Early functional impairment of sensory-motor connectivity in a mouse model of spinal muscular atrophy

To define alterations of neuronal connectivity that occur during motor neuron degeneration, we characterized the function and structure of spinal circuitry in spinal muscular atrophy (SMA) model mice. SMA motor neurons show reduced proprioceptive reflexes that correlate with decreased number and function of synapses on motor neuron somata and proximal dendrites. These abnormalities occur at an early stage of disease in motor neurons innervating proximal hindlimb muscles and medial motor neurons innervating axial muscles, but only at end-stage disease in motor neurons innervating distal hindlimb muscles. Motor neuron loss follows afferent synapse loss with the same temporal and topographical pattern. Trichostatin A, which improves motor behavior and survival of SMA mice, partially restores spinal reflexes, illustrating the reversibility of these synaptic defects. Deafferentation of motor neurons is an early event in SMA and may be a primary cause of motor dysfunction that is amenable to therapeutic intervention.     

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA.     

Selective Vulnerability of Spinal and Cortical Motor Neuron Subpopulations in delta7 SMA Mice

Loss of the survival motor neuron gene (SMN1) is responsible for spinal muscular atrophy (SMA), the most common inherited cause of infant mortality. Even though the SMA phenotype is traditionally considered as related to spinal motor neuron loss, it remains debated whether the specific targeting of motor neurons could represent the best therapeutic option for the disease. We here investigated, using stereological quantification methods, the spinal cord and cerebral motor cortex of ∆7 SMA mice during development, to verify extent and selectivity of motor neuron loss. We found progressive post-natal loss of spinal motor neurons, already at pre-symptomatic stages, and a higher vulnerability of motor neurons innervating proximal and axial muscles. Larger motor neurons decreased in the course of disease, either for selective loss or specific developmental impairment. We also found a selective reduction of layer V pyramidal neurons associated with layer V gliosis in the cerebral motor cortex. Our data indicate that in the ∆7 SMA model SMN loss is critical for the spinal cord, particularly for specific motor neuron pools. Neuronal loss, however, is not selective for lower motor neurons. These data further suggest that SMA pathogenesis is likely more complex than previously anticipated. The better knowledge of SMA models might be instrumental in shaping better therapeutic options for affected patients.     

DJ-1 Knockout Augments Disease Severity and Shortens Survival in a Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, neurodegenerative disorder, characterized by the degeneration of motor neurons. Oxidative stress plays a central role in the disease progression, in concert with an enhanced glutamate excitotoxicity and neuroinflammation. DJ-1 mutations, leading to the loss of functional protein, cause familial Parkinson’s disease and motor neuron disease in several patients. DJ-1 responds to oxidative stress and plays an important role in the cellular defense mechanisms. We aimed to investigate whether loss of functional DJ-1 alters the disease course and severity in an ALS mouse model. To this end we used mice that express the human SOD1^G93A mutation, the commonly used model of ALS and knockout of DJ-1 mice to generate SOD1 DJ-1 KO mice. We found that knocking out DJ-1in the ALS model led to an accelerated disease course and shortened survival time. DJ-1 deficiency was found to increase neuronal loss in the spinal cord associated with increased gliosis in the spinal cord and reduced antioxidant response that was regulated by the Nrf2 mechanism.The importance of DJ-1 in ALS was also illustrated in a motor neuron cell line that was exposed to glutamate toxicity and oxidative stress. Addition of the DJ-1 derived peptide, ND-13, enhanced the resistance to glutamate and SIN-1 induced toxicity. Thus, our results maintain that DJ-1 plays a role in the disease process and promotes the necessity of further investigation of DJ-1 as a therapeutic target for ALS.     

Molecular,genetic and stem cell‐mediated therapeutic strategies for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell‐mediated approaches for the treatment of SMA.     

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 153‐169, 2011     

The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein

Spinal muscular atrophy is an inherited motor neuron disease that results from a deficiency of the survival of motor neuron (SMN) protein. SMN is ubiquitinated and degraded through the ubiquitin proteasome system (UPS). We have previously shown that proteasome inhibition increases SMN protein levels, improves motor function, and reduces spinal cord, muscle, and neuromuscular junction pathology of spinal muscular atrophy (SMA) mice. Specific targets in the UPS may be more efficacious and less toxic. In this study, we show that the E3 ubiquitin ligase, mind bomb 1 (Mib1), interacts with and ubiquitinates SMN and facilitates its degradation. Knocking down Mib1 levels increases SMN protein levels in cultured cells. Also, knocking down the Mib1 orthologue improves neuromuscular function in Caenorhabditis elegans deficient in SMN. These findings demonstrate that Mib1 ubiquitinates and catalyzes the degradation of SMN, and thus represents a novel therapeutic target for SMA.     

The molecular bases of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common recessive autosomal disorder characterized by degeneration of motor neurons of the spinal cord. SMA is caused by mutations of the survival of motor neuron gene that encodes a multifunctional protein, and mouse models have been generated. These advances represent starting points towards an understanding of the pathophysiology of this disease and the design of therapeutic strategies in SMA.     

Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a genetic disorder caused by a deletion of the survival motor neuron 1 gene leading to motor neuron loss, muscle atrophy, paralysis, and death. We show here that induced pluripotent stem cell (iPSC) lines generated from two Type I SMA subjects-one produced with lentiviral constructs and the second using a virus-free plasmid-based approach-recapitulate the disease phenotype and generate significantly fewer motor neurons at later developmental time periods in culture compared to two separate control subject iPSC lines. During motor neuron development, both SMA lines showed an increase in Fas ligand-mediated apoptosis and increased caspase-8 and-3 activation. Importantly, this could be mitigated by addition of either a Fas blocking antibody or a caspase-3 inhibitor. Together, these data further validate this human stem cell model of SMA, suggesting that specific inhibitors of apoptotic pathways may be beneficial for patients.     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Reorganization of Cajal bodies and nucleolar targeting of coilin in motor neurons of type I spinal muscular atrophy

Type I spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by loss or mutations of the survival motor neuron 1 (SMN1) gene. The reduction in SMN protein levels in SMA leads to degeneration and death of motor neurons. In this study, we have analyzed the nuclear reorganization of Cajal bodies, PML bodies and nucleoli in type I SMA motor neurons with homozygous deletion of exons 7 and 8 of the SMN1 gene. Western blot analysis revealed a marked reduction of SMN levels compared to the control sample. Using a neuronal dissociation procedure to perform a careful immunocytochemical and quantitative analysis of nuclear bodies, we demonstrated a severe decrease in the mean number of Cajal bodies per neuron and in the proportion of motor neurons containing these structures in type I SMA. Moreover, most Cajal bodies fail to recruit SMN and spliceosomal snRNPs, but contain the proteasome activator PA28γ, a molecular marker associated with the cellular stress response. Neuronal stress in SMA motor neurons also increases PML body number. The existence of chromatolysis and eccentric nuclei in SMA motor neurons correlates with Cajal body disruption and nucleolar relocalization of coilin, a Cajal body marker. Our results indicate that the Cajal body is a pathophysiological target in type I SMA motor neurons. They also suggest the Cajal body-dependent dysfunction of snRNP biogenesis and, therefore, pre-mRNA splicing in these neurons seems to be an essential component for SMA pathogenesis.     

Maturation of spinal motor neurons derived from human embryonic stem cells

Our understanding of motor neuron biology in humans is derived mainly from investigation of human postmortem tissue and more indirectly from live animal models such as rodents. Thus generation of motor neurons from human embryonic stem cells and human induced pluripotent stem cells is an important new approach to model motor neuron function. To be useful models of human motor neuron function, cells generated in vitro should develop mature properties that are the hallmarks of motor neurons in vivo such as elaborated neuronal processes and mature electrophysiological characteristics. Here we have investigated changes in morphological and electrophysiological properties associated with maturation of neurons differentiated from human embryonic stem cells expressing GFP driven by a motor neuron specific reporter (Hb9::GFP) in culture. We observed maturation in cellular morphology seen as more complex neurite outgrowth and increased soma area over time. Electrophysiological changes included decreasing input resistance and increasing action potential firing frequency over 13 days in vitro. Furthermore, these human embryonic stem cell derived motor neurons acquired two physiological characteristics that are thought to underpin motor neuron integrated function in motor circuits; spike frequency adaptation and rebound action potential firing. These findings show that human embryonic stem cell derived motor neurons develop functional characteristics typical of spinal motor neurons in vivo and suggest that they are a relevant and useful platform for studying motor neuron development and function and for modeling motor neuron diseases.     

Human Motor Neurons Generated from Neural Stem Cells Delay Clinical Onset and Prolong Life in ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motor neuron disease. The etiology and pathogenic mechanisms of the disease remain unknown, and there is no effective treatment. Here we show that intrathecal transplantation of human motor neurons derived from neural stem cells (NSCs) in spinal cord of the SOD1G93A mouse ALS model delayed disease onset and extended life span of the animals. When HB1.F3.Olig2 (F3.Olig2) cells, stable immortalized human NSCs encoding the human Olig2 gene, were treated with sonic hedgehog (Shh) protein for 5–7 days, the cells expressed motor neuron cell type-specific phenotypes Hb9, Isl-1 and choline acetyltransferase (ChAT). These F3.Olig2-Shh human motor neurons were transplanted intrathecally in L5–L6 spinal cord of SOD1G93A mice, and at 4 weeks post-transplantation, transplanted F3.Olig2-Shh motor neurons expressing the neuronal phenotype markers NF, MAP2, Hb9, and ChAT were found in the ventral horn of the spinal cord. Onset of clinical signs in ALS mice with F3.Olig2-Shh motor neuron implants was delayed for 7 days and life span of animals was significantly extended by 20 days. Our results indicate that this treatment modality of intrathecal transplantation of human motor neurons derived from NSCs might be of value in the treatment of ALS patients without significant adverse effects.     

The contribution of mouse models to understanding the pathogenesis of spinal muscular atrophy

Spinal muscular atrophy (SMA), which is caused by inactivating mutations in the survival motor neuron 1 (SMN1) gene, is characterized by loss of lower motor neurons in the spinal cord. The gene encoding SMN is very highly conserved in evolution, allowing the disease to be modeled in a range of species. The similarities in anatomy and physiology to the human neuromuscular system, coupled with the ease of genetic manipulation, make the mouse the most suitable model for exploring the basic pathogenesis of motor neuron loss and for testing potential treatments. Therapies that increase SMN levels, either through direct viral delivery or by enhancing full-length SMN protein expression from the SMN1 paralog, SMN2, are approaching the translational stage of development. It is therefore timely to consider the role of mouse models in addressing aspects of disease pathogenesis that are most relevant to SMA therapy. Here, we review evidence suggesting that the apparent selective vulnerability of motor neurons to SMN deficiency is relative rather than absolute, signifying that therapies will need to be delivered systemically. We also consider evidence from mouse models suggesting that SMN has its predominant action on the neuromuscular system in early postnatal life, during a discrete phase of development. Data from these experiments suggest that the timing of therapy to increase SMN levels might be crucial. The extent to which SMN is required for the maintenance of motor neurons in later life and whether augmenting its levels could treat degenerative motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), requires further exploration.     

Analysis of SMN-neurite granules: Core Cajal body components are absent from SMN-cytoplasmic complexes

Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is a ubiquitously expressed house keeping protein that is involved in RNA production and processing. However, although SMN is expressed in every cell type, only the lower motor neurons of the spinal cord are degraded in SMA. It remains unclear why this is the case. Recently, SMN has been linked to the axonal transport of β-actin mRNA from the cell body down to the growth cones. β-Actin is transported actively in neurite granules (NGs). However, it remains unclear which known SMN-binding partners are present in these SMN-NGs. To address this we have analysed SMN-NGs in a human neuronal cell line, SH-SY5Y, using antibodies against the majority of reported SMN-binding partners, including: Gemin2, Gemin3, Gemin4, Gemin5, Gemin6, Gemin7, Sm core proteins, fibrillarin, EWS, PFNII, Unrip and ZPR1. The obtained results highlight the metamorphic nature of the SMN complex, suggesting that not all the “core” SMN-binding proteins are transported in SMN-NGs.     

PTEN regulates AMPA receptor-mediated cell viability in iPS-derived motor neurons

Excitatory transmission in the brain is commonly mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors. In amyotrophic lateral sclerosis (ALS), AMPA receptors allow cytotoxic levels of calcium into neurons, contributing to motor neuron injury. We have previously shown that oculomotor neurons resistant to the disease process in ALS show reduced AMPA-mediated inward calcium currents compared with vulnerable spinal motor neurons. We have also shown that PTEN (phosphatase and tensin homolog deleted on chromosome 10) knockdown via siRNA promotes motor neuron survival in models of spinal muscular atrophy (SMA) and ALS. It has been reported that inhibition of PTEN attenuates the death of hippocampal neurons post injury by decreasing the effective translocation of the GluR2 subunit into the membrane. In addition, leptin can regulate AMPA receptor trafficking via PTEN inhibition. Thus, we speculate that manipulation of AMPA receptors by PTEN may represent a potential therapeutic strategy for neuroprotective intervention in ALS and other neurodegenerative disorders. To this end, the first step is to establish a fibroblast–iPS–motor neuron in vitro cell model to study AMPA receptor manipulation. Here we report that iPS-derived motor neurons from human fibroblasts express AMPA receptors. PTEN depletion decreases AMPA receptor expression and AMPA-mediated whole-cell currents, resulting in inhibition of AMPA-induced neuronal death in primary cultured and iPS-derived motor neurons. Taken together, our results imply that PTEN depletion may protect motor neurons by inhibition of excitatory transmission that represents a therapeutic strategy of potential benefit for the amelioration of excitotoxicity in ALS and other neurodegenerative disorders.     

Programmed cell death and the gene behind spinal muscular atrophy.

A gene involved in the development of spinal muscular atrophy (SMA) has been found on human chromosome 5 after a 4-year search. Named the neuronal apoptosis inhibitor protein (NAIP) gene, it is believed to inhibit the normal process of apoptosis--the disintegration of single cells that results from programmed cell death--in motor neurons. The researchers who found the NAIP gene also discovered that healthy people carry one complete copy of the gene along with many other partial copies. Many children with SMA have the partial copies but not the complete gene. This discovery facilitates the accurate genetic diagnosis of SMA. But gene therapy for SMA will not be possible until researchers find a suitable vector to stably introduce activated and intact copies of the gene into the motor neurons of children with SMA in time to stop motor neuron loss.