Mechanisms of inherited neurodegenerative diseases

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

1. Disrupted RNA transport in ALS/FTD. In close collaboration with Jennifer Lippincott Schwartz (HHMI), our group made the surprising discovery that RNA granules are indirectly transported long distances in axons by hitchhiking on moving lysosomes (Liao et al, Cell, 2019), and that this process is disrupted by an ALS-associated mutant protein. Using proteomics, imaging, and biophysics, we showed that this hitchhiking is mediated by a newly-discovered ALS protein, ANXA11, which serves as a regulatable molecular tether between RNA granules and lysosomes during transport. ALS-associated mutations in ANXA11 block RNA transport and local translation of RNA in distal neuronal processes. These findings suggest that dysfunctional local translation may contribute to ALS, and that molecular convergences exist between lysosomal and RNA biology in ALS pathogenesis. 2. Dysregulated RNA splicing in ALS/FTD. As part of a multi-lab collaboration between my group, Pietro Frattas team (UCL), and Len Petrucellis team (Mayo), we have used a combination of iPSC cellular models and human datasets to identify and characterize pathological splicing events in ALS/FTD that occur in the setting of TDP-43 mislocalization. TDP-43 normally functions as a splicing repressor. However, TDP-43 becomes depleted from the nucleus in most cases of ALS and FTD, and the resulting loss of splicing repression leads to the inclusion of cryptic exons in numerous transcripts. We used our CRISPRi i3Neuron platform to deplete TDP-43 from cortical neurons, discovering hundreds of new cryptic-exon containing transcripts in addition to well-described cryptic exons in genes such as STMN2. One novel cryptic exon was found in UNC13A, a gene previously implicated in ALS through GWAS studies. We found that the risk-associated SNPs were near the cryptic exon, and interfered with TDP-43 binding, thereby increasing the pathologic inclusion of cryptic exons in the setting of TDP-43 mislocalization. In addition, with the Fratta and Petrucelli labs, we showed that a classic cryptic exon in STMN2 can be detected in post-mortem brains from FTLD-TDP and FTD-MND patients, and is specific for patients with TDP-43 pathology (i.e. not present in those with FUS/MAPT pathology). This study suggests that CSF/plasma-based identification of cryptic exon transcripts or protein products facilitates biomarkers of TDP-43 mislocalization in ALS/FTD. 3. iPSC neuron models to study neurodegenerative diseases. I co-developed an improved method to differentiate large numbers of human iPSCs into neurons (Wang & Ward, Stem Cell Reports, 2017). Through single-copy integration of a doxycycline-inducible neurogenin 2 (iNGN2), we created a clonal iSPC line that enables simple, rapid, reproducible, and scalable production of mature glutamatergic neurons. We termed this cellular platform i3Neurons (inducible x integrated x isogenic). i3Neurons can be produced less expensively and faster than primary rodent cultures, are genomically stable, and can be readily manipulated with genome-editing technologies. We have shared this technology broadly with the neuroscience community and have sent our cell lines to over 100 national and international labs. I was a co-first author on the original paper describing the technique, conceived of and developed the inducible NGN2 iPSC line, and optimized the methods for scalable i3Neuron production (Fernandopulle et al, Curr Protoc Cell Biol, 2018; also available at protocols.io). In collaboration with Martin Kampmann's team at UCSF, we expanded upon our iPSC neuron technology to develop a new CRISPR-inhibition forward genetics screening platform. Scalable production of iPSC-derived neurons that co-express dCas9 allowed us to discover a host of new neuron survival related pathways (Tian et al, Neuron, 2019), and will enable future synthetic lethality screens aimed at uncovering neurodegeneration-related pathways. Finally, I co-direct the largest-ever genome engineering project to date, the iPSC Neurodegenerative Disease Initiative (iNDI). iNDI is a multi-institute project funded by the NIA/NINDS that will genome-engineer >100 isogenic iPSC lines harboring familial mutations implicated in neurodegenerative diseases, and then phenotype disease-relevant differentiated cells such as neurons using multi-omic robotic platforms (Ramos et al, Neuron, 2021). iPSC lines are widely available to the research community and distributed by JAX since 2021, and phenotypic datasets will be uploaded to open central data repositories on a rolling basis. 4. Endogenous DNA break/repair at neuronal enhancers. In collaboration with Andre Nussenzweigs lab at the NCI, we recently discovered that neurons undergo high levels of constitutive endogenous DNA break/repair events. The Nussenzweig lab developed a new method to map endogenous DNA break/repair events genome-wide in post-mitotic cells, called SARseq through incorporation of a thymidine analog (EdU) followed by biotin enrichment and next generation sequencing. Our lab applied SARseq to our iPSC-derived i3Neuron system, and unexpectedly identified tens of thousands of recurrent DNA breaks throughout the genome. Further studies using Chip-seq, CRISPRi, and other molecular biology approaches identified the location of these DNA breaks (enhancers), the form of DNA break (single-stranded), and the mechanism of repair (base excision repair). We further showed that these ssDNA breaks occurred at genomic sites that under active demethylation events, suggesting that such ssDNA breaks are critical for chromatin remodeling and control of gene expression in neurons.