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1.
J Gen Physiol ; 152(9)2020 09 07.
Article in English | MEDLINE | ID: mdl-32633755

ABSTRACT

TRIP8b (tetratricopeptide repeat-containing Rab8b-interacting protein) is the neuronal regulatory subunit of HCN channels, a family of voltage-dependent cation channels also modulated by direct cAMP binding. TRIP8b interacts with the C-terminal region of HCN channels and controls both channel trafficking and gating. The association of HCN channels with TRIP8b is required for the correct expression and subcellular targeting of the channel protein in vivo. TRIP8b controls HCN gating by interacting with the cyclic nucleotide-binding domain (CNBD) and competing for cAMP binding. Detailed structural knowledge of the complex between TRIP8b and CNBD was used as a starting point to engineer a mutant channel, whose gating is controlled by cAMP, but not by TRIP8b, while leaving TRIP8b-dependent regulation of channel trafficking unaltered. We found two-point mutations (N/A and C/D) in the loop connecting the CNBD to the C-linker (N-bundle loop) that, when combined, strongly reduce the binding of TRIP8b to CNBD, leaving cAMP affinity unaltered both in isolated CNBD and in the full-length protein. Proof-of-principle experiments performed in cultured cortical neurons confirm that the mutant channel provides a genetic tool for dissecting the two effects of TRIP8b (gating versus trafficking). This will allow the study of the functional role of the TRIP8b antagonism of cAMP binding, a thus far poorly investigated aspect of HCN physiology in neurons.


Subject(s)
Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels , Neurons , Receptors, Cytoplasmic and Nuclear/genetics , Brain/metabolism , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Ion Channel Gating , Mutation , Neurons/metabolism
2.
Elife ; 72018 06 20.
Article in English | MEDLINE | ID: mdl-29923826

ABSTRACT

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented ß-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers.


Subject(s)
Cyclic AMP/chemistry , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Myocytes, Cardiac/drug effects , Peptides/pharmacology , Potassium Channels/chemistry , Animals , Binding Sites , Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/genetics , Calcium Channels, L-Type/metabolism , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/genetics , Cell-Penetrating Peptides/metabolism , Cyclic AMP/metabolism , Gene Expression , HEK293 Cells , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Molecular Docking Simulation , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Peptides/chemical synthesis , Peroxins/chemistry , Peroxins/genetics , Peroxins/metabolism , Potassium Channels/genetics , Potassium Channels/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Rabbits , Sinoatrial Node/cytology , Sinoatrial Node/drug effects , Sinoatrial Node/metabolism , tat Gene Products, Human Immunodeficiency Virus
3.
Proc Natl Acad Sci U S A ; 111(40): 14577-82, 2014 Oct 07.
Article in English | MEDLINE | ID: mdl-25197093

ABSTRACT

cAMP signaling in the brain mediates several higher order neural processes. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels directly bind cAMP through their cytoplasmic cyclic nucleotide binding domain (CNBD), thus playing a unique role in brain function. Neuronal HCN channels are also regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit that antagonizes the effects of cAMP by interacting with the channel CNBD. To unravel the molecular mechanisms underlying the dual regulation of HCN channel activity by cAMP/TRIP8b, we determined the NMR solution structure of the HCN2 channel CNBD in the cAMP-free form and mapped on it the TRIP8b interaction site. We reconstruct here the full conformational changes induced by cAMP binding to the HCN channel CNBD. Our results show that TRIP8b does not compete with cAMP for the same binding region; rather, it exerts its inhibitory action through an allosteric mechanism, preventing the cAMP-induced conformational changes in the HCN channel CNBD.


Subject(s)
Cyclic AMP/chemistry , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/chemistry , Ion Channel Gating , Receptors, Cytoplasmic and Nuclear/chemistry , Binding Sites , Crystallography, X-Ray , Cyclic AMP/metabolism , Cyclic Nucleotide-Gated Cation Channels/chemistry , Cyclic Nucleotide-Gated Cation Channels/metabolism , Electrophoresis, Polyacrylamide Gel , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/metabolism , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Structure , Potassium Channels/chemistry , Potassium Channels/metabolism , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Cytoplasmic and Nuclear/metabolism
4.
Sci Transl Med ; 4(165): 165ra162, 2012 Dec 19.
Article in English | MEDLINE | ID: mdl-23253609

ABSTRACT

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.


Subject(s)
Genetic Therapy , Muscular Atrophy, Spinal/therapy , Pluripotent Stem Cells/cytology , Cell Transplantation , Gene Expression , Genetic Vectors , Humans , Motor Neurons/metabolism , Motor Neurons/pathology , Motor Neurons/transplantation , Muscular Atrophy, Spinal/genetics , RNA Splicing , Survival of Motor Neuron 1 Protein/genetics
5.
Exp Cell Res ; 318(13): 1528-41, 2012 Aug 01.
Article in English | MEDLINE | ID: mdl-22426197

ABSTRACT

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.


Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Cell Dedifferentiation , Cell Transdifferentiation , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/cytology , Neurons/metabolism , Animals , Cell Dedifferentiation/genetics , Cell Dedifferentiation/physiology , Cell Transdifferentiation/genetics , Cell Transdifferentiation/physiology , Cells, Cultured , DNA Methylation , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Nanog Homeobox Protein , Neural Stem Cells/transplantation , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , SOXB1 Transcription Factors/genetics , SOXB1 Transcription Factors/metabolism , Transplantation, Heterologous
6.
J Cell Mol Med ; 16(7): 1353-64, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22129481

ABSTRACT

Muscular dystrophies (MDs) are a heterogeneous group of inherited disorders characterized by progressive muscle wasting and weakness likely associated with exhaustion of muscle regeneration potential. At present, no cures or efficacious treatments are available for these diseases, but cell transplantation could be a potential therapeutic strategy. Transplantation of myoblasts using satellite cells or other myogenic cell populations has been attempted to promote muscle regeneration, based on the hypothesis that the donor cells repopulate the muscle and contribute to its regeneration. Embryonic stem cells (ESCs) and more recently induced pluripotent stem cells (iPSCs) could generate an unlimited source of differentiated cell types, including myogenic cells. Here we review the literature regarding the generation of myogenic cells considering the main techniques employed to date to elicit efficient differentiation of human and murine ESCs or iPSCs into skeletal muscle. We also critically analyse the possibility of using these cellular populations as an alternative source of myogenic cells for cell therapy of MDs.


Subject(s)
Embryonic Stem Cells/transplantation , Induced Pluripotent Stem Cells/transplantation , Muscle Fibers, Skeletal/transplantation , Muscular Dystrophies/therapy , Animals , Cell Culture Techniques , Cell Differentiation , Disease Models, Animal , Embryonic Stem Cells/cytology , Humans , Induced Pluripotent Stem Cells/cytology , Regeneration , Satellite Cells, Skeletal Muscle/cytology , Satellite Cells, Skeletal Muscle/transplantation , Stem Cell Transplantation
7.
Exp Neurol ; 229(2): 214-25, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21295027

ABSTRACT

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 ß-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.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Ceftriaxone/therapeutic use , Motor Neurons/drug effects , Muscle, Skeletal/drug effects , Muscular Atrophy, Spinal/drug therapy , Neuroprotective Agents/therapeutic use , Analysis of Variance , Animals , Anti-Bacterial Agents/pharmacology , Blotting, Western , Ceftriaxone/pharmacology , Cell Count , Disease Models, Animal , Gene Expression/drug effects , Immunohistochemistry , Kaplan-Meier Estimate , Mice , Mice, Transgenic , Motor Neurons/metabolism , Motor Neurons/pathology , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/pathology , Neuroprotective Agents/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , SMN Complex Proteins/genetics , SMN Complex Proteins/metabolism , Spinal Cord/drug effects , Spinal Cord/metabolism , Spinal Cord/pathology , Treatment Outcome
9.
Hum Mol Genet ; 19(19): 3782-96, 2010 Oct 01.
Article in English | MEDLINE | ID: mdl-20650960

ABSTRACT

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.


Subject(s)
Amyotrophic Lateral Sclerosis/therapy , Disease Models, Animal , Proto-Oncogene Proteins c-kit/metabolism , Stem Cell Transplantation , Amino Acid Transport System X-AG/metabolism , Amyotrophic Lateral Sclerosis/enzymology , Animals , Biological Assay , Blood Vessels/pathology , Bone Marrow Cells/cytology , Cell Death , Cell Separation , Cell Survival , Coculture Techniques , Extracellular Space/metabolism , Glutamates/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Mice , Motor Neurons/pathology , Mutant Proteins/metabolism , Phenotype , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Survival Analysis
10.
Brain ; 133(Pt 2): 465-81, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20032086

ABSTRACT

Spinal muscular atrophy, characterized by selective loss of lower motor neurons, is an incurable genetic neurological disease leading to infant mortality. We previously showed that primary neural stem cells derived from spinal cord can ameliorate the spinal muscular atrophy phenotype in mice, but this primary source has limited translational value. Here, we illustrate that pluripotent stem cells from embryonic stem cells show the same potential therapeutic effects as those derived from spinal cord and offer great promise as an unlimited source of neural stem cells for transplantation. We found that embryonic stem cell-derived neural stem cells can differentiate into motor neurons in vitro and in vivo. In addition, following their intrathecal transplantation into spinal muscular atrophy mice, the neural stem cells, like those derived from spinal cord, survived and migrated to appropriate areas, ameliorated behavioural endpoints and lifespan, and exhibited neuroprotective capability. Neural stem cells obtained using a drug-selectable embryonic stem cell line yielded the greatest improvements. As with cells originating from primary tissue, the embryonic stem cell-derived neural stem cells integrated appropriately into the parenchyma, expressing neuron- and motor neuron-specific markers. Our results suggest translational potential for the use of pluripotent cells in neural stem cell-mediated therapies and highlight potential safety improvements and benefits of drug selection for neuroepithelial cells.


Subject(s)
Embryonic Stem Cells/transplantation , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/surgery , Neurons/transplantation , Phenotype , Animals , Cell Differentiation/genetics , Cell Line , Cell Movement/genetics , Cells, Cultured , Coculture Techniques , Disease Models, Animal , Embryonic Stem Cells/cytology , Humans , Mice , Mice, Knockout , Mice, Transgenic , Muscular Atrophy, Spinal/pathology , Neurons/cytology , Stem Cell Transplantation
11.
J Neurosci ; 29(38): 11761-71, 2009 Sep 23.
Article in English | MEDLINE | ID: mdl-19776263

ABSTRACT

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal form of infantile motoneuron disease. There is currently no effective treatment, although motor neuron replacement is a possible therapeutic strategy. We transplanted purified motor neurons into the spinal cord of nmd mice, an animal model of SMARD1. We also administered pharmacological treatment targeting the induction of axonal growth toward skeletal muscle target. At the end stage of the disease, donor-derived motor neurons were detected in the nmd anterior horns, extended axons into the ventral roots, and formed new neuromuscular junctions. These data correlated with improved neuromuscular function and increased life spans. The neuroprotective effect was associated with a reduction in proinflammatory molecules in treated spinal cords. This is the first report that functional restoration of motor units with transplanted motoneurons is feasible in an animal model of a human motoneuron disease, opening up new possibilities for therapeutic intervention.


Subject(s)
Motor Neurons/transplantation , Spinal Cord/surgery , Spinal Muscular Atrophies of Childhood/surgery , Animals , Axons/drug effects , Axons/physiology , Cytokines/metabolism , Disease Models, Animal , Longevity/drug effects , Longevity/physiology , Mice , Mice, Transgenic , Motor Neurons/drug effects , Motor Neurons/physiology , Neurogenesis , Neuromuscular Junction/drug effects , Neuromuscular Junction/physiology , Neuroprotective Agents/therapeutic use , Phenotype , Spinal Cord/drug effects , Spinal Cord/physiology , Spinal Muscular Atrophies of Childhood/drug therapy , Stem Cells/physiology
12.
Am J Hum Genet ; 84(5): 594-604, 2009 May.
Article in English | MEDLINE | ID: mdl-19409522

ABSTRACT

A disulfide relay system (DRS) was recently identified in the yeast mitochondrial intermembrane space (IMS) that consists of two essential components: the sulfhydryl oxidase Erv1 and the redox-regulated import receptor Mia40. The DRS drives the import of cysteine-rich proteins into the IMS via an oxidative folding mechanism. Erv1p is reoxidized within this system, transferring its electrons to molecular oxygen through interactions with cytochrome c and cytochrome c oxidase (COX), thereby linking the DRS to the respiratory chain. The role of the human Erv1 ortholog, GFER, in the DRS has been poorly explored. Using homozygosity mapping, we discovered that a mutation in the GFER gene causes an infantile mitochondrial disorder. Three children born to healthy consanguineous parents presented with progressive myopathy and partial combined respiratory-chain deficiency, congenital cataract, sensorineural hearing loss, and developmental delay. The consequences of the mutation at the level of the patient's muscle tissue and fibroblasts were 1) a reduction in complex I, II, and IV activity; 2) a lower cysteine-rich protein content; 3) abnormal ultrastructural morphology of the mitochondria, with enlargement of the IMS space; and 4) accelerated time-dependent accumulation of multiple mtDNA deletions. Moreover, the Saccharomyces cerevisiae erv1(R182H) mutant strain reproduced the complex IV activity defect and exhibited genetic instability of the mtDNA and mitochondrial morphological defects. These findings shed light on the mechanisms of mitochondrial biogenesis, establish the role of GFER in the human DRS, and promote an understanding of the pathogenesis of a new mitochondrial disease.


Subject(s)
Cataract/genetics , Cytochrome Reductases/physiology , Mitochondrial Diseases/genetics , Mitochondrial Myopathies/genetics , Mitochondrial Proteins/physiology , Adolescent , Cataract/congenital , Child , Child, Preschool , Consanguinity , Cytochrome Reductases/genetics , DNA, Mitochondrial/genetics , DNA, Mitochondrial/ultrastructure , Genetic Linkage , Hearing Loss/genetics , Humans , Intracellular Membranes/metabolism , Male , Mitochondrial Proteins/genetics , Mutation , Oxidoreductases Acting on Sulfur Group Donors
13.
J Neurol Sci ; 281(1-2): 85-92, 2009 Jun 15.
Article in English | MEDLINE | ID: mdl-19278689

ABSTRACT

The G8363A is a very rare mtDNA tRNA(Lys) gene mutation that has been associated to MERRF-like syndrome, cardiomyopathy or Leigh syndrome. Here, we describe the clinical and molecular features of a new large multigenerational family and we review the literature of cases with this mutation. In our family seven members presented a heterogeneous mitochondrial disease phenotype, from MERRF-like syndrome to isolated psychiatric disorder, associated with the G8363A mutation. The two probands are dizygotic twin sisters affected by mental retardation, neural deafness, myopathy, myoclonic epilepsy and ataxia. Twins' muscle biopsies showed a severe cytochrome c oxidase (COX) deficiency and ragged-red fibers. Their mitochondrial respiratory chain was defective in complexes I and IV in muscle. A severe reduction in complex IV activity was also observed in fibroblasts and myoblasts. Molecular analysis showed a G8363A transition in the mtDNA tRNA(Lys) gene. The mutation was almost homoplasmic (>90%) in muscle and blood of the twins and heteroplasmic (55+/-8%) in blood sample from affected maternal relatives. Based on our family data and the meta-analysis of the literature, we confirm that mutational load directly correlates with severity of the disease (severe vs mild/moderate phenotype; P=0.00168) and with disease onset (P<0.00001). However the presence of several exceptions and overlaps among patients with different clinical severity limits the clinical usefulness of this observation. Although the pathogenicity of the G8363A mutation is well established, counselling is a difficult task for clinicians because of the large phenotypical variability. Our study contributes further data on the clinical spectrum and its relation with the level of G8363A tRNA(Lys) mtDNA mutation.


Subject(s)
DNA, Mitochondrial/genetics , Mitochondrial Diseases/genetics , Mutation, Missense , RNA, Transfer, Lys/genetics , Adult , Age of Onset , Aged , Cells, Cultured , Electron Transport , Family , Female , Fibroblasts/enzymology , Humans , MERRF Syndrome/genetics , Mental Disorders/genetics , Middle Aged , Muscle, Skeletal/enzymology , Muscle, Skeletal/pathology , Myoblasts/enzymology , Pedigree , Phenotype
14.
J Neurol Sci ; 276(1-2): 170-4, 2009 Jan 15.
Article in English | MEDLINE | ID: mdl-19000626

ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.


Subject(s)
Amyotrophic Lateral Sclerosis , Genetic Predisposition to Disease , Mitochondria, Muscle/pathology , Mitochondrial Diseases/etiology , Mutation/genetics , Superoxide Dismutase/genetics , Adult , Amyotrophic Lateral Sclerosis/complications , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Arginine/genetics , Cytochromes c/metabolism , DNA Mutational Analysis , Family Health , Genetic Linkage/physiology , Glutamic Acid/genetics , Humans , Male , Mitochondria, Muscle/ultrastructure , Superoxide Dismutase-1
15.
J Clin Invest ; 118(10): 3316-30, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18769634

ABSTRACT

Spinal muscular atrophy (SMA), a motor neuron disease (MND) and one of the most common genetic causes of infant mortality, currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study, we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern, including changes in RNA metabolism proteins, cell cycle proteins, and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA.


Subject(s)
Disease Models, Animal , Fetal Stem Cells/transplantation , Muscular Atrophy, Spinal/surgery , Neurons/cytology , Stem Cell Transplantation , Animals , Female , Gene Expression Profiling , Gene Expression Regulation , Male , Mice , Mice, Knockout , Motor Neurons/cytology , Motor Neurons/metabolism , Muscular Atrophy, Spinal/metabolism , Muscular Atrophy, Spinal/mortality , Neurons/metabolism , Phenotype , Spinal Cord/cytology , Survival Analysis , Weight Loss
16.
Ann Neurol ; 62(1): 81-92, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17503505

ABSTRACT

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.


Subject(s)
Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/pathology , Motor Neurons/drug effects , RNA, Small Interfering/therapeutic use , fas Receptor/genetics , Amyotrophic Lateral Sclerosis/chemically induced , Animals , Cell Death/drug effects , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Embryo, Mammalian , Fas Ligand Protein/pharmacology , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/metabolism , Receptors, Nerve Growth Factor/metabolism , Spinal Cord/pathology , Superoxide Dismutase/genetics , Time Factors , Transfection
17.
Exp Neurol ; 205(2): 547-62, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17466977

ABSTRACT

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.


Subject(s)
Antigens, CD/biosynthesis , Glycoproteins/biosynthesis , Neurons/metabolism , Stem Cells/metabolism , AC133 Antigen , Animals , Cell Differentiation/physiology , Cell Separation , Female , Green Fluorescent Proteins/metabolism , Immunohistochemistry , Lateral Ventricles/cytology , Lateral Ventricles/physiology , Mice , Mice, Inbred C57BL , Neurons/transplantation , Peptides , Pregnancy , Reverse Transcriptase Polymerase Chain Reaction , Stem Cell Transplantation
18.
Brain ; 130(Pt 5): 1289-305, 2007 May.
Article in English | MEDLINE | ID: mdl-17439986

ABSTRACT

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.


Subject(s)
Amyotrophic Lateral Sclerosis/surgery , Lewis X Antigen/metabolism , Multipotent Stem Cells/transplantation , Receptors, CXCR4/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Axons/pathology , Biomarkers/analysis , Cell Count , Cell Differentiation , Clone Cells , Disease Models, Animal , Disease Progression , Enzyme-Linked Immunosorbent Assay , Glial Fibrillary Acidic Protein/analysis , Immunohistochemistry , Insulin-Like Growth Factor I/analysis , Lewis X Antigen/analysis , Lewis X Antigen/genetics , Mice , Mice, Transgenic , Microscopy, Confocal , Motor Neurons/pathology , Multipotent Stem Cells/metabolism , Nerve Regeneration , Receptors, CXCR4/analysis , Receptors, CXCR4/genetics , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/pathology , Superoxide Dismutase/genetics , Vascular Endothelial Growth Factor A/analysis
19.
Hum Mol Genet ; 15(2): 167-87, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16339214

ABSTRACT

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal-recessive motor neuron disease caused by mutations in the immunoglobulin micro-binding protein 2. We investigated the potential of a spinal cord neural stem cell population isolated on the basis of aldehyde dehydrogenase (ALDH) activity to modify disease progression of nmd mice, an animal model of SMARD1. ALDH(hi)SSC(lo) stem cells are self-renewing and multipotent and when intrathecally transplanted in nmd mice generate motor neurons properly localized in the spinal cord ventral horns. Transplanted nmd animals presented delayed disease progression, sparing of motor neurons and ventral root axons and increased lifespan. To further investigate the molecular events responsible for these differences, microarray and real-time reverse transcription-polymerase chain reaction analyses of wild-type, mutated and transplanted nmd spinal cord were undertaken. We demonstrated a down-regulation of genes involved in excitatory amino acid toxicity and oxidative stress handling, as well as an up-regulation of genes related to the chromatin organization in nmd compared with wild-type mice, suggesting that they may play a role in SMARD1 pathogenesis. Spinal cord of nmd-transplanted mice expressed high transcript levels for genes related to neurogenesis such as doublecortin (DCX), LIS1 and drebrin. The presence of DCX-expressing cells in adult nmd spinal cord suggests that both exogenous and endogenous neurogeneses may contribute to the observed nmd phenotype amelioration.


Subject(s)
Gene Expression Regulation , Motor Neurons/cytology , Spinal Cord/metabolism , Spinal Muscular Atrophies of Childhood/physiopathology , Stem Cell Transplantation , Stem Cells/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Aldehyde Dehydrogenase/metabolism , Animals , Blotting, Western , Cells, Cultured , Disease Progression , Doublecortin Domain Proteins , Doublecortin Protein , Immunohistochemistry , Mice , Mice, Mutant Strains , Microarray Analysis , Microtubule-Associated Proteins/metabolism , Neuropeptides/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/cytology , Stem Cells/cytology , Survival Analysis
20.
Stem Cells ; 24(4): 975-85, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16293577

ABSTRACT

Stem cells are undifferentiated cells defined by their ability to self-renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues, and an emerging idea is that they share common characteristics such as the presence of ATP-binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity, raising the hypothesis of a set of universal stem cell markers. In the present study, we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue, based on the expression of high levels of ALDH activity. Single-cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSC(lo)) and bright ALDH (ALDH(br)) activity was isolated. SSC(lo)ALDH(br) cells are capable of self-renewal and are able to generate new neurospheres and neuroepithelial stem-like cells. Furthermore, these cells are multipotent, differentiating both in neurons and macroglia, as determined by immunocytochemistry and real-time reverse transcription-polymerase chain reaction analysis. To evaluate the engraftment potential of SSC(lo)ALDH(br) cells in vivo, we transplanted them into mouse brain. Donor-derived neurons with mature morphology were detected in the cortex and subcortical areas, demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs, and improvement of the stem cell isolation protocol may be useful in the development of a cell-mediated therapeutic strategy for neurodegenerative diseases.


Subject(s)
Aldehyde Dehydrogenase/metabolism , Brain/cytology , Brain/enzymology , Multipotent Stem Cells/cytology , Multipotent Stem Cells/enzymology , Neurons/cytology , Neurons/enzymology , Animals , Biomarkers/metabolism , Cell Separation , Cerebral Ventricles , Flow Cytometry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multipotent Stem Cells/classification , Neurons/classification , Stem Cell Transplantation
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