C73 A MITOCHONDRIAL PARADIGM FOR AGE‐RELATED METABOLIC AND DEGENERATIVE DISEASES
Wallace DC
Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, USA
E‐mail address for correspondence: [email protected]
The mitochondrial (mt) genome encompasses approximated 1500 genes, 37 encoded by the maternally‐inherited mtDNA, which is present in thousands of copies per cell, and the remainder encoded by various chromosomal loci. The mitochondria produce most of the cellular energy, generate much of the endogenous reactive oxygen species (ROS) as a toxic by‐product, and can initiate apoptosis via activation of the mitochondrial permeability transition pore (mtPTP): when energy production declines, oxidative stress becomes excessive, and calcium accumulates in the cell and mitochondria. Because of its direct exposure to ROS, the mtDNA has a very high mutation rate resulting in the age‐related accumulation of somatic mtDNA mutations in post‐mitotic tissues. As mtDNA mutations accumulate, mitochondrial energy production declines until the mtPTP is activated and the cell with its defective mitochondria is destroyed. Therefore, the accumulation of somatic mtDNA mutations is the aging clock. Age‐related diseases result from the combined effects of an inherited predisposition to the disease, environmental insults, and the accumulating somatic mtDNA mutations. When these combined effects exceed the threshold of the mtPTP, cells are lost. When sufficient cells are lost within a tissue, clinical symptoms ensue.
C74 REGULATION OF MITOCHONDRIAL AND ER CELL DEATH PATHWAYS
Prehn JHM
Royal College of Surgeons in Ireland, Department of Physiology and Medical Physics, Dublin, Ireland
E‐mail address for correspondence: [email protected]
The activation of genetically and biochemically determined cell death programs are believed to contribute to pathophysiological cell death in acute and chronic neurological disorders. The identification of the individual genes and proteins mediating neuronal cell death in response to different stress conditions is therefore of major interest for current neuroscience research. The Bcl‐2 family proteins are key regulators of these cell death programs. They have been shown to be involved in the control of caspase‐dependent and ‐independent apoptosis. Caspase‐dependent apoptosis results from an increase in mitochondrial outer membrane permeability. It leads to the release of cytochrome C from mitochondria and the formation of a caspase‐activating multiprotein complex, the apoptosome. Caspase‐independent apoptosis can result from the mitochondrial release of a pro‐apoptotic nuclease, the apoptosis‐inducing factor (AIF). Caspase‐independent cell death may also occur through mitochondrial dysfunction secondary to the loss of cytochrome C, and has been shown to involve energy depletion, increased ROS production, and the activation of autophagy. Bcl‐2 and related anti‐apoptotic proteins such as Bcl‐xL protect neurons against caspase‐dependent and ‐independent cell death pathways by inhibiting the activation of pro‐apoptotic Bcl‐2 family members. Bax and Bak are pro‐apoptotic Bcl‐2 family members that are able to form a megachannel in the outer mitochondrial membrane large enough for the permeation of proteins such as cytochrome‐C. In order to cause this permeability increase, Bax and Bak undergo a conformational change and insert into the outer mitochondrial membrane.
In apoptotic cells, the transcriptional induction or post‐translational activation of Bcl‐2‐homolgy domain‐3 (BH3)‐only proteins triggers the activation of Bax and Bak. BH3‐only proteins either directly activate Bax and Bak, or interact with and neutralize the anti‐apoptotic activity of Bcl‐2 and Bcl‐xL. BH3‐only proteins are structurally diverse and couple specific upstream stress signals to the evolutionary conserved mitochondrial apoptosis pathways. BH3‐only proteins can be activated via transcriptional induction (PUMA, Noxa, Hrk, Bim, BNIP3L), phosphorylation (Bad, Bik, Bim, Bmf), or proteolytic cleavage (Bid). In the nervous system, induction of PUMA occurs in response to p53 activation and as a consequence of prolonged ER stress, while induction of BNIP3L occurs during hypoxia. Activation of Bim has been implicated in trophic factor withdrawal‐induced apoptosis in neurons, as well as in response to the activation of stress‐activated protein kinases. Finally, Bid has been shown to be required for excitotoxic and ischaemic nerve cell injury. Both Bcl‐2 and Bcl‐xL reside in the outer mitochondrial membrane, but are also localized to the ER membrane and nuclear envelope, facing the cytosol. Evidence has been presented that exclusive ER localization of Bcl‐2 is sufficient to protect cells against several apoptosis stimuli, possibly by sequestering pro‐apoptotic family members at the ER.
C75 IMPAIRED MITOCHONDRIAL ANTI‐OXIDANT DEFENCE IN SOD1‐RELATED FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Wood‐Allum CA1, Barber SC1, Kirby J1, Heath P1, Holden H1, Allen S1, Beaujeux T1, Alexson SEH2, Ince P3 & Shaw PJ1
1Academic Neurology Unit, Sheffield University Medical School, Sheffield, UK, 2Department of Medical Laboratory Sciences and Technology, Karolinska Institute, Huddinge, Sweden, and 3Academic Pathology Unit, Sheffield University Medical School, Sheffield, UK
E‐mail address for correspondence: [email protected]
Background: Mutations to Cu/Zn superoxide dismutase (SOD1) are responsible for ∼2% of amyotrophic lateral sclerosis (ALS). It remains unclear how mutant SOD1 injures motor neurons but increasing evidence suggests that mitochondrial dysfunction may be important in the pathogenesis of both the sporadic and familial forms of the disease.
Objectives: 1) To identify changes in mitochondrial protein expression attributable to the presence of mutant SOD1 in a motor neuronal cell‐culture model of SOD1‐related familial ALS; 2) To establish whether these changes in protein expression have functional consequences and demonstrate relevance to ALS in patients.
Methods: 2D‐SDS PAGE, MALDI‐TOF MS and online database searching were used to identify changes in mitochondrial protein expression brought about by the presence of mutant SOD1 in NSC34 cells. Confirmatory Western blotting was performed in NSC34 cells and key changes also confirmed in mitochondrial preparations of SOD1 transgenic mouse spinal cord. Q‐PCR of laser‐capture micro‐dissected spinal motor neurons from sporadic ALS and SOD1‐related FALS cases was then used to compare the mRNA levels of key protein changes with those of controls.
Results: The expression of 29 proteins changed in a mutant SOD1‐specific manner. These included anti‐oxidant enzymes, apoptotic effectors, and electron transport chain components. Peroxiredoxin 3 (Prx3), a thioredoxin‐dependent hydroperoxidase, was down‐regulated in mutant SOD1‐expressing cells and in SOD1 transgenic mouse spinal cord. Immunocytochemistry confirmed mitochondrial expression of Prx3 in NSC34 cells and immunohistochemistry was used to confirm expression of Prx3 within murine and human spinal motor neurons. Q‐PCR for Prx3 further suggested down‐regulation in SALS and SOD1‐related FALS cases compared to controls. Data from the pharmacological manipulation of mitochondrial anti‐oxidant defence arising from this work will also be presented.
Conclusions: Given the evidence for oxidative stress in ALS, it is interesting that Prx3, an anti‐oxidant mitochondrial matrix protein, is down‐regulated in the presence of mutant SOD1 in NSC34 cells, in SOD1 transgenic mice and in patients. Changes in mitochondrial anti‐oxidant defence may play a role in the death of motor neurons in SOD1‐related FALS and its modulation may offer therapeutic opportunities.
C76 CNS METABOLIC DEFECTS PRECEDE PATHOLOGIC ALTERATIONS IN A MOUSE MODEL OF FAMILIAL ALS
Browne SE, Yang L, Dimauro J‐PP, Fuller SW & Berger SE
Weill Medical College of Cornell University, New York, USA
E‐mail address for correspondence: [email protected]
Background: Multiple cell death pathways have been implicated in the selective loss of motor neurons in ALS, but the causal event remains enigmatic. One hypothesis implicates metabolic dysfunction, since alterations in energy metabolism and mitochondrial function occur in patients. In addition, expression of mutant Cu/Zn‐superoxide dismutase (SOD1), linked with ∼25% of familial ALS (FALS), can induce mitochondrial abnormalities.
Objectives: To determine if metabolic defects contribute to disease onset in vivo, we examined the association between CNS energetic defects and disease progression in G93A mutant SOD1 mice.
Methods: We used quantitative [14C]‐2‐deoxyglucose autoradiography to measure glucose use rates in nine spinal cord and 49 brain regions of conscious pre‐symptomatic (60‐day) and symptomatic (120‐day) G93A mice, in age‐matched wild‐type littermates, and in aged N1029 mice overexpressing human wild‐type SOD1. We also determined metabolite levels by HPLC in flash‐frozen brain and spinal cord tissue from G93A and wild‐type mice at multiple ages, in G93A mice treated with creatine (2% in diet) from symptom onset, and in aged N1029 SOD1 mice.
Results: Glucose use rates were impaired in multiple brain components of the motor system in G93A mice as early as 60 days of age, preceding the first detectable pathologic changes (∼70–80 days) and onset of hind‐limb weakness (∼90–100 days). At 60 days, glucose use was reduced in components of the corticospinal projection, notably primary motor cortex (Fr1), and several areas synaptically associated with Fr1 including the pontine nuclei and reticular formation of the bulbospinal pathway, and some thalamic nuclei. In the spinal cord, regarded as the crucial site of neuronal dysfunction in ALS, glucose metabolism was unaltered at 60 days, but was markedly impaired in cervical and thoracic grey matter by 120 days. Aged (21‐month‐old) N1029 mice showed no alterations in cerebral or spinal cord glucose use, implying that the changes detected in G93A mice are due to the SOD1 mutation rather than overexpression. HPLC revealed significant depletions in ATP levels in the cerebral cortex of G93A mice evident as early as 30 days of age, implying that reduced neuronal energy generation is an extremely early consequence of mSOD1 expression. Alterations in spinal cord did not reach significance. ATP depletion was partially rescued by creatine administration.
Conclusion: In conclusion, these studies demonstrate that energetic defects occur earlier than other pathogenic processes reported in G93A mice, and suggest that dysfunction within the corticospinal projection may precede alterations in spinal neurons in this FALS model. Overall, results support a critical role for metabolic dysfunction in the pathogenesis of ALS.