Santosh R. D'Mello

Chemotherapy for the Brain: The Antitumor Antibiotic Mithramycin Prolongs Survival in a Mouse Model of Huntington's Disease

Huntingtons disease (HD) is a fully penetrant autosomal-dominant inherited neurological disorder caused by expanded CAG repeats in the Huntingtin gene. Transcriptional dysfunction, excitotoxicity, and oxidative stress have all been proposed to play important roles in the pathogenesis of HD. This study was designed to explore the therapeutic potential of mithramycin, a clinically approved guanosine-cytosine-rich DNA binding antitumor antibiotic. Pharmacological treatment of a transgenic mouse model of HD (R6/2) with mithramycin extended survival by 29.1%, greater than any single agent reported to date. Increased survival was accompanied by improved motor performance and markedly delayed neuropathological sequelae. To identify the functional mechanism for the salubrious effects of mithramycin, we examined transcriptional dysfunction in R6/2 mice. Consistent with transcriptional repression playing a role in the pathogenesis of HD, we found increased methylation of lysine 9 in histone H3, a well established mechanism of gene silencing. Mithramycin treatment prevented the increase in H3 methylation observed in R6/2 mice, suggesting that the enhanced survival and neuroprotection might be attributable to the alleviation of repressed gene expression vital to neuronal function and survival. Because it is Food and Drug Administration-approved, mithramycin is a promising drug for the treatment of HD.

Akt Is a Downstream Target of NF-κB

The ubiquitously expressed transcription factor NF-κB and the serine-threonine kinase Akt both are involved in the promotion of cell survival. Although initially believed to operate as components of distinct signaling pathways, several studies have demonstrated that the NF-κB and Akt signaling pathways can converge. Indeed, IκB kinase, the kinase involved in NF-κB activation, is a substrate of Akt, and activation of Akt therefore stimulates NF-κB activity. Although these results place Akt upstream of NF-κB activation in the sequence of signaling events, we report that this may not necessarily be the case and that Akt is a downstream target of NF-κB. Treatment of NIH3T3 cells with the NF-κB activators, tumor necrosis factor (TNF) α and lipopolysaccharide, results in the stimulation of Akt phosphorylation. The stimulation of Akt is, however, detected only after IκB-α degradation is induced by these agents. The nuclear translocation of p65 and increased DNA binding activity of NF-κB also precede Akt phosphorylation. Treatment with two pharmacological inhibitors of NF-κB, SN50 andN-tosyl-l-phenylalanine chloromethyl ketone (TPCK), blocks TNF-induced Akt activation. On the other hand TNF-mediated NF-κB activation is not reduced by the phosphoinositide-3 kinase inhibitors wortmannin and LY294002, although these inhibitors completely block the activation of Akt. These results suggest that NF-κB is required for TNF-mediated Akt activation and that it lies upstream of the stimulation of Akt. Consistent with this conclusion is the finding that overexpression of p65/RelA leads to Akt phosphorylation in the absence of extracellular stimulatory factors, whereas overexpression of IκB-α reduces Akt phosphorylation below basal levels. Interestingly, in addition to stimulating the phosphorylation of Akt, overexpression of p65 causes an increase in the expression of Akt mRNA and protein.

Opposing Effects of Sirtuins on Neuronal Survival: SIRT1-Mediated Neuroprotection Is Independent of Its Deacetylase Activity

Background Growing evidence suggests that sirtuins, a family of seven distinct NAD-dependent enzymes, are involved in the regulation of neuronal survival. Indeed, SIRT1 has been reported to protect against neuronal death, while SIRT2 promotes neurodegeneration. The effect of SIRTs 3–7 on the regulation of neuronal survival, if any, has yet to be reported. Methodology and Principal Findings We examined the effect of expressing each of the seven SIRT proteins in healthy cerebellar granule neurons (CGNs) or in neurons induced to die by low potassium (LK) treatment. We report that SIRT1 protects neurons from LK-induced apoptosis, while SIRT2, SIRT3 and SIRT6 induce apoptosis in otherwise healthy neurons. SIRT5 is generally localized to both the nucleus and cytoplasm of CGNs and exerts a protective effect. In a subset of neurons, however, SIRT5 localizes to the mitochondria and in this case it promotes neuronal death. Interestingly, the protective effect of SIRT1 in neurons is not reduced by treatments with nicotinamide or sirtinol, two pharmacological inhibitors of SIRT1. Neuroprotection was also observed with two separate mutant forms of SIRT1, H363Y and H355A, both of which lack deacetylase activity. Furthermore, LK-induced neuronal death was not prevented by resveratrol, a pharmacological activator of SIRT1, at concentrations at which it activates SIRT1. We extended our analysis to HT-22 neuroblastoma cells which can be induced to die by homocysteic acid treatment. While the effects of most of the SIRT proteins were similar to that observed in CGNs, SIRT6 was modestly protective against homocysteic acid toxicity in HT-22 cells. SIRT5 was generally localized in the mitochondria of HT-22 cells and was apoptotic. Conclusions/Significance Overall, our study makes three contributions - (a) it represents the first analysis of SIRT3–7 in the regulation of neuronal survival, (b) it shows that neuroprotection by SIRT1 can be mediated by a novel, non-catalytic mechanism, and (c) that subcellular localization may be an important determinant in the effect of SIRT5 on neuronal viability.

HDAC4 inhibits cell-cycle progression and protects neurons from cell death

HDAC4 is a Class II histone deacetylase (HDAC) that is highly expressed in the brain, but whose functional significance in the brain is not known. We show that forced expression of HDAC4 in cerebellar granule neurons protects them against low potassium‐induced apoptosis. HDAC4 also protects HT22 neuroblastoma cells from death induced by oxidative stress. HDAC4‐mediated neuroprotection does not require its HDAC catalytic domain and cannot be inhibited by chemical inhibitors of HDACs. Neuroprotection by HDAC4 also does not require the Raf‐MEK‐ERK or the PI‐3 kinase‐Akt signaling pathways and occurs despite the activation of c‐jun, an event that is generally believed to condemn neurons to die. The protective action of HDAC4 occurs in the nucleus and is mediated by a region that contains the nuclear localization signal. HDAC4 inhibits the activity of cyclin‐dependent kinase‐1 (CDK1) and the progression of proliferating HEK293T and HT22 cells through the cell cycle. Mice‐lacking HDAC4 have elevated CDK1 activity and display cerebellar abnormalities including a progressive loss of Purkinje neurons postnatally in posterior lobes. Surviving Purkinje neurons in these lobes have duplicated soma. Furthermore, large numbers of cells within these affected lobes incorporate BrdU, indicating cell‐cycle progression. These abnormalities along with the ability of HDAC4 to inhibit CDK1 and cell‐cycle progression in cultured cells suggest that neuroprotection by HDAC4 is mediated by preventing abortive cell‐cycle progression.

Caspase‐3 is required for apoptosis‐associated DNA fragmentation but not for cell death in neurons deprived of potassium

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis‐inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase‐3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase‐3 (−/−CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from −/−CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in −/−CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase‐3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in −/−CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low‐K+‐mediated cell death and does so to the same extent in −/−CPP32 cultures, suggesting the involvement of a caspase other than caspase‐3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases ‐ 6 ,7, 8, 9 and 10) are also not involved in low‐K+‐mediated apoptosis. J. Neurosci. Res. 59:24–31, 2000

Neuroprotection by Histone Deacetylase-Related Protein

ABSTRACT The expression of histone deacetylase-related protein (HDRP) is reduced in neurons undergoing apoptosis. Forced reduction of HDRP expression in healthy neurons by treatment with antisense oligonucleotides also induces cell death. Likewise, neurons cultured from mice lacking HDRP are more vulnerable to cell death. Adenovirally mediated expression of HDRP prevents neuronal death, showing that HDRP is a neuroprotective protein. Neuroprotection by forced expression of HDRP is not accompanied by activation of the phosphatidylinositol 3-kinase-Akt or Raf-MEK-ERK signaling pathway, and treatment with pharmacological inhibitors of these pathways fails to inhibit the neuroprotection by HDRP. Stimulation of c-Jun phosphorylation and expression, an essential feature of neuronal death, is prevented by HDRP. We found that HDRP associates with c-Jun N-terminal kinase (JNK) and inhibits its activity, thus explaining the inhibition of c-Jun phosphorylation by HDRP. HDRP also interacts with histone deacetylase 1 (HDAC1) and recruits it to the c-Jun gene promoter, resulting in an inhibition of histone H3 acetylation at the c-Jun promoter. Although HDRP lacks intrinsic deacetylase activity, treatment with pharmacological inhibitors of histone deacetylases induces apoptosis even in the presence of ectopically expressed HDRP, underscoring the importance of c-Jun promoter deacetylation by HDRP-HDAC1 in HDRP-mediated neuroprotection. Our results suggest that neuroprotection by HDRP is mediated by the inhibition of c-Jun through its interaction with JNK and HDAC1.

The c-Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism

Cerebellar granule neurons undergo apoptosis when switched from a medium containing high potassium (HK) to one that has low potassium (LK). LK‐induced cell death is blocked by GW5074 {5‐Iodo‐3‐[(3,5‐dibromo‐4‐hydroxyphenyl) methylene]‐2‐indolinone}, a synthetic drug that inhibits c‐Raf activity in vitro. GW5074 has no direct effect on the activities of several apoptosis‐associated kinases when assayed in vitro. In contrast to its effect in vitro, treatment of neurons with GW5074 causes c‐Raf activation (when measured in vitro in the absence of the drug) and stimulates the Raf‐MEK‐ERK pathway. Treatment of neurons with GW5074 also leads to an increase in the activity of B‐Raf, which is not inhibited by GW5074 in vitro at concentrations at which the drug exerts its neuroprotective effect. PD98059 and U0126, two distinct inhibitors of MEK, block the activation of ERK by GW5074 but have no effect on its ability to prevent cell death. Overexpression of a dominant‐negative form of Akt does not reduce the efficacy of GW5074, demonstrating an Akt‐independent mechanism of action. Neuroprotection is inhibited by SN‐50, a specific inhibitor of nuclear factor‐kappa B (NF‐κB) and by the Ras inhibitor S‐trans, trans‐farnesylthiosalicylic acid (FTS) implicating NF‐κB and Ras in the neuroprotective signaling pathway activated by GW5074. In addition to preventing LK‐induced apoptosis, treatment with GW5074 protects against the neurotoxic effects of MPP+ and methylmercury in cerebellar granule neurons, and glutathione depletion‐induced oxidative stress in cortical neurons. Furthermore, GW5074 prevents neurodegeneration and improves behavioral outcome in an animal model of Huntingtons disease. Given its neuroprotective effect on distinct types of cultured neurons, in response to different neurotoxic stimuli, and in an animal model of neurodegeneration, GW5074 could have therapeutic value against neurodegenerative pathologies in humans.

NF-κB is involved in the survival of cerebellar granule neurons: association of Iκβ phosphorylation with cell survival: NF-κB in neuronal survival

The NF‐κB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c‐Rel. NF‐κB activity is tightly controlled by IκB proteins which bind to NF‐κB preventing its translocation to the nucleus. Activation of NF‐κB is most often mediated by IκB degradation, which permits NF‐κB to enter the nucleus. We investigated the role of NF‐κB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF‐κB activity: SN‐50, N‐tosyl‐l‐phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF‐κB is required for neuronal survival. Gel‐shift assays reveal three complexes that bind to the NF‐κB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium‐induced apoptosis, whereas overexpression of IκBα promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IκBα and IκBβ is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c‐Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IκBβ is, however, reduced by low potassium treatment. Besides being necessary for high potassium‐mediated neuronal survival, NF‐κB is also involved in the survival‐promoting effects of IGF‐1 and cAMP as judged by the ability of SN‐50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium‐induced alterations in the DNA‐binding activity of NF‐κB. Taken together, our results show that NF‐κB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF‐κB‐mediated survival of cerebellar granule neurons.

Histone Deacetylase-1 (HDAC1) Is a Molecular Switch between Neuronal Survival and Death

Background: The role of HDAC1 in the regulation of neuronal survival is unresolved. Results: In cooperation with HDRP, HDAC1 promotes neuronal survival, but when it interacts with HDAC3, HDAC1 promotes neuronal death. Conclusion: HDAC1 can protect neurons or promote neuronal death depending on whether it interacts with HDRP or HDAC3. Significance: Our results provide insight into the role of HDAC1 in the regulation of neuronal survival. Both neuroprotective and neurotoxic roles have previously been described for histone deacetylase-1 (HDAC1). Here we report that HDAC1 expression is elevated in vulnerable brain regions of two mouse models of neurodegeneration, the R6/2 model of Huntington disease and the Ca2+/calmodulin-dependent protein kinase (CaMK)/p25 double-transgenic model of tauopathic degeneration, suggesting a role in promoting neuronal death. Indeed, elevating HDAC1 expression by ectopic expression promotes the death of otherwise healthy cerebellar granule neurons and cortical neurons in culture. The neurotoxic effect of HDAC1 requires interaction and cooperation with HDAC3, which has previously been shown to selectively induce the death of neurons. HDAC1-HDAC3 interaction is greatly elevated under conditions of neurodegeneration both in vitro and in vivo. Furthermore, the knockdown of HDAC3 suppresses HDAC1-induced neurotoxicity, and the knockdown of HDAC1 suppresses HDAC3 neurotoxicity. As described previously for HDAC3, the neurotoxic effect of HDAC1 is inhibited by treatment with IGF-1, the expression of Akt, or the inhibition of glycogen synthase kinase 3β (GSK3β). In addition to HDAC3, HDAC1 has been shown to interact with histone deacetylase-related protein (HDRP), a truncated form of HDAC9, whose expression is down-regulated during neuronal death. In contrast to HDAC3, the interaction between HDRP and HDAC1 protects neurons from death, an effect involving acquisition of the deacetylase activity of HDAC1 by HDRP. We find that elevated HDRP inhibits HDAC1-HDAC3 interaction and prevents the neurotoxic effect of either of these two proteins. Together, our results suggest that HDAC1 is a molecular switch between neuronal survival and death. Its interaction with HDRP promotes neuronal survival, whereas interaction with HDAC3 results in neuronal death.

Isoform-Specific Toxicity of Mecp2 in Postmitotic Neurons: Suppression of Neurotoxicity by FoxG1

The methyl-CpG binding protein 2 (MeCP2) is a widely expressed protein, the mutations of which cause Rett syndrome. The level of MeCP2 is highest in the brain where it is expressed selectively in mature neurons. Its functions in postmitotic neurons are not known. The MeCP2 gene is alternatively spliced to generate two proteins with different N termini, designated as MeCP2-e1 and MeCP2-e2. The physiological significance of these two isoforms has not been elucidated, and it is generally assumed they are functionally equivalent. We report that in cultured cerebellar granule neurons induced to die by low potassium treatment and in Aβ-treated cortical neurons, Mecp2-e2 expression is upregulated whereas expression of the Mecp2-e1 isoform is downregulated. Knockdown of Mecp2-e2 protects neurons from death, whereas knockdown of the e1 isoform has no effect. Forced expression of MeCP2-e2, but not MeCP2-e1, promotes apoptosis in otherwise healthy neurons. We find that MeCP2-e2 interacts with the forkhead protein FoxG1, mutations of which also cause Rett syndrome. FoxG1 has been shown to promote neuronal survival and its downregulation leads to neuronal death. We find that elevated FoxG1 expression inhibits MeCP2-e2 neurotoxicity. MeCP2-e2 neurotoxicity is also inhibited by IGF-1, which prevents the neuronal death-associated downregulation of FoxG1 expression, and by Akt, the activation of which is necessary for FoxG1-mediated neuroprotection. Finally, MeCP2-e2 neurotoxicity is enhanced if FoxG1 expression is suppressed or in neurons cultured from FoxG1-haplodeficient mice. Our results indicate that Mecp2-e2 promotes neuronal death and that this activity is normally inhibited by FoxG1. Reduced FoxG1expression frees MecP2-e2 to promote neuronal death.