Spinal Muscular Atrophy: From Animal Model to Treatment
Date Issued
2010
Date
2010
Author(s)
Tsai, Li-Kai
Abstract
Spinal muscular atrophy (SMA) is characterized by the degeneration of spinal motor neurons, and it is associated with muscle paralysis and atrophy. Childhood SMA exhibits an autosomal recessive pattern of inheritance. Based on age of onset of symptoms and achievement of motor milestones, SMA has been subdivided into three clinical types. No curative treatment is available so far for SMA.
Several mouse models for SMA have been generated through different methods. Because of the disadvantage of short survival time, heterogeneous phenotype or the non-homologous genetic situation compared to human SMA in these mouse models, there is still no standard preclinical therapeutic testing system for SMA. Type III (mild-type) SMA mice have a lifespan longer than half a year, offering a chance to become a good therapeutic testing system. This study first characterized the type III SMA mice by a series of examinations.
For characterization of SMA mice, type III SMA mice (Smn−/−SMN2+/−) and wild-type mice (Smn+/+) were subdivided into four subgroups, according to their evaluated age: 3, 6, 9 and 12 months old. Each mouse underwent morphological, motor functional, electrophysiological and pathological studies.
The results revealed that motor neuron degeneration in type III SMA mice started at 6 months of age, and the number of residual motor neurons reduced to 71% of wild-type mice at the age of 9 months. At the same time, SMA mice exhibited clear morphological changes such as short tails and irregular ears, motor function decline demonstrated by four motor functional tests and muscle atrophy measured by gastrocnemius muscle weight. By 12 months old, the number of residual motor neurons reduced to 60% of wild-type mice. In addition, the amplitudes of compound muscle action potentials (CMAPs) in SMA mice had decreased significantly.
Valproic acid (VPA), a histone deacetylase inhibitor, increased SMN protein levels in cells derived from SMA patients. VPA also improved muscle strength in some SMA patients. SMA-related physicians and patients are becoming more interested in VPA; however, the therapeutic effects of VPA in treating SMA are still not fully understood. This study thus further investigated the treatment effects and therapeutic mechanisms of VPA in type III SMA mice.
Type III SMA mice were randomly assigned to treated and untreated groups. In treated mice, VPA was added to the daily drinking water at 0.2 mg/ml from 6 to 12 months of age. The therapeutic effects were evaluated using morphological tests, motor functional tests, electrophysiological studies, spinal pathological tests, and muscle pathological studies. For investigation of therapeutic mechanisms of VPA, Western blots, real-time PCR, and immunohistochemistry were used.
We found that VPA-treated SMA mice showed considerably reduced degeneration of spinal motor neurons compared to untreated SMA mice. As such, their motor function was better preserved with less muscle denervation and atrophy, greater CMAP amplitudes on sciatic nerves, and less morphological abnormalities than seen in untreated SMA mice. In addition, VPA exhibited multiple therapeutic effects in SMA. VPA elevated SMN protein levels in the spinal motor neurons. VPA also increased the levels of Bcl-2 and Bcl-xL proteins in the spinal neurons, which may reduce motor neuron apoptosis in SMA. Furthermore, VPA probably could induce neurogenesis and promoted astrocyte proliferation in the SMA mouse spinal cord, which might contribute to therapeutic effects by enhancing neuroprotection.
Moreover, Bcl-xL is an anti-apoptotic member of the Bcl-2 family and acts by inhibiting proapoptotic members of the Bcl-2 family through heterodimerization. Viral-mediated Bcl-xL expression can protect motor neuron death in primary motor neuron cultures. Since the degeneration of spinal motor neurons in SMA is mediated by apoptosis, over-expression of Bcl-xL may be of benefit in SMA. This study then investigated the benefits of Bcl-xL transgenes in type III SMA mice.
Crossing type III SMA mice with Bcl-xL transgenic mice created SMA/Bcl-xL mice. The Bcl-xL transgenic effects were evaluated using morphological tests, motor functional tests, electrophysiological studies, spinal pathological tests, and survival analysis. For investigation of SMN and Bcl-xL expression and degrees of spinal apoptosis, Western blots and immunohistochemistry were used.
The levels of Bcl-xL protein in spinal neurons of SMA/Bcl-xL mice were nearly double of that in SMA mice while the levels of SMN protein were similar between these two groups. The SMA/Bcl-xL mice showed preserved motor function, normalized electrophysiological tests, diminished muscle atrophy, and less motor neuron apoptosis and degeneration. Although the SMA/Bcl-xL mice still showed the typical SMA morphological phenotypes throughout their lives, they lived 1.5 times longer than SMA mice.
In summary, the above studies established a standard therapeutic testing system for SMA with type III SMA mice. The morphological, motor functional, electrophysiological and spinal pathological tests here could be used to evaluate treatment responses in both treated and non-treated mice between 9 and 12 months of age. In addition, by a combination of effects such as increased SMN protein levels, anti-apoptosis, and probable neuroprotection, VPA showed considerable therapeutic benefit in the mouse model of type III SMA. Over-expression of Bcl-xL has a potential for amelioration of SMA.
Subjects
spinal muscular atrophy
animal model
bcl-xL
apoptosis
Type
thesis
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