Taurine is a ubiquitous sulfur-containing amino acid found in high concentration in most tissues. Because of its involvement in fundamental physiological functions, such as regulating respiratory chain activity, modulating cation transport, controlling inflammation, altering protein phosphorylation and prolonging lifespan, taurine is an important nutrient whose deficiency leads to severe pathology and cell death. However, the mechanism by which taurine deficiency causes cell death is inadequately understood. Therefore, the present study examined the hypothesis that overproduction of reactive oxygen species (ROS) by complex I of the respiratory chain triggers mitochondria-dependent apoptosis in hearts of taurine transporter knockout (TauTKO) mice. In support of the hypothesis, a 60% decrease in mitochondrial taurine content of 3-month-old TauTKO hearts was observed, which was associated with diminished complex I activity and the onset of mitochondrial oxidative stress. Oxidative damage to stressed mitochondria led to activation of a caspase cascade, with stimulation of caspases 9 and 3 prevented by treatment of 3-month-old TauTKO mice with the mitochondria specific antioxidant, MitoTempo. In 12 month-old, but not 3-month-old, TauTKO hearts, caspase 12 activation contributes to cell death, revealing a pathological role for endoplasmic reticulum (ER) stress in taurine deficient, aging mice. Thus, taurine is a cytoprotective nutrient that ensures normal mitochondrial and ER function, which is important for the reduction of risk for apoptosis and premature death.

Previously, we have shown that the GABA synthesizing enzyme, L-glutamic acid decarboxylase 65 (GAD65) is cleaved to form its truncated form (tGAD65) which is 2–3 times more active than the full length form (fGAD65). The enzyme responsible for cleavage was later identified as calpain. Calpain is known to cleave its substrates either under a transient physiological stimulus or upon a sustained pathological insult. However, the precise role of calpain cleavage of fGAD65 is poorly understood. In this communication, we examined the cleavage of fGAD65 under diverse pathological conditions including rats under ischemia/reperfusion insult as well as rat brain synaptosomes and primary neuronal cultures subjected to excessive stimulation with high concentration of KCl. We have shown that the formation of tGAD65 progressively increases with increasing stimulus concentration both in rat brain synaptosomes and primary rat embryo cultures. More importantly, direct cleavage of synaptic vesicle - associated fGAD65 by calpain was demonstrated and the resulting tGAD65 bearing the active site of the enzyme was detached from the synaptic vesicles. Vesicular GABA transport of the newly synthesized GABA was found to be reduced in calpain treated SVs. Furthermore, we also observed that the levels of tGAD65 in the focal cerebral ischemic rat brain tissue increased corresponding to the elevation of local glutamate as indicated by microdialysis. Moreover, the levels of tGAD65 was also proportional to the degree of cell death when the primary neuronal cultures were exposed to high KCl. Based on these observations, we conclude that calpain-mediated cleavage of fGAD65 is pathological, presumably due to decrease in the activity of synaptic vesicle - associated fGAD65 resulting in a decrease in the GABA synthesis - packaging coupling process leading to reduced GABA neurotransmission.

Alzheimer's disease (AD) is the leading cause of mortality, disability, and long‐term care burden in the United States, with women comprising the majority of AD diagnoses. While AD‐related dementia is associated with tau and amyloid beta accumulation, concurrent derangements in cerebral blood flow have been observed alongside these proteinopathies in humans and rodent models. The homeostatic production of nitric oxide synthases (NOS) becomes uncoupled in AD which leads to decreased NO‐mediated vasodilation and oxidative stress via the production of peroxynitrite (ONOO−∙) superoxide species. Here, we investigate the role of the novel protein arginine methyltransferase 4 (PRMT4) enzyme function and its downstream product asymmetric dimethyl arginine (ADMA) as it relates to NOS dysregulation and cerebral blood flow in AD. ADMA (type‐1 PRMT product) has been shown to bind NOS as a noncanonic ligand causing enzymatic dysfunction. Our results from RT‐qPCR and protein analyses suggest that aged (9−12 months) female mice bearing tau‐ and amyloid beta‐producing transgenic mutations (3xTg‐AD) express higher levels of PRMT4 in the hippocampus when compared to age‐ and sex‐matched C57BL6/J mice. In addition, we performed studies to quantify the expression and activity of different NOS isoforms. Furthermore, laser speckle contrast imaging analysis was indicative that 3xTg‐AD mice have dysfunctional NOS activity, resulting in reduced production of NO metabolites, enhanced production of free‐radical ONOO−, and decreased cerebral blood flow. Notably, the aforementioned phenomena can be reversed via pharmacologic PRMT4 inhibition. Together, these findings implicate the potential importance of PRMT4 signaling in the pathogenesis of Alzheimer's‐related cerebrovascular derangement.

Background: Global ischemia is the resulting effect of a cardiopulmonary arrest (CPA). Presently there is no effective treatment to address neurological deficits in patients who survived a CPA. Granulocyte-colony stimulating factor is a growth factor (G-CSF) with a plethora of beneficial effects, including neuroprotection. Clinical application of human G-CSF (hG-CSF) is limited due to its plasma half-life of 4 h. Therefore, novel approaches need to be investigated that would (1) enable prolonged manifestation of hG-CSF and (2) demonstrate G-CSF efficacy from studying the underlying protective mechanisms of hG-CSF. In our previous work, we used the self-complementary adeno-associated virus (stereotype2: scAAV2) as a vector to transfect the hG-CSF gene into the global ischemic brain of a mouse. As an extension of that work, we now seek to elucidate the protective mechanisms of hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia. Method: A single drop of either AAV-CMV-hG-CSF or AAV-CMV-GFP was dropped into the conjunctival sac of the Swiss Webster mouse’s left eye, 30–60 min after bilateral common artery occlusion (BCAO). The efficacy of the expressed hG-CSF gene product was analyzed by monitoring the expression levels of endoplasmic reticulum stress (ER), mitochondrial dynamics and autophagic proteins over 4- and 7-days post-BCAO in vulnerable brain regions including the striatum, overlying cortex (frontal brain regions) and the hippocampus (middle brain regions). Statistical analysis was performed using mostly One-Way Analysis of variance (ANOVA), except for behavioral analysis, which used Repeated Measures Two-Way ANOVA, post hoc analysis was performed using the Tukey test. Results: Several biomarkers that facilitated cellular death, including CHOP and GRP78 (ER stress) DRP1 (mitochondrial dynamics) and Beclin 1, p62 and LC3-ll (autophagy) were significantly downregulated by hG-CSF gene transfer. hG-CSF gene therapy also significantly upregulated antiapoptotic Bcl2 while downregulating pro-apoptotic Bax. The beneficial effects of hG-CSF gene therapy resulted in an overall improvement in functional behavior. Conclusion: Taken together, this study has substantiated the approach of sustaining the protein expression of hG-CSF by eye drop administration of the hG-CSF gene. In addition, the study has validated the efficacy of using hG-CSF gene therapy against endoplasmic reticulum induced stress, mitochondrial dynamics and autophagy in global ischemia.