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Up-regulation of mitochondrial uncoupling protein 3 reveals an early muscular metabolic defect in amyotrophic lateral sclerosis¹

LUC DUPUIS, FRANCK DI SCALA, FRÉDÉRIQUE RENE, MARC DE TAPIA, HUGUES OUDART^*, PIERRE-FRANÇOIS PRADAT, VINCENT MEININGER and JEAN-PHILIPPE LOEFFLER²

Laboratoire de Signalisations Moléculaires et Neurodégénérescence, EA 3433, Université Louis Pasteur, Faculté de Médecine, 67085 Strasbourg Cedex, France;
^* CEPE, CNRS, 67087 Strasbourg, France; and
Service de Neurologie, Hôpital de la Pitié Salpêtrière, 75651 Paris Cedex 13, France

²Correspondence: Laboratoire de Signalisations Moléculaires et Neurodégénérescence, EA 3433, Université Louis Pasteur, Faculté de Médecine, 11 rue Humann, 67085 Strasbourg Cedex, France. E-mail: loeffler{at}neurochem.u-strasbg.fr

SPECIFIC AIMS

The specific aim of this study was to investigate the physiopathological function of the mitochondrial uncoupling proteins (UCPs) in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease involving oxidative stress, mitochondrial dysfunction, and cell death, these three pathological processes being dependent on UCPs. We monitored the expression levels of UCPs in a transgenic model of ALS carrying a mutated form of SOD1 and in human muscular biopsies of ALS patients.

PRINCIPAL FINDINGS

1. We systematically studied the expression levels of UCPs in various tissues of ALS affected mice carrying a mutated form of SOD1 (G86R mice) using semiquantitative RT-PCR. We found an increased expression of UCP2 and UCP3 expression in skeletal muscle of G86R mice compared with control littermates. UCP3 expression was increased 2 wk before disease onset (90 days of age) and sustained until death of the animals (105 days of age), whereas UCP2 up-regulation occurred later, close to disease onset (Fig. 1 ). We did not detect any variation of UCPs in other tissues (liver, spinal cord).

View larger version (61K): [in this window] [in a new window]   Figure 1. UCP2 and UCP3 are overexpressed in ALS mice muscles. A) Representative RT-PCR showing UCP2 and UCP3 mRNA levels in the gastrocnemius muscle of wild-type (+) and G86R (M) mice of 75 (75d), 90 (90d), and 105 days (105d) old. G3PDH mRNA levels were used as internal control. B) Quantitative analysis of RT-PCR. *P < 0,05 (vs. wt) **P < 0.01 (vs. wt).

2. UCP2 mRNA levels are up-regulated in mouse gastrocnemius muscles by experimental sciatic nerve axotomy or crush. On the contrary, UCP3 mRNA levels are down-regulated in experimentally denervated muscles, a situation opposite to the one observed in muscles of G86R transgenic mice.

3. UCP2 mRNA levels are unchanged in muscles of a transgenic mouse line overexpressing the wild-type allele of SOD1 (wt-SOD1). UCP3 mRNA levels are decreased in wt-SOD1 muscles. The early up-regulation of UCP3 mRNA in the skeletal muscle of G86R animals is thus specifically linked to the expression of the ALS-associated mutant enzyme.

4. UCP3 mRNA and protein levels were up-regulated in human muscular sporadic ALS biopsies compared with various neuromuscular and control patients.

5. Isolated mitochondria of G86R muscles displayed a lower respiratory control ratio while cytochrome c oxidase activity was found unchanged (Fig. 2 ), thus suggesting the occurrence of mitochondrial uncoupling. Furthermore, ATP levels are depleted in skeletal muscles of G86R mice (30% decrease at 90days of age; 38% decrease at 105 days of age) and unchanged in liver and spinal cord tissues, correlating with a UCP pattern of expression.

View larger version (19K): [in this window] [in a new window]   Figure 2. Mitochondrial dysfunction in ALS mice muscles. A) Respiratory control ratio (state 3/state 2 ratio) of isolated mitochondria from hindlimb muscles. Pooled results of 3 independent experiments. The hindlimb muscles of 2 wild-type (+) and G86R (M) mice (90–105 days old) and 2 control littermates were pooled for each experiment. *P< 0,05 as determined using paired Student’s t test. For statistical purposes, results of each experiment were paired. B) Cytochrome c oxidase activity in the same mitochondrial preparations. No significant differences were noted.

CONCLUSIONS AND SIGNIFICANCE

Our findings demonstrate that UCP3 is specifically overexpressed in skeletal muscles of ALS patients and an ALS animal model independent from the transcriptional response of the muscle to neuronal death. Furthermore, this UCP3 overexpression is specific to muscle tissue and correlates with a decrease in mitochondrial respiratory control ratio in ATP levels in this tissue. Our results thus suggest that UCPs trigger mitochondrial uncoupling, which lowers ATP levels and contributes to the ALS phenotype.

Indeed, UCP2 and UCP3 are both susceptible to trigger mitochondrial uncoupling in vitro and in vivo as shown by the decreased mitochondrial proton leak of isolated mitochondria from UCP3 -/- or UCP2 -/- animals. However, even if these proteins are susceptible to significantly uncouple mitochondria, their absence has no effect on whole animal metabolic responses: UCP3 and UCP2 knockout animals are neither obese nor show abnormal responses to fasting or cold-induced thermogenesis. This is probably due to the involvement of UCP2 and UCP3 in an oxidative stress-inducible proton leak rather than to basal proton leak. In physiological situations where oxidative stress occurs, such as exercise or fasting, UCP2 and UCP3 uncoupling activities are activated by superoxide anion, limiting ROS production by mitochondrial respiratory chain. This in turn decreases superoxide levels and UCP uncoupling activity by a feedback loop. This assumption is further supported by the regulation of UCP2 and 3 gene expression by oxidative stress and antioxidants. Oxidative stress is indeed a general condition in ALS and is not restricted to the nervous system: oxidative stress biomarkers have been detected in the plasma, increase over time, and correlate with the severity of the disease. Respiratory chain defects and abnormalities of mitochondrial DNA are present in muscle of ALS patients and have been related to oxidative stress damage. ALS is thus a condition in which UCP2 and UCP3 overexpressions are susceptible to triggering a massive mitochondrial uncoupling.

Although our data showing increased UCP levels do not formally prove mitochondrial uncoupling, its association with decreased RCR and ATP nevertheless favors this interpretation. Indirect evidences exists of uncoupling in vivo in ALS mice and ALS patients: mitochondrial swelling, an event associated with uncoupling is one of the hallmarks of ALS mice mitochondria. When mutated SOD1 is stably transfected in a cell line, it induces a decrease in mitochondrial membrane potential, a finding suggestive of mitochondrial uncoupling.

UCP3 up-regulation thus appears as a primary protective response against oxidative stress. Our experiments further suggest that in vivo muscular mitochondrial uncoupling is able to be deleterious for skeletal muscle cells. For example, a possible deleterious effect of a sharp UCP3 up-regulation is its potential effect on general metabolism. Although UCP3 levels have no effect on the basal metabolism in normal conditions, transgenic mice that overexpress UCP3 in muscles are lean and hyperphagic due to hypermetabolism through mitochondrial uncoupling. In ALS, weight loss is a common symptom, and a clinical study showed that ALS patients are hypermetabolic. Further studies are needed to determine whether up-regulation of UCP3 in muscle of ALS patients plays a role in weight loss and hypermetabolism through mitochondrial uncoupling.

This study demonstrates that UCP3 is abnormally up-regulated in skeletal muscles of ALS patients and ALS mice. Increased expression of UCP3 appears to be a protective response against oxidative stress through uncoupling of muscle mitochondria, but could also induce deleterious muscle and metabolic effects. Our results further suggest that skeletal muscle contributes to ALS phenotype, which is consistent with recently published studies showing that targeted overexpression of mutated SOD1 in neurons or glial cell does not trigger ALS phenotype (see Fig. 3 for further details).

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram. ALS results from neuronal and muscular events. Several neuronal events resulting from the gain of function of the mutated SOD1 are described. Glutamate excitotoxicity, slow and fast axonal transport impairment, or mitochondrial dysfunction are documented (right). Here we report the existence of specific alterations of the motoneuron target, namely, skeletal muscle. UCPs, molecular effectors of mitochondrial uncoupling, are overexpressed; possibly as a result, ATP levels are decreased. How the neuronal and muscular events relate and determine the course of ALS needs in-depth investigation. We favor a model where alterations at the neuromuscular junction chronically modify gene expression of a motor neuron and its muscular target. Such changes might ultimately lead to the severe dysfunction of the motor unit and to motoneuronal death.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1182fje; doi: 10.1096/fj.02-1182fje

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