Homozygous loss of SMN1 causes spinal muscular atrophy (SMA), the most common and devastating childhood genetic motor-neuron disease. The copy gene SMN2 produces only ∼10% functional SMN protein, insufficient to counteract development of SMA. In contrast, the human genetic modifier plastin 3 (PLS3), an actin-binding and -bundling protein, fully protects against SMA in SMN1-deleted individuals carrying 3-4 SMN2 copies. Here, we demonstrate that the combinatorial effect of suboptimal SMN antisense oligonucleotide treatment and PLS3 overexpression-a situation resembling the human condition in asymptomatic SMN1-deleted individuals-rescues survival (from 14 to >250 days) and motoric abilities in a severe SMA mouse model. Because PLS3 knockout in yeast impairs endocytosis, we hypothesized that disturbed endocytosis might be a key cellular mechanism underlying impaired neurotransmission and neuromuscular junction maintenance in SMA. Indeed, SMN deficit dramatically reduced endocytosis, which was restored to normal levels by PLS3 overexpression. Upon low-frequency electro-stimulation, endocytotic FM1-43 (SynaptoGreen) uptake in the presynaptic terminal of neuromuscular junctions was restored to control levels in SMA-PLS3 mice. Moreover, proteomics and biochemical analysis revealed CORO1C, another F-actin binding protein, whose direct binding to PLS3 is dependent on calcium. Similar to PLS3 overexpression, CORO1C overexpression restored fluid-phase endocytosis in SMN-knockdown cells by elevating F-actin amounts and rescued the axonal truncation and branching phenotype in Smn-depleted zebrafish. Our findings emphasize the power of genetic modifiers to unravel the cellular pathomechanisms underlying SMA and the power of combinatorial therapy based on splice correction of SMN2 and endocytosis improvement to efficiently treat SMA.

SUMMARY We investigated the properties of the extensor tibiae muscle of the stick insect (Carausius morosus) middle leg. Muscle geometry of the middle leg was compared to that of the front and hind legs and to the flexor tibiae,respectively. The mean length of the extensor tibiae fibres is 1.41±0.23 mm and flexor fibres are 2.11±0.30 mm long. The change of fibre length with joint angle was measured and closely follows a cosine function. Its amplitude gives effective moment arm lengths of 0.28±0.02 mm for the extensor and 0.56±0.04 mm for the flexor. Resting extensor tibiae muscle passive tonic force increased from 2 to 5 mN in the maximum femur–tibia (FT)-joint working range when stretched by ramps. Active muscle properties were measured with simultaneous activation (up to 200 pulses s–1) of all three motoneurons innervating the extensor tibiae, because this reflects most closely physiological muscle activation during leg swing. The force–length relationship corresponds closely to the typical characteristic according to the sliding filament hypothesis: it has a plateau at medium fibre lengths, declines nearly linearly in force at both longer and shorter fibre lengths, and the muscle's working range lies in the short to medium fibre length range. Maximum contraction velocity showed a similar relationship. The force–velocity relationship was the traditional Hill curve hyperbola, but deviated from the hyperbolic shape in the region of maximum contraction force close to the isometric contraction. Step-like changes in muscle length induced by loaded release experiments characterised the non-linear series elasticity as a quadratic spring.

Stick insect (Carausius morosus) leg muscles contract and relax slowly. Control of stick insect leg posture and movement could therefore differ from that in animals with faster muscles. Consistent with this possibility, stick insect legs maintained constant posture without leg motor nerve activity when the animals were rotated in air. That unloaded leg posture was an intrinsic property of the legs was confirmed by showing that isolated legs had constant, gravity-independent postures. Muscle ablation experiments, experiments showing that leg muscle passive forces were large compared with gravitational forces, and experiments showing that, at the rest postures, agonist and antagonist muscles generated equal forces indicated that these postures depended in part on leg muscles. Leg muscle recordings showed that stick insect swing motor neurons fired throughout the entirety of swing. To test whether these results were specific to stick insect, we repeated some of these experiments in cockroach (Periplaneta americana) and mouse. Isolated cockroach legs also had gravityindependent rest positions and mouse swing motor neurons also fired throughout the entirety of swing. These data differ from those in human and horse but not cat. These size-dependent variations in whether legs have constant, gravity-independent postures, in whether swing motor neurons fire throughout the entirety of swing, and calculations of how quickly passive muscle force would slow limb movement as limb size varies suggest that these differences may be caused by scaling. Limb size may thus be as great a determinant as phylogenetic position of unloaded limb motor control strategy.

Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties. J Neurophysiol 114: 1255-1271, 2015. First published June 10, 2015 doi:10.1152/jn.01010.2014.-We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K 2 SO 4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities Ն2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with 14 C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills. current injection; leech; neuron properties; stomatogastric IN WHOLE-CELL PATCH CLAMP, electrode fill solution ion composition is matched as closely as possible to neuron cytoplasmic composition because of the high leak and consequent cell dialysis that occur with these large-tip diameter electrodes (Hille 2003; Pusch and Neher 1988). Despite their much smaller-tip diameters, substantial leak of electrode contents (Fromm and Schultz 1981), sufficient to cause cell swelling (Stoner et al. 1984) and chloride loading (Eisen and Marder 1982), also occurs with sharp microelectrodes. Nonetheless, sharp electrodes filled with 3 M KCl (much greater than the ionic strength of neuron cytoplasm) solutions were used in the second paper, demonstrating that sharp electrode physiology was possible (Nastuk and Hodgkin 1950). Nastuk and Hodgkin (1950) made this choice to reduce the resistance of their electrodes, because it obviated the need to correct for liquid junctional potentials (see MATERIALS AND METHODS). They were aware of the danger of electrode leak using such a high ionic strength electrode fill solution but reasoned that it would have very little effect with the volumes of the muscle ...