Christopher E. Henderson

Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell types affected by that patients disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.

Neuronal Cell Death

We thank J.-F. Brunet, P. G. H. Clarke, O. deLapeyriere, P. Golstein, J.-C. Martinou, C. Mulle, and R. W. Oppenheim for thoughtful comments on the manuscript and C. Waldmann for artwork. In a short review such as this, it is not possible to establish precedence for all ideas or mechanisms, many of which originate from (or are still confined to) studies outside the nervous system. Wherever possible, we have therefore illustrated each topic by citing recent articles involving neurons. We refer the interested reader to the reference lists of those articles, or of the many excellent reviews we have cited, for more ample details. Studies from our laboratory were supported by INSERM, CNRS, Association Francaise contre les Myopathies (AFM), Institut pour la Recherche sur la Moelle Epiniere (IRME), and European Commission BIO4 contract CT960433.

Persephin, a Novel Neurotrophic Factor Related to GDNF and Neurturin

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS

Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1G93A transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.

Motoneuron Death Triggered by a Specific Pathway Downstream of Fas: Potentiation by ALS-Linked SOD1 Mutations

Death pathways restricted to specific neuronal classes could potentially allow for precise control of developmental neuronal death and also underlie the selectivity of neuronal loss in neurodegenerative disease. We show that Fas-triggered death of normal embryonic motoneurons requires transcriptional upregulation of neuronal NOS and involves Daxx, ASK1, and p38 together with the classical FADD/caspase-8 cascade. No evidence for involvement of this pathway was found in cells other than motoneurons. Motoneurons from transgenic mice overexpressing ALS-linked SOD1 mutants (G37R, G85R, or G93A) displayed increased susceptibility to activation of this pathway: they were more sensitive to Fas- or NO-triggered cell death but not to trophic deprivation or excitotoxic stimulation. Thus, triggering of a motoneuron-restricted cell death pathway by neighboring cells might contribute to motoneuron loss in ALS.

A functionally characterized test set of human induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) present exciting opportunities for studying development and for in vitro disease modeling. However, reported variability in the behavior of iPSCs has called their utility into question. We established a test set of 16 iPSC lines from seven individuals of varying age, sex and health status, and extensively characterized the lines with respect to pluripotency and the ability to terminally differentiate. Under standardized procedures in two independent laboratories, 13 of the iPSC lines gave rise to functional motor neurons with a range of efficiencies similar to that of human embryonic stem cells (ESCs). Although three iPSC lines were resistant to neural differentiation, early neuralization rescued their performance. Therefore, all 16 iPSC lines passed a stringent test of differentiation capacity despite variations in karyotype and in the expression of early pluripotency markers and transgenes. This iPSC and ESC test set is a robust resource for those interested in the basic biology of stem cells and their applications.

Cardiotrophin-1, a Cytokine Present in Embryonic Muscle, Supports Long-Term Survival of Spinal Motoneurons

The muscle-derived factors required for survival of embryonic motoneurons are not clearly identified. Cardiotrophin-1 (CT-1), a cytokine related to ciliary neurotrophic factor (CNTF), is expressed at high levels in embryonic limb bud and is secreted by differentiated myotubes. In vitro, CT-1 kept 43% of purified E14 rat motoneurons alive for 2 weeks (EC50 = 20 pM). In vivo, CT-1 protected neonatal sciatic motoneurons against the effects of axotomy. CT-1 action on motoneurons was inhibited by phosphatidylinositol-specific phospholipase C (PIPLC), suggesting that CT-1 may act through a GPI-linked component. Since no binding of CT-1 to CNTFR alpha was detected, CT-1 may use a novel cytokine receptor alpha subunit. CT-1 may be important in normal motoneuron development and as a potential tool for slowing motoneuron degeneration in human diseases.

Motor Neuron Diversity in Development and Disease

Although often considered as a group, spinal motor neurons are highly diverse in terms of their morphology, connectivity, and functional properties and differ significantly in their response to disease. Recent studies of motor neuron diversity have clarified developmental mechanisms and provided novel insights into neurodegeneration in amyotrophic lateral sclerosis (ALS). Motor neurons of different classes and subtypes--fast/slow, alpha/gamma--are grouped together into motor pools, each of which innervates a single skeletal muscle. Distinct mechanisms regulate their development. For example, glial cell line-derived neurotrophic factor (GDNF) has effects that are pool-specific on motor neuron connectivity, column-specific on axonal growth, and subtype-specific on survival. In multiple degenerative contexts including ALS, spinal muscular atrophy (SMA), and aging, fast-fatigable (FF) motor units degenerate early, whereas motor neurons innervating slow muscles and those involved in eye movement and pelvic sphincter control are strikingly preserved. Extrinsic and intrinsic mechanisms that confer resistance represent promising therapeutic targets in these currently incurable diseases.

ETS Gene Pea3 Controls the Central Position and Terminal Arborization of Specific Motor Neuron Pools

The projection of developing axons to their targets is a crucial step in the assembly of neuronal circuits. In the spinal cord, the differentiation of specific motor neuron pools is associated with the expression of ETS class transcription factors, notably PEA3 and ER81. Their initial expression coincides with the arrival of motor axons in the vicinity of muscle targets and depends on limb-derived signals. We show that in Pea3 mutant mice, the axons of specific motor neuron pools fail to branch normally within their target muscles, and the cell bodies of these motor neurons are mispositioned within the spinal cord. Thus, the induction of an intrinsic program of ETS gene expression by peripheral signals is required to coordinate the central position and terminal arborization of specific sets of spinal motor neurons.

Identification and characterization of cholest-4-en-3-one, oxime (TRO19622), a novel drug candidate for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1G93A transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.