Synaptic withdrawal following nerve injury is influenced by postnatal maturity, muscle-specific properties, and the presence of underlying pathology in mice.

Axonal and synaptic degeneration occur following nerve injury and during disease. Traumatic nerve injury results in rapid fragmentation of the distal axon and loss of synaptic terminals, in a process known as Wallerian degeneration (WD). Identifying and understanding factors that influence the rate of WD is of significant biological and clinical importance, as it will facilitate understanding of the mechanisms of neurodegeneration and identification of novel therapeutic targets. Here, we investigate levels of synaptic loss following nerve injury under a range of conditions, including during postnatal development, in a range of anatomically distinct muscles and in a mouse model of motor neuron disease. By utilising an ex vivo model of nerve injury, we show that synaptic withdrawal is slower during early postnatal development. Significantly more neuromuscular junctions remained fully innervated in the cranial nerve/muscle preparations analysed at P15 than at P25. Furthermore, we demonstrate variability in the level of synaptic withdrawal in response to injury in different muscles, with retraction being slower in abdominal preparations than in cranial muscles across all time points analysed. Importantly, differences between the cranial and thoracoabdominal musculature seen here are not consistent with differences in muscle vulnerability that have been previously reported in mouse models of the childhood motor neuron disease, spinal muscular atrophy (SMA), caused by depletion of survival motor neuron protein (Smn). To further investigate the relationship between synaptic degeneration in SMA and WD, we induced WD in preparations from the Smn2B/- mouse model of SMA. In a disease-resistant muscle (rostral band of levator auris longus), where there is minimal denervation, there was no change in the level of synaptic loss, which suggests that the process of synaptic withdrawal following injury is Smn-independent. However, in a muscle with ongoing degeneration (transvs. abdominis), the level of synaptic loss significantly increased, with the percentage of denervated endplates increasing by 33% following injury, compared to disease alone. We therefore conclude that the presence of a die-back can accelerate synaptic loss after injury in Smn2B/- mice.

Abnormal fatty acid metabolism is a core component of spinal muscular atrophy.

OBJECTIVE: Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder leading to paralysis and subsequent death in young children. Initially considered a motor neuron disease, extra-neuronal involvement is increasingly recognized. The primary goal of this study was to investigate alterations in lipid metabolism in SMA patients and mouse models of the disease. METHODS: We analyzed clinical data collected from a large cohort of pediatric SMA type I-III patients as well as SMA type I liver necropsy data. In parallel, we performed histology, lipid analysis, and transcript profiling in mouse models of SMA. RESULTS: We identify an increased susceptibility to developing dyslipidemia in a cohort of 72 SMA patients and liver steatosis in pathological samples. Similarly, fatty acid metabolic abnormalities were present in all SMA mouse models studied. Specifically, Smn2B/- mice displayed elevated hepatic triglycerides and dyslipidemia, resembling non-alcoholic fatty liver disease (NAFLD). Interestingly, this phenotype appeared prior to denervation. INTERPRETATION: This work highlights metabolic abnormalities as an important feature of SMA, suggesting implementation of nutritional and screening guidelines in patients, as such defects are likely to increase metabolic distress and cardiovascular risk. This study emphasizes the need for a systemic therapeutic approach to ensure maximal benefits for all SMA patients throughout their life.

Reduced P53 levels ameliorate neuromuscular junction loss without affecting motor neuron pathology in a mouse model of spinal muscular atrophy.

Spinal Muscular Atrophy (SMA) is a childhood motor neuron disease caused by mutations or deletions within the SMN1 gene. At endstages of disease there is profound loss of motor neurons, loss of axons within ventral roots and defects at the neuromuscular junctions (NMJ), as evidenced by pathological features such as pre-synaptic loss and swelling and post-synaptic shrinkage. Although these motor unit defects have been widely described, the time course and interdependancy of these aspects of motor unit degeneration are unclear. Recent reports have also revealed an early upregulation of transcripts associated with the P53 signalling pathway. The relationship between the upregulation of these transcripts and pathology within the motor unit is also unclear. In this study, we exploit the prolonged disease timecourse and defined pre-symptomatic period in the Smn2B/- mouse model to perform a temporal analysis of the different elements of motor unit pathology. We demonstrate that NMJ loss occurs prior to cell body loss, and coincides with the onset of symptoms. The onset of NMJ pathology also coincides with an increase in P53-related transcripts at the cell body. Finally, using a tamoxifen inducible P53 knockout, we demonstrate that post-natal reduction in P53 levels can reduce NMJ loss, but does not affect other aspects of NMJ pathology, motor neuron loss or the phenotype of the Smn2B/- mouse model. Together this work provides a detailed temporal description of pathology within motor units of an SMA mouse model, and demonstrates that NMJ loss is a P53-dependant process. This work supports the role for P53 as an effector of synaptic and axonal degeneration in a die-back neuropathy.

Altered mitochondrial bioenergetics are responsible for the delay in Wallerian degeneration observed in neonatal mice.

Neurodegenerative and neuromuscular disorders can manifest throughout the lifespan of an individual, from infant to elderly individuals. Axonal and synaptic degeneration are early and critical elements of nearly all human neurodegenerative diseases and neural injury, however the molecular mechanisms which regulate this process are yet to be fully elucidated. Furthermore, how the molecular mechanisms governing degeneration are impacted by the age of the individual is poorly understood. Interestingly, in mice which are under 3 weeks of age, the degeneration of axons and synapses following hypoxic or traumatic injury is significantly slower. This process, known as Wallerian degeneration (WD), is a molecularly and morphologically distinct subtype of neurodegeneration by which axons and synapses undergo distinct fragmentation and death following a range of stimuli. In this study, we first use an ex-vivo model of axon injury to confirm the significant delay in WD in neonatal mice. We apply tandem mass-tagging quantitative proteomics to profile both nerve and muscle between P12 and P24 inclusive. Application of unbiased in silico workflows to relevant protein identifications highlights a steady elevation in oxidative phosphorylation cascades corresponding to the accelerated degeneration rate. We demonstrate that inhibition of Complex I prevents the axotomy-induced rise in reactive oxygen species and protects axons following injury. Furthermore, we reveal that pharmacological activation of oxidative phosphorylation significantly accelerates degeneration at the neuromuscular junction in neonatal mice. In summary, we reveal dramatic changes in the neuromuscular proteome during post-natal maturation of the neuromuscular system, and demonstrate that endogenous dynamics in mitochondrial bioenergetics during this time window have a functional impact upon regulating the stability of the neuromuscular system.