Abstract:Disease modeling requires appropriate cellular models that best mimic the underlying pathophysiology. Human origin and an adequate expression of the disease protein are prerequisites that support information from a model to be meaningful. In this study we investigated expression profiles of (i) PBMCs and (ii) fibroblasts as patient derived cells as well as (iii) lymphoblasts and (iv) induced pluripotent stem cells (iPSC) as immortalized sources, and (v) iPSC-derived cortical neurons to assess their aptitude to… Show more
“…Several ligand–receptor pairs were found to be differentially expressed in SMA and control mice. Ncam and L1cam are shown downregulated in SMA patients or motor neuron disease cell models, consistent with our finding of weakened interactions between the two [ 69 – 71 ]. Although it is unclear whether these ligand-receptor pairs play a role in SMA pathogenesis, they still provide potential topics for follow-up research.…”
Although spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival of motor neuron (SMN) proteins, there is growing evidence that non-neuronal cells play important roles in SMA pathogenesis. However, transcriptome alterations occurring at the single-cell level in SMA spinal cord remain unknown, preventing us from fully comprehending the role of specific cells. Here, we performed single-cell RNA sequencing of the spinal cord of a severe SMA mouse model, and identified ten cell types as well as their differentially expressed genes. Using CellChat, we found that cellular communication between different cell types in the spinal cord of SMA mice was significantly reduced. A dimensionality reduction analysis revealed 29 cell subtypes and their differentially expressed gene. A subpopulation of vascular fibroblasts showed the most significant change in the SMA spinal cord at the single-cell level. This subpopulation was drastically reduced, possibly causing vascular defects and resulting in widespread protein synthesis and energy metabolism reductions in SMA mice. This study reveals for the first time a single-cell atlas of the spinal cord of mice with severe SMA, and sheds new light on the pathogenesis of SMA.
“…Several ligand–receptor pairs were found to be differentially expressed in SMA and control mice. Ncam and L1cam are shown downregulated in SMA patients or motor neuron disease cell models, consistent with our finding of weakened interactions between the two [ 69 – 71 ]. Although it is unclear whether these ligand-receptor pairs play a role in SMA pathogenesis, they still provide potential topics for follow-up research.…”
Although spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival of motor neuron (SMN) proteins, there is growing evidence that non-neuronal cells play important roles in SMA pathogenesis. However, transcriptome alterations occurring at the single-cell level in SMA spinal cord remain unknown, preventing us from fully comprehending the role of specific cells. Here, we performed single-cell RNA sequencing of the spinal cord of a severe SMA mouse model, and identified ten cell types as well as their differentially expressed genes. Using CellChat, we found that cellular communication between different cell types in the spinal cord of SMA mice was significantly reduced. A dimensionality reduction analysis revealed 29 cell subtypes and their differentially expressed gene. A subpopulation of vascular fibroblasts showed the most significant change in the SMA spinal cord at the single-cell level. This subpopulation was drastically reduced, possibly causing vascular defects and resulting in widespread protein synthesis and energy metabolism reductions in SMA mice. This study reveals for the first time a single-cell atlas of the spinal cord of mice with severe SMA, and sheds new light on the pathogenesis of SMA.
“…Several ligand–receptor pairs were found to be differentially expressed in SMA and control mice. Ncam and L1cam are shown downregulated in SMA patients or motor neuron disease cell models, consistent with our results [69–71]. Although it is unclear whether these ligand-receptor pairs play a role in SMA pathogenesis, they still provide potential topics for follow-up research.…”
Although spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival of motor neuron proteins, there is growing evidence that lesions in nonmotor neuron cells are involved in SMA pathogenesis. Transcriptome alterations occurring at the single-cell level in SMA spinal cord are unknown. Here, we performed single-cell RNA sequencing of the spinal cord of a severe SMA mouse model, and characterized ten cell types as well as their differentially expressed genes. Using CellChat, we found that cellular communication between different cell types in the spinal cord of SMA mice is substantially reduced. A dimensionality reduction analysis revealed 29 cell subtypes and differentially expressed genes for each subtype. Among them, the most significant change in the SMA spinal cord at the single-cell level was observed in a subpopulation of vascular fibroblasts. This subpopulation was greatly reduced, which may be responsible for vascular defects in SMA mice and may lead to widespread reductions in protein synthesis and energy metabolism. This study is the first to report a single-cell atlas of the spinal cord of mice with severe SMA, providing new insights into SMA pathogenesis.
“…In the next step, iPSCs were differentiated into iPSC-derived cortical neurons according to our previously published protocol. 38 , 39 After 36 days of differentiation, iPSC-derived neurons were then treated with ASOs targeting ATXN3 ( Figure 1 A). Figure 1 Establishment of an iPSC-based in vitro model to screen ASOs for SCA3 (A) Schematic representation of the experimental procedure to reprogram fibroblasts to iPSCs and differentiate them to iPSC-derived neurons for ASO treatments.…”
Section: Resultsmentioning
confidence: 99%
“…iPSCs were differentiated to neurons of cortical layers V and VI according to published protocols. 38 , 39 , 42 In brief, iPSCs were plated in Matrigel-coated plates at a density of 3 × 10 5 cells/cm 2 in E8 medium supplemented with 10 μM Y-27632 (Selleckchem). Cultivation in 3N medium with 10 μM SB431542 (Sigma-Aldrich) and 500 nM LDN-193189 (Sigma-Aldrich) for 9 days led to neural induction.…”
Spinocerebellar ataxia type 3 (SCA3) is caused by an expanded polyglutamine stretch in ataxin-3. While wild-type ataxin-3 has important functions, e.g., as a deubiquitinase, downregulation of mutant ataxin-3 is likely to slow down the course of this fatal disease. We established a screening platform with human neurons of patients and controls derived from induced pluripotent stem cells to test antisense oligonucleotides (ASOs) for their effects on ataxin-3 expression. We identified an ASO that suppressed mutant and wild-type ataxin-3 levels by >90% after a singular treatment. Next, we screened pairs of ASOs designed to selectively target the mutant or the wild-type allele by taking advantage of a SNP (c.987G > C) in
ATXN3
that is present in most SCA3 patients. We found ASOmut4 to reduce levels of mutant ataxin-3 by 80% after 10 days while leaving expression of wild-type ataxin-3 largely unaffected. In a long-term study we proved this effect to last for about 4 weeks after a single treatment without signs of neurotoxicity. This study provides proof of principle that allele-specific lowering of poly(Q)-expanded ataxin-3 by selective ASOs is feasible and long lasting, with sparing of wild-type ataxin-3 expression in a human cell culture model that is genetically identical to SCA3 patients.
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