Excitotoxic glutamate levels cause the secretion of resident endoplasmic reticulum proteins.

Dysregulation of synaptic glutamate levels can lead to excitotoxicity such as that observed in stroke, traumatic brain injury, and epilepsy. The role of increased intracellular calcium (Ca2+ ) in the development of excitotoxicity is well established. However, less is known regarding the impact of glutamate on endoplasmic reticulum (ER)-Ca2+ -mediated processes such as proteostasis. To investigate this, we expressed a secreted ER Ca2+ modulated protein (SERCaMP) in primary cortical neurons to monitor exodosis, a phenomenon whereby ER calcium depletion causes the secretion of ER-resident proteins that perform essential functions to the ER and the cell. Activation of glutamatergic receptors (GluRs) led to an increase in SERCaMP secretion indicating that normally ER-resident proteins are being secreted in a manner consistent with ER Ca2+ depletion. Antagonism of ER Ca2+ channels attenuated the effects of glutamate and GluR agonists on SERCaMP release. We also demonstrate that endogenous proteins containing an ER retention/retrieval sequence (ERS) are secreted in response to GluR activation supporting that neuronal activation by glutamate promotes ER exodosis. Ectopic expression of KDEL receptors attenuated the secretion of ERS-containing proteins caused by GluR agonists. Taken together, our data indicate that excessive GluR activation causes disruption of neuronal proteostasis by triggering the secretion of ER-resident proteins through ER Ca2+ depletion and describes a new facet of excitotoxicity.

Transcription-associated DNA DSBs activate p53 during hiPSC-based neurogenesis.

Neurons are overproduced during cerebral cortical development. Neural progenitor cells (NPCs) divide rapidly and incur frequent DNA double-strand breaks (DSBs) throughout cortical neurogenesis. Although half of the neurons born during neurodevelopment die, many neurons with inaccurate DNA repair survive leading to brain somatic mosaicism. Recurrent DNA DSBs during neurodevelopment are associated with both gene expression level and gene length. We used imaging flow cytometry and a genome-wide DNA DSB capture approach to quantify and map DNA DSBs during human induced pluripotent stem cell (hiPSC)-based neurogenesis. Reduced p53 signaling was brought about by knockdown (p53KD); p53KD led to elevated DNA DSB burden in neurons that was associated with gene expression level but not gene length in neural progenitor cells (NPCs). Furthermore, DNA DSBs incurred from transcriptional, but not replicative, stress lead to p53 activation in neurotypical NPCs. In p53KD NPCs, DNA DSBs accumulate at transcription start sites of genes that are associated with neurological and psychiatric disorders. These findings add to a growing understanding of how neuronal genome dynamics are engaged by high transcriptional or replicative burden during neurodevelopment.

Stability of advanced life support drugs in the field.

PURPOSE: The effects of wide temperature variations on the stability of atropine, epinephrine, and lidocaine stored under field conditions in advanced life support (ALS) paramedic units were evaluated. METHODS: Vehicles from various ALS paramedic units were selected throughout Los Angeles County, California, including desert, marine, and helicopter-based divisions. A temperature-recording device was placed in the compartment where drugs are stored and used to record and store temperature data at 15-minute intervals. Three autoinjector-style syringes of atropine, epinephrine, and lidocaine were taken from stock for each ALS unit and placed in each vehicle, while three control syringes were stored in the laboratory under controlled conditions. Six samples of each drug were withdrawn at time 0 and on days 5, 10, 15, 30, and 45. Samples were analyzed using high-performance liquid chromatography. Stock solutions, created using analytical grade atropine, epinephrine, and lidocaine, were used to construct 5-point standard curves to determine the drug concentration of each sample. RESULTS: Seven sites exceeded 104 degrees F (40 degrees C) for as little as 30 minutes and as long as 795 minutes. Ten of the sites achieved a mean kinetic temperature (MKT) above 77 degrees F (25 degrees C), with the highest MKT calculated being 84.1 degrees F (28.9 degrees C) over a 45-day period. There was no evidence of drug degradation at any site, at any temperature, or at any time point. CONCLUSION: Atropine, epinephrine, and lidocaine can be stored at temperatures of up to 84.1 degrees F (28.9 degrees C) for up to 45 days and tolerate temperature spikes of up to 125 degrees F (51.7 degrees C) for a cumulative time of 795 minutes (13.25 hours) without undergoing degradation.