Biomedical subjects

R Posmantur

Publications and source records attributed to R Posmantur.

4 recordsLinked to original sources

PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Analysis of Variance↗

PubMed1996

Calpain inhibitors protect against depolarization-induced neurofilament protein loss of septo-hippocampal neurons in culture.

We examined the effect of a 6 min depolarization with 60 mM KCl and 1.8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF-H), medium (NF-M) and low (NF-L) molecular weight in primary septohippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to approximately 80% of control values in the absence of cell death. Calcium-dependent losses of the neurofilament proteins correlated with calcium-dependent increases in calpain 1-mediated breakdown products of alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to approximately 14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗

PubMed1995

Calpain inhibitors reduce depolarization induced loss of tau protein in primary septo-hippocampal cultures.

We studied the effects of a 6-min potassium depolarization injury produced by 60 mM KCl and 1.8 mM or 5.8 mM extracellular CaCl2 on tau protein levels in primary rat septo-hippocampal cultures. One day after injury, Western blot analyses revealed a calcium dependent loss of tau protein of approximately 50% of control values. Loss of tau protein was associated with calpain 1 mediated breakdown products to alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization injury and available to cultures for 24 h reduced depolarization induced degradation of tau protein to approximately 35% or 25% of control values, respectively. We propose that brief potassium depolarization causes degradation of tau protein, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗

PubMed1994

Neurofilament 68 and neurofilament 200 protein levels decrease after traumatic brain injury.

We have examined the effect of lateral cortical impact injury on the levels of axonal cytoskeletal proteins in adult rats. Traumatic brain injury (TBI) causes a significant decrease in the protein levels of two prominent neurofilament (NF) proteins, NF68 and NF200. We employed quantitative immunoreactivity measurements on Western blots to examine NF68 and NF200 levels in homogenates of hippocampal and cortical tissue taken at several intervals postinjury. Sham injury had no effect on NF protein levels. However, injury was associated with a significant loss of NF68, restricted to the cortex ipsilateral to the injury site. NF68 loss was detectable as early as 3 h and lasted at least 2 weeks postinjury. Similarly, TBI induced a decrease in NF200 protein, although losses were observed both ipsilateral and contralateral to the injury site. No loss of NF68 or NF200 protein was detected in hippocampal samples obtained from the same injured animals. An increase in the presence of lower molecular weight (MW) NF68 immunopositive bands was associated with the decrease of NF68 in the ipsilateral cortex. This NF68 antigenicity pattern suggests the production of NF68 breakdown products caused by the pathologic activation of neuronal proteases, such as calpain. Putative NF68 breakdown products increase significantly until 1 day postinjury, suggesting that NF degradation may be ongoing until that time and indicating that a potential therapeutic window may exist within the first 24 h postinjury. In summary, these data identify specific biochemical alterations of the neuronal cytoskeleton following TBI and lay a foundation for further investigation of postinjury cytoskeletal changes in neuronal processes.

Animals↗