Stem cell-derived motor neurons from spinal and bulbar muscular atrophy patients

Abstract

Spinal and bulbar muscular atrophy (SBMA, Kennedy’s disease), is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy.

p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration

“…Mature neurons were derived from iPSC (Supplementary Figure S5C) and confirmation of neuronal phenotypes was achieved through expression of sequential markers of differentiation: iPSCs expressed TRA-1-81 demonstrating pluripotency (Supplementary Figure S5D); neural stem cells (NSC) expressed Nestin and Sox1 (Supplementary Figure S5E); and neurons expressed neuronal nuclei marker (NeuN) (Supplementary Figure S5F). Motor neurons, a mature neuronal subtype that is primarily lost in ALS, were also derived from iPSC 49 and their differentiation was confirmed by expression of non-phosphorylated neurofilament marker, SMI-32 (Supplementary Figure S5G).…”

“…Although both AD and ALS are associated with increased cellular senescence, qRT-PCR using brain tissue RNA samples showed that SASP cytokine IL-6 and a p53-inducible senescence Figure 5 Increased neuronal death upon co-culture with Δ133p53-knocked-down or p53β-overexpressing astrocytes. Early-passage primary astrocytes (P5) with Δ133p53 siRNA and control siRNA (generated as in Supplementary Figure S3) (a, b, e, and f) and those with p53β -overexpression or control vector (generated as in Supplementary Figure S4) (c, d, g, and h) were used in co-culture for 48 h with motor neurons 49 or less specialized neurons (as generated as in Supplementary Figures S5C-F Data are presented as mean ± S.E.M. NS indicates P40.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 by unpaired two-tailed Student's t test p53 isoforms in neurodegeneration C Turnquist et al regulator p21 WAF1 were upregulated more remarkably in ALS, while NOS2 upregulation was more evident in AD (Figure 7c), possibly reflecting the different disease pathologies.…”

Nucleocytoplasmic transport defect in a North American patient with ALS8

“…Fibroblasts were stained on coverslips as previously described 14. Antibodies used for staining can be found in Table S2.…”

Clinical Trials in a Dish: The Potential of Pluripotent Stem Cells to Develop Therapies for Neurodegenerative Diseases

“…Instability can be particularly troubling when reprogramming patient samples from diseases, such as HD, spinal-bulbar muscular dystrophy, C9ORF72-associated ALS, and FTD, that are caused by expansion of repeat regions within the disease-causing gene (Table 1). In these lines, the repeat region is often not stable during and after reprogramming, and the instability can continue during differentiation (85, 86). For example, in one study, six iPSC lines with spinal-bulbar muscular atrophy were derived, but only three were used in which the disease associated poly-Q-androgen receptor expansion was stable over multiple passages (86).…”