Annual Report of Activities CNC 2008 - Center for Neuroscience and ...

Mitochondrial Dysfuntion and Cell Death | Head: Ana Cristina RegoObjectivesSelective neurodegeneration in irreversible disordersof the CNS has been largely attributed to proteinmisfolding, excitotoxicity and mitochondrialimpairment. However, how modified or mutantproteins interfere with mitochondrial and neuronalfunction is largely unclear. Furthermore, severalneurotoxic substances cause neuronal death throughchanges in mitochondrial activity, deregulation ofintracellular calcium homeostasis and oxidativestress. Thus, the main objective of our group is tostudy defective intracellular signaling mechanismsand identify molecular targets for therapeuticintervention underlying excitotoxicity, mitochondrialdysfunction, oxidative stress and neuronal death inneurodegenerative diseases, including polyglutamineexpansion (Huntington’s (HD) and Machado‐Joseph’s (MJD) disorders), Parkinson’s (PD) andAlzheimer’s (AD) diseases, and in the neuropathologycaused by drug addiction. We also aim to evaluatethe efficacy of neuroprotective strategies (pharmacologicaland cell replacement therapies) that help recoveringcell function and thus cell survival in animal andcellular models of neurodegenerative disorders.The protective effect of growth factors that act astrophic signalling molecules assumes a highimportance in neurodegeneration. Therefore, wewill examine the influence of brain‐derivedneurotrophic (BDNF) and glial cell‐derivedneurotrophic factor (GDNF) either directly or aftertransduction into stem cells acquiring a neuralphenotype, and the neuroprotective effect ofinsulin‐growth factor (IGF‐1), insulin and histonedeacetylase modulators in PD and HD models.derived from HD knock‐in mice. Mutant cellsrevealed increased ROS formation, decreased GSHsynthesis and in the activity of antioxidantenzymes. We further studied markers of apoptosisand mitochondrial dysfunction in peripheral bloodcells of HD patients (Almeida et al, 2008). Wefound increased Bax expression in B and Tlymphocytes, and monocytes from HD patients. Blymphocytes also showed decreased mitochondrialpotential, suggesting that these cells may reflectchanges observed in HD brain. In the context ofMJD, we observed an increase in ataxin‐3deubiquitinating activity in vitro after long periodsof incubation with valosin‐containing protein(VCP/p97). Using human SH‐SY5Y cells transfectedwith WT and mutant A53T alpha‐synuclein (a‐syn)subjected to iron and rotenone as models of PD, wedetected increased P‐Ser129‐a‐syn, decreasedmitochondrial potential and ROS production, inthe absence of a‐syn aggregates. Within the scopeof AD pathogenesis, we observed that amyloidbetapeptide (Abeta) oligomers increase NR2A denovo protein synthesis, but enhance membranesurface of NR2B NMDAR subunits in maturehippocampal neurons. Abeta evoked calcium risesensitive to NMDAR antagonists and decreasedtubulin‐beta III levels, suggesting the involvementof NMDAR subunits on neuronal dysfunctioncaused by Abeta oligomers. Regarding research ondrugs of abuse we showed that combination ofcocaine and heroin enhances the neurotoxicity ofthe drugs alone and that cocaine‐morphine adductsshift cell death pathways towards necrosis.25Main AchievementsIn the context of HD, our group showed thatactivation of BDNF signaling pathways prevent 3‐NP toxicity and transcriptional deregulation inneurons. We also observed alterations in themitochondrial proteome of HD mice brain and incomplex I activity, which were prevented byBDNF. These data implicate expression ofmitochondrial proteins and molecular activity inthis organelle as HD therapeutic targets. Moreover,we demonstrated insulin neuroprotection againstoxidative stress through increased expression ofproteins involved in antioxidant defense, glucosemetabolism and prevention against apoptosis(Duarte et al, 2008). We also observed insulinprotection against oxidative stress in striatal cellsInsulin‐mediated mRNA expression of glutathione peroxidase‐1 (A), hexokinase‐II (B), Bcl‐2 (C) and caspase‐3 (D) uponoxidative stress in cultured cortical neurons labeling theinsulin receptor (IR) (E) [Duarte et al., 2008].