Biology subjects

Nitsch, R.

Publications and source records attributed to Nitsch, R..

3 recordsLinked to original sources

bioRxiv2018

Mutant Plasticity Related Gene 1 (PRG1) acts as a potential modifier in SCN1A related epilepsy

Plasticity related gene 1 encodes a cerebral neuron-specific synaptic transmembrane protein that modulates hippocampal excitatory transmission on glutamatergic neurons. In mice, homozygous Prg1-deficiency results in juvenile epilepsy. Screening a cohort of 18 patients with infantile spasms (West syndrome), we identified one patient with a heterozygous mutation in the highly conserved third extracellular phosphatase domain (p.T299S). The functional relevance of this mutation was verified by in-utero electroporation of a mutant Prg1 construct into neurons of Prg1-knockout embryos, and the subsequent inability of hippocampal neurons to rescue the knockout phenotype on the single cell level. Whole exome sequencing revealed the index patient to additionally harbor a novel heterozygous SCN1A variant (p.N541S) that was inherited from her healthy mother. Only the affected child carried both heterozygous PRG1 and SCN1A mutations. The aggravating effect of Prg1-haploinsufficiency on the epileptic phenotype was verified using the kainate-model of epilepsy. Double heterozygous Prg1-/+|Scn1awt/p.R1648Hmice exhibited higher seizure susceptibility than either wildtype, Prg1-/+, or Scn1awt/p.R1648H littermates. Our study provides evidence that PRG1-mutations have a potential modifying influence on SCN1A-related epilepsy in humans.

neuroscience↗

bioRxiv2018

In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1-5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8-12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

molecular biology↗

bioRxiv2017

Targeting xCT-Mediated Glutamate Release Normalizes Tumor Angiogenesis In The Brain

Brain tumors are among the most malignant primary tumors, hallmarked by angiogenesis, neuronal destruction and brain swelling. Inhibition of the glutamate-cystein antiporter xCT (system xc-/SLC7A11) alleviates seizures, neuronal cell death and tumor-associated brain edema. Here we show enhanced tumor vessel growth and increased brain edema in xCT-expressing brain tumors. Furthermore, xCT-mediated glutamate impacts directly on endothelial cells in an N-methyl-D-aspartate receptor (NMDAR) dependent manner with intracellular Ca2+ release. Cerebral intravital microscopy revealed that xCT-driven tumor vessels are functional and display increased permeability. Endothelial-cell-specific NMDAR1 knockout mice (GRINi{Delta}EC) show suppressed endothelial sprouting and vascular density compared to control littermates. In addition, implanted gliomas in GRINi{Delta}EC mice display reduced tumor vessels in contrast to gliomas in wildtype animals. Moreover, therapeutic targeting of xCT in gliomas alleviates tumor angiogenesis to normalized levels comparable to controls. Our data reveal that xCT and its substrate glutamate specifically operate on endothelial cells and promote neoangiogenesis. Thus, targeting xCT expression and glutamate secretion in gliomas provides a novel therapeutic roadmap for normalizing tumor angiogenesis.

cell biology↗