Presynaptic Mitochondria Communicate With Release Sites for Spatio-Temporal Regulation of Exocytosis at the Motor Nerve Terminal.

Presynaptic Ca2+ regulation is critical for accurate neurotransmitter release, vesicle reloading of release sites, and plastic changes in response to electrical activity. One of the main players in the regulation of cytosolic Ca2+ in nerve terminals is mitochondria, which control the size and spread of the Ca2+ wave during sustained electrical activity. However, the role of mitochondria in Ca2+ signaling during high-frequency short bursts of action potentials (APs) is not well known. Here, we studied spatial and temporal relationships between mitochondrial Ca2+ (mCa2+) and exocytosis by live imaging and electrophysiology in adult motor nerve terminals of transgenic mice expressing synaptophysin-pHluorin (SypHy). Our results show that hot spots of exocytosis and mitochondria are organized in subsynaptic functional regions and that mitochondria start to uptake Ca2+ after a few APs. We also show that mitochondria contribute to the regulation of the mode of fusion (synchronous and asynchronous) and the kinetics of release and replenishment of the readily releasable pool (RRP) of vesicles. We propose that mitochondria modulate the timing and reliability of neurotransmission in motor nerve terminals during brief AP trains.

Calcium is reduced in presynaptic mitochondria of motor nerve terminals during neurotransmission in SMA mice.

Spinal muscular atrophy (SMA) is an autosomal recessive degenerative motor neuron disease characterized by symmetrical muscle weakness and atrophy of limb and trunk muscles being the most severe genetic disease in children. In SMA mouse models, motor nerve terminals display neurotransmitter release reduction, endocytosis decrease and mitochondria alterations. The relationship between these changes is, however, not well understood. In the present study, we investigated whether the endocytosis impairment could be related to the functional alteration of the presynaptic mitochondria during action potential (AP) firing. To this aim, we generated a Synaptophysin-pHluorin (SypHy) transgenic mouse, crossed it with Taiwanese SMA mice, and recorded exo- and endocytosis and mitochondria Ca2+ signaling in real-time at ex vivo motor nerve terminals of Taiwanese-SypHy mice. The experiments were performed at the beginning of the motor symptoms to get an integrated view of the nerve terminal's functional state before degeneration. Our electrophysiological and live imaging results demonstrated that the mitochondria's capacity to increase matrix-free Ca2+ in SMA mice was significantly limited during nerve AP firing, except when the rate of Ca2+ entry to the cytosol was considerably reduced. These results indicate that both the mitochondrial Ca2+ signaling alterations and the secretion machinery defects are significant players in the dysfunction of the presynaptic terminal in SMA.

The Mouse Levator Auris Longus Muscle: An Amenable Model System to Study the Role of Postsynaptic Proteins to the Maintenance and Regeneration of the Neuromuscular Synapse.

The neuromuscular junction (NMJ) is the peripheral synapse that controls the coordinated movement of many organisms. The NMJ is also an archetypical model to study synaptic morphology and function. As the NMJ is the primary target of neuromuscular diseases and traumatic injuries, the establishment of suitable models to study the contribution of specific postsynaptic muscle-derived proteins on NMJ maintenance and regeneration is a permanent need. Considering the unique experimental advantages of the levator auris longus (LAL) muscle, here we present a method allowing for efficient electroporation-mediated gene transfer and subsequent detailed studies of the morphology and function of the NMJ and muscle fibers. Also, we have standardized efficient facial nerve injury protocols to analyze LAL muscle NMJ degeneration and regeneration. Our results show that the expression of a control fluorescent protein does not alter either the muscle structural organization, the apposition of the pre- and post-synaptic domains, or the functional neurotransmission parameters of the LAL muscle NMJs; in turn, the overexpression of MuSK, a major regulator of postsynaptic assembly, induces the formation of ectopic acetylcholine receptor clusters. Our NMJ denervation experiments showed complete reinnervation of LAL muscle NMJs four weeks after facial nerve injury. Together, these experimental strategies in the LAL muscle constitute effective methods to combine protein expression with accurate analyses at the levels of structure, function, and regeneration of the NMJ.

Synaptotagmin-2, and -1, linked to neurotransmission impairment and vulnerability in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is the most frequent genetic cause of infant mortality. The disease is characterized by progressive muscle weakness and paralysis of axial and proximal limb muscles. It is caused by homozygous loss or mutation of the SMN1 gene, which codes for the Survival Motor Neuron (SMN) protein. In mouse models of the disease, neurotransmitter release is greatly impaired, but the molecular mechanisms of the synaptic dysfunction and the basis of the selective muscle vulnerability are unknown. In the present study, we investigated these open questions by comparing the molecular and functional properties of nerve terminals in severely and mildly affected muscles in the SMNΔ7 mouse model. We discovered that synaptotagmin-1 (Syt1) was developmentally downregulated in nerve terminals of highly affected muscles but not in low vulnerable muscles. Additionally, the expression levels of synaptotagmin-2 (Syt2), and its interacting protein, synaptic vesicle protein 2 (SV2) B, were reduced in proportion to the degree of muscle vulnerability while other synaptic proteins, such as syntaxin-1B (Stx1B) and synaptotagmin-7 (Syt7), were not affected. Consistently with the extremely low levels of both Syt-isoforms, and SV2B, in most affected neuromuscular synapses, the functional analysis of neurotransmission revealed highly reduced evoked release, altered short-term plasticity, low release probability, and inability to modulate normally the number of functional release sites. Together, we propose that the strong reduction of Syt2 and SV2B are key factors of the functional synaptic alteration and that the physiological downregulation of Syt1 plays a determinant role in muscle vulnerability in SMA.