A relationship between cerebellar Purkinje cells and spatial working memory demonstrated in a lurcher/chimera mouse model system

New emphasis has been placed upon cerebellar research because of recent reports demonstrating involvement of the cerebellum in non-motor cognitive behaviors. Included in the growing list of cognitive functions associated with cerebellar activation is working memory. In this study, we explore the potential role of the cerebellum in spatial working memory using a mouse model of Purkinje cell loss. Specifically, we make aggregation chimeras between heterozygous lurcher (Lc/+) mutant embryos and +/+ (wildtype) embryos and tested them in the delayed matching-to-position (DMTP) task. Lc/+ mice lose 100% of their Purkinje cells postnatally due to a cell-intrinsic gain-of-function mutation. Lc/+<->+/+ chimeras therefore have Purkinje cells ranging from 0 to normal numbers. Through histological examination of chimeric mice and observations of motor ability, we showed that ataxia is dependent upon both the number and distribution of Purkinje cells in the cerebellum. In addition, we found that Lc/+ mice, with a complete loss of Purkinje cells, have a generalized deficit in DMTP performance that is probably associated with their motor impairment. Finally, we found that Lc/+<->+/+ chimeric mice, as a group, did not differ from control mice in this task. Rather, surprisingly, analysis of their total Purkinje cells and performance in the DMTP task revealed a significant negative relationship between these two variables. Together, these findings indicate that the cerebellum plays a minor or indirect role in spatial working memory.     

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death

p53 is a potent inhibitor of cell growth and an inducer of apoptosis. During embryonic development, Mdm2 and Mdm4 inhibit the growth suppressive activities of p53. However, whether tight surveillance of p53 activity is required in quiescent cells is unknown. To test this, conditional inactivation of mdm2 and mdm4 was carried out in smooth muscle cells (SMCs). Upon SMC-specific inactivation of mdm2, and not of mdm4, mice rapidly became ill and died. Necropsy showed small intestinal dilation, and histological analyses indicated a severe reduction in the number of intestinal SMCs. Increased p53 levels and activity were detected in the remaining SMCs, and the phenotype was completely rescued on a p53-null background. Interestingly, intestinal SMCs are caspase-3-negative and therefore did not undergo caspase-3-dependent apoptotic cell death. Together, Mdm2, but not Mdm4, prevents accumulation of active p53 in quiescent SMCs and thereby the induction of p53-mediated caspase-3-independent cell death.     

Muscarinic receptor activation protects cells from apoptotic effects of DNA damage,oxidative stress,and mitochondrial inhibition

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50-200 microm H(2)O(2) caused the activation of caspase-3 beginning after 2-3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H(2)O(2)-induced caspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H(2)O(2) or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss.     

Oxidative stress, nitric oxide, and the mechanisms of cell death in Lurcher Purkinje cells

Oxidative stress is postulated to play a role in cell death in many neurodegenerative diseases. As a model of neonatal neuronal cell death, we have examined the role of oxidative stress in Purkinje cell death in the heterozygous Lurcher mutant (+/Lc). Lurcher is a gain of function mutation in the delta2 glutamate receptor (GluRdelta2) that turns the receptor into a leaky membrane channel, resulting in chronic depolarization of +/Lc Purkinje cells starting around the first week of postnatal development. Virtually, all +/Lc Purkinje cells die by the end of the first postnatal month. To investigate the role of oxidative stress in +/Lc Purkinje cell death, we have examined nitric oxide synthase (NOS) activity and the expression of two markers for oxidative stress, nitrotyrosine and manganese super oxide dismutase (MnSOD), in wild type and +/Lc Purkinje cells at P10, P15, and P25. The results show that NOS activity and immunolabeling for nitrotyrosine and MnSOD are increased in +/Lc Purkinje cells. To determine whether peroxynitrite formation is a prerequisite for +/Lc Purkinje cell death, +/Lc mutants were crossed with an alpha-nNOS knockout mutant (nNOSalpha(-/-)) to reduce the production of NO. Analysis of the double mutants showed that blocking alpha-nNOS expression does not rescue +/Lc Purkinje cells. However, we present evidence for sustained NOS activity and nitrotyrosine formation in the GluRdelta2(+/Lc):nNOS(-/-) double mutant Purkinje cells, which suggests that the failure to rescue GluRdelta2(+/Lc):nNOS(-/-) Purkinje cells may be explained by the induction of alternative nNOS isoforms.     

LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21(WAF1/Cip1) expression

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21(WAF1/Cip1) and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21(WAF1/Cip1), as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21(WAF1/Cip1) and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53-p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.     

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver ( wv ) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation ( pcd ) destroys the vast majority of Purkinje cells. The staggerer ( sg ) and lurcher ( Lc ) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of G_D1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of G_TIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of G_D3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg , and Lc /+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that G_DT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for G_Tla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.     

Bax and Bak independently promote cytochrome C release from mitochondria

Pro-apoptotic Bax and Bak have been implicated in the regulation of p53-dependent apoptosis. We assessed the ability of primary baby mouse kidney (BMK) epithelial cells from bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice to be transformed by E1A alone or in conjunction with dominant-negative p53 (p53DD). Although E1A alone transformed BMK cells from p53-deficient mice, E1A alone did not transform BMK cells from bax(-/-), bak(-/-), or bax(-/-) bak(-/-) mice. Thus, the loss of both Bax and Bak was not sufficient to relieve p53-dependent suppression of transformation in epithelial cells. To test the requirement for Bax and Bak in other death signaling pathways, stable E1A plus p53DD-transformed BMK cell lines were derived from the bax(-/-), bak(-/-), and bax(-/-) bak(-/-) mice and characterized for their response to tumor necrosis factor-alpha (TNF-alpha)-mediated apoptosis. The loss of both Bax and Bak severely impaired TNF-alpha-mediated apoptosis, but the presence of either Bax or Bak alone was sufficient for cell death. Cytochrome c was released from mitochondria, and caspase-9 was activated in Bax- or Bak-deficient cells in response to TNF-alpha but not in cells deficient in both. Thus, either Bax or Bak is required for death signaling through mitochondria in response to TNF-alpha, but both are dispensable for p53-dependent transformation inhibition.     

Involvement of activation of PI3K/Akt pathway in the protective effects of puerarin against MPP+-induced human neuroblastoma SH-SY5Y cell death

In an attempt to clarify the protective effect of puerarin on toxin-insulted dopaminergic neuronal death, this present study was carried out by using a typical Parkinson's disease (PD) model - 1-methyl-4-phenylpyridinium iodide (MPP(+))-induced dopaminergic SH-SY5Y cellular model. Data are presented, which showed that puerarin up-regulated Akt phosphorylation in both of MPP(+)-treated and non-MPP(+)-treated cells. The presence of PI3K inhibitor LY294002 completely blocked puerarin-induced activation of Akt phosphorylation. Moreover, puerarin decreased MPP(+)-induced cell death, which was blocked by phosphoinositide 3-kinase (PI3K) inhibitor LY294002. We further demonstrated that puerarin protected against MPP(+)-induced p53 nuclear accumulation, Puma (p53-upregulated mediator of apoptosis) and Bax expression and caspase-3-dependent programmed cell death (PCD). This protection was blocked by applying a PI3K/Akt inhibitor. Additionally, it was Pifithrin-α, but not Pifithrin-μ, which blocked MPP(+)-induced Puma and Bax expression, caspase-3 activation and cell death. Collectively, these data suggest that the activation of PI3K/Akt pathway is involved in the protective effect of puerarin against MPP(+)-induced neuroblastoma SH-SY5Y cell death through inhibiting nuclear p53 accumulation and subsequently caspase-3-dependent PCD. Puerarin might be a potential therapeutic agent for PD.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Characterization of the cell death induced by cadmium in HaCaT and C6 cell lines

Cell death resulting from cadmium (Cd) intoxication has been confirmed to induce both necrosis and apoptosis. The ratio between both types of cell death is dose- and cell-type-dependent. This study used the human keratinocytes HaCaT expressing a mutated p53 and the rat glial cells C6 expressing a wild p53 as models to characterize Cd-induced apoptosis, using sub-lethal and lethal doses. At these concentrations, features of apoptosis were observed 24 h after C6 cell treatment: apoptotic DNA fragmentation and caspase-9 activation, whereas Cd did not induce caspase-3. In HaCaT, Cd did not induce apoptotic DNA fragmentation or caspase-9 and -3 activation. The results also showed that the inhibition of p53 led to a resistance of the C6 cells to 20 µm Cd, decreased the apoptosis and increased the metallothioneins in these cells. p53 restoration increased the sensitivity of HaCaT cells to Cd but did not affect the MT expression. The results suggest that Cd induced apoptosis in C6 cells but a non-apoptotic cellular death in HaCaT cells.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997     

p53-dependent caspase-2 activation in mitochondrial release of apoptosis-inducing factor and its role in renal tubular epithelial cell injury

We demonstrate the role of p53-mediated caspase-2 activation in the mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-treated renal tubular epithelial cells. Gene silencing of AIF with its small interfering RNA (siRNA) suppressed cisplatin-induced AIF expression and provided a marked protection against cell death. Subcellular fractionation and immunofluorescence studies revealed cisplatin-induced translocation of AIF from the mitochondria to the nuclei. Pancaspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone or p53 inhibitor pifithrin-alpha markedly prevented mitochondrial release of AIF, suggesting that caspases and p53 are involved in this release. Caspase-2 and -3 that were predominantly activated in response to cisplatin provided a unique model to study the role of these caspases in AIF release. Cisplatin-treated caspase-3 (+/+) and caspase-3 (-/-) cells exhibited similar AIF translocation to the nuclei, suggesting that caspase-3 does not affect AIF translocation, and thus, caspase-2 may be involved in the translocation. Caspase-2 inhibitor benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethylketone or down-regulation of caspase-2 by its siRNA significantly prevented translocation of AIF. Caspase-2 activation was a critical response from p53, which was markedly induced and phosphorylated in cisplatin-treated cells. Overexpression of p53 not only resulted in caspase-2 activation but also mitochondrial release of AIF. The p53 inhibitor pifithrin-alpha or p53 siRNA prevented both cisplatin-induced caspase-2 activation and mitochondrial release of AIF. Caspase-2 activation was dependent on the p53-responsive gene, PIDD, a death domain-containing protein that was induced by cisplatin in a p53-dependent manner. These results suggest that caspase-2 activation mediated by p53 is an important pathway involved in the mitochondrial release of AIF in response to cisplatin injury.     

Curcumin induces Apaf-1-dependent,p21-mediated caspase activation and apoptosis

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.     

Hairless expression attenuates apoptosis in a mouse model and the COS cell line; involvement of p53

Background

Neurons are more likely to die through apoptosis in the immature brain after injury whereas adult neurons in the mature brain die by necrosis. Several studies have suggested that this maturational change in the mechanism of cell death is regulated, in part, by thyroid hormone. We examined the involvement of the hairless (Hr) gene which has been suspected of having a role in cell cycle regulation and apoptosis in the hair follicle and is strongly regulated by the thyroid hormone in the brain.

Methodology

Forced expression of Hr by transfection decreased the number of apoptotic nuclei, levels of caspase-3 activity, and cytosolic cytochrome C in COS cells exposed to staurosporine and tunicamycin. Similarly, capsase-3 activity was lower and the decrease in mitochondrial membrane potential was smaller in cultures of adult cerebellar granule neurons from wild type mice compared to Hr knockout mice induced to undergo apoptosis. In vivo, apoptosis as detected by positive TUNEL labeling and caspase 3 activity was lower in wild-type mice compared to Hr knockouts after exposure to trimethyltin. Hr expression lowered levels of p53, p53 mediated reporter gene activity, and lower levels of the pro-apoptotic Bcl2 family member Bax in COS cells. Finally, Hr expression did not attenuate apoptosis in mouse embryonic fibroblasts from p53 knockout mice but was effective in mouse embryonic fibroblasts from wild type mice.

Conclusions/Significance

Overall, our studies demonstrate that Hr evokes an anti-apoptotic response by repressing expression of p53 and pro-apoptotic events regulated by p53.     

p53-dependent Fas expression is critical for Ginsenoside Rh2 triggered caspase-8 activation in HeLa cells

We have recently reported that Ginsenoside Rh2 (G-Rh2) induces the activation of two initiator caspases, caspase-8 and caspase-9 in human cancer cells. However, the molecular mechanism of its death-inducing function remains unclear. Here we show that G-Rh2 stimulated the activation of both caspase-8 and caspase-9 simultaneously in HeLa cells. Under G-Rh2 treatment, membrane death receptors Fas and TNFR1 are remarkably upregulated. However, the induced expression of Fas but not TNFR1 was contributed to the apoptosis process. Moreover, significant increases in Fas expression and caspase-8 activity temporally coincided with an increase in p53 expression in p53-nonmutated HeLa and SK-HEP-1 cells upon G-Rh2 treatment. In contrast, Fas expression and caspase-8 activity remained constant with G-Rh2 treatment in p53-mutated SW480 and PC-3 cells. In addition, siRNA-mediated knockdown of p53 diminished G-Rh2-induced Fas expression and caspase-8 activation. These results indicated that G-Rh2-triggered extrinsic apoptosis relies on p53-mediated Fas over-expression. In the intrinsic apoptotic pathway, G-Rh2 induced strong and immediate translocation of cytosolic BAK and BAX to the mitochondria, mitochondrial cytochrome c release, and subsequent caspase-9 activation both in HeLa and in SW480 cells. p53-mediated Fas expression and subsequent downstream caspase-8 activation as well as p53-independent caspase-9 activation all contribute to the activation of the downstream effector caspase-3/-7, leading to tumor cell death. Taken together, we suggest that G-Rh2 induces cancer cell apoptosis in a multi-path manner and is therefore a promising candidate for antitumor drug development.     

Caspase-2 triggers Bax-Bak-dependent and -independent cell death in colon cancer cells treated with resveratrol

Polyphenol phytoalexin (resveratrol), found in grapes and red wine is a strong chemopreventive agent with promising safety records with human consumption and unique forms of cell death induction in a variety of tumor cells. However, the mechanism of resveratrol-induced apoptosis upstream of mitochondria is still not defined. The results from this study suggest that caspase-2 activation occurs upstream of mitochondria in resveratrol-treated cells. The upstream activation of caspase-2 is not dependent on its antioxidant property or NF-kappaB inhibition. The activated caspase-2 triggers mitochondrial apoptotic events by inducing conformational changes in Bax/Bak with subsequent release of cytochrome c, apoptosis-inducing factor, and endonuclease G. Caspase-8 activation seems to be independent of these events and does not appear to be mediated by classical death receptor processing or downstream caspases. Both caspase-2 and caspase-8 contribute toward the mitochondrial translocation of Bid, since neither caspase-8 inhibition nor caspase-2 inhibition could prevent translocation of Bid DsRed into mitochondria. Caspase-2 inhibitors or antisense silencing of caspase-2 prevented cell death induced by resveratrol and partially prevented processing of downstream caspases, including caspase-9, caspase-3, and caspase-8. Studies using mouse embryonic fibroblasts deficient for both Bax and Bak indicate the contribution of both Bax and Bak in mediating cell death induced by resveratrol and the existence of Bax/Bak-independent cell death possibly through caspase-8- or caspase-2-mediated mitochondria-independent downstream caspase processing.