Genetic evidence for splicing-dependent structural and functional plasticity in CASK protein

Journal of Medical Genetics · 2024-04-26 · 3 citations

Background Pontocerebellar hypoplasia (PCH) may present with supratentorial phenotypes and is often accompanied by microcephaly. Damaging mutations in the X-linked gene CASK produce self-limiting microcephaly with PCH in females but are often lethal in males. CASK deficiency leads to early degeneration of cerebellar granule cells but its role in other regions of the brain remains uncertain. Method We generated a conditional Cask knockout mice and deleted Cask ubiquitously after birth at different times. We examined the clinical features in several subjects with damaging mutations clustered in the central part of the CASK protein. We have performed phylogenetic analysis and RT-PCR to assess the splicing pattern within the same protein region and performed in silico structural analysis to examine the effect of splicing on the CASK’s structure. Result We demonstrate that deletion of murine Cask after adulthood does not affect survival but leads to cerebellar degeneration and ataxia over time. Intriguingly, damaging hemizygous CASK mutations in boys who display microcephaly and cerebral dysfunction but without PCH are known. These mutations are present in two vertebrate-specific CASK exons. These exons are subject to alternative splicing both in forebrain and hindbrain. Inclusion of these exons differentially affects the molecular structure and hence possibly the function/s of the CASK C-terminus. Conclusion Loss of CASK function disproportionately affects the cerebellum. Clinical data, however, suggest that CASK may have additional vertebrate-specific function/s that play a role in the mammalian forebrain. Thus, CASK has an ancient function shared between invertebrates and vertebrates as well as novel vertebrate-specific function/s.

Residency Program Directors’ Views on Research Conducted During Medical School: A National Survey

Academic Medicine · 2023 · 67 citations

• Computer Science
• Medical education
• Psychology

PURPOSE: With the United States Medical Licensing Examination Step 1 transition to pass/fail in 2022, uncertainty exists regarding how other residency application components, including research conducted during medical school, will inform interview and ranking decisions. The authors explore program director (PD) views on medical student research, the importance of disseminating that work, and the translatable skill set of research participation. METHOD: Surveys were distributed to all U.S. residency PDs and remained open from August to November 2021 to query the importance of research participation in assessing applicants, whether certain types of research were more valued, productivity measures that reflect meaningful research participation, and traits for which research serves as a proxy. The survey also queried whether research would be more important without a numeric Step 1 score and the importance of research vs other application components. RESULTS: A total of 885 responses from 393 institutions were received. Ten PDs indicated that research is not considered when reviewing applicants, leaving 875 responses for analysis. Among 873 PDs (2 nonrespondents), 358 (41.0%) replied that meaningful research participation will be more important in offering interviews. A total of 164 of 304 most competitive specialties (53.9%) reported increased research importance compared with 99 of 282 competitive (35.1%) and 95 of 287 least competitive (33.1%) specialties. PDs reported that meaningful research participation demonstrated intellectual curiosity (545 [62.3%]), critical and analytical thinking skills (482 [55.1%]), and self-directed learning skills (455 [52.0%]). PDs from the most competitive specialties were significantly more likely to indicate that they value basic science research vs PDs from the least competitive specialties. CONCLUSIONS: This study demonstrates how PDs value research in their review of applicants, what they perceive research represents in an applicant, and how these views are shifting as the Step 1 exam transitions to pass/fail.

Flexibility of the Rotavirus NSP2 C-Terminal Region Supports Factory Formation via Liquid-Liquid Phase Separation

Journal of Virology · 2023-02-07 · 24 citations

Viruses often condense the materials needed for their replication into discrete intracellular factories. For rotaviruses, agents of severe gastroenteritis in children, factory formation is mediated in part by an octameric protein called NSP2. A flexible C-terminal region of NSP2 has been proposed to link several NSP2 octamers together, a feature that might be important for factory formation. Here, we created a change in NSP2 that reduced C-terminal flexibility and analyzed the impact on rotavirus factories. We found that the change caused the formation of smaller and more numerous factories that could not readily fuse together like those of the wild-type virus. The altered NSP2 protein also had a reduced capacity to form factory-like condensates in a test tube. Together, these results add to our growing understanding of how NSP2 supports rotavirus factory formation-a key step of viral replication.

Phosphorylation of guanosine monophosphate reductase triggers a GTP-dependent switch from pro- to anti-oncogenic function of EPHA4

Cell chemical biology · 2022-02-10 · 15 citations

Complete loss of the X-linked gene <i>CASK</i> causes severe cerebellar degeneration

Journal of Medical Genetics · 2022-02-11 · 15 citations

Background Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes developmental delay in girls, while in boys, loss of the only allele of these genes leads to epileptic encephalopathy. The mechanism for these disorders remains unknown. CASK -linked cerebellar hypoplasia is presumed to result from defects in Tbr1-reelin-mediated neuronal migration. Method Here we report clinical and histopathological analyses of a deceased 2-month-old boy with a CASK -null mutation. We next generated a mouse line where CASK is completely deleted (hemizygous and homozygous) from postmigratory neurons in the cerebellum. Result The CASK -null human brain was smaller in size but exhibited normal lamination without defective neuronal differentiation, migration or axonal guidance. The hypoplastic cerebellum instead displayed astrogliosis and microgliosis, which are markers for neuronal loss. We therefore hypothesise that CASK loss-induced cerebellar hypoplasia is the result of early neurodegeneration. Data from the murine model confirmed that in CASK loss, a small cerebellum results from postdevelopmental degeneration of cerebellar granule neurons. Furthermore, at least in the cerebellum, functional loss from CASK deletion is secondary to degeneration of granule cells and not due to an acute molecular functional loss of CASK . Intriguingly, female mice with heterozygous deletion of CASK in the cerebellum do not display neurodegeneration. Conclusion We suggest that X-linked neurodevelopmental disorders like CASK mutation and Rett syndrome are pathologically neurodegenerative; random X-chromosome inactivation in heterozygous mutant girls, however, results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.

The Non-Linear Path from Gene Dysfunction to Genetic Disease: Lessons from the MICPCH Mouse Model

Cells · 2022-03-28 · 7 citations

Most human disease manifests as a result of tissue pathology, due to an underlying disease process (pathogenesis), rather than the acute loss of specific molecular function(s). Successful therapeutic strategies thus may either target the correction of a specific molecular function or halt the disease process. For the vast majority of brain diseases, clear etiologic and pathogenic mechanisms are still elusive, impeding the discovery or design of effective disease-modifying drugs. The development of valid animal models and their proper characterization is thus critical for uncovering the molecular basis of the underlying pathobiological processes of brain disorders. MICPCH (microcephaly and pontocerebellar hypoplasia) is a monogenic condition that results from variants of an X-linked gene, CASK (calcium/calmodulin-dependent serine protein kinase). CASK variants are associated with a wide range of clinical presentations, from lethality and epileptic encephalopathies to intellectual disabilities, microcephaly, and autistic traits. We have examined CASK loss-of-function mutations in model organisms to simultaneously understand the pathogenesis of MICPCH and the molecular function/s of CASK. Our studies point to a highly complex relationship between the potential molecular function/s of CASK and the phenotypes observed in model organisms and humans. Here we discuss the implications of our observations from the pathogenesis of MICPCH as a cautionary narrative against oversimplifying molecular interpretations of data obtained from genetically modified animal models of human diseases.

Complete Loss of CASK Causes Severe Ataxia Through Cerebellar Degeneration

Research Square · 2021-05-25 · 1 citations

Complete loss of CASK causes severe ataxia through cerebellar degeneration

Research Square · 2021-05-04

Abstract Background: Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes neurodevelopmental disorders (NDD) in girls, while in boys loss of the only allele of these genes leads to profound encephalopathy. The cellular basis for these disorders remains unknown. CASK is presumed to work through the Tbr1-reelin pathway in neuronal migration. Methods: Here we report clinical and histopathological analysis of a deceased 2-month-old boy with a CASK-null mutation. We first analyze in vivo data from the subject including genetic characterization, magnetic resonance imaging (MRI) findings, and spectral characteristics of the electroencephalogram (EEG). We next compare features of the cerebellum to an-age matched control. Based on this, we generate a murine model where CASK is completely deleted from post-migratory neurons in the cerebellum. Results: Although smaller, the CASK-null human brain exhibits normal lamination without defective neuronal differentiation, migration, or axonal guidance, excluding the role of reelin. The hypoplastic cerebellum instead displayed astrogliosis, a marker for neuronal loss. We therefore hypothesized that cerebellar hypoplasia with CASK loss is a result of early neurodegeneration. Data from our murine model confirm that a small cerebellum in the context of CASK-loss results from post-developmental degeneration of cerebellar granule neurons. We further demonstrate that at least in the cerebellum the functional loss with CASK deletion results secondary to degeneration of granule cells rather that any acute molecular functional loss of CASK. Intriguingly, female mice with heterozygous deletion of CASK in the cerebellum did not display any neurodegeneration. Conclusions: Herein we demonstrate in human that complete loss of CASK is not associated with any dysfunction in the Tbr1-reelin pathway. Further, immunohistochemistry indicates a degenerative cell death which is mechanistically confirmed in our murine model. In the absence of CASK in cerebellar cells, the cerebellum develops normally but degenerates over a period of several weeks. We suggest that NDDs like CASK mutation and Rett syndrome are pathologically neurodegenerative; however, random X-chromosome inactivation in the heterozygous mutant girls results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy.

Complete loss of CASK causes severe ataxia through cerebellar degeneration in human and mouse

bioRxiv (Cold Spring Harbor Laboratory) · 2021-03-22 · 2 citations

Abstract Heterozygous loss of X-linked genes like CASK and MeCP2 (Rett syndrome) causes neurodevelopmental disorders (NDD) in girls, while in boys such loss leads to profound encephalopathy. The cellular basis for these disorders remains unknown. CASK is presumed to work through the Tbr1-reelin pathway in neuronal migration during brain development. Here we report our clinical and histopathological analysis of a deceased 2-month-old boy with a CASK-null mutation. We demonstrate that although smaller in size, the CASK-null human brain exhibits normal lamination without defective neuronal differentiation, migration, or axonal guidance, excluding the role of reelin in CASK-linked pathology. The disproportionately hypoplastic cerebellum in humans without CASK expression is associated with cerebellar astrogliosis, a marker for neuronal loss. Cerebellum-specific deletion in mouse confirms a post-developmental degeneration of cerebellar granular neurons that results in a small cerebellum. Mechanistically, cerebellar hypoplasia in CASK mutation thus results from neurodegeneration rather that developmental defects. Zygosity-pathology correlation suggests that NDDs like CASK mutation and Rett syndrome are pathologically neurodegenerative; however, random X-chromosome inactivation in the typical heterozygous mutant girls results in 50% of cells expressing the functional gene, resulting in a non-progressive pathology, whereas complete loss of the only allele in boys leads to unconstrained degeneration and encephalopathy. One sentence summary of study CASK loss causes cerebellar degeneration. The authors have declared that no conflict of interest exists.

Reverse genetic engineering of simian rotaviruses with temperature-sensitive lesions in VP1, VP2, and VP6

Virus Research · 2021-06-17 · 7 citations