[Show abstract][Hide abstract] ABSTRACT: Huntington’s disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca2+ levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca2+ accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial dysfunction has been implicated in Huntington's disease (HD) pathogenesis. We analysed the activity of mitochondrial complexes (Cx) I-IV, protein levels of selected Cx subunits and adenine nucleotides in platelet mitochondria from pre-symptomatic versus symptomatic HD human carriers and age-matched control individuals. Mitochondrial platelets exhibited reduced activity of citrate synthase in pre-symptomatic and Cx-I in pre-symptomatic and symptomatic HD carriers. Positive correlation between Cx activity and protein subunits was observed for Cx-I in symptomatic HD patient's mitochondria. Moreover, AMP increased in mitochondria from pre-symptomatic HD carriers. Results highlight mitochondrial changes occurring before the onset of HD clinical symptoms.
[Show abstract][Hide abstract] ABSTRACT: Machado-Joseph disease (MJD), also known as Spinocerebellar Ataxia type 3, is an inherited dominant autosomal neurodegenerative disorder. An expansion of Cytosine-Adenine-Guanine (CAG) repeats in the ATXN3 gene is translated as an expanded polyglutamine domain in the disease protein, ataxin-3. Selective neurodegeneration in MJD is evident in several subcortical brain regions including the cerebellum. Mitochondrial dysfunction has been proposed as a mechanism of neurodegeneration in polyglutamine disorders. In this study, we used different cell models and transgenic mice to assess the importance of mitochondria on cytotoxicity observed in MJD. Transiently transfected HEK cell lines with expanded (Q84) ataxin-3 exhibited a higher susceptibility to 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II. Increased susceptibility to 3-NP was also detected in stably transfected PC6-3 cells that inducibly express expanded (Q108) ataxin-3 in a tetracycline-regulated manner. Moreover, cerebellar granule cells from MJD transgenic mice were more sensitive to 3-NP inhibition than wild-type cerebellar neurons. PC6-3 (Q108) cells differentiated into a neuronal-like phenotype with nerve growth factor (NGF) exhibited a significant decrease in mitochondrial complex II activity. Mitochondria from MJD transgenic mouse model and lymphoblast cell lines derived from MJD patients also showed a trend toward reduced complex II activity. Our results suggest that mitochondrial complex II activity is moderately compromised in MJD, which may designate a common feature in polyglutamine toxicity.
Biochimica et Biophysica Acta 02/2012; 1822(2):139-49. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alterations in the ubiquitin-proteasome system (UPS) have been reported in several neurodegenerative disorders characterized by protein misfolding and aggregation, including the polylgutamine diseases. Machado-Joseph disease (MJD) or Spinocerebellar Ataxia type 3 is caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene, which encodes a 42 kDa deubiquitinating enzyme (DUB), ataxin-3. We investigated ataxin-3 deubiquitinating activity and the functional relevance of ataxin-3 interactions with two proteins previously described to interact with ataxin-3, hHR23A and valosin-containing protein (VCP/p97). We confirmed ataxin-3 affinity for both hHR23A and VCP/p97. hHR23A and ataxin-3 were shown to co-localize in discrete nuclear foci, while VCP/p97 was primarily cytoplasmic. hHR23A and VCP/p97 recombinant proteins were added, separately or together, to normal and expanded ataxin-3 in in vitro deubiquitination assays to evaluate their influence on ataxin-3 activity. VCP/p97 was shown to be an activator specifically of wild-type ataxin-3, exhibiting no effect on expanded ataxin-3, In contrast, we observed no significant alterations in ataxin-3 enzyme kinetics or substrate preference in the presence of hHR23A alone or in combination with VCP. Based on our results we propose a model where ataxin-3 normally functions with its interactors to specify the cellular fate of ubiquitinated proteins.
PLoS ONE 01/2012; 7(9):e43563. · 3.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3-Nitropropionic acid (3-NP), an inhibitor of mitochondrial complex II, leads to metabolic impairment and neurodegeneration.
In this study, we investigated the roles of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the
dysregulation of transcription factors and histone modifying enzymes induced by 3-NP in primary cortical neurons. BDNF prevented
the 3-NP-induced decrease in cAMP response-element binding protein (CREB) phosphorylation and CREB-binding protein levels.
Both NGF and BDNF counteracted the increase in the levels of histone H3 and H4 acetylations and reduced histone deacetylase
(HDAC) activity induced by 3-NP. BDNF further led to hyperphosphorylation of HDAC2. Our results support an important role
for neurotrophins, particularly BDNF, in preventing detrimental changes in transcription factors and histone acetylation states
in cortical neurons that have been subjected to selective mitochondrial inhibition.
KeywordsCREB-binding protein (CBP)-cAMP response-element binding protein (CREB)-Histone acetylation-Mitochondria-3-Nitropropionic acid-Cortical neurons
Neurotoxicity Research 01/2010; 17(4):399-405. · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase that has been used to explore the primary mechanisms of cell death associated with mitochondrial dysfunction and neurodegeneration in Huntington's disease. In this study we investigated the ability of brain-derived neurotrophic factor (BDNF) to suppress mitochondrial-dependent cell death induced by 3-NP in primary cortical neurons. This neurotrophin prevented 3-NP-induced release of cytochrome c and Smac/Diablo, caspase-3-like activity and nuclear condensation/fragmentation. Furthermore, it greatly increased phosphorylation of Akt and MAPK, suggesting the involvement of these signalling pathways in BDNF neuroprotection. Interestingly, BDNF decreased the levels of the pro-apoptotic protein Bim in mitochondrial and total cell lysates through the activation of the MEK1/2 pathway. This effect was due to an increase in the degradation rates of Bim. Our data support an important role for BDNF, in protecting cortical neurons against apoptotic cell death caused by inhibition of mitochondrial complex II.
Neurobiology of Disease 07/2009; 35(3):448-56. · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ataxin-3, a deubiquitinating enzyme, is the disease protein in spinocerebellar ataxia type 3, one of many neurodegenerative disorders caused by polyglutamine expansion. Little is known about the cellular regulation of ataxin-3. This is an important issue, since growing evidence links disease protein context to pathogenesis in polyglutamine disorders. Expanded ataxin-3, for example, is more neurotoxic in fruit fly models when its active site cysteine is mutated (1). We therefore sought to determine the influence of ataxin-3 enzymatic activity on various cellular properties. Here we present evidence that the catalytic activity of ataxin-3 regulates its cellular turnover, ubiquitination, and subcellular distribution. Cellular protein levels of catalytically inactive ataxin-3 were much higher than those of active ataxin-3, in part reflecting slower degradation. In vitro studies revealed that inactive ataxin-3 was more slowly degraded by the proteasome and that this degradation occurred independent of ubiquitination. Slower degradation of inactive ataxin-3 correlated with reduced interaction with the proteasome shuttle protein, VCP/p97. Enzymatically active ataxin-3 also showed a greater tendency to concentrate in the nucleus, where it colocalized with the proteasome in subnuclear foci. Taken together, these and other findings suggest that the catalytic activity of this disease-linked deubiquitinating enzyme regulates several of its cellular properties, which in turn may influence disease pathogenesis.
Journal of Biological Chemistry 11/2007; 282(40):29348-58. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Repeated use of drugs of abuse, namely opiates, has been shown to affect glutamate-releasing neurons. Moreover, blockade of N-methyl-D-aspartate (NMDA) receptors (NMDAR) prevents cell death by apoptosis induced by morphine, a heroin metabolite. Thus, in this article we investigated the involvement of different NMDAR subunits in heroin cytotoxicity. Human embryonic kidney (HEK293) cells, which do not express native NMDAR, were transfected with NR1/NR2A or NR1/NR2B subunits. As a control, cells were transfected with NR1 alone, which does not form functional channels. Incubation with heroin for 24 h induced a dose-dependent decrease in cell viability both in NR1-transfected and nontransfected cells. The loss of membrane integrity induced by heroin was more evident in cells transfected with NR1/NR2B than in cells transfected with NR1 alone or NR1/NR2A. This decrease in cell viability was blocked by MK-801, a selective and noncompetitive antagonist of NMDAR. Nevertheless, no significant changes in intracellular adenosine 5'-triphosphate (ATP) were observed in cells treated with heroin. These data implicate NR2B-composed NMDAR as important mediators of heroin neurotoxicity.
Annals of the New York Academy of Sciences 09/2006; 1074:458-65. · 4.38 Impact Factor