Excitoprotective effects of conditional tau reduction in excitatory neurons and in adulthood.

Tau reduction is a promising therapeutic strategy for Alzheimer's disease. In numerous models, tau reduction via genetic knockout is beneficial, at least in part due to protection against hyperexcitability and seizures, but the underlying mechanisms are unclear. Here we describe the generation and initial study of a new conditional Tau flox model to address these mechanisms. Given the protective effects of tau reduction against hyperexcitability, we compared the effects of selective tau reduction in excitatory or inhibitory neurons. Tau reduction in excitatory neurons mimicked the protective effects of global tau reduction, while tau reduction in inhibitory neurons had the opposite effect and increased seizure susceptibility. Since most prior studies used knockout mice lacking tau throughout development, we crossed Tau flox mice with inducible Cre mice and found beneficial effects of tau reduction in adulthood. Our findings support the effectiveness of tau reduction in adulthood and indicate that excitatory neurons may be a key site for its excitoprotective effects. SUMMARY: A new conditional tau knockout model was generated to study the protective effects of tau reduction against hyperexcitability. Conditional tau reduction in excitatory, but not inhibitory, neurons was excitoprotective, and induced tau reduction in adulthood was excitoprotective without adverse effects.

Neuronal MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Tauopathies are a group of neurodegenerative diseases defined by abnormal aggregates of tau, a microtubule-associated protein encoded by MAPT. MAPT expression is near absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression could be controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding disease risk and pathogenesis. Here, we performed chromatin conformation assays (HiC & Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27ac and CTCF in NPCs and differentiated neurons to nominate candidate cis-regulatory elements (cCREs). We assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in neurodegeneration-affected individuals and control subjects. We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the H1/H2 haplotype inversion breakpoint. We also found that rare and predicted damaging genetic variation in nominated CREs was nominally depleted in dementia-affected individuals relative to control subjects, consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduced MAPT expression, may be protective against neurodegenerative disease. Overall, this study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

MAPT expression is mediated by long-range interactions with cis-regulatory elements.

Background: Tauopathies are a group of neurodegenerative diseases driven by abnormal aggregates of tau, a microtubule associated protein encoded by the MAPT gene. MAPT expression is absent in neural progenitor cells (NPCs) and increases during differentiation. This temporally dynamic expression pattern suggests that MAPT expression is controlled by transcription factors and cis-regulatory elements specific to differentiated cell types. Given the relevance of MAPT expression to neurodegeneration pathogenesis, identification of such elements is relevant to understanding genetic risk factors. Methods: We performed HiC, chromatin conformation capture (Capture-C), single-nucleus multiomics (RNA-seq+ATAC-seq), bulk ATAC-seq, and ChIP-seq for H3K27Ac and CTCF in NPCs and neurons differentiated from human iPSC cultures. We nominated candidate cis-regulatory elements (cCREs) for MAPT in human NPCs, differentiated neurons, and pure cultures of inhibitory and excitatory neurons. We then assayed these cCREs using luciferase assays and CRISPR interference (CRISPRi) experiments to measure their effects on MAPT expression. Finally, we integrated cCRE annotations into an analysis of genetic variation in AD cases and controls. Results: Using orthogonal genomics approaches, we nominated 94 cCREs for MAPT, including the identification of cCREs specifically active in differentiated neurons. Eleven regions enhanced reporter gene transcription in luciferase assays. Using CRISPRi, 5 of the 94 regions tested were identified as necessary for MAPT expression as measured by RT-qPCR and RNA-seq. Rare and predicted damaging genetic variation in both nominated and confirmed CREs was depleted in AD cases relative to controls (OR = 0.40, p = 0.004), consistent with the hypothesis that variants that disrupt MAPT enhancer activity, and thereby reduce MAPT expression, may be protective against neurodegenerative disease. Conclusions: We identified both proximal and distal regulatory elements for MAPT and confirmed the regulatory function for several regions, including three regions centromeric to MAPT beyond the well-described H1/H2 haplotype inversion breakpoint. This study provides compelling evidence for pursuing detailed knowledge of CREs for genes of interest to permit better understanding of disease risk.

An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression.

The expression of genetic material governs brain development, differentiation, and function, and targeted manipulation of gene expression is required to understand contributions of gene function to health and disease states. Although recent improvements in CRISPR/dCas9 interference (CRISPRi) technology have enabled targeted transcriptional repression at selected genomic sites, integrating these techniques for use in non-dividing neuronal systems remains challenging. Previously, we optimized a dual lentivirus expression system to express CRISPR-based activation machinery in post-mitotic neurons. Here we used a similar strategy to adapt an improved dCas9-KRAB-MeCP2 repression system for robust transcriptional inhibition in neurons. We find that lentiviral delivery of a dCas9-KRAB-MeCP2 construct driven by the neuron-selective human synapsin promoter enabled transgene expression in primary rat neurons. Next, we demonstrate transcriptional repression using CRISPR sgRNAs targeting diverse gene promoters, and show superiority of this system in neurons compared to existing RNA interference methods for robust transcript specific manipulation at the complex Brain-derived neurotrophic factor (Bdnf) gene. Our findings advance this improved CRISPRi technology for use in neuronal systems for the first time, potentially enabling improved ability to manipulate gene expression states in the nervous system.