurposed brain-penetrant ACE inhibitors and AT1R blockers may protect against AD.