Calpain-Independent Intracellular Protease Activity Is Elevated in Excitotoxic Cortical Neurons Prior to Delayed Calcium Deregulation and Mitochondrial Dysfunction.

Glutamate excitotoxicity contributes to many neurodegenerative diseases. Excessive glutamate receptor-mediated calcium entry causes delayed calcium deregulation (DCD) that coincides with abrupt mitochondrial depolarization. We developed cA-TAT, a live-cell protease activity reporter based on a vimentin calpain cleavage site, to test whether glutamate increases protease activity in neuronal cell bodies prior to DCD. Treatment of rat cortical neurons with excitotoxic (100 µM) glutamate increased the low baseline rate of intracellular cA-TAT proteolysis by approximately three-fold prior to DCD and by approximately seven-fold upon calcium deregulation. The glutamate-induced rate enhancement prior to DCD was suppressed by glutamate receptor antagonists, but not by calpain or proteasome inhibitors, whereas DCD-stimulated proteolysis was partly attenuated by the proteasome inhibitor MG132. Further suggesting that cA-TAT cleavage is calpain-independent, cA-TAT fluorescence was observed in immortalized Capn4 knockout fibroblasts lacking the regulatory calpain subunit. About half of the neurons lost calcium homeostasis within two hours of a transient, 20 min glutamate receptor stimulation. These neurons had a significantly (49%) higher mean baseline cA-TAT proteolysis rate than those maintaining calcium homeostasis, suggesting that the unknown protease(s) cleaving cA-TAT may influence DCD susceptibility. Overall, the results indicate that excitotoxic glutamate triggers the activation of calpain-independent neuronal protease activity prior to the simultaneous loss of calcium homeostasis and mitochondrial bioenergetic function.

Vitamin D Promotes Protein Homeostasis and Longevity via the Stress Response Pathway Genes skn-1, ire-1, and xbp-1.

Vitamin D has multiple roles, including the regulation of bone and calcium homeostasis. Deficiency of 25-hydroxyvitamin D, the major circulating form of vitamin D, is associated with an increased risk of age-related chronic diseases, including Alzheimer's disease, Parkinson's disease, cognitive impairment, and cancer. In this study, we utilized Caenorhabditis elegans to examine the mechanism by which vitamin D influences aging. We found that vitamin-D3-induced lifespan extension requires the stress response pathway genes skn-1, ire-1, and xbp-1. Vitamin D3 (D3) induced expression of SKN-1 target genes but not canonical targets of XBP-1. D3 suppressed an important molecular pathology of aging, that of widespread protein insolubility, and prevented toxicity caused by human ß-amyloid. Our observation that D3 improves protein homeostasis and slows aging highlights the importance of maintaining appropriate vitamin D serum levels and may explain why such a wide variety of human age-related diseases are associated with vitamin D deficiency.

Dimethyl sulfoxide and dimethyl formamide increase lifespan of C. elegans in liquid.

Lifespan extension through pharmacological intervention may provide valuable tools to understanding the mechanisms of aging and could uncover new therapeutic approaches for the treatment of age-related disease. Although the nematode Caenorhabditis elegans is well known as a particularly suitable model for genetic manipulations, it has been recently used in a number of pharmacological studies searching for compounds with anti-aging activity. These compound screens are regularly performed in amphipathic solvents like dimethyl sulfoxide (DMSO), the solvent of choice for high-throughput drug screening experiments performed throughout the world. In this work, we report that exposing C. elegans to DMSO in liquid extends lifespan up to 20%. Interestingly, another popular amphipathic solvent, dimethyl formamide (DMF), produces a robust 50% increase in lifespan. These compounds work through a mechanism independent of insulin-like signaling and dietary restriction (DR). Additionally, the mechanism does not involve an increased resistance to free radicals or heat shock suggesting that stress resistance does not play a major role in the lifespan extension elicited by these compounds. Interestingly, we found that DMSO and DMF are able to decrease the paralysis associated with amyloid-ß3-42 aggregation, suggesting a role of protein homeostasis for the mechanism elicited by these molecules to increase lifespan.

Real-time visualization of cytoplasmic calpain activation and calcium deregulation in acute glutamate excitotoxicity.

Although calpain (EC 3.4.22) protease activation was suggested to contribute to excitotoxic delayed calcium deregulation (DCD) via proteolysis of Na+/Ca2+ exchanger 3 (NCX3), cytoplasmic calpain activation in relation to DCD has never been visualized in real-time. We employed a calpain fluorescence resonance energy transfer substrate to simultaneously image calpain activation and calcium deregulation in live cortical neurons. A calpain inhibitor-sensitive decline in fluorescence resonance energy transfer was observed at 39 +/- 5 min after the occurrence of DCD in neurons exposed to continuous glutamate (100 microM). Inhibition of calpain by calpeptin did not delay the onset of DCD, recovery from DCD-like reversible calcium elevations, or cell death despite inhibiting alpha-spectrin processing by > 90%. NCXs reversed during glutamate exposure, the NCX antagonist KB-R7943 prolonged the time to DCD, and significant NCX3 cleavage following 90 min of glutamate exposure was not observed. Our findings suggest that robust calpain activation associated with acute glutamate toxicity occurs only after a sustained loss in calcium homeostasis. Processing of NCX3 or other calpain substrates is unlikely to be the primary cause of acute excitotoxicity in cortical neurons. However, a role for calpain as a contributing factor or in response to milder glutamate insults is not excluded.

Dynamic changes in vesicular glutamate transporter 1 function and expression related to methamphetamine-induced glutamate release.

The vesicular glutamate (GLU) transporter (VGLUT1) is a critical component of glutamatergic neurons that regulates GLU release. Despite the likely role of GLU release in drug abuse pathology, there is no information that links VGLUT1 with drugs of abuse. This study provides the first evidence that methamphetamine (METH) alters the dynamic regulation of striatal VGLUT1 function and expression through a polysynaptic pathway. METH increases cortical VGLUT1 mRNA, striatal VGLUT1 protein in subcellular fractions, and the Vmax of striatal vesicular GLU uptake. METH also increases glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in the crude vesicle fraction. METH-induced increases in cortical VGLUT1 mRNA, as well as striatal VGLUT1 and GAPDH, are GABA(A) receptor-dependent because they are blocked by GABA(A) receptor antagonism in the substantia nigra. These results show that VGLUT1 can be dynamically regulated via a polysynaptic pathway to facilitate vesicular accumulation of GLU for subsequent release after METH.

High-dose methamphetamine acutely activates the striatonigral pathway to increase striatal glutamate and mediate long-term dopamine toxicity.

Methamphetamine (METH) has been shown to increase the extracellular concentrations of both dopamine (DA) and glutamate (GLU) in the striatum. Dopamine, glutamate, or their combined effects have been hypothesized to mediate striatal DA nerve terminal damage. Although it is known that METH releases DA via reverse transport, it is not known how METH increases the release of GLU. We hypothesized that METH increases GLU indirectly via activation of the basal ganglia output pathways. METH increased striatonigral GABAergic transmission, as evidenced by increased striatal GAD65 mRNA expression and extracellular GABA concentrations in substantia nigra pars reticulata (SNr). The METH-induced increase in nigral extracellular GABA concentrations was D1 receptor-dependent because intranigral perfusion of the D1 DA antagonist SCH23390 (10 microm) attenuated the METH-induced increase in GABA release in the SNr. Additionally, METH decreased extracellular GABA concentrations in the ventromedial thalamus (VM). Intranigral perfusion of the GABA-A receptor antagonist, bicuculline (10 microm), blocked the METH-induced decrease in extracellular GABA in the VM and the METH-induced increase in striatal GLU. Intranigral perfusion of either a DA D1 or GABA-A receptor antagonist during the systemic administrations of METH attenuated the striatal DA depletions when measured 1 week later. These results show that METH enhances D1-mediated striatonigral GABAergic transmission (1), which in turn activates GABA-A receptors in the SNr (2), leading to a decrease in GABAergic nigrothalamic activity (3), an increase in corticostriatal GLU release (4), and a consequent long-term depletion of striatal DA content (5).