Altered active zones, vesicle pools, nerve terminal conductivity, and morphology during experimental MuSK myasthenia gravis.

Recent studies demonstrate reduced motor-nerve function during autoimmune muscle-specific tyrosine kinase (MuSK) myasthenia gravis (MG). To further understand the basis of motor-nerve dysfunction during MuSK-MG, we immunized female C57/B6 mice with purified rat MuSK ectodomain. Nerve-muscle preparations were dissected and neuromuscular junctions (NMJs) studied electrophysiologically, morphologically, and biochemically. While all mice produced antibodies to MuSK, only 40% developed respiratory muscle weakness. In vitro study of respiratory nerve-muscle preparations isolated from these affected mice revealed that 78% of NMJs produced endplate currents (EPCs) with significantly reduced quantal content, although potentiation and depression at 50 Hz remained qualitatively normal. EPC and mEPC amplitude variability indicated significantly reduced number of vesicle-release sites (active zones) and reduced probability of vesicle release. The readily releasable vesicle pool size and the frequency of large amplitude mEPCs also declined. The remaining NMJs had intermittent (4%) or complete (18%) failure of neurotransmitter release in response to 50 Hz nerve stimulation, presumably due to blocked action potential entry into the nerve terminal, which may arise from nerve terminal swelling and thinning. Since MuSK-MG-affected muscles do not express the AChR γ subunit, the observed prolongation of EPC decay time was not due to inactivity-induced expression of embryonic acetylcholine receptor, but rather to reduced catalytic activity of acetylcholinesterase. Muscle protein levels of MuSK did not change. These findings provide novel insight into the pathophysiology of autoimmune MuSK-MG

Cardiac specific expression of threonine 5 to alanine mutant sarcolipin results in structural remodeling and diastolic dysfunction.

The functional importance of threonine 5 (T5) in modulating the activity of sarcolipin (SLN), a key regulator of sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) pump was studied using a transgenic mouse model with cardiac specific expression of threonine 5 to alanine mutant SLN (SLNT5A). In these transgenic mice, the SLNT5A protein replaces the endogenous SLN in atria, while maintaining the total SLN content. The cardiac specific expression of SLNT5A results in severe cardiac structural remodeling accompanied by bi-atrial enlargement. Biochemical analyses reveal a selective downregulation of SR Ca2+ handling proteins and a reduced SR Ca2+ uptake both in atria and in the ventricles. Optical mapping analysis shows slower action potential propagation in the transgenic mice atria. Doppler echocardiography and hemodynamic measurements demonstrate a reduced atrial contractility and an impaired diastolic function. Together, these findings suggest that threonine 5 plays an important role in modulating SLN function in the heart. Furthermore, our studies suggest that alteration in SLN function can cause abnormal Ca2+ handling and subsequent cardiac remodeling and dysfunction

Altered Differentiation Potential of Gaucherâs Disease iPSC Neuronal Progenitors due to Wnt/β-Catenin Downregulation

Summary: Gaucherâs disease (GD) is an autosomal recessive disorder caused by mutations in the GBA1 gene, which encodes acid β-glucocerebrosidase (GCase). Severe GBA1 mutations cause neuropathology that manifests soon after birth, suggesting that GCase deficiency interferes with neuronal development. We found that neuronopathic GD induced pluripotent stem cell (iPSC)-derived neuronal progenitor cells (NPCs) exhibit developmental defects due to downregulation of canonical Wnt/β-catenin signaling and that GD iPSCsâ ability to differentiate to dopaminergic (DA) neurons was strikingly reduced due to early loss of DA progenitors. Incubation of the mutant cells with the Wnt activator CHIR99021 (CHIR) or with recombinant GCase restored Wnt/β-catenin signaling and rescued DA differentiation. We also found that GD NPCs exhibit lysosomal dysfunction, which may be involved in Wnt downregulation by mutant GCase. We conclude that neuronopathic mutations in GCase lead to neurodevelopmental abnormalities due to a critical requirement of this enzyme for canonical Wnt/β-catenin signaling at early stages of neurogenesis. : In this article, Feldman and colleagues describe a new mechanism linking severe GBA1 mutations to neurodevelopmental defects through Wnt/β-catenin downregulation. Using GD iPSCs as a model, the authors show that the ability of neuronopathic GD NPCs to differentiate to DA neurons is strikingly reduced due to early loss of DA progenitors and that lysosomal dysfunction may be directly involved in canonical Wnt downregulation. Keywords: Gaucherâs disease, GBA1, glucocerebrosidase, neuronal progenitors, dopaminergic development, iPSCs, Wnt/β-catenin, lysosomal storage disease, neurodegeneratio

Reduced size of the functional vesicle pool within motor-nerve terminals contributes to lowered quantal content of MuSK-MG-affected TS preparations.

<p>(A) Representative time course of quantal content for EPCs in control or MuSK-MG-affected TS muscles that were stimulated at 50 Hz; X axis is the stimulus number (log 2 scale). (B) Representative graph showing the summation of quantal content during the 2 minute, 50 Hz train. The Y axis value is the summation of all quantal contents prior to the EPC number plotted on the X axis. The final value, or Y axis endpoint, of each curve is the total number of vesicles released during the 2 minute, 50 Hz stimulus train. (C) The total number of quanta released during the 2 minute, 50 Hz stimulus train is significantly (* P<0.05) less for MuSK-MG-affected (8 NMJs, 4 mice) muscles than that for vehicle-injected control muscles (7 NMJs, 3 mice). (D) Distribution of mEPC amplitudes as a percent of the total mEPCs used to calculate the mean amplitude shown in (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110571#pone-0110571-g002" target="_blank">Figure 2B</a>). While the modal value remains equivalent to that of control, reduced numbers of mEPCs larger than 2–3 nA accounts for the decline of mean mEPC of MuSK-MG-affected TS preparations.</p

MuSK-MG-affected TS preparations have altered motor-nerve structure that may contribute to EPC failure.

<p>Maximum intensity projection images obtained from confocal optical slices of NMJs in the TS of MuSK-MG-affected (A-C) and control (D-F) mice. The AChR-enriched endplate area is stained red, while motor nerves are stained green. Morphological abnormalities observed for NMJs of MuSK-MG-affected mice include: double innervation (white arrow heads), neurofilament positive swellings (white arrows), and axonal thinning (blue arrows) in B and C. Representative 3D-reconstruction images derived from confocal optical slices of control (G) and MuSK-MG-affected (H) nerve terminals. (I) Graph showing mean (+ SEM) values of maximum to minimum motor-nerve terminal diameter within 25 µm of branch point in control and MuSK-MG-affected TS preparations; P<0.0001. Calibration bars represent 20 µm.</p

Kinetics of neurotransmitter release are qualitatively normal for non-failing NMJs of MuSK-MG-affected mice.

<p>Representative EPCs that do not fail in response to 50 Hz stimulus trains (40 stimuli) applied to TS preparations removed from control (A) and MuSK-MG-affected (B) mice. (C and D) EPC amplitudes were normalized to the first EPC in 50-Hz trains to demonstrate potentiation (1) and depression (2) of neurotransmitter release to a steady-state level (3). At 1 second after the end of the stimulus train (arrow), a challenge stimulus tested for recovery of neurotransmitter release (4). Data points are shown as the mean ± SEM.</p

Number of vesicle release sites and probability of vesicle release are reduced for MuSK-MG-affected TS preparations.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110571#pone.0110571.e001" target="_blank">Equations 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110571#pone.0110571.e002" target="_blank">2</a> were used to calculate the number of vesicle release sites (A) and the probability of neurotransmitter release (B), respectively. Bars are shown as the mean + SEM for control (34 NMJs, 12 mice) and MuSK-MG-affected (31 NMJs, 8 mice) preparations; * and *** denote P<0.05 and P<0.0001, respectively.</p

Neurotransmitter release is reduced for respiratory muscles of MuSK-MG-affected mice.

<p>(A) Representative mEPCs (A) and mean mEPC amplitude (B) of <i>Triangularis sterni</i> (TS) nerve-muscle preparations removed from control (black trace) and MuSK-MG-affected (grey trace) mice. Representative EPCs (C), mean EPC amplitude (D), and mean quantal content (E) of <i>Triangularis sterni</i> (TS) nerve-muscle preparations removed from control (black trace) and MuSK-MG-affected (grey trace) mice. Data bars for B, D, and E are mean + SEM of 40 endplates, in 8 MuSK_MG affected mice and for 41 endplates in 12 vehicle injected control mice; *** denotes P<0.0001.</p

Reduced motor-nerve terminal excitability, but not prolonged synaptic delay, is responsible for high-frequency EPC failures of MuSK-MG-affected NMJs.

<p>(A) Representative extracellular recordings at NMJ of TS preparations isolated from control and MuSK-MG-affected mice lacking EPC failures. Each trace is the average of 120 recordings acquired at 50 Hz. Synaptic delay was measured as the time from the beginning of the nerve terminal current to the time at which the EPC amplitude was 10% of its maximum value, as indicated by the vertical lines following the stimulus artifact. (B) Synaptic delay is equivalent for control (70 NMJs, 4 mice) and MuSK-MG-affected (60 NMJs, 4 mice) muscles. (C) Series of 120 extracellular recordings made at an endplate of a MuSK-MG-affected NMJ, which exhibited partial failure in response to 50 Hz nerve stimulation. Double circled records clearly possessed EPCs, while dot circled records were failures of EPC initiation. (D) Average of double-circled and dot circled records of panel C show that failures of EPC initiation resulted from lack of nerve impulse entry into the nerve terminal; the boxed portion of the averaged records is amplified in the inset with an arrow indicating nerve terminal current in association with the EPC.</p