Systematic Literature Review of the Natural History of Spinal Muscular Atrophy: Motor Function, Scoliosis, and Contractures.

BACKGROUND AND OBJECTIVES: Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder associated with continuous motor function loss and complications, such as scoliosis and contractures. Understanding the natural history of SMA is key to demonstrating the long-term outcomes of SMA treatments. This study reviews the natural history of motor function, scoliosis, and contractures in patients with SMA. METHODS: Electronic databases were searched from inception to June 27, 2022 (Embase, MEDLINE, and Evidence-Based Medicine Reviews). Observational studies, case-control studies, cross-sectional studies, and case series reporting on motor function (i.e., sitting, standing, and walking ability), scoliosis, and contracture outcomes in patients with types 1-3 SMA were included. Data on study design, baseline characteristics, and treatment outcomes were extracted. Data sets were generated from studies that reported Kaplan-Meier (KM) curves and pooled to generate overall KM curves. RESULTS: Ninety-three publications were included, of which 68 reported on motor function. Of these, 10 reported KM curves (3 on the probability of sitting in patients with types 2 and 3 SMA and 8 on the probability of walking/ambulation in patients with type 3 SMA). The median time to loss of sitting (95% CI) was 14.5 years (14.1-31.5) for the type 2 SMA sitter population (their maximum ability was independent sitting). The median time to loss of ambulation (95% CI) was 13.4 years (12.5-14.5) for type 3a SMA (disease onset at age younger than 3 years) and 44.2 years (43.0-49.4) for type 3b SMA (disease onset at age 3 years or older). Studies including scoliosis and contracture outcomes mostly reported non-time-to-event data. DISCUSSION: The results demonstrate that a high degree of motor function loss is inevitable, affecting patients of all ages. In addition, data suggest that untreated patients with types 2 and 3 SMA remain at risk of losing motor milestones during late adulthood, and patients with types 3a and 3b SMA are at risk of loss of ambulation over time. These findings support the importance of stabilization of motor function development even at older ages. Natural history data are key for the evaluation of SMA treatments as they contextualize the assessment of long-term outcomes.

How does risdiplam compare with other treatments for Types 1-3 spinal muscular atrophy: a systematic literature review and indirect treatment comparison.

Aim: To conduct indirect treatment comparisons between risdiplam and other approved treatments for spinal muscular atrophy (SMA). Patients & methods: Individual patient data from risdiplam trials were compared with aggregated data from published studies of nusinersen and onasemnogene abeparvovec, accounting for heterogeneity across studies. Results: In Type 1 SMA, studies of risdiplam and nusinersen included similar populations. Indirect comparison results found improved survival and motor function with risdiplam versus nusinersen. Comparison with onasemnogene abeparvovec in Type 1 SMA and with nusinersen in Types 2/3 SMA was challenging due to substantial differences in study populations; no concrete conclusions could be drawn from the indirect comparison analyses. Conclusion: Indirect comparisons support risdiplam as a superior alternative to nusinersen in Type 1 SMA.

Preferences and Utilities for Treatment Attributes in Type 2 and Non-ambulatory Type 3 Spinal Muscular Atrophy in the United Kingdom.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare neuromuscular disease that affects motor neurons, resulting in progressive skeletal muscle weakness and atrophy. OBJECTIVE: The aim was to understand the value patients with SMA and caregivers place on treatment attributes and to estimate health utilities for SMA treatment outcomes from a general public sample. METHODS: Two discrete choice experiments were designed to elicit treatment preferences and health utilities, respectively. Patients with Type 2 and non-ambulatory Type 3 SMA, caregivers of patients with SMA and a general public sample in the UK completed the surveys. Patients and caregiver participants were recruited through patient associations. General public participants were recruited via a survey recruitment panel. Attributes included motor function, breathing function, treatment administration, treatment reactions, eyesight monitoring, contraception (patients only) and overall survival (general public only). Clustered conditional logit models were used to estimate treatment preferences, and marginal rates of substitution were used to estimate disutilities. RESULTS: Adult patients (n = 84) were twice as likely to choose a treatment with improved (vs. stable) motor and breathing function and four to five times less likely to choose a treatment with deteriorated (vs. stable) motor and breathing function as a treatment outcome. Caregivers (n = 83) were three to nine times more likely to choose improved and two to four times less likely to choose deteriorated (vs. stable) motor and breathing function. Both patients and caregivers preferred oral over intrathecal treatment. Treatment reactions, eyesight monitoring or contraception had no significant effect on patient choices. Conversely, caregivers preferred avoidance of treatment reactions. General public data (n = 506) yielded disutilities for unable to sit (- 0.408), need for > 16 h daily mechanical breathing support (- 0.304) and intrathecal therapy (- 0.071). CONCLUSIONS: Study results show the importance of motor and breathing function to patients and caregivers, and an oral treatment preference. Disutilities (decrements to utility) were substantial for SMA disease outcomes and care aspects.

Patient and Caregiver Treatment Preferences in Type 2 and Non-ambulatory Type 3 Spinal Muscular Atrophy: A Discrete Choice Experiment Survey in Five European Countries.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare neuromuscular disease that affects motor neurons, resulting in progressive skeletal muscle weakness and atrophy. OBJECTIVE: The aim of this study was to examine treatment preferences of patients and caregivers of patients with Type 2 and non-ambulatory Type 3 SMA in the Netherlands, Belgium, Finland, Ireland and Portugal. METHODS: A discrete choice experiment (DCE) survey was developed to elicit the preferences of adult patients and caregivers regarding different treatment aspects of SMA. This survey built on the design of a similar study undertaken in the UK. The DCE described choice questions in terms of attributes and levels combined using a D-efficient design. The attributes described improvements or worsening in motor and breathing function. The mode of treatment administration (intrathecal injection, single intravenous infusion or regular oral therapy) was described. Treatment risks and side effects related to currently available treatments including risk of liver injury, fatigue, headache, nausea, diarrhoea and rash were described. Lastly, an attribute described whether a treatment had evidence of treatment effectiveness in different SMA types. Participants were recruited via patient advocacy associations to complete an online survey. A clustered conditional logit model was used to estimate treatment preferences. RESULTS: Participants (n = 65) were 4.8 times and 8.1 times more likely to choose a treatment with stable or improved (vs worse) motor function, respectively. Similarly, participants were 4.3 times and 5.8 times more likely to choose stable or improved (vs worse) breathing function, respectively. Treatments with a risk of liver injury, fatigue, headache and nausea were 1.6 times less likely to be chosen than treatments with a risk of diarrhoea and rash. Treatments with demonstrated effectiveness in Type 1 SMA only were 2.3 times less likely to be chosen than those with demonstrated effectiveness in Types 1-3 SMA. Treatments administered via intrathecal injections were also 1.8 times less likely to be chosen than daily oral treatments. DISCUSSION: Study results show the importance of improvement as well as stabilisation of motor and breathing function to patients and caregivers, and a preference for oral treatments, treatments with demonstrated effectiveness in Types 2-3 SMA, and avoidance of liver injury risk.

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.

Human dopamine transporter: the first implementation of a combined in silico/in vitro approach revealing the substrate and inhibitor specificities.

Parkinson's disease (PD) is characterized by the loss of dopamine-generating neurons in the substantia nigra and corpus striatum. Current treatments alleviate PD symptoms rather than exerting neuroprotective effect on dopaminergic neurons. New drugs targeting the dopaminergic neurons by specific uptake through the human dopamine transporter (hDAT) could represent a viable strategy for establishing selective neuroprotection. Molecules able to increase the bioactive amount of extracellular dopamine, thereby enhancing and compensating a loss of dopaminergic neurotransmission, and to exert neuroprotective response because of their accumulation in the cytoplasm, are required. By means of homology modeling, molecular docking, and molecular dynamics simulations, we have generated 3D structure models of hDAT in complex with substrate and inhibitors. Our results clearly reveal differences in binding affinity of these compounds to the hDAT in the open and closed conformations, critical for future drug design. The established in silico approach allowed the identification of promising substrate compounds that were subsequently analyzed for their efficiency in inhibiting hDAT-dependent fluorescent substrate uptake, through in vitro live cell imaging experiments. Taken together, our work presents the first implementation of a combined in silico/in vitro approach enabling the selection of promising dopaminergic neuron-specific substrates.

Publisher Correction: Purification of replicating pancreatic ß-cells for gene expression studies.

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Purification of replicating pancreatic ß-cells for gene expression studies.

ß-cell proliferation is a rare event in adult pancreatic islets. To study the replication-related ß-cell biology we designed a replicating ß-cells sorting system for gene expression experiments. Replicating ß-cells were identified by EdU incorporation and purified by flow cytometry. For ß-cell separation islet cells were sorted by size, granularity and Newport Green fluorescence emission that was combined with emitted fluorescence for EdU-labelled replicating cells sorting. The purity of the resulting sorted populations was evaluated by insulin staining and EdU for ß-cell identification and for replicating cells, respectively. Total RNA was isolated from purified cell-sorted populations for gene expression analysis. Cell sorting of dispersed islet cells resulted in 96.2% purity for insulin positivity in the collected ß-cell fraction and 100% efficiency of the EdU-based cell separation. RNA integrity was similar between FACS-sorted replicating and quiescent ß-cells. Global transcriptome analysis of replicating vs quiescent ß-cells showed the expected enrichment of categories related to cell division and DNA replication. Indeed, key genes in the spindle check-point were the most upregulated genes in replicating ß-cells. This work provides a method that allows for the isolation of replicating ß-cells, a very scarce population in adult pancreatic islets.

Methyl-4-phenylpyridinium (MPP+) differentially affects monoamine release and re-uptake in murine embryonic stem cell-derived dopaminergic and serotonergic neurons.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) is known to selectively damage dopaminergic (DA) cells in the substantia nigra and to produce symptoms which are alike to those observed in Parkinson's disease (PD). Based on the similarity between MPTP-induced neurotoxicity and PD-related neuropathology, application of MPTP or its metabolite methyl-4-phenylpyridinium (MPP+) was successfully established in experimental rodent models to study PD-related neurodegenerative events. MPP+ is taken up by the dopamine transporter (DAT) into DA neurons where it exerts its neurotoxic action on mitochondria by affecting complex I of the respiratory chain. MPP+ is also a high affinity substrate for the serotonin transporter (SERT), however little is known about possible toxic effects of MPP+ on serotonergic (5-HT) neurons. In order to compare cell type-specific effects of MPP+ treatment, we have differentiated mouse embryonic stem (ES) cells into DA and 5-HT neurons and studied the impact of MPP+ treatment on both types of monoaminergic neurons in vitro. MPP+ treatment impacts on mitochondrial membrane potential in DA as well as 5-HT ES cell-derived neurons. Although mitochondria metabolisms are similarly affected, synaptic vesicle cycling is only impaired in DA ES cell-derived neurons. Most importantly we show that MPP+ induces DAT externalization in DA neurons, but internalization of SERT in 5-HT neurons. This diverse MPP+-induced transporter trafficking is reflected by elevated substrate uptake in DA neurons, and diminished substrate uptake in 5-HT neurons. In summary, our experimental data point toward differential effects of MPP+ intoxication on neurotransmitter release and re-uptake in different types of monoaminergic neurons.

The allosteric citalopram binding site differentially interferes with neuronal firing rate and SERT trafficking in serotonergic neurons.

Citalopram is a clinically applied selective serotonin re-uptake inhibitor for antidepressant pharmacotherapy. It consists of two enantiomers, S-citalopram (escitalopram) and R-citalopram, of which escitalopram exerts the antidepressant therapeutic effect and has been shown to be one of the most efficient antidepressants, while R-citalopram antagonizes escitalopram via an unknown molecular mechanism that may depend on binding to a low-affinity allosteric binding site of the serotonin transporter. However, the precise mechanism of antidepressant regulation of the serotonin transporter by citalopram enantiomers still remains elusive. Here we investigate escitalopram׳s acute effect on (1) serotonergic neuronal firing in transgenic mice that express the human serotonin transporter without and with a mutation that disables the allosteric binding site, and (2) regulation of the serotonin transporter׳s cell surface localization in stem cell-derived serotonergic neurons. Our results demonstrate that escitalopram inhibited neuronal firing less potently in the mouse line featuring a mutation that abolishes the function of the allosteric binding site and induced serotonin transporter internalization independently of the allosteric binding site mechanism. Furthermore, citalopram enantiomers dose-dependently induced serotonin transporter internalization. In conclusion, this study provides new insight into antidepressant effects exerted by citalopram enantiomers in presence and absence of a functional allosteric binding site.

ß-Cell dedifferentiation, reduced duct cell plasticity, and impaired ß-cell mass regeneration in middle-aged rats.

Limitations in ß-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated ß-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px + V) and gastrin administration (Px + G). Pancreatic remnants were analyzed 3 and 14 days after surgery. ß-Cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, ß-cell mass did not change after Px or after gastrin treatment in middle-aged rats. ß-Cell replication and individual ß-cell size were similarly increased after Px in young and middle-aged animals, and ß-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet ß-cells of middle-aged rats and further increased after Px. The percentage of chromogranin A+/hormone islet cells was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory ß-cell hyperplasia and hypertrophy was retained in middle-aged rats, but ß-cell dedifferentiation and impaired duct cell plasticity limited ß-cell regeneration.