Mitochondrial uncoupling leads to muscle weakness but preserves muscle structure
To achieve a chronic muscle-restricted uncoupling, we used mice overexpressing UCP1 under the control of the promoter of the muscle creatine kinase (MCK) gene. Previous work on these MCK-UCP1 mice had shown slight decreases in both mitochondrial membrane potential (from 170 mV to 160 mV) and respiratory control ratio (from 3.3 to 2.5), which resulted in a mild phenotype primarily characterized by diminished muscle mass and fast-to-slow muscle fiber type switching in muscles such as the gastrocnemius 
. As a first step toward the characterization of the neuromuscular phenotype in these animals, we sought to replicate and extend these results. Consistent with this, we found that MCK-UCP1 mice were lighter (), and the cross-sectional surface of gastrocnemius myofibers showed a dramatic age-dependent reduction (), which could account for their decreased grip strength (). Since Han and collaborators 
had previously reported that mice overexpressing high levels of UCP1 suffer from a prominent myopathic disorder, we checked whether the reduction in muscle fiber size observed in our animals could reflect a degenerative process. Serum concentrations of creatine kinase and lactate, an increase of which is indicative of muscular dystrophy 
, were not increased in MCK-UCP1 mice but rather tended to decrease, as compared to wild-type littermates (). In addition, we did not detect the presence of myofibers containing centrally located nuclei (data not shown), that would be a sign of the regenerative process observed in muscular dystrophy 
. In agreement with these findings, MCK-UCP1 muscles did not upregulate the satellite cell marker M-Cadherin, which is typically found in such regenerative processes (). Necrotic myofibers, another hallmark of muscular dystrophy 
, can be easily identified by their permeability to Evans blue dye. In control experiments, a huge amount of Evans blue positive myofibers were observed in wild-type muscles following cardiotoxin injury, but only very few atrophic myofibers (less than 1 per field) were detected in 7-month-old MCK-UCP1 mice (). Finally, mRNA levels of atrogin-1, an ubiquitin-ligase whose expression is strongly upregulated upon muscle wasting 
, appeared unchanged in MCK-UCP1 mice, as compared to wild-type littermates (). Altogether, these findings confirm that mild mitochondrial uncoupling in MCK-UCP1 mice does not cause a dystrophic or massive proteolytic degeneration in skeletal muscle.
Muscle phenotype of MCK-UCP1 mice.
MCK-UCP1 do not display muscle dystrophy.
Muscle mitochondrial uncoupling affects the integrity of AchR clustering
We next studied in detail the morphology of the NMJ in both 1- and 7-month-old mice. In wild-type muscle fibers, AchR clusters were organized in a classical pretzel-shaped structure, either at 1 (, picture a and b) or 7 months of age (, picture c and d). In contrast, the postsynaptic primary gutters in MCK-UCP1 fibers appeared less ramified already at 1 month of age (, pictures e-h), and, in very rare cases, fragmented (, picture h). At 7 months of age, fragmentation was very frequent, and the size of the postsynaptic apparatus had decreased, as compared to wild-type mice (, pictures i–l). Moreover, in the few NMJs presenting with normal primary gutters at this age, thinner cisternae (arrowhead in , picture i) and neighboring ectopic AchR clusters (asterisks in , picture i) could be detected. Quantitative analysis of these phenomena corroborated the histological observations and revealed a significant deterioration of NMJ morphology in MCK-UCP1 mice (). In all, these findings indicate that NMJ morphology progressively deteriorates with age upon chronic muscle mitochondrial uncoupling.
Aging MCK-UCP1 mice show progressive NMJ deterioration.
Muscle mitochondrial uncoupling triggers abnormal muscle electrical activity
Next we asked whether the morphological abnormalities of the postsynaptic apparatus in MCK-UCP1 mice could be secondary to muscle denervation. To this end, we performed longitudinal electromyography studies. Before 6 weeks of age, MCK-UCP1 mice showed normal electrical activity recordings, as compared to wild-type animals (), suggesting that NMJs were functional and properly innervated at that age. Later on, however, a subset of mice started to exhibit fasciculations, which gradually progressed in older animals to strongly altered patterns characterized by fibrillations, fasciculations and myotonic discharges (). After 7 months of age, all the mice were affected (). To test whether this abnormal electrical activity was the reflect of altered neurotransmission at the NMJ, as occurs for example in congenital myasthenic syndromes, we recorded the muscle evoked response to repetitive nerve stimulation. If neuromuscular transmission was affected, a sharp decrease (>10%) in the amplitude of the electrical response should be observed after repeated stimulations, but no differences were detected between MCK-UCP1 and wild-type mice (), indicating that neuromuscular transmission did not appear compromised. Additionally, we measured caudal sensory nerve velocity to determine whether sensory abnormalities could be observed, but found no changes in MCK-UCP1 mice ().
Electromyographic features of MCK-UCP1 mice.
Muscle mitochondrial uncoupling triggers endplate denervation and distal axonal degeneration
The presence of abnormal electromyography patterns together with the observed morphological changes in the postsynaptic apparatus from old MCK-UCP1 mice could reflect denervation. Immunohistochemical analysis of the pre- and postsynaptic compartments of the NMJ revealed complete overlapping of synaptophysin and alpha-bungarotoxin staining in 1-month-old MCK-UCP1 mice, whereas 60% of the alpha-bungarotoxin positive NMJs in 7-month-old MCK-UCP1 mice lacked synaptophysin labeling (), thus suggesting loss of the presynaptic button. To further confirm these observations, muscle sections were stained with an antibody directed against p75NTR, which is typically overexpressed in Schwann cells surrounding degenerating motor endings 
. p75NTR positive nerve fibers were systematically detected in old, but not young, MCK-UCP1 mice (). Altogether, these findings show that MCK-UCP1 mice are afflicted by a distal, age-related axonopathy.
Aging MCK-UCP1 mice show progressive denervation and distal axonal degeneration.
Muscle mitochondrial uncoupling triggers spinal cord astrocytosis and mild motor neuron degeneration
The existence of a NMJ pathology in aging MCK-UCP1 mice prompted us to determine whether any abnormalities could be detected in the ventral root axons and their corresponding spinal cord motor neuronal cell bodies. Ventral roots from MCK-UCP1 mice appeared grossly normal, but showed sparse axonal degeneration (). A more detailed analysis of the distribution of axonal calibers revealed an almost 50% reduction in the amount of large (>5 micrometers in diameter) caliber axons, together with a concomitant increase in the amount of very small caliber, presumably degenerating axons (). Motor neuron counting was performed in the ventral horns of the lumbar spinal cord at 3 and 7 months of age, by means of a morphometric approach based on the cross-sectional area of the cells (, left) or by direct identification of choline acetyltransferase positive motor neurons (, right). Both methods demonstrated a significant 20–30% decrease in the number of motor neurons in 7-month-old MCK-UCP1 mice, as compared to 3-month-old MCK-UCP1 or wild-type animals. The presence of astrocytosis in the lumbar spinal cord, which is typically associated with neuronal suffering 
, was assessed using an antibody directed against the astrocyte marker GFAP. An increase of GFAP positive astrocytes was observed in 7-month-old MCK-UCP1 mice, as compared to wild-type littermates (). In all, these findings strongly suggest that chronic muscle-restricted mitochondrial uncoupling is able to induce a mild but clear motor neuron pathology, likely as a result of the progressive degenerative process initiated at the NMJ.
Mild late onset motor neuron degeneration in MCK-UCP1 mice.
Muscle mitochondrial uncoupling strongly increases agrin-induced AchR clustering
The first pathological event observed in MCK-UCP1 mice appeared to be the dismantlement of the post-synaptic apparatus. Since acetylcholine receptor (AchR) clustering is likely to be an energy-dependent process, we hypothesized that mitochondrial uncoupling occuring in MCK-UCP1 muscles lead to an inability of these muscles to maintain AchR clusters with age. To test this hypothesis, we determined whether old MCK-UCP1 mice were still able to newly form ectopic AchR clusters. Neural agrin, via activation of muscle specific kinase (MuSK), is responsible for NMJ formation and stability 
, and is known to trigger ectopic AchR clustering upon intramuscular injection, the response being further enhanced by muscle denervation. In both innervated and denervated muscle, intramuscular injection of agrin stimulated ectopic AchR clustering much more efficiently in MCK-UCP1 mice than in wild-type animals, the largest clusters being observed in innervated MCK-UCP1 muscle (). In particular, large AchR clusters were frequently detected in the extra junctional area in MCK-UCP1 muscle fibers, whereas only small AchR clusters could be observed and were closely confined to the synaptic region in wild-type fibers. These findings show that, contrary to our initial hypothesis, aged MCK-UCP1 mice are still able to efficiently respond to NMJ-inducing stimulatory nerve factors and suggest that the NMJ defects of these mice are rather presynaptic.
Old MCK-UCP1 mice show increased AchR clustering in response to agrin.
Muscle mitochondrial uncoupling delays motor recovery after lesion
Since AchR clustering is efficiently maintained in MCK-UCP1 mice, we assessed whether axonal regeneration upon injury was normal. To this aim, we performed sciatic nerve crush studies. This experimental model is typically characterized by axonal degeneration and subsequent reinnervation, generally completed within 2 weeks post-lesion. After nerve crush, motor recovery was evaluated by the sciatic functional index (SFI), a scale based on the shape of footprints, and grip strength 
. In wild-type mice, the SFI decreased to a minimum 3 days post-operation and then progressively recovered to reach a normal value at day 15. In MCK-UCP1 mice, the decrease of the SFI was much more pronounced, and recovery was greatly delayed and hardly completed 30 days after lesion (). Consistent with this, grip strength of the operated limb showed a quite similar evolution (). In addition, the accompanying recovery of muscle mass, which was complete 1 month post-operation in wild-type mice, appeared significantly impaired in MCK-UCP1 animals (). To determine whether the retardation in functional recovery was due to delayed reinnervation, we performed double neurofilament/synaptophysin immunostaining along with alpha-bungarotoxin labeling to examine the aspect of the NMJs after nerve crush. NMJs from operated wild-type mice had almost completely recovered 8 days after lesion, whereas most NMJs in MCK-UCP1 mice remained denervated and appeared smaller (). Altogether, these findings show that mitochondrial uncoupling in skeletal muscle limits considerably axonal regeneration after lesion.
MCK-UCP1 mice show delayed functional recovery after nerve crush.
Muscle mitochondrial uncoupling exacerbates motor neuron disease in mice
To further explore the negative influence of muscle mitochondrial uncoupling on motor neuron degeneration, we analysed whether muscle UCP1 overexpression could change the pattern of disease in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). To this end, we crossbred MCK-UCP1 mice with SOD1(G86R) mice, a transgenic line harboring an ALS-linked mutated form of Cu/Zn-superoxide dismutase 
. To monitor disease progression, we followed the characteristic decline in peak body mass observed in these animals as the earliest symptom that coincides with initial axonal retraction from NMJs. Then, we defined an early stage of disease as the time from onset until maximals body mass has decreased by 10% (
and our own observations on the SOD1(G86R) mouse line). Based on these criteria, no differences were detected in disease onset (). However, both progression from onset through early disease () and total survival () were significantly shortened in heterozygous SOD1(G86R)/MCK-UCP1 mice, as compared to SOD1(G86R) mice (). Altogether, these data indicate that muscle-specific mitochondrial uncoupling is, in itself, sufficient to deteriorate NMJ stability and hence contribute to the progression of motor neuron disease.
UCP1 overexpression exacerbates motor neuron disease in mice.