Spinal muscular atrophy (SMA) is a common pediatric neuromuscular disorder caused by insufficient levels of the survival of motor neuron (SMN) protein. Studies involving SMA patients and animal models expressing the human SMN2 gene have yielded relatively little information about the earliest cellular consequences of reduced SMN protein. In this study, we have used severe- and mild-SMN2 expressing mouse models of SMA as well as material from human patients to understand the initial stages of neurodegeneration in the human disease. We show that the earliest structural defects appear distally and involve the neuromuscular synapse. Insufficient SMN protein arrests the post-natal development of the neuromuscular junction (NMJ), impairing the maturation of acetylcholine receptor (AChR) clusters into 'pretzels'. Pre-synaptic defects include poor terminal arborization and intermediate filament aggregates which may serve as a useful biomarker of the disease. These defects are reflected in functional deficits at the NMJ characterized by intermittent neurotransmission failures. We suggest that SMA might best be described as a NMJ synaptopathy and that one promising means of treating it could involve maintaining function at the NMJ.

Spinal muscular atrophy (SMA) is a common (ϳ1:6400) autosomal recessive neuromuscular disorder caused by a paucity of the survival of motor neuron (SMN) protein. Although widely recognized to cause selective spinal motor neuron loss when deficient, the precise cellular site of action of the SMN protein in SMA remains unclear. In this study we sought to determine the consequences of selectively depleting SMN in the motor neurons of model mice. Depleting but not abolishing the protein in motor neuronal progenitors causes an SMA-like phenotype. Neuromuscular weakness in the model mice is accompanied by peripheral as well as central synaptic defects, electrophysiological abnormalities of the neuromuscular junctions, muscle atrophy, and motor neuron degeneration. However, the disease phenotype is more modest than that observed in mice expressing ubiquitously low levels of the SMN protein, and both symptoms as well as early electrophysiological abnormalities that are readily apparent in neonates were attenuated in an age-dependent manner. We conclude that selective knock-down of SMN in motor neurons is sufficient but may not be necessary to cause a disease phenotype and that targeting these cells will be a requirement of any effective therapeutic strategy. This realization is tempered by the relatively mild SMA phenotype in our model mice, one explanation for which is the presence of normal SMN levels in non-neuronal tissue that serves to modulate disease severity.

A tight balance between synaptic vesicle exocytosis and endocytosis is fundamental to maintaining synaptic structure and function. Calcium influx through voltage-gated Ca2+ channels is crucial in regulating synaptic vesicle exocytosis. However, much less is known about how Ca2+ regulates vesicle endocytosis or how the endocytic machinery becomes enriched at the nerve terminal. We report here a direct interaction between voltage-gated Ca2+ channels and endophilin, a key regulator of clathrin-mediated synaptic vesicle endocytosis. Formation of the endophlin-Ca2+ channel complex is Ca2+ dependent. The primary Ca2+ binding domain resides within endophilin and regulates both endophilin-Ca2+ channel and endophilin-dynamin complexes. Introduction into hippocampal neurons of a dominant-negative endophilin construct, which constitutively binds to Ca2+ channels, significantly reduces endocytosis-mediated uptake of FM 4-64 dye without abolishing exocytosis. These results suggest an important role for Ca2+ channels in coordinating synaptic vesicle recycling by directly coupling to both exocytotic and endocytic machineries.

Multiple protein kinase C (PKC) isozymes are present in neurons, where they regulate a variety of cellular functions. Due to the lack of specific PKC isozyme inhibitors, it remains unknown how PKC acts on its selective target(s) and achieves its specific actions. Here we show that a PKC binding protein, enigma homolog (ENH), interacts specifically with both PKCepsilon and N-type Ca2+ channels, forming a PKCepsilon-ENH-Ca2+ channel macromolecular complex. Coexpression of ENH facilitated modulation of N-type Ca2+ channel activity by PKC. Disruption of the complex reduced the potentiation of the channel activity by PKC in neurons. Thus, ENH, by interacting specifically with both PKCepsilon and the N-type Ca2+ channel, targets a specific PKC to its substrate to form a functional signaling complex, which is the molecular mechanism for the specificity and efficiency of PKC signaling.