Use of the Assessment of Caregiver Experience with Neuromuscular Disease (ACEND) in Spinal Muscular Atrophy.

Background: Spinal muscular atrophy (SMA) has a remarkable impact on function and participation. Subsequently, the caregivers of individuals with SMA are impacted as well. Providers and the SMA community should be aware of the presence of and likely expectations for the existence of caregiver burden. Methods: The Assessment of Caregiver Experience with Neuromuscular Disease (ACEND) quantifies caregivers' perceptions of function and quality of life pertaining to time, finance and emotion. Analyses were conducted among SMA types and ambulatory and ventilatory status. Participants with SMA had varying ranges of function and were on pharmaceutical treatment. Total ACEND score, longitudinal change in total ACEND score, total quality of life (QOL) score, change in total QOL score and subdomains for QOL, including time, emotion and finance, were all explored. Results: Overall, the ACEND demonstrated discriminant validity and some observed trends. Total ACEND scores improved for caregivers of those with SMA 2, remained stable longitudinally for caregivers of those with SMA 1 and 3 and were not influenced by ventilation status. The caregivers of individuals with SMA 1 had the lowest total quality of life (QOL) score, as did the caregivers of non-ambulatory individuals and those requiring assisted ventilation. Longitudinally, there were no changes in total QOL between caregivers of individuals with different SMA types or ambulatory or ventilation status. There were some differences in emotional needs, but no differences in financial impact between the caregivers of individuals with different types of SMA or ambulatory and ventilatory status. Conclusions: With this information enlightening the presence of caregiver burden and expected changes in burden with pharmaceutical treatment, providers, third party payors and the SMA community at large can better assist, equip and empower those providing the necessary assistance to enable the lives of those with SMA.

Development of an International SMA Bulbar Assessment for Inter-professional Administration.

BACKGROUND: Progressive weakness can affect bulbar muscles in individuals with moderate to severe forms of spinal muscular atrophy (SMA). The paucity of standardized, valid bulbar assessments capturing clinically significant deficits in SMA impedes the ability to monitor function, facilitate intervention, or detect treatment response. OBJECTIVE: To fill this void, an international multidisciplinary team gathered to develop an agreed upon consensus-derived assessment of bulbar function in SMA for inter-professional administration to enhance our ability to monitor disease progression, support clinical management, and evaluate treatment effects. METHODS: Fifty-six international clinicians experienced in SMA were invited and engaged using the Delphi method over multiple rounds of web-based surveys to establish consensus. RESULTS: Serial virtual meetings occurred with 42 clinicians (21 speech and language therapists, 11 physical therapists, 5 neurologists, 4 occupational therapists, and 1 dentist). Seventy-two validated assessments of bulbar function were identified for potential relevance to individuals with SMA (32 accessible objective, 11 inaccessible objective, 29 patient-reported outcomes). Delphi survey rounds (n = 11, 15, 15) achieved consensus on individual items with relevance and wording discussed. Key aspects of bulbar function identified included: oral intake status, oral facial structure and motor strength, swallowing physiology, voice & speech, and fatigability. CONCLUSIONS: Multidisciplinary clinicians with expertise in bulbar function and SMA used Delphi methodology to reach consensus on assessments/items considered relevant for SMA across all age groups. Future steps include piloting the new scale moving towards validation/reliability. This work supports the advancement of assessing bulbar function in children and adults with SMA by a variety of professionals.

Longitudinal Assessment of Timed Function Tests in Ambulatory Individuals with SMA Treated with Nusinersen.

BACKGROUND: Ambulatory individuals with spinal muscular atrophy experience weakness and impairments of speed and endurance. This leads to decreased motor skill performance required for daily living including transitioning from floor to stand, climbing stairs, and traversing short and community distances. Motor function improvements have been reported in individuals receiving nusinersen, but changes in timed functional tests (TFTs) which assess shorter distance walking and transitions have not been well documented. OBJECTIVE: To evaluate changes in TFT performance over the course of nusinersen treatment in ambulatory individuals with SMA and identify potential factors [age, SMN2 copy number, BMI, Hammersmith Functional Motor Scale Expanded (HFMSE score), Peroneal Compound Motor Action Potential (CMAP) amplitude] associated with TFT performance. METHODS: Nineteen ambulatory participants receiving nusinersen were followed from 2017 through 2019 (range: 0-900 days, mean 624.7 days, median 780 days); thirteen of 19 (mean age = 11.5 years) completed TFTs. The 10-meter walk/run test, time-to-rise from supine, time-to-rise from sitting, 4-stair climb, 6-minute walk test (6MWT), Hammersmith Expanded and peroneal CMAP were assessed at each visit. Linear mixed-effects models were used to evaluate unadjusted and adjusted changes in these outcomes over time. RESULTS: Apart from time to rise from sitting and from supine, all TFTs were found to improve over the course of treatment after adjusting for baseline age and BMI. CONCLUSIONS: Improvement in TFTs over time in patients with SMA treated with nusinersen suggests that shorter TFTs may have value to assess individuals with SMA who have or later gain ambulatory function during treatment.

Retrograde activation of CB1R by muscarinic receptors protects against central organophosphorus toxicity.

The acute toxicity of organophosphorus-based compounds is primarily a result of acetylcholinesterase inhibition in the central and peripheral nervous systems. The resulting cholinergic crisis manifests as seizure, paralysis, respiratory failure and neurotoxicity. Though overstimulation of muscarinic receptors is the mechanistic basis of central organophosphorus (OP) toxicities, short-term changes in synapse physiology that precede OP-induced seizures have not been investigated in detail. To study acute effects of OP exposure on synaptic function, field excitatory postsynaptic potentials (fEPSPs) were recorded from Schaffer collateral synapses in the mouse hippocampus CA1 stratum radiatum during perfusion with various OP compounds. Administration of the OPs paraoxon, soman or VX rapidly and stably depressed fEPSPs via a presynaptic mechanism, while the non-OP proconvulsant tetramethylenedisulfotetramine had no effect on fEPSP amplitudes. OP-induced presynaptic long-term depression manifested prior to interictal spiking, occurred independent of recurrent firing, and did not require NMDA receptor currents, suggesting that it was not mediated by activity-dependent calcium uptake. Pharmacological dissection revealed that the presynaptic endocannabinoid type 1 receptor (CB1R) as well as postsynaptic M1 and M3 muscarinic acetylcholine receptors were necessary for OP-LTD. Administration of CB1R antagonists significantly reduced survival in mice after a soman challenge, revealing an acute protective role for endogenous CB1R signaling during OP exposure. Collectively these data demonstrate that the endocannabinoid system alters glutamatergic synaptic function during the acute response to OP acetylcholinesterase inhibitors.

Lynx Prototoxins: Roles of Endogenous Mammalian Neurotoxin-Like Proteins in Modulating Nicotinic Acetylcholine Receptor Function to Influence Complex Biological Processes.

The cholinergic system modulates many biological functions, due to the widespread distribution of cholinergic neuronal terminals, and the diffuse release of its neurotransmitter, acetylcholine. Several layers of regulation help to refine and control the scope of this excitatory neurotransmitter system. One such regulatory mechanism is imparted through endogenous toxin-like proteins, prototoxins, which largely control the function of nicotinic receptors of the cholinergic system. Prototoxins and neurotoxins share the distinct three finger toxin fold, highly effective as a receptor binding protein, and the former are expressed in the mammalian brain, immune system, epithelium, etc. Prototoxins and elapid snake neurotoxins appear to be related through gene duplication and divergence from a common ancestral gene. Protein modulators can provide a graded response of the cholinergic system, and within the brain, stabilize neural circuitry through direct interaction with nicotinic receptors. Understanding the roles of each prototoxin (e.g., lynx1, lynx2/lypd1, PSCA, SLURP1, SLURP2, Lypd6, lypd6b, lypdg6e, PATE-M, PATE-B, etc.), their binding specificity and unique expression profile, has the potential to uncover many fascinating cholinergic-dependent mechanisms in the brain. Each family member can provide a spatially restricted level of control over nAChR function based on its expression in the brain. Due to the difficulty in the pharmacological targeting of nicotinic receptors in the brain as a result of widespread expression patterns and similarities in receptor sequences, unique interfaces between prototoxin and nicotinic receptor could provide more specific targeting than nicotinic receptors alone. As such, this family is intriguing from a long-term therapeutic perspective.

In vitro and ex vivo screening of candidate therapeutics to restore neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A1.

Botulinum neurotoxins (BoNTs) are exceedingly potent neurological poisons that block cholinergic release in the peripheral nervous system and cause death by asphyxiation. While post-exposure prophylaxis can effectively eliminate toxin in the bloodstream, there are no clinically effective treatments to prevent or reverse disease once BoNT has entered the neuron. To address the need for post-symptomatic countermeasures, we designed and developed an in vitro assay based on whole-cell, patch-clamp electrophysiological monitoring of miniature excitatory post-synaptic currents in synaptically active murine embryonic stem cell-derived neurons. This synaptic function-based assay was used to assess the efficacy of rationally selected drugs to restore neurotransmission in neurons comprehensively intoxicated by BoNT/A. Based on clinical reports suggesting that elevated Ca2+ signaling promotes symptomatic relief from botulism, we identified seven candidate drugs that modulate presynaptic Ca2+ signaling and assessed their ability to reverse BoNT/A-induced synaptic blockade. The most effective drugs from the screen were found to phasically agonize voltage-gated calcium channel (VGCC) activity. Lead candidates were then applied to ex vivo studies in BoNT/A-paralyzing mouse phrenic nerve-hemidiaphragm (PND) preparations. Treatment of PNDs with VGCC agonists after paralytic onset transiently potentiated nerve-elicited muscle contraction and delayed progression to neuromuscular failure. Collectively, this study suggests that Ca2+-modulating drugs represent a novel symptomatic treatment for neuromuscular paralysis following BoNT/A poisoning.

Use-dependent potentiation of voltage-gated calcium channels rescues neurotransmission in nerve terminals intoxicated by botulinum neurotoxin serotype A.

Botulinum neurotoxins (BoNTs) are highly potent toxins that cleave neuronal SNARE proteins required for neurotransmission, causing flaccid paralysis and death by asphyxiation. Currently, there are no clinical treatments to delay or reverse BoNT-induced blockade of neuromuscular transmission. While aminopyridines have demonstrated varying efficacy in transiently reducing paralysis following BoNT poisoning, the precise mechanisms by which aminopyridines symptomatically treat botulism are not understood. Here we found that activity-dependent potentiation of presynaptic voltage-gated calcium channels (VGCCs) underlies 3,4-diaminopyridine (3,4-DAP)-mediated rescue of neurotransmission in central nervous system synapses and mouse diaphragm neuromuscular junctions fully intoxicated by BoNT serotype A. Combinatorial treatments with 3,4-DAP and VGCC agonists proved synergistic in restoring suprathreshold endplate potentials in mouse diaphragms fully intoxicated by BoNT/A. In contrast, synapses fully intoxicated by BoNT serotypes D or E were refractory to synaptic rescue by any treatment. We interpret these data to propose that increasing the duration or extent of VGCC activation prolongs the opportunity for low-efficiency fusion by fusogenic complexes incorporating BoNT/A-cleaved SNAP-25. The identification of VGCC agonists that rescue neurotransmission in BoNT/A-intoxicated synapses provides compelling evidence for potential therapeutic utility in some cases of human botulism.

Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

Identifying neurotransmitter spill-over in hippocampal field recordings.

A model of synaptic and extra-synaptic excitatory signaling in the hippocampus is presented. The model is used to analytically evaluate the potential contributions of homosynaptic and heterosynaptic glutamate spill-over to receptor signaling during an electrophysiological experiment in which glutamate transporters are pharmacologically blocked. Inhibition of glutamate uptake selectively prolongs the decay kinetics of the second field excitatory postsynaptic potential evoked by paired pulse stimulation of Schaffer collateral axons in area CA1. The model includes AMPA and NMDA glutamate receptors, and the removal of glutamate by transporters and diffusion. We establish analytically that the prolongation cannot be caused by local effects, i.e., the transporters acting within or near the synapse. In contrast, a time profile of glutamate consistent with spill-over from adjacent synapses can explain the effect. The different reaction kinetics of AMPA and NMDA receptors have a significant role in reproducing the experimental results, as explained by analysis of the ODEs governing the reactions.

Specificity and actions of an arylaspartate inhibitor of glutamate transport at the Schaffer collateral-CA1 pyramidal cell synapse.

In this study we characterized the pharmacological selectivity and physiological actions of a new arylaspartate glutamate transporter blocker, L-threo-ß-benzylaspartate (L-TBA). At concentrations up to 100 µM, L-TBA did not act as an AMPA receptor (AMPAR) or NMDA receptor (NMDAR) agonist or antagonist when applied to outside-out patches from mouse hippocampal CA1 pyramidal neurons. L-TBA had no effect on the amplitude of field excitatory postsynaptic potentials (fEPSPs) recorded at the Schaffer collateral-CA1 pyramidal cell synapse. Excitatory postsynaptic currents (EPSCs) in CA1 pyramidal neurons were unaffected by L-TBA in the presence of physiological extracellular Mg(2+) concentrations, but in Mg(2+)-free solution, EPSCs were significantly prolonged as a consequence of increased NMDAR activity. Although L-TBA exhibited approximately four-fold selectivity for neuronal EAAT3 over glial EAAT1/EAAT2 transporter subtypes expressed in Xenopus oocytes, the L-TBA concentration-dependence of the EPSC charge transfer increase in the absence of Mg(2+) was the same in hippocampal slices from EAAT3 +/+ and EAAT3 -/- mice, suggesting that TBA effects were primarily due to block of glial transporters. Consistent with this, L-TBA blocked synaptically evoked transporter currents in CA1 astrocytes with a potency in accord with its block of heterologously expressed glial transporters. Extracellular recording in the presence of physiological Mg(2+) revealed that L-TBA prolonged fEPSPs in a frequency-dependent manner by selectively increasing the NMDAR-mediated component of the fEPSP during short bursts of activity. The data indicate that glial glutamate transporters play a dominant role in limiting extrasynaptic transmitter diffusion and binding to NMDARs. Furthermore, NMDAR signaling is primarily limited by voltage-dependent Mg(2+) block during low-frequency activity, while the relative contribution of transport increases during short bursts of higher frequency signaling.

Differential role of beta-arrestin ubiquitination in agonist-promoted down-regulation of M1 vs M2 muscarinic acetylcholine receptors.

BACKGROUND: Sustained agonist-promoted ubiquitination of beta-arrestin has been correlated with increased stability of the GPCR - beta-arrestin complex. Moreover, abrogation of beta-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation. RESULTS: Herein we report that agonist activation of M1 mAChRs produces a sustained beta-arrestin ubiquitination but no stable co-localization with beta-arrestin. In contrast, sustained ubiquitination of beta-arrestin by activation of M2 mAChRs does result in stable co-localization between the M2 mAChR and beta-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of beta-arrestin following agonist treatment, we sought to determine the effects of beta-arrestin ubiquitination on M1 and M2 mAChR down-regulation. A constitutively ubiquitinated beta-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of beta-arrestin 2 (YFP-beta-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M1 and M2 mAChRs, with the effect substantially higher on the M2 mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the beta-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M2 mAChR was not affected by expression of beta-arrestin lysine mutants lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, while down-regulation and stable co-localiztion of the receptor with this beta-arrestin lysine mutant were significantly reduced. Interestingly, expression of beta-arrestin 2K18R, K107R, K108R, K207R, K296R increased the agonist-promoted down-regulation of the M1 mAChR but did not result in a stable co-localiztion of the receptor with this beta-arrestin lysine mutant. CONCLUSION: These findings indicate that ubiquitination of beta-arrestin has a distinct role in the differential trafficking and degradation of M1 and M2 mAChRs.