Plastin 3 is upregulated in iPSC-derived motoneurons from asymptomatic SMN1-deleted individuals

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier

Evaluating the SERCA2 and VEGF mRNAs as Potential Molecular Biomarkers of the Onset and Progression in Huntington's Disease

Abnormalities of intracellular Ca2+ homeostasis and signalling as well as the down-regulation of neurotrophic factors in several areas of the central nervous system and in peripheral tissues are hallmarks of Huntington\u2019s disease (HD). As there is no therapy for this hereditary, neurodegenerative fatal disease, further effort should be made to slow the progression of neurodegeneration in patients through the definition of early therapeutic interventions. For this purpose, molecular biomarker(s) for monitoring disease onset and/or progression and response to treatment need to be identified. In the attempt to contribute to the research of peripheral candidate biomarkers in HD, we adopted a multiplex real-time PCR approach to analyse the mRNA level of targeted genes involved in the control of cellular calcium homeostasis and in neuroprotection. For this purpose we recruited a total of 110 subjects possessing the HD mutation at different clinical stages of the disease and 54 sex- and agematched controls. This study provides evidence of reduced transcript levels of sarco-endoplasmic reticulum-associated ATP2A2 calcium pump (SERCA2) and vascular endothelial growth factor (VEGF) in peripheral blood mononuclear cells (PBMCs) of manifest and premanifest HD subjects. Our results provide a potentially new candidate molecular biomarker for monitoring the progression of this disease and contribute to understanding some early events that might have a role in triggering cellular dysfunctions in HD

Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.

<p>Primers used to evaluate the number of CAG repeats for the molecular diagnosis of HD.</p

RNA quantification of PMCA1, SERCA2, SERCA3 and VEGF in PBMCs from healthy controls and HD patients divided according to sex.

<p>Subjects analysed: 10 healthy male controls and 10 healthy female controls age-matched ± 3 years; 10 males HD, mean age of 44.5 ± 10.1; 10 females HD, mean age of 53.4 ± 9.6. *** p<0.005. Each column represents the relative mean value of three independent experiments ± SD.</p

Primer and probe sequences used for the real-time detection of β-actin.

<p>Primer and probe sequences used for the real-time detection of β-actin.</p

mRNA quantification of VEGF relative to the age and clinical conditions of the patients.

<p>(A) Comparison of the level of VEGF mRNA in PBMCs from HD pre-manifest, manifest patients and age- and sex-matched healthy controls.Age 27–40: Controls = 15; pre-manifest = 12; manifest = 13; Age 43–58: Controls = 26; pre-manifest = 8; manifest = 42; Age 59–84: Controls = 13; manifest = 35; ** p<0.01; *** p<0.001. (B) Comparison of the level of VEGF mRNA in PBMCs of patients with different stages of the disease: pre-manifest = 20; early-manifest = 17; manifest = 55; late-manifest = 18;* p<0.05. The mean values ± SD of three independent assays are shown.</p