Research & Scholarship
CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases.
View details for DOI 10.1038/s41588-018-0070-7
View details for PubMedID 29507424
Raise the Roof: Boosting the Efficacy of a Spinal Muscular Atrophy Therapy.
2017; 93 (1): 3-5
Spinal muscular atrophy is the most common genetic killer of infants. A therapy shows promise in the clinic, but there is a potential limit to its efficacy. In this issue of Neuron, d'Ydewalle et al. (2017) devise a new way to make it more effective.
View details for DOI 10.1016/j.neuron.2016.12.029
View details for PubMedID 28056344
Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.
2016; 353 (6300): 708-712
An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately.
View details for DOI 10.1126/science.aaf7791
View details for PubMedID 27516603
- Regrowing axons with alternative splicing. eLife 2016; 5
Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS.
2015; 18 (9): 1226-1229
C9orf72 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) produced by unconventional translation of the C9orf72 repeat expansions cause neurodegeneration in cell culture and in animal models. We performed two unbiased screens in Saccharomyces cerevisiae and identified potent modifiers of DPR toxicity, including karyopherins and effectors of Ran-mediated nucleocytoplasmic transport, providing insight into potential disease mechanisms and therapeutic targets.
View details for DOI 10.1038/nn.4085
View details for PubMedID 26308983
View details for PubMedCentralID PMC4552077
Altered lymphopoiesis and immunodeficiency in miR-142 null mice.
2015; 125 (24): 3720-3730
MicroRNAs (miRNAs) are a class of powerful posttranscriptional regulators implicated in the control of diverse biological processes, including regulation of hematopoiesis and the immune response. To define the biological functions of miR-142, which is preferentially and abundantly expressed in immune cells, we created a mouse line with a targeted deletion of this gene. Our analysis of miR-142(-/-) mice revealed a critical role for this miRNA in the development and homeostasis of lymphocytes. Marginal zone B cells expand in the knockout spleen, whereas the number of T and B1 B cells in the periphery is reduced. Abnormal development of hematopoietic lineages in miR-142(-/-) animals is accompanied by a profound immunodeficiency, manifested by hypoimmunoglobulinemia and failure to mount a productive immune response to soluble antigens and virus. miR-142(-/-) B cells express elevated levels of B-cell-activating factor (BAFF) receptor (BAFF-R) and as a result proliferate more robustly in response to BAFF stimulation. Lowering the BAFF-R gene dose in miR-142(-/-) mice rescues the B-cell expansion defect, suggesting that BAFF-R is a bona fide miR-142 target through which it controls B-cell homeostasis. Collectively, our results uncover miR-142 as an essential regulator of lymphopoiesis, and suggest that lesions in this miRNA gene may lead to primary immunodeficiency.
View details for DOI 10.1182/blood-2014-10-603951
View details for PubMedID 25931583
Parkinson's Disease Genes VPS35 and EIF4G1 Interact Genetically and Converge on a-Synuclein.
2015; 85 (1): 76-87
Parkinson's disease (PD) is a common neurodegenerative disorder. Functional interactions between some PD genes, like PINK1 and parkin, have been identified, but whether other ones interact remains elusive. Here we report an unexpected genetic interaction between two PD genes, VPS35 and EIF4G1. We provide evidence that EIF4G1 upregulation causes defects associated with protein misfolding. Expression of a sortilin protein rescues these defects, downstream of VPS35, suggesting a potential role for sortilins in PD. We also show interactions between VPS35, EIF4G1, and ?-synuclein, a protein with a key role in PD. We extend our findings from yeast to an animal model and show that these interactions are conserved in neurons and in transgenic mice. Our studies reveal unexpected genetic and functional interactions between two seemingly unrelated PD genes and functionally connect them to ?-synuclein pathobiology in yeast, worms, and mouse. Finally, we provide a resource of candidate PD genes for future interrogation.
View details for DOI 10.1016/j.neuron.2014.11.027
View details for PubMedID 25533483
View details for PubMedCentralID PMC4289081
Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae
2013; 7 (4): 267-279
Infantile-onset neuronal ceroid lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate group from its substrate proteins, which may include presynaptic proteins like SNAP-25, cysteine string protein (CSP), dynamin, and synaptotagmin. The fruit fly, Drosophila melanogaster, has been a powerful model system for studying the functions of these proteins and the molecular basis of neurological disorders like the NCLs. Genetic modifier screens and tracer uptake studies in Ppt1 mutant larval garland cells have suggested that Ppt1 plays a role in endocytic trafficking. We have extended this analysis to examine the involvement of Ppt1 in synaptic function at the Drosophila larval neuromuscular junction (NMJ). Mutations in Ppt1 genetically interact with temperature sensitive mutations in the Drosophila dynamin gene shibire, accelerating the paralytic behavior of shibire mutants at 27 °C. Electrophysiological work in NMJs of Ppt1-deficient larvae has revealed an increase in miniature excitatory junctional potentials (EJPs) and a significant depression of evoked EJPs in response to repetitive (10 hz) stimulation. Endocytosis was further examined in Ppt1-mutant larvae using FM1-43 uptake assays, demonstrating a significant decrease in FM1-43 uptake at the mutant NMJs. Finally, Ppt1-deficient and Ppt1 point mutant larvae display defects in locomotion that are consistent with alterations in synaptic function. Taken together, our genetic, cellular, and electrophysiological analyses suggest a direct role for Ppt1 in synaptic vesicle exo- and endocytosis at motor nerve terminals of the Drosophila NMJ.
View details for DOI 10.4161/fly.26630
View details for Web of Science ID 000330379200010
View details for PubMedID 24091420
View details for PubMedCentralID PMC3896500