Matrix metalloproteinases (MMPs) are implicated in the pathogenesis of neurodegenerative diseases and stroke. However, the mechanism of MMP activation remains unclear. We report that MMP activation involves S-nitrosylation. During cerebral ischemia in vivo, MMP-9 colocalized with neuronal nitric oxide synthase. S-Nitrosylation activated MMP-9 in vitro and induced neuronal apoptosis. Mass spectrometry identified the active derivative of MMP-9, both in vitro and in vivo, as a stable sulfinic or sulfonic acid, whose formation was triggered by S-nitrosylation. These findings suggest a potential extracellular proteolysis pathway to neuronal cell death in which S-nitrosylation activates MMPs, and further oxidation results in a stable posttranslational modification with pathological activity.

Mitochondria are present as tubular organelles in neuronal projections. Here, we report that mitochondria undergo profound fission in response to nitric oxide (NO) in cortical neurons of primary cultures. Mitochondrial fission by NO occurs long before neurite injury and neuronal cell death. Furthermore, fission is accompanied by ultrastructural damage of mitochondria, autophagy, ATP decline and generation of free radicals. Fission is occasionally asymmetric and can be reversible. Strikingly, mitochondrial fission is also an early event in ischemic stroke in vivo. Mitofusin 1 (Mfn1) or dominant-negative Dynamin related protein 1 (Drp1 K38A ) inhibits mitochondrial fission induced by NO, rotenone and Amyloid-b peptide. Conversely, overexpression of Drp1 or Fis1 elicits fission and increases neuronal loss. Importantly, NO-induced neuronal cell death was mitigated by Mfn1 and Drp1 K38A . Thus, persistent mitochondrial fission may play a causal role in NO-mediated neurotoxicity.

The N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) serves critical functions in physiological and pathological processes in the central nervous system, including neuronal development, plasticity and neurodegeneration. Conventional heteromeric NMDARs composed of NR1 and NR2A-D subunits require dual agonists, glutamate and glycine, for activation. They are also highly permeable to Ca2+, and exhibit voltage-dependent inhibition by Mg2+. Coexpression of NR3A with NR1 and NR2 subunits modulates NMDAR activity. Here we report the cloning and characterization of the final member of the NMDAR family, NR3B, which shares high sequence homology with NR3A. From in situ and immunocytochemical analyses, NR3B is expressed predominantly in motor neurons, whereas NR3A is more widely distributed. Remarkably, when co-expressed in Xenopus oocytes, NR3A or NR3B co-assembles with NR1 to form excitatory glycine receptors that are unaffected by glutamate or NMDA, and inhibited by D-serine, a co-activator of conventional NMDARs. Moreover, NR1/NR3A or -3B receptors form relatively Ca2+-impermeable cation channels that are resistant to Mg2+, MK-801, memantine and competitive antagonists. In cerebrocortical neurons containing NR3 family members, glycine triggers a burst of firing, and membrane patches manifest glycine-responsive single channels that are suppressible by D-serine. By itself, glycine is normally thought of as an inhibitory neurotransmitter. In contrast, these NR1/NR3A or -3B 'NMDARs' constitute a type of excitatory glycine receptor.

Electrophilic compounds are a newly recognized class of redox‐active neuroprotective compounds with electron deficient, electrophilic carbon centers that react with specific cysteine residues on targeted proteins via thiol (S‐)alkylation. Although plants produce a variety of physiologically active electrophilic compounds, the detailed mechanism of action of these compounds remains unknown. Catechol ring‐containing compounds have attracted attention because they become electrophilic quinones upon oxidation, although they are not themselves electrophilic. In this study, we focused on the neuroprotective effects of one such compound, carnosic acid (CA), found in the herb rosemary obtained from Rosmarinus officinalis. We found that CA activates the Keap1/Nrf2 transcriptional pathway by binding to specific Keap1 cysteine residues, thus protecting neurons from oxidative stress and excitotoxicity. In cerebrocortical cultures, CA‐biotin accumulates in non‐neuronal cells at low concentrations and in neurons at higher concentrations. We present evidence that both the neuronal and non‐neuronal distribution of CA may contribute to its neuroprotective effect. Furthermore, CA translocates into the brain, increases the level of reduced glutathione in vivo, and protects the brain against middle cerebral artery ischemia/reperfusion, suggesting that CA may represent a new type of neuroprotective electrophilic compound.

. We determined that MMP-9 degrades the extracellular matrix protein laminin and that this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice. We also determined that the highly specific thiirane gelatinase inhibitor SB-3CT blocks MMP-9 activity, including MMP-9-mediated laminin cleavage, thus rescuing neurons from apoptosis. We conclude that MMP-9 is a highly promising drug target and that SB-3CT derivatives have significant therapeutic potential in stroke patients.

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. ␤-Amyloid peptide (A␤), which is derived from the ␤-amyloid precursor protein (APP) by sequential proteolytic cleavages from ␤-secretase (BACE1) and presenilin-1 (PS1)/␥-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-␤ (␤CTF) and A␤. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-␣-converting enzyme (TACE, an enzyme known to cleave APP at the ␣-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1␣ increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1␣ reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1␣ overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1␣. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1␣ conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1␣ in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.An important pathologic feature of Alzheimer disease (AD) 4 is formation of extracellular senile plaques in the brain, whose major components are small peptides called ␤-amyloid (A␤) derived from ␤-amyloid precursor protein (APP). APP is sequentially cleaved first by the ␤-secretase (␤-site amyloid precursor protein cleaving enzyme, BACE) and then by the ␥-secretase complex (including presenilin, nicastrin, APH-1, and PEN-2) to generate the heterogeneous A␤ species, mostly A␤40 but also the more deleterious A␤42. Alternatively, APP can be cleaved by ␣-secretase within the A␤ domain to generate non-amyloidogenic soluble APP␣ (sAPP␣) (1-3). The exact ␣-secretase is not known, but a disintegrin and metalloprotease domain 10 (ADAM10) and TNF-␣-converting enzyme (TACE) are two likely candidates (4, 5). It is widely believed that A␤ overproduction directly or indirectly initiates a cascade of neurodegenerative steps resulting in formation of senile plaques, neurofibrillary tangles, and neuronal loss, which characterize AD (6). Hence analysis of cellular regulation affecting A␤ generation, including identification of factors regulating the level/ activity of APP cleavage enzymes, should provide invaluable information for AD therapeutic intervention.BACE is a membrane-bound aspartic protease whose activity is t...

Electrophilic neurite outgrowth-promoting prostaglandin (NEPP) compounds protect neurons from oxidative insults. At least part of the neuroprotective action of NEPPs lies in induction of hemeoxygenase-1 (HO-1), which, along with other phase II enzymes, serve as a defense system against oxidative stress. Here, we found that, by using fluorescent tags and immunoprecipitation assays, NEPPs are taken up preferentially into neurons and bind in a thiol-dependent manner to Keap1, a negative regulator of the transcription factor Nrf2. By binding to Keap1, NEPPs prevent Keap1-mediated inactivation of Nrf2 and, thus, enhance Nrf2 translocation into the nucleus of cultured neuronal cells. In turn, Nrf2 binds to antioxidant͞ electrophile-responsive elements of the HO-1 promoter to induce HO-1 expression. Consistent with this notion, NEPP induction of an HO-1 reporter construct is prevented if the antioxidant-responsive elements are mutated. We show that NEPPs are neuroprotective both in vitro from glutamate-related excitotoxicity and in vivo in a model of cerebral ischemia͞reperfusion injury (stroke). Our results suggest that NEPPs prevent excitotoxicity by activating the Keap1͞ Nrf2͞HO-1 pathway. Because NEPPs accumulate preferentially in neurons, they may provide a category of neuroprotective compounds, distinct from other electrophilic compounds such as tertbutylhydroquinone, which activates the antioxidant-responsive element in astrocytes. NEPPs thus represent a therapeutic approach for stroke and neurodegenerative disorders.hemeoxygenase-1 ͉ middle cerebral artery occlusion ͉ neurite outgrowth-promoting prostaglandin ͉ stroke ͉ neurodegenerative diseases

. Characterization and comparison of the NR3A subunit of the NMDA receptor in recombinant systems and primary cortical neurons. J Neurophysiol 87: 2052-2063, 2002; 10.1152/jn.00531.2001. Recently, we cloned and began to characterize a new N-methyl-D-aspartate receptor (NMDAR) subunit, NR3A. Here we extend our earlier findings by showing that recombinantly expressed NR3A in COS cells is biochemically associated with both NR1 and NR2 subunits. In the oocyte or HEK 293 cell expression systems, co-injection of NR3A with NR1/NR2 subunits acts in a dominant-interfering manner, resulting in a decrease in NMDAR unitary conductance, decrease in Ca 2ϩ permeability, decrease in Mg 2ϩ sensitivity, and slight increase in mean open time compared with NR1/NR2 channels. The smaller unitary conductance channel has also been observed in primary cortical neurons cultured from wild-type rodent on postnatal day 8 (P8) and similarly found to be relatively insensitive to Mg 2ϩ block. Consistent with these findings, whole cell NMDA-evoked currents are larger in NR3A-deficient mice compared with wild-type mice, and this effect follows a developmental pattern similar to that of NR3A protein expression on Western blots, with peak expression at P8. Finally, a new longer splice variant of NR3A has been cloned and found to be expressed in rodent cortical neurons by single-cell RT-PCR and in situ hybridization.