PubMed Health⌕ Search

Biomedical subjects

Stephen F Larner

Publications and source records attributed to Stephen F Larner.

10 recordsLinked to original sources
PubMed2006

Calpain- and caspase-mediated alphaII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure.

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Amino Acid Chloromethyl Ketones↗
PubMed2006

Amino acid starvation induced autophagic cell death in PC-12 cells: evidence for activation of caspase-3 but not calpain-1.

While the apoptotic and necrotic cell death pathways have been well studied, there lacks a comprehensive understanding of the molecular events involving autophagic cell death. We examined the potential roles of the apoptosis-linked caspase-3 and the necrosis/apoptosis-linked calpain-1 after autophagy induction under prolonged amino acid (AA) starvation conditions in PC-12 cells. Autophagy induction was observed as early as three hours following amino acid withdrawal. Cell death, measured by lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays occurred within 24 h following starvation and was accompanied by an upregulation in caspase-3 activity but not calpain-1. The cell death that occurred following AA starvation was significantly alleviated by treatment with the autophagy inhibitor 3-methyl adenine but not with the broad spectrum caspase inhibitors. Thus, this study demonstrates that 3-methyladenine-sensitive autophagic cell death due to AA starvation in PC-12 cells is mechanistically and biochemically similar to, yet distinct from, classic caspase dependent apoptosis.

Amino Acids↗
PubMed2006

Unfolded protein response after neurotrauma.

The endoplasmic reticulum (ER) lumen, which actively monitors the synthesis, folding, and modification of newly synthesized transmembrane and secretory proteins as well as lipids, is quite sensitive to homeostatic perturbations. The biochemical, molecular, and physiological events that elevate cellular ER stress levels and disrupt Ca2+ homeostasis trigger secondary reactions. These reactions are factors in the ongoing neurological pathology contributing to the continual tissue loss. However, the cells are not without defensive systems. One of the reactive mechanisms, the unfolded protein response (UPR), when evoked, provides some measure of protection, unless the stress conditions become prolonged or overwhelming. UPR activation occurs when key ER membrane-bound sensor proteins detect the excess accumulation of misfolded or unfolded proteins within the ER lumen. The activation of these sensors leads to a general protein translation shut-down, transcriptional induction, and translation of select proteins to deal with the difficult and miscreant protein or to encourage their degradation so they will do no harm. If the stress is prolonged, caspase-12, along with other apoptotic proteins, are activated, triggering programmed cell death. UPR, once considered to be a rather simple response, can now be characterized as a multifaceted labyrinth of reactions that continues expanding as research intensifies. This review will examine what has been learned to date about how this highly efficient and specific signaling pathway copes with ER stress, by centering on the basic components, their roles, and the complex interactions engendered. Finally, the UPR impact in various central nervous system injuries is summarized.

Animals↗
PubMed2006

Neuroprotection targets after traumatic brain injury.

PURPOSE OF REVIEW: The scarcity of pharmacological neuroprotective treatments for traumatic brain injury is a concern being targeted on various fronts. This review examines the latest treatments under investigation. RECENT FINDINGS: In the last 12-18 months, no drug has completed phase III clinical trials as a clearly proven method to treat traumatic brain injury. While the drugs work in rodents, when they make it to clinical trial they have failed primarily due to negative side-effects. Those still in trial show promise, and even those rejected have undergone modifications and now show potential, e.g. second-generation N-methyl-D-aspartic acid and alpha-amino-3-hydroxy-methyl-4-isoxazolyl-propionic acid receptor antagonists, calpain inhibitors, and cyclosporine A analogues. Also, several drugs not previously given much attention, such as the antibiotic minocycline, estrogen and progesterone, and a drug already approved for other diseases, erythropoietin, are being examined. Finally, a treatment generating some controversy, but showing potential, is the application of hypothermia to the patients. SUMMARY: Clearly, finding treatments for traumatic brain injury is not going to be easy and is evidently going to require numerous trials. The good news is that we are closer to finding one or more methods for treating traumatic brain injury patients.

Animals↗
PubMed2006

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury.

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.

Amino Acid Sequence↗
PubMed2006

Direct Rho-associated kinase inhibition [correction of inhibiton] induces cofilin dephosphorylation and neurite outgrowth in PC-12 cells.

Axons fail to regenerate in the adult central nervous system (CNS) following injury. Developing strategies to promote axonal regeneration is therapeutically attractive for various CNS pathologies such as traumatic brain injury, stroke and Alzheimer's disease. Because the RhoA pathway is involved in neurite outgrowth, Rho-associated kinases (ROCKs), downstream effectors of GTP-bound Rho, are potentially important targets for axonal repair strategies in CNS injuries. We investigated the effects and downstream mechanisms of ROCK inhibition in promoting neurite outgrowth in a PC-12 cell model. Robust neurite outgrowth (NOG) was induced by ROCK inhibitors Y-27632 and H-1152 in a time-and dose-dependent manner. Dramatic cytoskeletal reorganization was noticed upon ROCK inhibition. NOG initiated within 5 to 30 minutes followed by neurite extension between 6 and 10 hours. Neurite processes were then sustained for over 24 hours. Rapid cofilin dephosphorylation was observed within 5 minutes of Y-27632 and H-1152 treatment. Re-phosphorylation was observed by 6 hours after Y-27632 treatment, while H-1152 treatment produced sustained cofilin dephosphorylation for over 24 hours. The results suggest that ROCK-mediated dephosphorylation of cofilin plays a role in the initiation of NOG in PC-12 cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2005

Caspase 7: increased expression and activation after traumatic brain injury in rats.

Caspases, a cysteine proteinase family, are required for the initiation and execution phases of apoptosis. It has been suggested that caspase 7, an apoptosis executioner implicated in cell death proteolysis, is redundant to the main executioner caspase 3 and it is generally believed that it is not present in the brain or present in only minute amounts with highly restricted activity. Here we report evidence that caspase 7 is up-regulated and activated after traumatic brain injury (TBI) in rats. TBI disrupts homeostasis resulting in pathological apoptotic activation. After controlled cortical impact TBI of adult male rats we observed, by semiquantitative real-time PCR, increased mRNA levels within the traumatized cortex and hippocampus peaking in the former about 5 days post-injury and in the latter within 6-24 h of trauma. The activation of caspase 7 protein after TBI, demonstrated by immunoblot by the increase of the active form of caspase 7 peaking 5 days post-injury in the cortex and hippocampus, was found to be up-regulated in both neurons and astrocytes by immunohistochemistry. These findings, the first to document the up-regulation of caspase 7 in the brain after acute brain injury in rats, suggest that caspase 7 activation could contribute to neuronal cell death on a scale not previously recognized.

Animals↗
PubMed2004

Increased expression and processing of caspase-12 after traumatic brain injury in rats.

Traumatic brain injury (TBI) disrupts tissue homeostasis resulting in pathological apoptotic activation. Recently, caspase-12 was reported to be induced and activated by the unfolded protein response following excess endoplasmic reticulum (ER) stress. This study examined rat caspase-12 expression using the controlled cortical impact TBI model. Immunoblots of fractionalized cell lysates found elevated caspase-12 proform (approximately 60 kDa) and processed form (approximately 12 kDa), with peak induction observed within 24 h post-injury in the cortex (418% and 503%, respectively). Hippocampus caspase-12 proform induction peaked at 24 h post-injury (641%), while processed form induction peaked at 6 h (620%). Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed elevated caspase-12 mRNA levels after TBI. Injury severity (1.0, 1.2 or 1.6 mm compression) was associated with increased caspase-12 mRNA expression, peaking at 5 days in the cortex (657%, 651% and 1259%, respectively) and 6 h in the hippocampus (435%, 451% and 460%, respectively). Immunohistochemical analysis revealed caspase-12 induction in neurons in both the cortex and hippocampus as well as in astrocytes at the contusion site. This is the first report of increased expression of caspase-12 following TBI. Our results suggest that the caspase-12-mediated ER apoptotic pathway may play a role in rat TBI pathology independent of the receptor- or mitochondria-mediated apoptotic pathways.

Animals↗