Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair

Neurodegeneration is a common hallmark of individuals with hereditary defects in DNA single-strand break repair; a process regulated by poly(ADP-ribose) metabolism. Recently, mutations in the ARH3 (ADPRHL2) hydrolase that removes ADP-ribose from proteins have been associated with neurodegenerative disease. Here, we show that ARH3-mutated patient cells accumulate mono(ADP-ribose) scars on core histones that are a molecular memory of recently repaired DNA single-strand breaks. We demonstrate that the ADP-ribose chromatin scars result in reduced endogenous levels of important chromatin modifications such as H3K9 acetylation, and that ARH3 patient cells exhibit measurable levels of deregulated transcription. Moreover, we show that the mono(ADP-ribose) scars are lost from the chromatin of ARH3-defective cells in the prolonged presence of PARP inhibition, and concomitantly that chromatin acetylation is restored to normal. Collectively, these data indicate that ARH3 can act as an eraser of ADP-ribose chromatin scars at sites of PARP activity during DNA single-strand break repair

Committing curriculum time to science literacy: The benefits from science based media resources

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT), which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells

Elucidating the Genetic and Molecular Mechanisms of Recessive Pediatric Brain Disease

The structural organization and maturation of the brain are the result of a precisely orchestrated series of developmental processes with complex genetic regulation that occur during embryonic gestation and are critical for neuronal identity, wiring and connectivity. Mutations affecting any of the cellular processes involved in proper human brain development can lead to altered neurodevelopment and result in a number of different neurological diseases. Such diseases arising as a consequence of a biallelic mutation in a single gene are much more prevalent among offspring of consanguineous marriages, increasing the odds that a deleterious mutation will be inherited on both chromosomes. The identification of a disease-causing gene in families with inherited brain disorders is one of the most useful methods for gaining insight into human brain development, function, and pathology. This study largely focuses on identifying novel mutations in both known and novel recessive pediatric brain diseases using next-generation sequencing approaches in combination with in vitro and in vivo modeling. By taking advantage of the large number of consanguineous families in our cohort, we are not only able to explore the cause of disease directly in humans, but also able to determine genetic disease risk and identify novel disease-causing variants with a high level of certainty. A total of 88 affected individuals were identified and studied from 46 families displaying a range of SBDs including neurodegeneration (i.e. cortical and cerebellar atrophy), microlissencephaly, PCH, and Joubert syndrome, among other symptoms. A total of six disease-causing genes, ADPRHL2, TMX2, TRAPPC4, HEATR5B, HPDL, and ARMC9 were identified, four of which had never been implicated in disease prior to this study. We were further able to model disease in vivo for three out of the five genes (ADPRHL2, HEATR5B, and HPDL) using either fly or mouse models. Taken together, this study not only provides further knowledge in identifying novel causes of both previously unknown diseases as well as known diseases with unknown cause, but also provides further promise to the application of NGS in revealing the full spectrum of mutations associated with these diseases and in further delineating the relevant molecular pathways involved

Elucidating the Genetic and Molecular Mechanisms of Recessive Pediatric Brain Disease

The structural organization and maturation of the brain are the result of a precisely orchestrated series of developmental processes with complex genetic regulation that occur during embryonic gestation and are critical for neuronal identity, wiring and connectivity. Mutations affecting any of the cellular processes involved in proper human brain development can lead to altered neurodevelopment and result in a number of different neurological diseases. Such diseases arising as a consequence of a biallelic mutation in a single gene are much more prevalent among offspring of consanguineous marriages, increasing the odds that a deleterious mutation will be inherited on both chromosomes. The identification of a disease-causing gene in families with inherited brain disorders is one of the most useful methods for gaining insight into human brain development, function, and pathology. This study largely focuses on identifying novel mutations in both known and novel recessive pediatric brain diseases using next-generation sequencing approaches in combination with in vitro and in vivo modeling. By taking advantage of the large number of consanguineous families in our cohort, we are not only able to explore the cause of disease directly in humans, but also able to determine genetic disease risk and identify novel disease-causing variants with a high level of certainty. A total of 88 affected individuals were identified and studied from 46 families displaying a range of SBDs including neurodegeneration (i.e. cortical and cerebellar atrophy), microlissencephaly, PCH, and Joubert syndrome, among other symptoms. A total of six disease-causing genes, ADPRHL2, TMX2, TRAPPC4, HEATR5B, HPDL, and ARMC9 were identified, four of which had never been implicated in disease prior to this study. We were further able to model disease in vivo for three out of the five genes (ADPRHL2, HEATR5B, and HPDL) using either fly or mouse models. Taken together, this study not only provides further knowledge in identifying novel causes of both previously unknown diseases as well as known diseases with unknown cause, but also provides further promise to the application of NGS in revealing the full spectrum of mutations associated with these diseases and in further delineating the relevant molecular pathways involved

Motoneuron expression profiling identifies an association between an axonal splice variant of HDGF-related protein 3 and peripheral myelination

Disorders that disrupt myelin formation during development or in adulthood, such as multiple sclerosis and peripheral neuropathies, lead to severe pathologies, illustrating myelin's crucial role in normal neural functioning. However, although our understanding of glial biology is increasing, the signals that emanate from axons and regulate myelination remain largely unknown. To identify the core components of the myelination process, here we adopted a microarray analysis approach combined with laser-capture microdissection of spinal motoneurons during the myelinogenic phase of development. We identified neuronal genes whose expression was enriched during myelination and further investigated hepatoma-derived growth factor-related protein 3 (HRP3 or HDGFRP3). HRP3 was strongly expressed in the white matter fiber tracts of the peripheral (PNS) and central (CNS) nervous systems during myelination and remyelination in a cuprizone-induced demyelination model. The dynamic localization of HPR3 between axons and nuclei during myelination was consistent with its axonal localization during neuritogenesis. To study this phenomenon, we identified two splice variants encoded by theHRP3gene: the canonical isoform HRP3-I and a newly recognized isoform, HRP3-II. HRP3-I remained solely in the nucleus, whereas HRP3-II displayed distinct axonal localization both before and during myelination. Interestingly, HRP3-II remained in the nuclei of unmyelinated neurons and glial cells, suggesting the existence of a molecular machinery that transfers it to and retains it in the axons of neurons fated for myelination. Overexpression of HRP3-II, but not of HRP3-I, increased Schwann cell numbers and myelination in PNS neuron-glia co-cultures. However, HRP3-II overexpression in CNS co-cultures did not alter myelination.Christopher and Dana Reeve Foundation Roche Foundation Swiss National Science Foundation (SNSF) American Heart Association Paul G. Allen Frontiers Group Grant Leona M. and Harry B. Helmsley Charitable Trust Grant Grace Foundation JPB Foundation Robert and Mary Jane Engman Foundation Ray and Dagmar Dolby Family Fund Crick-Jacob Center for Theoretical and Computational Biology Neurosurgery Neuroscience Consortium Fellowshi

Loss of NARS1 impairs progenitor proliferation in cortical brain organoids and leads to microcephaly

Asparaginyl-tRNA synthetase1 (NARS1) is a member of the ubiquitously expressed cytoplasmic Class IIa family of tRNA synthetases required for protein translation. Here, we identify biallelic missense and frameshift mutations in NARS1 in seven patients from three unrelated families with microcephaly and neurodevelopmental delay. Patient cells show reduced NARS1 protein, impaired NARS1 activity and impaired global protein synthesis. Cortical brain organoid modeling shows reduced proliferation of radial glial cells (RGCs), leading to smaller organoids characteristic of microcephaly. Single-cell analysis reveals altered constituents of both astrocytic and RGC lineages, suggesting a requirement for NARS1 in RGC proliferation. Our findings demonstrate that NARS1 is required to meet protein synthetic needs and to support RGC proliferation in human brain development

Author Correction: Loss of NARS1 impairs progenitor proliferation in cortical brain organoids and leads to microcephaly (Nature Communications, (2020), 11, 1, (4038), 10.1038/s41467-020-17454-4)

The original version of this Article omitted a reference to another publication which included overlapping genetic and MRI data for a research participant. This has been added as reference 59 at the end of the Discussion: ‘While this paper was under review, a separate paper appeared reporting that mutations in NARS1 associate with neurodevelopmental delay through either biallelic loss or dominant negative effects, impairing NARS1 enzyme activity. This article contained genetic data on family MIC-1433 which overlaps this study59.’ Accordingly, reference 59 has now been included in the References section as ‘Manole, A. et al. De novo and bi-allelic pathogenic variants in NARS1 cause neurodevelopmental delay due to toxic gain-of-function and partial loss-of-function effects. Am. J. Hum. Genet. 107(2), 311–324 (2020).’ In addition, the original version of this Article contained an error in Fig. 1A, where there was an error in the family depiction in generation 1 of pedigree MIC-1433; this has been revised to correctly depict the family. Figure 1D also contained MRI scans that had been previously published in Ref 59; this has been revised to include different and unpublished MRI scans from the same individual. (Figure presented.)