Background Rett syndrome (RTT) is a progressive neurodevelopmental disease that is characterized by abnormalities in cognitive, social, and motor skills. RTT is often caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). The mechanism by which impaired MeCP2 induces the pathological abnormalities in the brain is not understood. Both patients and mouse models have shown abnormalities at molecular and cellular level before typical RTT-associated symptoms appear. This implies that underlying mechanisms are already affected during neurodevelopmental stages. Methods To understand the molecular mechanisms involved in disease onset, we used an RTT patient induced pluripotent stem cell (iPSC)-based model with isogenic controls and performed time-series of proteomic analysis using in-depth high-resolution quantitative mass spectrometry during early stages of neuronal development. Results We provide mass spectrometry-based quantitative proteomic data, depth of about 7000 proteins, at neuronal progenitor developmental stages of RTT patient cells and isogenic controls. Our data gives evidence of proteomic alteration at early neurodevelopmental stages, suggesting alterations long before the phase that symptoms of RTT syndrome become apparent. Significant changes are associated with the GO enrichment analysis in biological processes cell-cell adhesion, actin cytoskeleton organization, neuronal stem cell population maintenance, and pituitary gland development, next to protein changes previously associated with RTT, i.e., dendrite morphology and synaptic deficits. Differential expression increased from early to late neural stem cell phases, although proteins involved in immunity, metabolic processes, and calcium signaling were affected throughout all stages analyzed. Limitations The limitation of our study is the number of RTT patients analyzed. As the aim of our study was to investigate a large number of proteins, only one patient was considered, of which 3 different RTT iPSC clones and 3 isogenic control iPSC clones were included. Even though this approach allowed the study of mutation-induced alterations due to the usage of isogenic controls, results should be validated on different RTT patients to suggest common disease mechanisms. Conclusions During early neuronal differentiation, there are consistent and time-point specific proteomic alterations in RTT patient cells carrying exons 3–4 deletion in MECP2. We found changes in proteins involved in pathway associated with RTT phenotypes, including dendrite morphology and synaptogenesis. Our results provide a valuable resource of proteins and pathways for follow-up studies, investigating common mechanisms involved during early disease stages of RTT syndrome.
Neuronal development is a complex multistep process that shapes neurons by progressing though several typical stages, including axon outgrowth, dendrite formation, and synaptogenesis. Knowledge of the mechanisms of neuronal development is mostly derived from the study of animal models. Advances in stem cell technology now enable us to generate neurons from human induced pluripotent stem cells (iPSCs). Here we provide a mass spectrometry-based quantitative proteomic signature of human iPSC-derived neurons, i.e., iPSC-derived induced glutamatergic neurons and iPSC-derived motor neurons, throughout neuronal differentiation. Tandem mass tag 10-plex labeling was carried out to perform proteomic profiling of cells at different time points. Our analysis reveals significant expression changes (FDR < 0.001) of several key proteins during the differentiation process, e.g., proteins involved in the Wnt and Notch signaling pathways. Overall, our data provide a rich resource of information on protein expression during human iPSC neuron differentiation.
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder characterized by loss of motor neurons (MN) in the spinal cord leading to progressive muscle atrophy and weakness. SMA is caused by mutations in the survival motor neuron 1 ( SMN1 ) gene, resulting in reduced levels of survival motor neuron (SMN) protein. The mechanisms that link SMN deficiency to selective motor neuron dysfunction in SMA remain largely unknown. We present here, for the first time, a comprehensive quantitative TMT-10plex proteomics analysis that covers the development of induced pluripotent stem cell-derived MNs from both healthy individuals and SMA patients. We show that the proteomes of SMA samples segregate from controls already at early stages of neuronal differentiation. The altered proteomic signature in SMA MNs is associated with mRNA splicing, ribonucleoprotein biogenesis, organelle organization, cellular biogenesis, and metabolic processes. We highlight several known SMN-binding partners and evaluate their expression changes during MN differentiation. In addition, we compared our study to human and mouse in vivo proteomic studies revealing distinct and similar signatures. Altogether, our work provides a comprehensive resource of molecular events during early stages of MN differentiation, containing potentially therapeutically interesting protein expression profiles for SMA.
Background Mesenchymal stromal/stem cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) hold promise as a disease modifying treatment in osteoarthritis (OA). Obesity, and its associated inflammation, contribute to OA development and metabolic OA represents a specific and significant group of the OA patient population. Given their immunomodulatory properties, MSC and MSC-EVs are especially interesting for this group of patients as a therapeutic option. Here, we were the first to compare the therapeutic efficacy of MSCs and MSC-EVs in a mild OA model taking these metabolic aspects into consideration. Methods Male Wistar-Han rats (Crl:WI(Han) (n = 36) were fed a high fat diet for 24 weeks, with unilateral induction of OA by groove surgery after 12 weeks. Eight days after surgery rats were randomized in three treatment groups receiving MSCs, MSC-EVs or vehicle injection. Pain-associated behavior, joint degeneration, and local and systemic inflammation were measured. Results We demonstrated that despite not having a significant therapeutic effect, MSC-EV treatment results in lower cartilage degeneration, less pain behaviour, osteophytosis and joint inflammation, than MSC treatment. Suggesting that MSC-EVs could be a more promising therapeutic strategy than MSCs in this mild metabolic OA model. Conclusion In summary, we find that MSC treatment has negative effects on the joint in metabolic mild OA. This is an essential finding for the significant group of patients with metabolic OA phenotype, and might help to understand why clinical translation of MSC treatment shows varying therapeutic efficacy thus far. Our results also suggest that MSC-EV-based treatment might be a promising option for these patients, however MSC-EV therapeutic efficacy will need improvement.
Rett syndrome (RTT) is a progressive neurodevelopmental disease often caused by mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MeCP2). The mechanisms by which impaired MeCP2 induces the pathological abnormalities in the brain are not understood. To understand the molecular mechanisms involved in disease, we used an RTT patient induced pluripotent stem cell (iPSC)-based model and applied an in-depth high-resolution quantitative mass spectrometry-based analysis during early stages of neuronal development. Our data provide evidence of proteomic alteration at developmental stages long before the phase that symptoms of RTT syndrome become apparent. Differences in expression profiles became more pronounced from early to late neural stem cell phases, although proteins involved in immunity, metabolic processes and calcium signaling were already affected at initial stages. These results can help development of new biomarkers and therapeutic approaches by selectively target the affected proteins in RTT syndrome.
Neuronal development is a multistep process with different regulatory programs that shapes neurons to form dendrites, axons and synapses. To date, knowledge on neuronal development is largely based on murine data and largely restricted to the genomic and transcriptomic level. Advances in stem cell differentiation now enable the study of human neuronal development, and here we provide a mass spectrometry-based quantitative proteomic signature, at high temporal resolution, of human stem cellderived neurons. To reveal proteomic changes during neuronal development we make use of two differentiation approaches, either by expression of neurogenin-2 (Ngn2) leading to glutamatergic induced neurons (iN) or via small molecule manipulations, leading to patterned motor neurons. Our analysis revealed key proteins that show significant expression changes (FDR <0.001) during neuronal differentiation. We overlay our proteomics data with available transcriptomic data during neuronal differentiation and show distinct, datatype-specific, signatures. Overall, we provide a rich resource of information on proteins associated with human neuronal development, and moreover, highlight several signaling pathways involved, such as Wnt and Notch. 2 KEYWORDS iPSC; human; neuron differentiation; quantitative mass spectrometry; TMT-10plex
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