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F. Junyent, L. de Lemos, E. Verdaguer, M. Pallàs, J. Folch, C. Beas-Zárate, A. Camins and C. Auladell (2012) Neuropathology and Applied Neurobiology 38, 311-321 Lack of Jun-N-terminal kinase 3 (JNK3) does not protect against neurodegeneration induced by 3-nitropropionic acid Aims: 3-Nitropropionic acid (3-NP) is a toxin that replicates most of the clinical and pathophysiological symptoms of Huntington's disease, inducing neurodegeneration in the striatum due to the inhibition of mitochondrial succinate dehydrogenase. Different pathways have been implicated in the cell death induced by 3-NP in rodents. One of them is the Jun-N-terminal kinase (JNK) pathway, which may play a role in the neurodegenerative process in different diseases. Moreover, the lack of one isoform of JNK (JNK3) has been associated with neuroprotection in different experimental models of neurodegeneration. Therefore, in the present study the role of JNK3 in the experimental Huntington's model induced by 3-NP administration was evaluated. Methods: 3-NP was intraperitoneally administered once a day for 3 days to wild-type and Jnk3-null mice. Coronal brain sections were used to determine cell death and astrogliosis in striatum. Western blots were performed to determine the involvement of different pathways in both wild-type and Jnk3-null mice. Results: Although JNK activation was observed following 3-NP administration, the results indicate that the lack of JNK3 does not confer neuroprotection against 3-NP toxicity. Thus, other pathways must be involved in the neurodegeneration induced in this model. One of the possible pathways towards 3-NP-induced apoptosis could involve the calpains, as their activity was increased in wild-type and Jnk3-null mice. Conclusion: Although JNK3 is a key protein involved in cell death in different neurodegenerative diseases, the present study demonstrates that the lack of JNK3 does not confer neuroprotection against 3-NP-induced neuronal death. [ABSTRACT FROM AUTHOR]

Abstract: Increasing evidence implicates the c-Jun NH2-terminal kinase (JNK) pathway in the regulation of apoptosis in neurodegenerative diseases. In this study, we examined the neuroprotective effect of SP600125, a selective JNK inhibitor, in cerebellar granule cells (CGNs) deprived of serum and potassium (S/K withdrawal). S/K withdrawal-induced apoptosis occurs via activation of multiple pro-apoptotic pathways, including re-entry into the cell cycle, activation of glycogen synthase kinase-3 beta (GSK-3β), cyclin-dependent kinase 5 (cdk5/p35) breakdown, formation of cdk5/p25 and JNK activation. Here we demonstrate that SP600125 is able to inhibit all these pro-apoptotic pathways via the inhibition of JNK. Further, we found that JNK inhibition maintains the phosphorylation/activation of Akt after S/K withdrawal. For further confirmation of this result, we studied several targets downstream of Akt including GSK-3β, p-FOXO1, p-CREB and p35. In addition, the specific PI3K/Akt inhibitor LY294002 greatly diminished the antiapoptotic effects of SP600125 upon S/K withdrawal, confirming that Akt is involved in the neuroprotection achieved by SP600125. These results suggest that the maintenance of the PI3-kinase/Akt pathway by inhibition of JNK contributes to the prevention of apoptosis in rat cerebellar granule neurons mediated by S/K withdrawal. Furthermore, we propose that JNK may regulate the cell cycle re-entry by a novel mechanism that involves Akt, GSK-3β and Rb phosphorylation. [Copyright &y& Elsevier]

Abstract: Recent studies have demonstrated that neuronal reentry in the cell cycle and specifically the expression of the transcription factor E2F-1, constitutes a pathway that may be involved in neuronal apoptosis after serum and potassium withdrawal. Other enzymes such as glycogen synthase kinase-3β (GSK-3β) are also involved in this apoptotic stimulus, and thus in the process of neuronal cell death. Primary cerebellar granule cells (CGNs) were used in this study to determine whether pharmacological inhibition of GSK-3β is involved in neuronal modulation of the cell cycle, and specifically in the regulation of E2F-1 and retinoblastoma protein (Rb). CGNs showed a dramatic increase in GSK-3β activity after 2h of serum and potassium deprivation. Immunoblot and activity assays revealed that lithium and SB415286 inhibit fully the activation of GSK-3β and attenuate the expression of cyclin D, cyclin E, pRb phosphorylation and the transcription factor E2F-1. These data were confirmed using AR-014418, a selective GSK-3β inhibitor that prevents the expression of cell-cycle proteins. Our data indicate that GSK-3β inhibition regulates, in part, the cell cycle in CGNs by inhibiting Rb phosphorylation and thus inhibiting E2F-1 activity. However, the selective inhibition of GSK-3β with AR-A014418 had not effect on cell viability or apoptosis mediated by S/K withdrawal. Furthermore, our results suggest that selective GSK-3β inhibition is not sufficient to protect against apoptosis in this S/K withdrawal model, indicating that Li+ and SB415286 neuroprotective effects are mediated by the inhibition of additional targets to GSK3β. Therefore, there is a connection between cell cycle and GSK-3β activation and that these, along with other mechanisms, are involved in the molecular paths leading to the apoptotic process of rat CGNs triggered by S/K withdrawal. [Copyright &y& Elsevier]

Abstract: The biochemical pathways involved in neuronal cell death in Parkinson’s disease are not completely characterized. Mitochondrial dysfunction, specifically alteration of the mitochondrial complex I, is the primary target of the parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP+) induced apoptosis in neurons. In the present study, we examine the role of caspase-dependent and -independent routes in MPP+-induced apoptosis in rat cerebellar granule neurons (CGNs). We show a distinct increase in the expression of the cell cycle proteins cyclin D, cyclin E, cdk2, cdk4 and the transcription factor E2F-1 following a MPP+ treatment of CGNs. Flavopiridol (FLAV), a broad inhibitor of cyclin-dependent kinases (CDKs), attenuated the neurotoxic effects of MPP+ and significantly attenuates apoptosis mediated by MPP+ 200 μM. Likewise, the antioxidant vitamin E (vit E) increases neuronal cell viability and attenuates apoptosis induced by MPP+. Moreover, the expression levels of cyclin D and E2F-1 induced by this parkinsonian neurotoxin were also attenuated by vit E. Since, the broad-spectrum caspase inhibitor zVAD-fmk did not attenuate MPP+-induced apoptosis in CGNs, our data provide a caspase-independent mechanism mediated by neuronal reentry in the cell cycle and increased expression of the pro-apoptotic transcription factor E2F-1. Our results also suggest a potential role of oxidative stress in neuronal reentry in the cell cycle mediated by MPP+. Finally, our data further support the therapeutic potential of flavopiridol, for the treatment of Parkinson’s disease. [Copyright &y& Elsevier]

Li+ exerts protective effect against several neurotoxins in neuronal cell preparations. Here we examined the antiapoptotic effects of GSK3β in cerebellar granule neurons (CGNs) in the presence of several neurotoxins. Acute treatment with Li+ protected neurons against nocodazole and serum/potassium (S/K) deprivation, but were ineffective against kainic acid and MPP+. Li+ 5 mM also decreased caspase-3 activation induced by nocodazole and S/K deprivation as measured by Ac-DEVD-p-nitroaniline and the breakdown of α-spectrin. All the neurotoxins used in the present study activated GSK3β, evaluated with a specific antibody phospho-GSK-3β (Ser9) by Western-blot and immunocytochemistry and were always inhibited by Li+ 5 mM. Our results implicate Li+ in the regulation of apoptosis mediated by caspase activation (Type I). Furthermore inhibition of GSK3β by acute treatment with Li+ 5 mM is not an indicator of neuroprotection. The acute antiapoptotic function of Li+ is discussed in terms of its inhibition of Type I pathway, the intrinsic (mitochondrial) apoptotic pathway in cerebellar granule cells. [ABSTRACT FROM AUTHOR]

Abstract: Although numerous studies have demonstrated a neuroprotective and anti-apoptotic role of lithium in neuronal cell cultures, the precise mechanism by which this occurs, remains to be elucidated. In this study, we evaluated the lithium-mediated neuroprotection against colchicine-induced apoptosis in cultured cerebellar granule neurons. Previously, it has been demonstrated that colchicine mediates apoptosis in cerebellar granule neurons through cytoskeletal alteration and activation of an intrinsic pro-apoptotic pathway. Recently we also demonstrated a potential role of cyclin-dependent kinase 5 (cdk5) in this pathway. Here we report that colchicine induces dephosphorylation in Ser-9 and phosphorylation in Tyr-216, and thus activation, of glycogen synthase kinase-3β in cerebellar granule neurons, and that this modification is inhibited by the presence of 5mM lithium. However, the selective glycogen synthase kinase-3β inhibitors SB-415286 and SB-216763 were unable to prevent colchicine-induced apoptosis in these cells, suggesting that the anti-apoptotic activity of lithium is not mediated by glycogen synthase kinase-3β under these conditions. On the other hand, 5mM lithium prevented the colchicine-induced increase in cdk5 expression and breakdown of cdk5/p35 to cdk5/p25. In addition, we show that up-regulation of cdk5/p25 is unrelated to inhibition of the activity of myocyte enhancer factor 2, a pro-survival transcription factor. These data suggest a previously undescribed neuroprotective mechanism of lithium associated with the modulation of cdk5/p35 or cdk5/p25 expression. [Copyright &y& Elsevier]

Acetylcholinesterase inhibitors (AChEI) are among the drugs most widely used in the treatment of Alzheimer's disease. They increase the levels of acetylcholine and thus improve the cognitive symptoms that are impaired. We tested whether specific AChEI show additional neuroprotective properties against colchicine-induced apoptosis in cerebellar granule neurons (CGNs), a well established apoptotic model mediated by neuronal cytoskeleton alteration. Colchicine-induced apoptosis is due to an increase in the activity of GSK-3β and CDK5, two enzymes involved in cytoskeletal alteration. Furthermore, the intrinsic apoptotic pathway is activated by colchicines, as revealed by cytochrome c release and Bax translocation. Tacrine, (-)-huperzine A and (±)-huprine Y, the AChEI tested in the study, did not reverse the loss of neuronal viability induced by colchicine. Moreover, the increase in apoptotic features induced by colchicine treatment, as measured by flow cytometry and nuclear chromatin condensation, was not prevented by these AChEI. Although some of these drugs are of interest to treat Alzheimer's disease, their lack of efficacy in the prevention of colchicine-induced apoptosis in CGNs suggests that they cannot prevent neuronal loss due to cytoskeleton alteration. [ABSTRACT FROM AUTHOR]

Jordà EG, Verdaguer E, Morano A, Jiménez A, Canudas AM, Camins A, Pallàs M. Lithium prevents colchicine-induced apoptosis in rat cerebellar granule neurons. Bipolar Disord 2004: 6: 144–149. © Blackwell Munksgaard, 2004 Here we evaluated the neuroprotective effects of two well-known mood stabilizers, lithium and valproic acid (VPA), against colchicine neurotoxicity in cerebellar granule cells (CGNs). The CGNs were differentiated for 7 days, pretreated with lithium or VPA for 24 h and after colchicine 1 μM was added. Cellular damage was assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) method and apoptosis in CGNs was characterized by chromatin condensation and DNA fragmentation. Incubation with lithium (1–5 mM) attenuated this apoptosis markedly, in a dose–dependent way however, the addition of VPA (0.5–2 mM) did not protect CGNs. Colchicine-induced apoptosis is mediated through the activation of caspase-3. An increase in caspase-3 activity was detected within 18 h and was blocked in presence of lithium 5 mM. Our data indicate that lithium treatment is selectively neuroprotective; however, in our experimental conditions VPA did not protect CGNs from apoptosis induced by colchicine. Our results support the hypothesis that distinct pathways mediate the neuroprotective effects of lithium and VPA. [ABSTRACT FROM AUTHOR]