Roberto Zayas | St. Olaf College (original) (raw)
Papers by Roberto Zayas
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in... more Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in some hereditary epilepsies, in familial hyperekplexia, and the slow-channel congenital myasthenic syndrome (SCCMS) may perturb the kinetics of synaptic currents, leading to significant clinical consequences. Although at least 12 acetylcholine receptor (AChR) mutations have been identified in the SCCMS, the altered channel properties critical for disease pathogenesis in the SCCMS have not been identified. To approach this question, we investigated the effect of different AChR subunit mutations on muscle weakness and the function and viability of neuromuscular synapses in transgenic mice. Targeted expression of distinct mutant AChR subunits in skeletal muscle prolonged the decay phases of the miniature endplate currents (MEPCs) over a broad range. In addition, both muscle strength and the amplitude of MEPCs were lower in transgenic lines with greater MEPC duration. SCCMS is associated wit...
Synapse, 2006
The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progre... more The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progressive weakness, and degeneration of the neuromuscular junction. The SCS is caused by missense mutations in the four subunits of the skeletal muscle acetylcholine receptor (AChR), which leads to altered channel gating, prolonged neuromuscular postsynaptic currents, and impaired neuromuscular transmission. Although a diverse set of mutations in different functional domains of the AChR appear to be associated with symptoms of widely ranging severity, there is as yet no mutant channel property or combination that explains the variations in disease severity. By observing the recovery time of AChR from desensitization, the authors determined that this process is significantly enhanced in SCS channels. In addition, as expected, the authors found that SCS macroscopic decay currents in transfected HEK293 cells are slower than wild type currents. While slight differences in relative Ca(2+) permeability between some SCS mutations were identified, they did not correlate with apparent disease severity. These results suggest that of the different AChR kinetic features studied, only recovery from desensitization and slow postsynaptic currents correlate with the severity observed in the different mutations of this syndrome.
Neurobiology of Disease, 2006
In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myo... more In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myonuclear degeneration at the neuromuscular junction (NMJ), without muscle fiber death. Activated caspases are present at SCS motor endplates. We hypothesized that SCS represents a limited form of apoptosis. We found condensed chromatin and occasional single-strand DNA nicks in degenerating synaptic nuclei. Cleaved forms of caspases-3 and-9 were present in mouse SCS muscle homogenates and were specifically localized to NMJs. Finally, interruption of cholinergic activity by axotomy markedly reduced NMJ caspase activity and improved the morphological features of apoptosis at NMJs. These results demonstrate that in SCS processes leading to apoptosis may remain compartmentalized and reversible. Use of cysteine protease inhibitors may aid in treatment of this and other dystrophic muscle and excitotoxic disorders. Identification of extrasynaptic factors that prevent the spread of apoptosis in SCS muscle fibers may aid in developing treatments for neurological disorders characterized by excitotoxicity or apoptosis.
Molecular and Cellular Neuroscience, 2006
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V Y F substitution in the M1 domain of the b subunit (bV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation bV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the bV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the bV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the bV229F mutation on the miniature endplate currents and thus are direct evidence that the bV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
Journal of Clinical Investigation, 2007
The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor... more The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca 2+ overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.
Cell Calcium, 2007
Strict control of calcium entry through excitatory synaptic receptors is important for shaping sy... more Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca 2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca 2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca 2+ overload in SCS we studied the role of sarcoplasmic Ca 2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca 2+ accumulates around wild type NMJ and discovered that Ca 2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca 2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca 2+ and that inositol-1,4,5-triphosphate receptors (IP 3 R), and to a lesser degree L-type voltage gated Ca 2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca 2+ , and IP 3 R is responsible for localized Ca 2+ signals observed in muscle fibers and the Ca 2+ overload observed in SCS.
Molecular and Cellular …, 2006
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2002
Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in... more Mutations affecting the gating and channel properties of ionotropic neurotransmitter receptors in some hereditary epilepsies, in familial hyperekplexia, and the slow-channel congenital myasthenic syndrome (SCCMS) may perturb the kinetics of synaptic currents, leading to significant clinical consequences. Although at least 12 acetylcholine receptor (AChR) mutations have been identified in the SCCMS, the altered channel properties critical for disease pathogenesis in the SCCMS have not been identified. To approach this question, we investigated the effect of different AChR subunit mutations on muscle weakness and the function and viability of neuromuscular synapses in transgenic mice. Targeted expression of distinct mutant AChR subunits in skeletal muscle prolonged the decay phases of the miniature endplate currents (MEPCs) over a broad range. In addition, both muscle strength and the amplitude of MEPCs were lower in transgenic lines with greater MEPC duration. SCCMS is associated wit...
Synapse, 2006
The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progre... more The slow-channel syndrome (SCS) is a neuromuscular disorder characterized by fatigability, progressive weakness, and degeneration of the neuromuscular junction. The SCS is caused by missense mutations in the four subunits of the skeletal muscle acetylcholine receptor (AChR), which leads to altered channel gating, prolonged neuromuscular postsynaptic currents, and impaired neuromuscular transmission. Although a diverse set of mutations in different functional domains of the AChR appear to be associated with symptoms of widely ranging severity, there is as yet no mutant channel property or combination that explains the variations in disease severity. By observing the recovery time of AChR from desensitization, the authors determined that this process is significantly enhanced in SCS channels. In addition, as expected, the authors found that SCS macroscopic decay currents in transfected HEK293 cells are slower than wild type currents. While slight differences in relative Ca(2+) permeability between some SCS mutations were identified, they did not correlate with apparent disease severity. These results suggest that of the different AChR kinetic features studied, only recovery from desensitization and slow postsynaptic currents correlate with the severity observed in the different mutations of this syndrome.
Neurobiology of Disease, 2006
In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myo... more In the slow-channel syndrome (SCS) mutant acetylcholine receptors elicit calcium overload and myonuclear degeneration at the neuromuscular junction (NMJ), without muscle fiber death. Activated caspases are present at SCS motor endplates. We hypothesized that SCS represents a limited form of apoptosis. We found condensed chromatin and occasional single-strand DNA nicks in degenerating synaptic nuclei. Cleaved forms of caspases-3 and-9 were present in mouse SCS muscle homogenates and were specifically localized to NMJs. Finally, interruption of cholinergic activity by axotomy markedly reduced NMJ caspase activity and improved the morphological features of apoptosis at NMJs. These results demonstrate that in SCS processes leading to apoptosis may remain compartmentalized and reversible. Use of cysteine protease inhibitors may aid in treatment of this and other dystrophic muscle and excitotoxic disorders. Identification of extrasynaptic factors that prevent the spread of apoptosis in SCS muscle fibers may aid in developing treatments for neurological disorders characterized by excitotoxicity or apoptosis.
Molecular and Cellular Neuroscience, 2006
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V Y F substitution in the M1 domain of the b subunit (bV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation bV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the bV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the bV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the bV229F mutation on the miniature endplate currents and thus are direct evidence that the bV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
Journal of Clinical Investigation, 2007
The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor... more The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca 2+ overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.
Cell Calcium, 2007
Strict control of calcium entry through excitatory synaptic receptors is important for shaping sy... more Strict control of calcium entry through excitatory synaptic receptors is important for shaping synaptic responses, gene expression, and cell survival. Disruption of this control may lead to pathological accumulation of Ca 2+. The slow-channel congenital myasthenic syndrome (SCS), due to mutations in muscle acetylcholine receptor (AChR), perturbs the kinetics of synaptic currents, leading to post-synaptic Ca 2+ accumulation. To understand the regulation of calcium signaling at the neuromuscular junction (NMJ) and the etiology of Ca 2+ overload in SCS we studied the role of sarcoplasmic Ca 2+ stores in SCS. Using fura-2 loaded dissociated fibers activated with acetylcholine puffs, we confirmed that Ca 2+ accumulates around wild type NMJ and discovered that Ca 2+ accumulates significantly faster around the NMJ of SCS transgenic dissociated muscle fibers. Additionally, we determined that this process is dependant on the activation, altered kinetics, and movement of Ca 2+ ions through the AChR, although, surprisingly, depletion of intracellular stores also prevents the accumulation of this cation around the NMJ. Finally, we concluded that the sarcoplasmic reticulum is the main source of Ca 2+ and that inositol-1,4,5-triphosphate receptors (IP 3 R), and to a lesser degree L-type voltage gated Ca 2+ channels, are responsible for the efflux of this cation from intracellular stores. These results suggest that a signaling system mediated by the activation of AChR, Ca 2+ , and IP 3 R is responsible for localized Ca 2+ signals observed in muscle fibers and the Ca 2+ overload observed in SCS.
Molecular and Cellular …, 2006
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V --> F substitution in the M1 domain of the beta subunit (betaV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation betaV229F expressed normal amounts of AChRs and decreased the ACh EC50 by 10-fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the betaV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the betaV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the betaV229F mutation on the miniature endplate currents and thus are direct evidence that the betaV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.