Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases that cause infant mortality. Induced pluripotent stem cells (iPSCs) generated from skin fibroblasts from SMA patients and genetically corrected have been proposed to be useful for autologous cell therapy. We generated iPSCs from SMA patients (SMA-iPSCs) using nonviral, nonintegrating episomal vectors and used a targeted gene correction approach based on single-stranded oligonucleotides to convert the survival motor neuron 2 (SMN2) gene into an SMN1-like gene. Corrected iPSC lines contained no exogenous sequences. Motor neurons formed by differentiation of uncorrected SMA-iPSCs reproduced disease-specific features. These features were ameliorated in motor neurons derived from genetically corrected SMA-iPSCs. The different gene splicing profile in SMA-iPSC motor neurons was rescued after genetic correction. The transplantation of corrected motor neurons derived from SMA-iPSCs into an SMA mouse model extended the life span of the animals and improved the disease phenotype. These results suggest that generating genetically corrected SMA-iPSCs and differentiating them into motor neurons may provide a source of motor neurons for therapeutic transplantation for SMA.

Objective: To describe the correction of human spinal muscular atrophy (SMA)-induced pluripotent stem cells (iPSCs) and motoneurons using targeted gene correction with single-stranded oligonucleotides. Background Spinal muscular atrophy (SMA) is among the most common genetic neurological diseases causing infant mortality. Reprogramming adult human cells to induced pluripotent stem cells (iPSCs) allows obtaining patient-specific cells possible. However, the use of SMA iPSCs will require their genetic correction in a manner that is compatible with clinical applications. Design/Methods: We generated iPSCs from fibroblasts from a patient with SMA and his father using a non-viral method. Cells were nucleofected with oriP/EBNA1 vectors encoding six reprogramming factors. We used SMN2 sequence-specific ODNs to direct the exchange of a T to C at position +6 of exon 7, thus converting SMN2 into SMN1 in the SMA-iPSCs. The iPSCs were fully characterized and differentiated using a protocol to promote motoneuron commitment. The phenotype of cells was tested by morphological, gene expression, and protein analysis. iPSC-purified motoneurons were transplanted into the spinal cords of SMA mice. Histochemical and neuropathological analyses were conducted. Survival and neuromuscular function were investigated. Results: SMA corrected cell lines contained no exogenous sequences and appeared indistinguishable from healthy iPSCs. Non-viral SMA-iPSC-derived motoneurons reproduced disease-specific features (reductions in cell number, cell size, and axon length) while corrected SMA-specific-iPSCs gave rise to phenotypically rescued motoneurons in vitro and in vivo after transplantation in SMA spinal cord. Different splicing profiles, detected in vitro by microarray analysis in SMA motoneurons compared to wild-type, were normalized after the genetic correction. Transplantation of wild-type and corrected SMA motoneurons extended lifespan (>50%) and ameliorated the phenotype of SMA mice significantly more than SMA motoneurons and untreated animals. Conclusions: These results offer proof-of-concept that generating patient-specific corrected iPSCs and motoneurons free of exogenous elements may be possible, with potential for research and clinical applications. Disclosure: Dr. Corti has nothing to disclose. Dr. Nizzardo has nothing to disclose. Dr. Simone has nothing to disclose. Dr. Falcone has nothing to disclose. Dr. Nardini has nothing to disclose. Dr. Ronchi has nothing to disclose. Dr. Donadoni has nothing to disclose. Dr. Salani has nothing to disclose. Dr. Riboldi has nothing to disclose. Dr. Menozzi has nothing to disclose. Dr. Bonaglia has nothing to disclose. Dr. Magri has nothing to disclose. Dr. Bresolin has nothing to disclose. Dr. Comi has received research support from Telethon Italy and SMA Europe.

Spinal muscular atrophy (SMA) is a devastating genetic motoneuron disease leading to infant death. No effective therapy is currently available. It has been suggested that β-lactam antibiotics such as ceftriaxone may offer neuroprotection in motoneuron diseases. Here, we investigate the therapeutic effect of ceftriaxone in a murine model of SMA. Treated animals present a modest, but significant ameliorated neuromuscular phenotype and increased survival, which correlate with protection of neuromuscular units. Whole gene expression profiling in treated mice demonstrates modifications in several genes including those involved in RNA metabolism toward wild-type. The neuroprotective effect seems to be mediated by multiple mechanisms that encompass the increase of the glutamate transporter Glt1, the transcription factor Nrf2, as well as SMN protein. This study provides the first evidence of a potential positive effect of this class of molecules in SMA. Further investigation of analogs with increased and more specific therapeutic effects warrants the development of useful therapies for SMA.

Generating neural stem cells and neurons from reprogrammed human astrocytes is a potential strategy for neurological repair. Here we show dedifferentiation of human cortical astrocytes into the neural stem/progenitor phenotype to obtain progenitor and mature cells with a neural fate. Ectopic expression of the reprogramming factors OCT4, SOX2, or NANOG into astrocytes in specific cytokine/culture conditions activated the neural stem gene program and induced generation of cells expressing neural stem/precursor markers. Pure CD44+ mature astrocytes also exhibited this lineage commitment change and did not require passing through a pluripotent state. These astrocyte-derived neural stem cells gave rise to neurons, astrocytes, and oligodendrocytes and showed in vivo engraftment properties. ASCL1 expression further promoted neuronal phenotype acquisition in vitro and in vivo. Methylation analysis showed that epigenetic modifications underlie this process. The restoration of multipotency from human astrocytes has potential in cellular reprogramming of endogenous central nervous system cells in neurological disorders.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disease characterized by the loss of motor neurons. Motor neuron degeneration is probably both a cell autonomous and a non-autonomous event. Therefore, manipulating the diseased microenvironment via non-neural cell replacement could be a therapeutic strategy. We investigated a cell therapy approach using intravascular injection to transplant a specific population of c-kit(+) stem/progenitor cells from bone marrow into the SOD1G93A mouse model of ALS. Transplanted cells engrafted within the host spinal cord. Cell transplantation significantly prolonged disease duration and lifespan in superoxide dismutase 1 mice, promoted the survival of motor neurons and improved neuromuscular function. Neuroprotection was mediated by multiple effects, in particular by the expression of primary astrocyte glutamate transporter GLT1 and by the non-mutant genome. These findings suggest that this type of somatic cell transplantation strategy merits further investigation as a possible effective therapy for ALS and other neurodegenerative diseases.

Phototransduction begins when a photon activates a rhodopsin molecule. Activated rhodopsin activates the G-protein transducin, which activates phosphodiesterase, leading to decreased cGMP concentration and closure of cyclic-nucleotide-gated channels. Termination of this process requires inactivation of rhodopsin and transducin. The former is mediated by arrestin, which separates the bleached chromophore from the opsin. The affinity of arrestin for rhodopsin is increased by triple phosphorylation of the rhodopsin by the G-proteincoupled-receptor kinase GRK1. But arrestin can also bind to unphosphorylated and semiphosphorylated rhodopsin, leading Doan et al. to hypothesize that competition for rhodopsin binding between GRK1 and arrestin influences the rate of rhodopsin inactivation. Analysis of single-photon responses in mice that expressed reduced levels of either GRK1 or arrestin provided support for this hypothesis. In addition, the results suggested that, contrary to previous reports, the active time of rhodopsin was longer than that of transducin. The authors show this depends on recording conditions.

To investigate the role of erythropoietin (EPO) as genetic determinant in the susceptibility to sporadic amyotrophic lateral sclerosis (SALS). We sequenced a 259-bp region spanning the 3′hypoxia-responsive element of the EPO gene in 222 Italian SALS patients and 204 healthy subjects, matched for age and ethnic origin. No potentially causative variation was detected in SALS subjects; in addition, two polymorphic variants (namely C3434T and G3544T) showed the same genotype and haplotype frequencies in patients and controls. Conversely, a weak but significant association between G3544T and age of disease onset was observed (p=0.04). Overall, our data argue against the hypothesis of EPO as a genetic risk factor for motor neuron dysfunction, at least in Italian population. However, further studies on larger cohort of patients are needed to confirm the evidence of EPO gene as modifier factor.

OBJECTIVE:Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective motoneuron death. Understanding of the molecular mechanisms that trigger and regulate motoneuron degeneration could be relevant to ALS and other motoneuron disorders. This study investigates the role of Fas-linked motoneuron death in the pathogenesis of ALS.METHODS:We performed in vitro and in vivo small interfering RNA-mediated interference, by silencing the Fas receptor on motoneurons that carry the superoxide dismutase-1 (SOD1)-G93A mutation.RESULTS:We observed a significant reduction in Fas expression at messenger RNA (p < 0.001) and protein levels. Treated motoneurons demonstrated an increase in survival and a reduction in cytochrome c release from mitochondria. In vivo, continuous intrathecal administration of Fas small interfering RNA by an osmotic minipump improved motor function and survival in SOD1-G93A mice (mean increase, 18 days; p < 0.0001). Treated mice showed a significant reduction in Fas and Fas mediators p38 mitogen-activated protein kinase, neuronal nitric oxide synthase, and caspase-8.INTERPRETATION:Fas silencing interferes with motoneuron-specific downstream death pathways and results in increased motoneuron survival and amelioration of the SOD1-G93A phenotype, suggesting new possible strategies for molecular therapy of ALS.

The identification of strategies for the isolation of neural stem cells (NSCs) has important implications for the understanding of their biology and the development of therapeutic applications. It has been previously described that human neural stem and progenitor cells (NSPCs) can be isolated from the central nervous system (CNS) using antibodies to prominin (CD133) and fluorescence-activated cell sorting (FACS). Although this antigen displayed an identical membrane topology in several human and murine tissues there was uncertainty as to the relationship between human and mouse prominin because of the low level of amino acid identity.Here we show that prominin expression can be used to identify and isolate also murine NSPCs from the developing or adult brain. Prominin is co-expressed with known neural stem markers like SOX 1-2, Musashi and Nestin. Moreover, neurosphere-forming cells with multipotency and self-renewal capacity reside within the prominin-positive fraction. Transplantation experiments show that CD133-positive cells give rise to neurons and glial cells in vivo, and that many neurons display appropriate phenotypic characteristics of the recipient tissues.The demonstration that CD133 is a stem cell antigen for murine NSPCs as it is for human NSPCs is useful for the investigation of mammal neurogenesis and development of preclinical tests of NSPCs transplantation in mouse analogues of human diseases. (c) 2007 Elsevier Inc. All rights reserved.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of the motor neurons. We tested whether treatment of superoxide dismutase (SOD1)-G93A transgenic mouse, a model of ALS, with a neural stem cell subpopulation double positive for Lewis X and the chemokine receptor CXCR4 (LeX+CXCR4+) can modify the disease's progression. In vitro, after exposure to morphogenetic stimuli, LeX+CXCR4+ cells generate cholinergic motor neuron-like cells upon differentiation. LeX+CXCR4+ cells deriving from mice expressing Green Fluorescent Protein in all tissues or only in motor neurons, after a period of priming in vitro, were grafted into spinal cord of SOD1-G93A mice. Transplanted transgenic mice exhibited a delayed disease onset and progression, and survived significantly longer than non-treated animals by 23 days. Examination of the spinal cord revealed integration of donor-derived cells that differentiated mostly in neurons and in a lower proportion in motor neuron-like cells. Quantification of motor neurons of the spinal cord suggests a significant neuroprotection by LeX+CXCR4+ cells. Both VEGF- and IGF1-dependent pathways were significantly modulated in transplanted animals compared to controls, suggesting a role of these neurotrophins in MN protection. Our results support the therapeutic potential of neural stem cell fractions through both neurogenesis and growth factors release in motor neuron disorders.

To investigate the role of vascular endothelial growth factor (VEGF) and angiogenin (ANG) as genetic determinants in the susceptibility to sporadic ALS in Italian patients. VEGF genotype and haplotype analysis revealed no association between any variants and the risk of ALS. Regarding ANG gene, no mutation was detected and the rs11701 polymorphism, previously described as associated with ALS, was not differently distributed between patients and controls. Overall, our data argue against the hypothesis of both genes as risk factors for motoneuron neurodegeneration, at least in an Italian population.