Huntington's Disease: Genome-wide Neuroprotection Screening Goes Viral
Authors: C.Y. Daniel Lee, X. William Yang
Journal: Neuron (2020)

Forward genetic screen, typically performed in invertebrates or mammalian cell lines, has been instrumental in discovering genes essential for neural function. In this issue of Neuron, Wertz et al., 2020Wertz M.H. Mitchem M.R. Pineda S.S. Hachigian L.J. Lee H. Lau V. Powers A. Kulicke R. Madan G.K. Colic M. et al.Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.Neuron. 2020; 106 (this issue): 76-89Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar demonstrate the first viral-mediated, genome-wide screen to identify neuroprotective genes in wild-type and Huntington's disease (HD) mouse brains. Forward genetic screen, typically performed in invertebrates or mammalian cell lines, has been instrumental in discovering genes essential for neural function. In this issue of Neuron, Wertz et al., 2020Wertz M.H. Mitchem M.R. Pineda S.S. Hachigian L.J. Lee H. Lau V. Powers A. Kulicke R. Madan G.K. Colic M. et al.Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.Neuron. 2020; 106 (this issue): 76-89Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar demonstrate the first viral-mediated, genome-wide screen to identify neuroprotective genes in wild-type and Huntington's disease (HD) mouse brains. An overarching goal of molecular neuroscience is to establish causal links between genes expressed in the brain and their precise function from cellular to organismal levels. Such a functional molecular map for the mammalian brain is contended to be essential in gaining insights into normal brain function and diseases pathogenesis. The pace to study gene function in the mammalian brain has been crawling at a speed that has changed very little over the past two decades—mostly one gene at a time. The challenge to the field is how to incorporate the tremendous advances in the genetic and genomic technologies to assess gene functions in mammalian brain at a large scale and, ideally, at the genome-scale. In this issue of Neuron, Heiman and colleagues reported a major advance in the technology to perform genome-wide genetic screening in the mammalian central nervous system (CNS) and identified a large number of genes that are essential for the survival of striatal medium spiny neurons (MSNs; also known as spiny projection neurons) in normal brains and those of Huntington's disease (HD) mouse models (Wertz et al., 2020Wertz M.H. Mitchem M.R. Pineda S.S. Hachigian L.J. Lee H. Lau V. Powers A. Kulicke R. Madan G.K. Colic M. et al.Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.Neuron. 2020; 106 (this issue): 76-89Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Forward genetic screens in vivo to study gene functions in the nervous system have been widely performed in invertebrates (e.g., C. elegans, Drosophila) or lower vertebrates (e.g., zebrafish). More recently, powerful genetic tools such as knockdown (KD) with short hairpin RNA (shRNA) and genome editing with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) have been readily applied in these lower organisms to reduce or inactivate gene expression at the genome-wide scale in order to gain functional insights (Shalem et al., 2015Shalem O. Sanjana N.E. Zhang F. High-throughput functional genomics using CRISPR-Cas9.Nat. Rev. Genet. 2015; 16: 299-311Crossref PubMed Scopus (750) Google Scholar). In the mammalian system, such in vivo genome-wide loss-of-function screenings have been limited to proliferating cells, such as immune cells or cancer cells (Shalem et al., 2015). Due to the complexity of the mammalian brain, the difficulty to deliver genetic perturbagens into the brain, and the scarcity of tools for scalable evaluation of neuronal phenotypes, a forward genetic screen to unbiasedly identify genes for brain cell phenotypes seems to be an insurmountable task. HD is a common inherited neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat (to >35 CAGs), encoding a polyglutamine tract, in mutant huntingtin (mHTT). The hallmark of HD neuropathology is the massive degeneration of the striatal MSNs and, to a lesser extent, the degeneration of deep layer cortical pyramidal neurons. The molecular basis for how the nearly ubiquitously expressed mHTT causes relatively selective degeneration of striatal MSNs remains poorly understood. However, emerging evidence suggests that the pathological interaction of mHTT toxicities in cortical and striatal neurons (Wang et al., 2014Wang N. Gray M. Lu X.H. Cantle J.P. Holley S.M. Greiner E. Gu X. Shirasaki D. Cepeda C. Li Y. et al.Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of Huntington's disease.Nat. Med. 2014; 20: 536-541Crossref PubMed Scopus (151) Google Scholar) and selective mHTT CAG-length-dependent transcriptional dysregulation, especially suppression of MSN identity gene expression (Langfelder et al., 2016Langfelder P. Cantle J.P. Chatzopoulou D. Wang N. Gao F. Al-Ramahi I. Lu X.H. Ramos E.M. El-Zein K. Zhao Y. et al.Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.Nat. Neurosci. 2016; 19: 623-633Crossref PubMed Scopus (228) Google Scholar), could contribute to the striatal neuronal vulnerability in HD. Moreover, human genetic studies yielded 21 chromosomal loci that are genome-wide significant (GWAS) in modifying HD age of onset, and remarkably, the majority of these loci contain genes involved in DNA handling (GeM-HD Consortium, 2019GeM-HD ConsortiumCAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset.Cell. 2019; 178: 887-900.e14Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). How these DNA repair genes might contribute to HD pathogenesis remains elusive, but they are shown to modify the somatic instability of mHTT CAG repeats in the striatum (Massey and Jones, 2018Massey T.H. Jones L. The central role of DNA damage and repair in CAG repeat diseases.Dis. Model. Mech. 2018; 11: 1-14Crossref Scopus (63) Google Scholar). With a myriad of candidate molecular mechanisms being proposed for HD pathogenesis, scalable tools are in need to systematically and unbiasedly evaluate these mechanisms and to prioritize the molecular targets for therapeutic research. In their elegantly designed study, Heiman and colleagues devised a genome-wide perturbation approach to address a question that is central to understanding HD pathogenesis: the cell-intrinsic vulnerability of striatal MSNs to degeneration. Their study built upon the concept that reduction of gene expression by a virally delivered genetic perturbagen (i.e., shRNA or CRISPR/Cas9), either alone or in synergy with the expression of mHTT, might lead to the loss of MSNs and hence a depletion of the given perturbagens in the surviving neurons, which can be readily identified by sequencing. As a proof of concept, the Heiman group stereotaxically injected 95 shRNAs targeting 28 candidate genes into the striatum of R6/2 mice, an HD mouse model expressing mHTT fragments, and wild-type (WT) controls (Shema et al., 2015Shema R. Kulicke R. Cowley G.S. Stein R. Root D.E. Heiman M. Synthetic lethal screening in the mammalian central nervous system identifies Gpx6 as a modulator of Huntington's disease.Proc. Natl. Acad. Sci. USA. 2015; 112: 268-272Crossref PubMed Scopus (21) Google Scholar). After a few weeks, they amplified and sequenced the resulting viral shRNA targeting sequences in the striatum and identified a subset of genes that were selectively depleted only in the HD mouse striatum, a method they termed synthetic lethality in CNS (or SLIC). To expand the viral-mediated loss-of-function screening for neuronal-essential genes to a genome-wide scale, the authors needed to first define optimal screening parameters and develop appropriate bioinformatic tools. They used two well-characterized vesicular stomatitis virus G protein (VSV-G)-pseudotyped lentiviral genome-wide murine pooled libraries, one with 92,000 shRNA elements and the other with 80,000 sgRNA elements, with each library targeting about 15,000 to 20,000 murine genes. By bilateral injection of a control GFP virus into the striatum, they determined the viral spread to be about 120,000 cells per hemisphere. To assess the number of biological replicates and sequencing depth needed for a genome-wide library, they injected a sgRNA "sub-library" with increasing complexity (e.g., 1,000, 3,100, and 92,000 elements) into the striata of 20 WT mice and waited for four weeks before sequencing to assess integrated lentiviral sequence recovery. Using such an "insert library" (i.e., sgRNAs should not affect target gene expression without Cas9), they determined that at least 40 biological replicates are needed for a genome-wide screen of 80,000 and 92,000 elements in their sgRNA and shRNA libraries. Finally, to detect the loss of sgRNA or shRNA elements targeting specific genes as a result of perturbation-related MSN cell death, an inherently low signal-to-noise situation in such pooled library screenings, they employed DrugZ, an optimized algorithm for negative screens (Colic et al., 2019Colic M. Wang G. Zimmermann M. Mascall K. McLaughlin M. Bertolet L. Lenoir W.F. Moffat J. Angers S. Durocher D. Hart T. Identifying chemogenetic interactions from CRISPR screens with drugZ.Genome Med. 2019; 11: 52Crossref PubMed Scopus (64) Google Scholar). Together, the study has defined key parameters and established an analytical framework for a lentiviral-mediated loss-of-function genetic screening in the mouse brain. A main biological insight from this study is to define essential genes and molecular pathways for the survival of MSNs in the adult mouse brain. The authors identified 3,875 and 668 essential genes with their targeting shRNA and sgRNA sequences depleted in the recovered lentiviral genomes, respectively, an indication of neuronal cell death due to partial or complete loss of function of these genes. The relatively fewer neuronal essential genes identified with the CRISPR/Cas9 screen compared to the shRNA screen was somewhat unexpected and attributed to the relatively low efficiency of CRISPR/Cas9 in non-dividing neurons. This notion needs further validation and could be an important factor to consider when choosing libraries for genome-wide CNS screening in the future. Nonetheless, 214 genes were common in both screens, constituting a validated gene set essential for MSN survival. Pathway enrichment analyses reveal that MSN essential genes were enriched with essential genes and pathways previously found in Drosophila and C. elegans or defined as vital genes in human cancer cell lines. The enrichment gene terms include mRNA processing, proteostasis, and oxidative phosphorylation. Other neuronal essential genes, such as neurotrophin signaling and synaptic vesicle function, overlap with previously found neuronal viability genes. An interesting new finding is that a subset of striatal MSN marker genes (i.e., MSN identity genes) are required for MSN survival in the adult mouse striatum. These genes are targets of transcription factor Rarb and are also selectively downregulated in a mHtt CAG-length dependent manner in the striatum of the allelic series knockin mice (Langfelder et al., 2016Langfelder P. Cantle J.P. Chatzopoulou D. Wang N. Gao F. Al-Ramahi I. Lu X.H. Ramos E.M. El-Zein K. Zhao Y. et al.Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice.Nat. Neurosci. 2016; 19: 623-633Crossref PubMed Scopus (228) Google Scholar). Hence, the study suggests that the transcriptional downregulation of the MSN identity genes that are newly identified to be essential for neuronal survival could contribute to selective vulnerability in HD. Another major advance made by Wertz et al., 2020Wertz M.H. Mitchem M.R. Pineda S.S. Hachigian L.J. Lee H. Lau V. Powers A. Kulicke R. Madan G.K. Colic M. et al.Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.Neuron. 2020; 106 (this issue): 76-89Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar is the bold application of the genome-wide screening approach to identify synthetic lethality genes in two HD mouse models: one is R6/2, and the other is a full-length mHtt knockin model (zQ175). By comparing the screening results of HD mice versus input controls, they identified 564 genes as neuroprotective (i.e., essential for MSN survival) in the R6/2 screen and 436 such genes in the zQ175 screen. Interestingly, 263 genes in R6/2 and 253 in zQ175 are found to confer loss-of-function synthetic lethality to MSNs in HD mice only. Of note, 13 of them are shared in both HD mice. Some of the HD-specific neuronal essential genes belong to the same KEGG and Gene Ontology (GO) terms of neuronal essential genes found in WT mice (e.g., enriched with MAPK and neurotrophin signaling and cell-cycle-associated terms), suggesting that the mHTT toxicity further sensitized striatal MSNs to the perturbation of these functions. More interestingly, the HD-specific neuronal essentail gene set, with enrichment terms such as dopaminergic synapse and methylation-dependent chromatin silencing, reveals gene pathways that are uniquely modifying neuronal survival in HD mice. A deeper dive into the rich genetic perturbation datasets from HD mice provides many tantalizing insights that should inspire new studies into HD pathogenesis. First, Wertz et al. (2020) performed overlap analysis and showed that only 33 modifier genes identified in this screen were found in previous screenings of mHTT aggregation or toxicity in invertebrate models, suggesting that many of the genes found in the mammalian brain are indeed novel for the field. Second, several MSN essential genes in the current HD mouse screenings are also genome-wide significant or near significant in HD GWAS studies, e.g., Tacc3, Tcerg1, Farp, Hdlbp, Slc3a2, and Polq (GeM-HD Consortium, 2019GeM-HD ConsortiumCAG Repeat Not Polyglutamine Length Determines Timing of Huntington's Disease Onset.Cell. 2019; 178: 887-900.e14Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). These genes constitute a shortlist of prioritized genes for establishing possible links between human genetic findings and pathogenesis of striatal neuronal loss in HD. Another molecular insight in the study is the functional study of the Nme gene family (Nme1/3/4) as modifiers of mHTT-mediated neuronal toxicity. Nucleoside diphosphate kinase 1 (Nme1) is of particular interest due to its multitude of functions, including protein chaperone, nucleoside and protein kinase function, and DNA repair (Puts et al., 2018Puts G.S. Leonard M.K. Pamidimukkala N.V. Snyder D.E. Kaetzel D.M. Nuclear functions of NME proteins.Lab. Invest. 2018; 98: 211-218Crossref PubMed Scopus (38) Google Scholar). Its expression is decreased in HD patient brains (Hodges et al., 2006Hodges A. Strand A.D. Aragaki A.K. Kuhn A. Sengstag T. Hughes G. Elliston L.A. Hartog C. Goldstein D.R. Thu D. et al.Regional and cellular gene expression changes in human Huntington's disease brain.Hum. Mol. Genet. 2006; 15: 965-977Crossref PubMed Scopus (589) Google Scholar). The authors first validated that Nme1 shRNA led to the loss of striatal neurons, and furthermore, it elicited asymmetrical turning behavior only in the R6/2 mouse model background. Conversely, AAV9-Nme1 overexpression improved behavioral phenotypes and reduced mHTT aggregation in this HD model. Together, these results suggest that Nme1 could modify mHTT fragment toxicity in a mouse model of HD, but further studies in a full-length mHtt model could strengthen such observations. As the first genome-wide screen in the mammalian brain, the study by Wertz et al., 2020Wertz M.H. Mitchem M.R. Pineda S.S. Hachigian L.J. Lee H. Lau V. Powers A. Kulicke R. Madan G.K. Colic M. et al.Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.Neuron. 2020; 106 (this issue): 76-89Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar also raises intriguing questions that could to be further pursued. First, it is interesting that by comparing the screening results of HD mice versus the WT control mice, they surprisingly found that KD of 426 genes in R6/2 mice and 494 genes in zQ175 mice appear "protective" in HD mice, i.e., the shRNA elements were recovered more frequently in HD mice compared to WT controls. This list contains interesting genes, including complement factor C3, dopamine receptor D2, etc. However, since the screen is designed to recover shRNA or sgRNA depletion due to neuronal loss, and there is no known mechanism to amplify such elements. Indeed, about 270 of the 494 genes found as "protective" in zQ175 appear to be essential for neuronal survival in WT mice, but not in zQ175 mice. Thus, an alternative explanation is that the HD mouse backgrounds might dampen the sensitivities to the KD of a distinct subset of genes that are essential to MSNs in WT mice and further investigating the underlying mechanisms could be a fruitful pursuit. Another issue is our lack of understanding of how the striatal MSNs degenerate in the HD patient brains, and studies in existing HD mouse models provide few clues to this mystery due to the minimal or lack of neuronal cell loss in these models. Thus, much work remains to be done to define the precise mechanisms of SLIC-mediated striatal neuronal loss and to validate them in the HD brains. In neuroscience, new tools often spur the pace of discovery. It is our rational optimism to anticipate that these powerful new genome-wide screening tools in the mammalian brain will be impactful in the field of molecular neuroscience and brain disease research. X.W.Y. is on the Scientific Advisory Board of Mitokinin and was a paid consultant for Roche, Nuredis, and Voyager Therapeutics. Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT ToxicityWertz et al.NeuronJanuary 30, 2020In BriefWertz et al. report a method for unbiased genome-wide genetic screening in the CNS to identify neuronal essential genes in vivo. They also apply this approach to uncover modifiers of mutant huntingtin toxicity in vivo and show that Nme1 expression can modulate Huntington's disease model phenotypes. Full-Text PDF Open Archive