RESUMEN
Brain somatic variants in SLC35A2, an intracellular UDP-galactose transporter, are commonly identified mutations associated with drug-resistant neocortical epilepsy and developmental brain malformations, including focal cortical dysplasia type I and mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). However, the causal effects of altered SLC35A2 function on cortical development remain untested. We hypothesized that focal Slc35a2 knockout (KO) or knockdown (KD) in the developing mouse cortex would disrupt cortical development and change network excitability. Through two independent studies, we used in utero electroporation (IUE) to introduce CRISPR/Cas9/targeted guide RNAs or short-hairpin RNAs into the embryonic mouse brain at day 14.5-15.5 to achieve Slc35a2 KO or KD, respectively, from neural precursor cells. Slc35a2 KO or KD caused disrupted radial migration of electroporated neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, importantly suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects of Slc35a2 mutations. Adult KO mice were implanted with EEG electrodes for 72-hour continuous recording. Spontaneous seizures were not observed in focal Slc35a2 KO mice, but there was reduced seizure threshold following pentylenetetrazol injection. Here we demonstrate that focal Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration and that KO leads to reduced seizure threshold. Together these results demonstrate a direct causal role for SLC35A2 in cortical development.
Asunto(s)
Corteza Cerebral , Proteínas de Transporte de Monosacáridos , Animales , Corteza Cerebral/metabolismo , Ratones , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/deficiencia , Ratones Noqueados , Neuronas/metabolismo , Oligodendroglía/metabolismo , Femenino , Epilepsia/genética , Epilepsia/patología , Movimiento CelularRESUMEN
Brain somatic variants in SLC35A2 are associated with clinically drug-resistant epilepsy and developmental brain malformations, including mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). SLC35A2 encodes a uridine diphosphate galactose translocator that is essential for protein glycosylation; however, the neurodevelopmental mechanisms by which SLC35A2 disruption leads to clinical and histopathological features remain unspecified. We hypothesized that focal knockout (KO) or knockdown (KD) of Slc35a2 in the developing mouse cortex would disrupt cerebral cortical development through altered neuronal migration and cause changes in network excitability. We used in utero electroporation (IUE) to introduce CRISPR/Cas9 and targeted guide RNAs or short-hairpin RNAs to achieve Slc35a2 KO or KD, respectively, during early corticogenesis. Following Slc35a2 KO or KD, we observed disrupted radial migration of transfected neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects. Spontaneous seizures were not observed, but intracranial EEG recordings after focal KO showed a reduced seizure threshold following pentylenetetrazol injection. These results demonstrate that Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration.
RESUMEN
The goal of this study was to document if lakes in National Parks in Washington have exceeded critical levels of nitrogen (N) deposition, as observed in other Western States. We measured atmospheric N deposition, lake water quality, and sediment diatoms at our study lakes. Water chemistry showed that our study lakes were ultra-oligotrophic with ammonia and nitrate concentrations often at or below detection limits with low specific conductance (<100 µS/cm), and acid neutralizing capacities (<400 µeq/L). Rates of summer bulk inorganic N deposition at all our sites ranged from 0.6 to 2.4 kg N ha-1 year-1 and were variable both within and across the parks. Diatom assemblages in a single sediment core from Hoh Lake (Olympic National Park) displayed a shift to increased relative abundances of Asterionella formosa and Fragilaria tenera beginning in the 1969-1975 timeframe, whereas these species were not found at the remaining (nine) sites. These diatom species are known to be indicative of N enrichment and were used to determine an empirical critical load of N deposition, or threshold level, where changes in diatom communities were observed at Hoh Lake. However, N deposition at the remaining nine lakes does not seem to exceed a critical load at this time. At Milk Lake, also in Olympic National Park, there was some evidence that climate change might be altering diatom communities, but more research is needed to confirm this. We used modeled precipitation for Hoh Lake and annual inorganic N concentrations from a nearby National Atmospheric Deposition Program station, to calculate elevation-corrected N deposition for 1980-2009 at Hoh Lake. An exponential fit to this data was hindcasted to the 1969-1975 time period, and we estimate a critical load of 1.0 to 1.2 kg N ha-1 year-1 for wet deposition for this lake.