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.     

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.     

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.     

Infantile spinale Muskelatrophie: mehr als eine Motoneuronerkrankung?

Infantile spinal muscular atrophy (SMA) is characterized by loss of motor neurons in the ventral horn of the spinal cord leading to weakness and muscle atrophy and occurs as a result of homozygous deletions or mutations in the survival motor neuron (SMN 1) gene. Loss of SMN 1 leads to a dramatic reduction in survival motor neuron (SMN) protein in the motor neurons of the spinal cord and of the brain stem. The SMA disease severity ranges from extremely severe to a relatively mild adult onset form of proximal muscle atrophy. More recently, clinical case reports in patients and studies in animal models provided evidence that severe SMN protein deficiency not only results in loss of motor neurons but also to additional organ manifestations. These include the peripheral, central and autonomic nervous system, development and function of the heart and the digestive tract and metabolic deficiencies. Therefore, to develop the most efficient therapeutic approach and also prevent further complications in patients that may arise with extended survival following therapeutic interventions, it is necessary to investigate in detail the specific damage to every system independently. The comparison of the defects in SMA mouse models will provide valuable insights; however, phenotypic differences between mice and men still remain.     

The Wallerian degeneration slow (Wld(s)) gene does not attenuate disease in a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a severe neuromuscular disease characterized by loss of spinal α-motor neurons, resulting in the paralysis of skeletal muscle. SMA is caused by deficiency of survival motor neuron (SMN) protein levels. Recent evidence has highlighted an axon-specific role for SMN protein, raising the possibility that axon degeneration may be an early event in SMA pathogenesis. The Wallerian degeneration slow (Wld^s) gene is a spontaneous dominant mutation in mice that delays axon degeneration by approximately 2-3 weeks. We set out to examine the effect of Wld^s on the phenotype of a mouse model of SMA. We found that Wld^s does not alter the SMA phenotype, indicating that Wallerian degeneration does not directly contribute to the pathogenesis of SMA development.     

Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs

Spinal muscular atrophy (SMA) is a motor neuron disease caused by reduced levels of the survival motor neuron (SMN) protein. SMN together with Gemins2-8 and unrip proteins form a macromolecular complex that functions in the assembly of small nuclear ribonucleoproteins (snRNPs) of both the major and the minor splicing pathways. It is not known whether the levels of spliceosomal snRNPs are decreased in SMA. Here we analyzed the consequence of SMN deficiency on snRNP metabolism in the spinal cord of mouse models of SMA with differing phenotypic severities. We demonstrate that the expression of a subset of Gemin proteins and snRNP assembly activity are dramatically reduced in the spinal cord of severe SMA mice. Comparative analysis of different tissues highlights a similar decrease in SMN levels and a strong impairment of snRNP assembly in tissues of severe SMA mice, although the defect appears smaller in kidney than in neural tissue. We further show that the extent of reduction in both Gemin proteins expression and snRNP assembly activity in the spinal cord of SMA mice correlates with disease severity. Remarkably, defective SMN complex function in snRNP assembly causes a significant decrease in the levels of a subset of snRNPs and preferentially affects the accumulation of U11 snRNP--a component of the minor spliceosome--in tissues of severe SMA mice. Thus, impairment of a ubiquitous function of SMN changes the snRNP profile of SMA tissues by unevenly altering the normal proportion of endogenous snRNPs. These findings are consistent with the hypothesis that SMN deficiency affects the splicing machinery and in particular the minor splicing pathway of a rare class of introns in SMA.     

Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.     

Time Is Motor Neuron: Therapeutic Window and Its Correlation with Pathogenetic Mechanisms in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by the degeneration of lower motor neurons (MNs) in the spinal cord and brain stem, which results in relentless muscle weakness and wasting, leading to premature death due to respiratory complications. The identification of the specific mutations in the survival motor neuron 1 (SMN1) gene that causes SMA has led to the development of experimental therapeutic strategies to increase SMN protein expression, including antisense oligonucleotides, small molecules, and gene therapy, which have so far shown promising results. The timing of therapeutic intervention is crucial since most of the degeneration in MNs occurs in the first months of life in patients with SMA type 1, which is the most severe and common form of SMA. Nevertheless, a precise temporal window for therapeutic intervention has not yet been identified. Evidence from in vivo studies in mice and large animals suggested that early therapeutic intervention for SMA correlated with better motor performance, longer survival, and, occasionally, rescue of the pathological phenotype. Indeed, the need to compensate for the loss of SMN protein function seemed to diminish during adulthood (even though repair ability after nerve injury remained impaired), suggesting the possibility of tapering the therapy administration late in the disease course. Moreover, recent clinical trials on children afflicted with SMA type 1 have shown a more rapid achievement of motor milestones and diminished disease severity when therapy was administered at an early age and earlier in the disease course. Finally, these results highlight the importance of newborn screening for SMA to facilitate early diagnosis and present the patient with available treatments while they are still in the presymptomatic stage.     

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.     

Haploinsufficiency for Translation Elongation Factor eEF1A2 in Aged Mouse Muscle and Neurons Is Compatible with Normal Function

Translation elongation factor isoform eEF1A2 is expressed in muscle and neurons. Deletion of eEF1A2 in mice gives rise to the neurodegenerative phenotype "wasted" (wst). Mice homozygous for the wasted mutation die of muscle wasting and neurodegeneration at four weeks post-natal. Although the mutation is said to be recessive, aged heterozygous mice have never been examined in detail; a number of other mouse models of motor neuron degeneration have recently been shown to have similar, albeit less severe, phenotypic abnormalities in the heterozygous state. We therefore examined the effects of ageing on a cohort of heterozygous +/wst mice and control mice, in order to establish whether a presumed 50% reduction in eEF1A2 expression was compatible with normal function. We evaluated the grip strength assay as a way of distinguishing between wasted and wild-type mice at 3-4 weeks, and then performed the same assay in older +/wst and wild-type mice. We also used rotarod performance and immunohistochemistry of spinal cord sections to evaluate the phenotype of aged heterozygous mice. Heterozygous mutant mice showed no deficit in neuromuscular function or signs of spinal cord pathology, in spite of the low levels of eEF1A2.     

IPLEX Administration Improves Motor Neuron Survival and Ameliorates Motor Functions in a Severe Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disorder and the first genetic cause of death in childhood. SMA is caused by low levels of survival motor neuron (SMN) protein that induce selective loss of α-motor neurons (MNs) in the spinal cord, resulting in progressive muscle atrophy and consequent respiratory failure. To date, no effective treatment is available to counteract the course of the disease. Among the different therapeutic strategies with potential clinical applications, the evaluation of trophic and/or protective agents able to antagonize MNs degeneration represents an attractive opportunity to develop valid therapies. Here we investigated the effects of IPLEX (recombinant human insulinlike growth factor 1 [rhIGF-1] complexed with recombinant human IGF-1 binding protein 3 [rhIGFBP-3]) on a severe mouse model of SMA. Interestingly, molecular and biochemical analyses of IGF-1 carried out in SMA mice before drug administration revealed marked reductions of IGF-1 circulating levels and hepatic mRNA expression. In this study, we found that perinatal administration of IPLEX, even if does not influence survival and body weight of mice, results in reduced degeneration of MNs, increased muscle fiber size and in amelioration of motor functions in SMA mice. Additionally, we show that phenotypic changes observed are not SMN-dependent, since no significant SMN modification was addressed in treated mice. Collectively, our data indicate IPLEX as a good therapeutic candidate to hinder the progression of the neurodegenerative process in SMA.     

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.     

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     

Modeling spinal muscular atrophy in Drosophila

Spinal Muscular Atrophy (SMA), a recessive hereditary neurodegenerative disease in humans, has been linked to mutations in the survival motor neuron (SMN) gene. SMA patients display early onset lethality coupled with motor neuron loss and skeletal muscle atrophy. We used Drosophila, which encodes a single SMN ortholog, survival motor neuron (Smn), to model SMA, since reduction of Smn function leads to defects that mimic the SMA pathology in humans. Here we show that a normal neuromuscular junction (NMJ) structure depends on SMN expression and that SMN concentrates in the post-synaptic NMJ regions. We conducted a screen for genetic modifiers of an Smn phenotype using the Exelixis collection of transposon-induced mutations, which affects approximately 50% of the Drosophila genome. This screen resulted in the recovery of 27 modifiers, thereby expanding the genetic circuitry of Smn to include several genes not previously known to be associated with this locus. Among the identified modifiers was wishful thinking (wit), a type II BMP receptor, which was shown to alter the Smn NMJ phenotype. Further characterization of two additional members of the BMP signaling pathway, Mothers against dpp (Mad) and Daughters against dpp (Dad), also modify the Smn NMJ phenotype. The NMJ defects caused by loss of Smn function can be ameliorated by increasing BMP signals, suggesting that increased BMP activity in SMA patients may help to alleviate symptoms of the disease. These results confirm that our genetic approach is likely to identify bona fide modulators of SMN activity, especially regarding its role at the neuromuscular junction, and as a consequence, may identify putative SMA therapeutic targets.     

Induction of cyclooxygenase-2 in reactive glial cells by the CD40 pathway: relevance to amyotrophic lateral sclerosis

An inflammatory process in association with reactive gliosis has been suggested to play an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). One of the key findings is a marked increase in the level of cyclooxygenase-2 (COX-2), a therapeutic target of ALS. We investigated the expression of CD40 in the spinal cord of a transgenic mouse model of ALS (G93A mice), and its relevance to COX-2 upregulation. CD40 was predominantly expressed in neurons in normal spinal cord and upregulated in reactive glial cells in spinal cord injury. In the spinal cord of G93A mice, the expression of CD40 was increased in both reactive microglia and astrocytes, where COX-2 was especially increased. The level of COX-2 was upregulated in microglia and astrocytes by CD40 stimulation in vitro. CD40 stimulation in primary spinal cord cultures caused motor neuron loss that was protected by selective COX-2 inhibitor. These results suggest that CD40, which is upregulated in reactive glial cells in ALS, participates in motor neuron loss via induction of COX-2.