Regulation Of Neuronal Cell Death Research Articles

Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

Enhanced translocation of TRIM32 to mitochondria sensitizes dopaminergic neuronal cells to apoptosis during stress conditions in Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopamine-producing neurons from the substantia nigra region of the brain. Mitochondrial dysfunction is one of the major causes of oxidative stress and neuronal cell death in PD. E3 ubiquitin ligases such as Parkin (PRKN) modulate mitochondrial quality control in PD; however, the role of other E3 ligases associated with mitochondria in the regulation of neuronal cell death in PD has not been explored. The current study investigated the role of TRIM32, RING E3 ligase, in sensitization to oxidative stress-induced neuronal apoptosis. The expression of TRIM32 sensitizes SH-SY5Y dopaminergic cells to rotenone and 6-OHDA-induced neuronal death, whereas the knockdown increased cell viability under PD stress conditions. The turnover of TRIM32 is enhanced under PD stress conditions and is mediated by autophagy. TRIM32 translocation to mitochondria is enhanced under PD stress conditions and localizes on the outer mitochondrial membrane. TRIM32 decreases complex-I assembly and activity as well as mitochondrial reactive oxygen species (ROS) and ATP levels under PD stress. Deletion of the RING domain of TRIM32 enhanced complex I activity and rescued ROS levels and neuronal viability under PD stress conditions. TRIM32 decreases the level of XIAP, and co-expression of XIAP with TRIM32 rescued the PD stress-induced cell death and mitochondrial ROS level. In conclusion, turnover of TRIM32 increases during stress conditions and translocation to mitochondria is enhanced, regulating mitochondrial functions and neuronal apoptosis by modulating the level of XIAP in PD.

Signaling pathways regulating mitochondrial calcium efflux – a commentary on Rozenfeld et al. “Essential role of the mitochondrial Na+/Ca2+ exchanger NCLX in mediating PDE2-dependent neuronal survival and learning”

Mitochondrial calcium (mCa2+) is a critical regulator of neuronal cell death, bioenergetics, and signaling pathways. Although the regulatory machinery governing mCa2+ uptake via the mitochondrial calcium uniporter (mtCU) has been identified and functionally characterized, regulation of the mitochondrial Na+/Ca2+ exchanger (NCLX), the primary means of mCa2+ efflux, is poorly understood. Rozenfeld et al. report that inhibition of phosphodiesterase 2 (PDE2) enhances mCa2+efflux via increased NCLX phosphorylation by protein kinase A (PKA) [1]. The authors demonstrate that enhancing NCLX activity by pharmacologic inhibition of PDE2 improves neuronal survival in response to excitotoxic insult in vitro and enhances cognitive performance. Here we contextualize this discovery within existing literature and provide conjecture to add clarity to the proposed novel regulatory mechanism.

Elezanumab, a clinical stage human monoclonal antibody that selectively targets repulsive guidance molecule A to promote neuroregeneration and neuroprotection in neuronal injury and demyelination models

Repulsive guidance molecule A (RGMa) is a potent inhibitor of axonal growth and a regulator of neuronal cell death. It is up-regulated following neuronal injury and accumulates in chronic neurodegenerative diseases. Neutralizing RGMa has the potential to promote neuroregeneration and neuroprotection. Previously we reported that a rat anti-N terminal RGMa (N-RGMa) antibody r5F9 and its humanized version h5F9 (ABT-207) promote neuroprotection and neuroregeneration in preclinical neurodegenerative disease models. However, due to its cross-reactivity to RGMc/hemojuvelin, ABT-207 causes iron accumulation in vivo, which could present a safety liability. Here we report the generation and characterization of a novel RGMa-selective anti-N-RGMa antibody elezanumab, which is currently under Phase 2 clinical evaluation in multiple disease indications. Elezanumab, a human monoclonal antibody generated by in vitro PROfusion mRNA display technology, competes with ABT-207 in binding to N-RGMa but lacks RGMc cross-reactivity with no impact on iron metabolism. It neutralizes repulsive activity of soluble RGMa in vitro and blocks membrane RGMa mediated BMP signaling. In the optic nerve crush and optic neuritis models, elezanumab promotes axonal regeneration and prevents retinal nerve fiber layer degeneration. In the spinal targeted experimental autoimmune encephalomyelitis (EAE) model, elezanumab promotes axonal regeneration and remyelination, decreases inflammatory lesion area and improves functional recovery. Finally, in the mouse cuprizone model, elezanumab reduces demyelination, which is consistent with its inhibitory effect on BMP signaling. Taken together, these preclinical data demonstrate that elezanumab has neuroregenerative and neuroprotective activities without impact on iron metabolism, thus providing a compelling rationale for its clinical development in neurodegenerative diseases.

A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia–reperfusion injury in mice

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30min) with subsequent reperfusion (48h). Mice were treated with IQ-1S (25mg/kg) suspended in 10% solutol or with vehicle alone 30min before and 24h after middle cerebral artery (MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30min of MCAO provoked by a filament and during the first 30min of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48h of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury.

Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress.

Epigenetic regulations including DNA methylation and demethylation play critical roles in neural development. However, whether DNA methylation and demethylation may play a role in neuronal cell death remains largely unclear. Here we report that the blockade of DNA methyltransferase inhibits apoptosis of cerebellar granule cells and cortical neurons in response to oxidative stress. We found that knockdown of ten-eleven translocation methylcytosine dioxygenase (Tet1), a critical enzyme for DNA demethylation, significantly increase apoptosis of cerebellar granule cells induced by hydrogen peroxide. Moreover, cerebellar granule cells from tet1null mice appeared to be more sensitive to oxidative stress, suggesting the critical role of Tet1 in neuronal cell death. We further showed that the expression of Klotho, an antiaging protein, in cerebellar granule cells is tightly regulated by DNA methylation. Finally, we found that knockdown of Klotho diminished the rescue effects of DNA methyltransferase inhibitors and Tet1 on neuronal cell death induced by oxidative stress. Our work revealed the role of Tet1-mediated DNA demethylation on neuronal protection against oxidative stress and provided the molecular mechanisms underlying the epigenetic regulation of neuronal cell death, suggesting the role of Klotho in regulating neuronal cell death in response to oxidative stress.

Calbindin-D28K Prevents Staurosporin-induced Bax Cleavage and Membrane Permeabilization.

Calbindin-D28K has been implicated in the regulation of neuronal cell death. Previously, we demonstrated that calbindin-D28K prevents staurosporine (STS)-induced caspase activation and subsequent apoptosis in a neuronal cell line. However, the role of calbindin-D28K in STS-induced activation of calpain and necrotic cell death was not identified. Staurosporine induced the elevation of intracellular calcium after 1 hr of treatment. Overexpression of calbindin-D28K and presence of a calcium chelator, BAPTA, prevented the increase of calcium in STS-treated cells. Cleavage of Bax by calpain was prevented by the overexpressed calbindin-D28K. Permeabilization of the plasma membrane, a factor in necrosis, as well as apoptotic change of the nucleolus induced by STS, was prevented by calbindin-D28K. Thus, our study suggests that calbindin-D28K may exert its protective functions by preventing calpain activation in necrotic cell death, in addition to its effect on the caspase-apoptosis pathway.

Abstract WP255: The Novel C-Jun N-terminal Kinase Inhibitor IQ-1S Protects Against Cerebral Ischemia-reperfusion Injury In Mice

The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Recently, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated it was a high-affinity inhibitor of the JNK family, with preference for JNK3. We hypothesized that IQ-S1 could decrease stroke vulnerability in vivo by altering JNK activity, apoptosis, and inflammation. To test our hypothesis, we subjected wild-type (C57BL6, male, 8-12 weeks old) mice to a stroke model of temporal focal ischemia (30 minutes) with subsequent reperfusion (48 hours). The mice were anesthetized with 1.5 % of aerrane in 30% oxygen/70% nitrous oxide, and body temperature was maintained at 36.5-37.0° C. Mice were treated intraperitoneally with IQ-1S (25 mg/kg) suspended in 10% solutol (100 μl) or with vehicle alone (10% solutol, 100 μl) 30 minutes before and 12 and 24 hours after middle cerebral artery occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the middle cerebral artery during 30 minutes of MCAO provoked by a filament and during the first 30 minutes of subsequent reperfusion. In mice treated with IQ-S1, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-S1 treated mice demonstrated markedly reduced infarct volumes (35 ± 23 mm3, n=6; Mean ± SD, standard tetrazolium staining) when compared with vehicle-treated mice (68 ± 23 mm3, n=5, p< 0.05) after 48 hours of reperfusion. Neurological deficit after 48 hours of reperfusion was improved in IQ-S1 treated mice (1.0 point deficit on a 5 point scale) as compared with vehicle treated mice (1.8 point deficit). Our results indicate that the novel JNK inhibitor improved stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-S1 can serve as specific small-molecule modulator for mechanistic studies of JNK function in cerebral ischemia and reperfusion injury.

Regulation of Neuronal Cell Death by c-Abl-Hippo/MST2 Signaling Pathway

BackgroundMammalian Ste20-like kinases (MSTs) are the mammalian homologue of Drosophila hippo and play critical roles in regulation of cell death, organ size control, proliferation and tumorigenesis. MSTs exert pro-apoptotic function through cleavage, autophosphorylation and in turn phosphorylation of downstream targets, such as Histone H2B and FOXO (Forkhead box O). Previously we reported that protein kinase c-Abl mediates oxidative stress-induced neuronal cell death through phosphorylating MST1 at Y433, which is not conserved among mammalian MST2, Drosophila Hippo and C.elegans cst-1/2.Methodology/Principal FindingsUsing immunoblotting, in vitro kinase and cell death assay, we demonstrate that c-Abl kinase phosphorylates MST2 at an evolutionarily conserved site, Y81, within the kinase domain. We further show that the phosphorylation of MST2 by c-Abl leads to the disruption of the interaction with Raf-1 proteins and the enhancement of homodimerization of MST2 proteins. It thereby enhances the MST2 activation and induces neuronal cell death.Conclusions/SignificanceThe identification of the c-Abl tyrosine kinase as a novel upstream activator of MST2 suggests that the conserved c-Abl-MST signaling cascade plays an important role in oxidative stress-induced neuronal cell death.

Regulation by lipocalin‐2 of neuronal cell death, migration, and morphology

A secreted protein, lipocalin-2 (LCN2), has been previously shown to regulate a variety of cellular phenotypes such as cell death, migration, and morphology. The role of LCN2, however, appears to be different depending on the cellular context. Here, we investigated how LCN2 influences neuronal phenotypes by using primary cortical neuronal cell cultures and neuroblastoma cell lines as a model. When exposed to LCN2 protein, neurons and neuroblastoma cells were sensitized to cell death evoked by nitric oxide, oxidative stress, and tumor necrosis factor-α (TNF-α). A forced expression of lcn2 in glia enhanced neuronal cell death in cocultures of glia and neurons, indicating that both exogenous protein addition and endogenous expression of lcn2 give rise to similar results. Iron and BCL2-interacting mediator of cell death (BIM) protein were involved in LCN2-induced cell death sensitization, based on the studies using iron donor, chelator, siderophore, and short hairpin RNA (shRNA)-mediated knockdown of bim expression. Furthermore, cell migration assay and immunofluorescence microscopic observation revealed that LCN2 accelerated neuronal motility and process extension, suggesting multiple roles for LCN2 in the regulation of neuronal cell death, migration, and morphology.

Knockdown of growth‐arrest‐specific gene 7b (gas7b) using short‐hairpin RNA desensitizes neuroblastoma cells to cisplatin: Implications for preventing apoptosis of neurons

Efficient control of cell survival and cell proliferation is critical for the development of neuron cells. Earlier, we observed that growth arrest-specific gene 7 (Gas7) plays a role in controlling neuritogenesis in mammals. In the present study, we report that the Gas7b isoform is involved in controlling growth arrest and apoptosis of neuroblastoma cells in response to various stimuli. Accordingly, knockdown of Gas7b using small-hairpin RNA (shRNA) was shown to reduce apoptosis induced either by serum starvation or by the antineoplastic agents cisplatin and nocodazole in human neuroblastoma SH-SY5Y cells. Gas7b knockdown also enhanced the ability of the treated cells to form clones in response to cisplatin. On the other hand, forced expression of Gas7a or Gas7b isoform in mouse neuroblastoma Neuro2A cells, which express a defective Gas7 gene, rendered the cells proapoptotic and vulnerable to cisplatin-induced apoptosis. In addition, Neuro2A cells that overexpressed Gas7 showed a reduced ability to form clones. Overexpression of Gas7 produced similar but less extensive effects in nonneuronal HEK293 cells. Taken together, our observations suggest that Gas7b is involved not only in neuritogenesis but also in the regulation of neuronal cell death.

Mitochondria in neurodegenerative disorders: regulation of the redox state and death signaling leading to neuronal death and survival

In Parkinson's disease, impaired function of mitochondrial complex I is involved in selective degeneration of dopamine neurons in the substantia nigra. Mitochondria are now considered to play an active role in neuronal death process through activating "intrinsic" apoptotic signaling, in addition to production of reactive oxygen species. This paper presents our recent findings on new functions of mitochondria in regulation of their redox state and function through reversible "S-glutathionylation", a mixed disulfide binding between sulfhydryl groups of GSH and protein cysteine in complex I subunits. Type A monoamine oxidase (MAO-A) localized at the mitochondrial outer membrane is a binding site of neurotoxins leading to apoptosis. Rasagiline and (-)deprenyl, type B MAO inhibitors of propagylamine-derivatives, bind to MAO-A to protect neuronal cells against apoptosis through induction of pro-survival Bcl-2 and neurotrophic factors. This review discusses the new role of mitochondria in regulation of neuronal cell death of neurodegenerative disorders.

Regulation of Neuronal Cell Death by MST1-FOXO1 Signaling

The protein kinase mammalian Sterile 20-like kinase 1 (MST1) plays a critical role in the regulation of cell death. Recent studies suggest that MST1 mediates oxidative stress-induced neuronal cell death by phosphorylating the transcription factor FOXO3 at serine 207, a site that is conserved in other FOXO family members. Here, we show that MST1-induced phosphorylation of FOXO1 at serine 212, corresponding to serine 207 in FOXO3, disrupts the association of FOXO1 with 14-3-3 proteins. Accordingly, MST1 mediates the nuclear translocation of FOXO1 in primary rat cerebellar granule neurons that are deprived of neuronal activity. We also find a requirement for MST1 in cell death of granule neurons upon withdrawal of growth factors and neuronal activity, and MST1 induces cell death in a FOXO1-dependent manner. Finally, we show that the MST1-regulatory, scaffold protein Nore1 is required for survival factor deprivation induced neuronal death. Collectively, these findings define MST1-FOXO1 signaling as an important link survival factor deprivation-induced neuronal cell death with implications for our understanding of brain development and neurological diseases.

Class IIA HDACs in the regulation of neurodegeneration

Neurodegenerative diseases affect millions of patients annually and are a significant burden on the health care systems around the world. While there are symptomatic remedies for patients suffering from various neurodegenerative diseases, there are no cures as of today. Cell death by apoptosis is a common hallmark of neurodegeneration. Therefore, deciphering the molecular pathways regulating this process is of significant value to scientists' endeavor to understand neurodegenerative disorders. Efforts along these lines have uncovered a number of molecular pathways that regulate neuronal apoptosis. Recently, a family of proteins known as histone deacetylases (HDACs) has been linked to regulation of cell survival as well as death. The focus of this review is to summarize our current understanding of the role of HDACs and in particular a subgroup of proteins in this family classified as class IIa HDACs in the regulation of neuronal cell death. It is apparent based on the information presented in this review that although very similar in their primary sequence, members of this family of proteins often have distinct roles in orchestrating apoptotic cell death in the brain.

The Neurodegenerative Disease Protein Ataxin-1 Antagonizes the Neuronal Survival Function of Myocyte Enhancer Factor-2

Ataxin-1 is a neurodegenerative disorder protein whose mutant form causes spinocerebellar ataxia type-1 (SCA1). Evidence suggests that ataxin-1 may function as a transcription repressor. However, neither the importance of this putative transcriptional repression activity in neural cytotoxicity nor the transcriptional targets of ataxin-1 are known. Here we identify the MEF2-HDAC4 transcriptional complex involved in neuron survival as a target of ataxin-1. We show that ataxin-1 binds specifically to histone deacetylase-4 (HDAC4) and MEF2 and colocalizes with them in nuclear inclusion bodies. Significantly, these interactions are greatly reduced by the S776A mutation, which largely abrogates the cytotoxicity of ataxin-1. Supporting the importance of these interactions, we show that wild type ataxin-1 represses MEF2-dependent transcription, whereas the S776A mutant is less potent. Furthermore, overexpression of MEF2 can partially reverse cytotoxicity caused by ataxin-1. Our results identify the MEF2-HDAC4 complex as a target for ataxin-1 transcriptional repression activity and suggest a novel pathogenic mechanism whereby ataxin-1 sequesters and inhibits the neuronal survival factor MEF2.

Phosphorylation of Huntingtin by Cyclin-Dependent Kinase 5 Is Induced by DNA Damage and Regulates Wild-Type and Mutant Huntingtin Toxicity in Neurons

Huntingtin is an antiapoptotic protein that becomes toxic when its polyglutamine stretch is expanded, resulting in Huntington's disease (HD). Protein context and posttranslational modifications regulate huntingtin toxicity. Identifying signaling pathways that act on huntingtin is, therefore, key to understanding huntingtin function in normal and pathological conditions. We show here that huntingtin is phosphorylated by the cyclin-dependent kinase 5 (Cdk5) at serines 1181 and 1201. Phosphorylation can be induced by DNA damage in vitro and in vivo. The state of huntingtin phosphorylation is a crucial regulator of neuronal cell death. Absence of phosphorylation of huntingtin at serines 1181 and 1201 confers toxic properties to wild-type huntingtin in a p53-dependent manner in striatal neurons and accelerates neuronal death induced by DNA damage. In contrast, phosphorylation at serines 1181 and 1201 protects against polyQ-induced toxicity. Finally, we show in late stages of HD a sustained DNA damage that is associated with a decrease in Cdk5/p35 levels. We propose that wild-type huntingtin is a component of the DNA damage response signal in neurons and that the Cdk5/DNA damage pathway is dysregulated in HD.

Mitochondrial dynamics in the regulation of neuronal cell death

Mitochondria undergo continuous fission and fusion events in physiological situations. Fragmentation of mitochondria during cell death has been shown to play a key role in cell death progression, including release of the mitochondrial apoptotic proteins. Ultrastructural changes in mitochondria, such as cristae remodeling, is also involved in cell death initiation. Here, we emphasize the important role of mitochondrial fission/fusion machinery in neuronal cell death. Unlike many other cell types such as immortalized cell lines, neurons are distinct morphologically and functionally. We will discuss how this uniqueness presents special challenges in the cellular response to neurotoxic stresses, and how this affects the mitochondrial dynamics in the regulation of cell death in neurons.

Expression of Bcl-2 and Bax protein in normal pineal gland in children and young adult

The Bcl family contains both pro and antiapoptotic proteins participating in the regulation of neuronal cell death in several pathological conditions. However, very little is known about physiological profiles of Bcl-2/Bax expression in normal brain. In this study, we examined expression profile of Bcl-2 and Bax proteins in normal pineal gland in children. The material for analysis was obtained by biopsy of pineal parenchyma during surgery of pineal cysts. All specimens were labeled immunohistochemically and analyzed by means of confocal laser scanning microscope. We found only few Bcl-2 expressing (0.7%) and no Bax-immunopositive (0.0%) pinealocytes. Bcl-2-positive cells were mature neurons, neither young ones nor glia.

Caspase-3 deficiency during development increases vulnerability to hypoxic–ischemic injury through caspase-3-independent pathways

Neonatal hypoxia-ischemia (H-I) is a common cause of perinatal morbidity and mortality leading to prominent activation of caspase-3 in the brain. Previous studies have shown that acute inhibition of caspase-3 can protect against neonatal H-I in rats. In this study, we investigated brain injury following neonatal H-I in mice deficient in caspase-3. Wild-type, caspase-3+/- and caspase-3-/- mice underwent unilateral carotid ligation at postnatal day (P) 7, followed by 45 min of exposure to 8% oxygen. Surprisingly, tissue loss at P14 was significantly higher in caspase-3-/- mice when compared to wild-type littermates. As in rats, we found that acute inhibition of caspase-3 in mice leads to decrease in tissue loss at P14. There was no difference in nuclear morphology, DNA laddering or calpain activation between caspase-3-/-caspase-3+/- and wild-type mice subjected to H-I, and there was no evidence for compensatory activation of other caspases in caspase-3-/- mice. Also, all genotypes showed evidence of mitochondrial dysfunction after H-I, suggesting that this is a critical point in regulation of neuronal cell death following neonatal H-I. Our results suggest that long-term inhibition of caspase-3 during development, unlike acute inhibition, leads to upregulation of caspase-3-independent cell death pathways and increases the vulnerability of the developing brain to neonatal H-I injury.

The Role of CDK5/P25 Formation/Inhibition in Neurodegeneration

Cdk5 is an atypical cyclin-dependent kinase localized in the brain, and its activity is dependent upon binding to p35/p39. In addition, while cdk5 has important physiological functions related to brain development, the breakdown of cdk5/p35 into cdk5/p25 increases its kinase activity and neurotoxicity. Interestingly, in recent years increased cdk5/p25 expression has been demonstrated in the brains of patients with Alzheimer's and Parkinson's diseases. Experimental studies performed in neuronal cell cultures indicate that cdk5/p25 plays a prominent role in apoptosis. Moreover, an apoptotic pathway, via an intracellular calcium increase following calpain activation and cdk5/p25 formation, has been postulated. Cdk5/p25 subsequently phosphorylates the nuclear transcription factor myocyte enhancer factor (MEF2), thereby inhibiting its prosurvival activity. However, cdk5/p25 could phosphorylate other substrates such as tau and p53, as well as the retinoblastoma protein pRb. All these data lend credence to the hypothesis that cdk5/p25 acts as a master regulator of neuronal cell death. In addition, cdk5/p25 might also interact with other pathways such as glycogen synthetase kinase 3beta (GSK3beta) and c-JUN kinase. Drugs like roscovitine, flavopiridol, calpain inhibitors, kenpaullone and induribins, which inhibit cdk5/p25 formation, constitute potential drugs for the treatment of neurological disorders. Furthermore, the dual inhibitory effect of some of these drugs on cdk5 and GSK3beta could be beneficial.