Decay in survival motor neuron and plastin 3 levels during differentiation of iPSC-derived human motor neurons

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in Survival Motor Neuron 1 (SMN1), leading to degeneration of alpha motor neurons (MNs) but also affecting other cell types. Induced pluripotent stem cell (iPSC)-derived human MN models from severe SMA patients have shown relevant phenotypes. We have produced and fully characterized iPSCs from members of a discordant consanguineous family with chronic SMA. We differentiated the iPSC clones into ISL-1+/ChAT+ MNs and performed a comparative study during the differentiation process, observing significant differences in neurite length and number between family members. Analyses of samples from wild-type, severe SMA type I and the type IIIa/IV family showed a progressive decay in SMN protein levels during iPSC-MN differentiation, recapitulating previous observations in developmental studies. PLS3 underwent parallel reductions at both the transcriptional and translational levels. The underlying, progressive developmental decay in SMN and PLS3 levels may lead to the increased vulnerability of MNs in SMA disease. Measurements of SMN and PLS3 transcript and protein levels in iPSC-derived MNs show limited value as SMA biomarkers

Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy

The childhood motor neuron disease spinal muscular atrophy (SMA) results from reduced expression of the survival motor neuron (SMN) gene. Previous studies using in vitro model systems and lower organisms have suggested that low levels of Smn protein disrupt prenatal developmental processes in lower motor neurons, influencing neuronal outgrowth, axon branching and neuromuscular connectivity. The extent to which these developmental pathways contribute to selective vulnerability and pathology in the mammalian neuromuscular system in vivo remains unclear. Here, we have investigated the pre-symptomatic development of neuromuscular connectivity in differentially vulnerable motor neuron populations in Smn(-/-);SMN2 mice, a model of severe SMA. We show that reduced Smn levels have no detectable effect on morphological correlates of pre-symptomatic development in either vulnerable or stable motor units, indicating that abnormal pre-symptomatic developmental processes are unlikely to be a prerequisite for subsequent pathological changes to occur in vivo. Microarray analyses of spinal cord from two different severe SMA mouse models demonstrated that only minimal changes in gene expression were present in pre-symptomatic mice. In stark contrast, microarray analysis of late-symptomatic spinal cord revealed widespread changes in gene expression, implicating extracellular matrix integrity, growth factor signalling and myelination pathways in SMA pathogenesis. Taken together, these data suggest that reduced Smn levels induce SMA pathology by instigating rapidly progressive neurodegenerative pathways in lower motor neurons around the time of disease onset rather than by modulating pre-symptomatic neurodevelopmental pathways

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Molecular Determinants of Survival Motor Neuron (SMN) Protein Cleavage by the Calcium-Activated Protease, Calpain

Spinal muscular atrophy (SMA) is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN) protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs). It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V), reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S), abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294) resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN

PaLM 2 Technical Report

We introduce PaLM 2, a new state-of-the-art language model that has better multilingual and reasoning capabilities and is more compute-efficient than its predecessor PaLM. PaLM 2 is a Transformer-based model trained using a mixture of objectives. Through extensive evaluations on English and multilingual language, and reasoning tasks, we demonstrate that PaLM 2 has significantly improved quality on downstream tasks across different model sizes, while simultaneously exhibiting faster and more efficient inference compared to PaLM. This improved efficiency enables broader deployment while also allowing the model to respond faster, for a more natural pace of interaction. PaLM 2 demonstrates robust reasoning capabilities exemplified by large improvements over PaLM on BIG-Bench and other reasoning tasks. PaLM 2 exhibits stable performance on a suite of responsible AI evaluations, and enables inference-time control over toxicity without additional overhead or impact on other capabilities. Overall, PaLM 2 achieves state-of-the-art performance across a diverse set of tasks and capabilities. When discussing the PaLM 2 family, it is important to distinguish between pre-trained models (of various sizes), fine-tuned variants of these models, and the user-facing products that use these models. In particular, user-facing products typically include additional pre- and post-processing steps. Additionally, the underlying models may evolve over time. Therefore, one should not expect the performance of user-facing products to exactly match the results reported in this report

Prevalence and sociodemographic determinants of tobacco use among adults in Pakistan: findings of a nationwide survey conducted in 2012.

BACKGROUND: Smoking is one of the leading causes of preventable mortality. The World Health Organization recommends that countries should monitor tobacco use regularly. In Pakistan, the last national study on smoking in the general population was conducted in 2002 to 2003. METHODS: We conducted a cross-sectional survey of a nationally representative sample of men and women living in rural and urban areas of four main provinces of Pakistan from March through April 2012. Face-to-face in-house interviews were undertaken using a pre-tested structured questionnaire that asked about smoking and other forms of tobacco use. Multistage stratified random area probability sampling was used. To determine the national prevalence of tobacco use, the sample was weighted to correspond to rural-urban population proportions in each of the four provinces as in the 1998 census conducted by Pakistan's Population Census Organization. Associations between sociodemographic variables and tobacco use were investigated using multivariable robust regression. RESULTS: Out of 2,644 respondents (1,354 men and 1,290 women), 354 men and 4 women reported being current cigarette smokers. The weighted prevalence of current cigarette smoking was 15.2% (95% confidence interval [CI]; 11.2, 19.3) overall, 26.6% (95% CI: 19.1, 34.1) among males, and 0.4% (95% CI: -0.2, 1.0) among females. Among females, 1.8% (95% CI: 0.4, 3.1) used any smoked tobacco and 4.6% (95% CI: 1.8, 7.4) used any smokeless tobacco daily or on some days of the week. Among males, odds of current cigarette smoking decreased with increasing level of education (OR = 0.75; 95% CI: 0.68, 0.84) and increased with having a father who used tobacco (OR = 2.11; 95% CI: 1.39, 3.22) after adjusting for other sociodemographic characteristics. Lower household income was associated with current cigarette smoking among rural males only (odds ratio [OR] = 0.67; 95% CI: 0.48, 0.92 per category increase in monthly household income). CONCLUSION: A large proportion of males smoked cigarettes. Cigarette use was negligible among females, but they used other forms of tobacco. Low education was a determinant of cigarette smoking among males irrespective of socioeconomic status and area of residence. Tobacco control campaigns should target uneducated and rural poor men and monitor all forms of tobacco used by the population