ABSTRACT
Glycogen synthase kinase-3beta (GSK-3beta) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain-mediated cleavage. Calpain mediates in cell death induced by 3-nitropropionic acid (3-NP), but GSK-3beta regulation has not been demonstrated. Here we studied changes in total GSK-3beta protein levels and GSK-3beta phosphorylation at Ser-9 in this model. The 3-NP treatment induced GSK-3beta truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3-NP-induced neuronal loss, inhibition of GSK-3beta by SB-415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3-NP-induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross-talk between calpain and GSK-3beta in the 3-NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3-NP-induced neuronal loss.
Subject(s)
Calpain/metabolism , Convulsants/toxicity , Glycogen Synthase Kinase 3/metabolism , Neurodegenerative Diseases , Neurons/drug effects , Nitro Compounds/toxicity , Propionates/toxicity , Amino Acid Chloromethyl Ketones/pharmacology , Aminophenols/pharmacology , Animals , Calpain/pharmacology , Caspases/metabolism , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Glycogen Synthase Kinase 3 beta , Hippocampus/cytology , Male , Maleimides/pharmacology , Mice , Neurodegenerative Diseases/chemically induced , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neuroprotective Agents/pharmacology , Purines/pharmacology , Rats , Roscovitine , Signal Transduction/drug effects , Time FactorsABSTRACT
Lithium reduced striatal neurodegeneration induced in rats by 3-nitropropionic acid inhibiting calpain activation. Lithium prevented an increase in cdk5 activity, as shown by the levels of the co-activator p35. Myocite enhancer factor 2 (MEF2), a downstream substrate for cdk5 with pro-survival activity, showed increased phosphorylation. In primary cultures of neurons treated with 3-NP, lithium also reduced protease activity mediated by calpain, cdk5 activation and cellular death. These observations indicate that lithium has a neuroprotective effect. Lithium treatment also reduced the intracellular increase in calcium induced by 3-NP. The finding that lithium mediates the modulation of the calpain/cdk5 pathway further supports its use in the treatment of neurodegenerative diseases.
Subject(s)
Calpain/metabolism , Cyclin-Dependent Kinase 5/metabolism , Huntington Disease , Lithium Chloride/therapeutic use , Neuroprotective Agents/therapeutic use , Nitro Compounds , Propionates , Signal Transduction/drug effects , Animals , Calcium/metabolism , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Interactions , Embryo, Mammalian , Gene Expression Regulation/drug effects , Hippocampus/cytology , Huntington Disease/chemically induced , Huntington Disease/drug therapy , Huntington Disease/metabolism , Male , Mice , Neurons/drug effects , Rats , Rats, Sprague-Dawley , Succinate Dehydrogenase/metabolismABSTRACT
We examined the expression of SIRT1 in several experimental paradigms of human pathologies. We used a neuroblastoma cell line (B65), neuronal primary cultures (hippocampus and cerebellar granule cells) and in vivo approaches in rat and senescence murine models (SAM). Cell cultures and rats were treated with several well-know neurotoxins, i.e. rotenone, MPP(+), kainate and 3-nitropropionic acid. Subsequently, SIRT1 expression was compared in these different paradigms of neurotoxicity. The pattern of expression of SIRT1 in proliferating cell cultures (B65) was different to that in quiescent cell cultures. In the murine model of senescence (senescence-accelerated mice prone, SAMP8), SIRT1 expression progressively decreased, while in the control strain (senescence-accelerated mice resistant, SAMR1) it increased. Finally, we studied human samples of Parkinson's disease (PD), dementia with Lewy bodies (DLB) and Huntington's diseases (HD). SIRT1 expression decreased dramatically in HD, but there were no significant changes in Parkinson-related illnesses. In conclusion, SIRT1 expression may be a good sensor of toxic neuronal processes.
Subject(s)
Aging/metabolism , Neurodegenerative Diseases/metabolism , Sirtuins/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Blotting, Western , Cells, Cultured , Flow Cytometry , Humans , Male , Mice , Neurons/drug effects , Neurons/metabolism , Neurotoxins/pharmacology , Rats , Rats, Sprague-Dawley , Sirtuin 1 , Sirtuins/drug effectsABSTRACT
Acute treatment with kainate 30 mg/kg (KA) produced behavioral alterations and reactive gliosis. However, it did not produce major death of mouse hippocampal neurons, indicating that concentrations were not cytotoxic. KA caused rapid and temporal Erk phosphorylation (at 6h) and Akt dephosphorylation (1-3 days). Concomitantly, the activation of GSK3beta was increased 1-3 days after KA. After 7 days, a reduction in GSK3beta activation was observed. Caspase-3 activity increased, but to a lesser extent than calpain activation (measured by fluorimetry and calpain-cleaved alpha-spectrin). As calpain is involved in cdk5 activation, and cdk5 is related to GSK3beta, the cdk5/p25 pathway was examined. Results showed that the p25/p35 ratio in KA-injected mice for 3 days was 73.6% higher than control levels. However, no changes in cdk5 expression were detected. Both Western blot and immunohistochemistry against p-Tau(Thr(231)) indicated an increase at this phosphorylated site of tau protein. Indeed an increase in p-Tau(Ser(199)) and p-Tau(Ser(396)) was observed by Western blot. Our results demonstrate that tau hyperphosphorylation, induced by KA, is due to an increase in GSK3beta/cdk5 activity in combination with an inactivation of Akt. This indicates that the calpain/cdk5 pathway for tau phosphorylation has a potential role in delayed apoptotic death evoked by excitotoxicity. Moreover, the subsequent activation of caspase and calpain proteases leads to dephosphorylation of tau, thus increasing microtubular destructuration. Taken together, our results provide new insights in the activation of several kinase-pathways implicated in cytoskeletal alterations that are a common feature of neurodegenerative diseases.
