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The thesis deals with the role of a family in particular life phases of a person suffering from the rare disease, spinal muscular atrophy. There are three main parts. The first one defines the term family, namely its system, features and functions, and explores the particularities of a family living with a handicapped member. The second part focuses on the spinal muscular atrophy, primarily on the development objectives of individual life phases of the people suffering from the disease. Thirdly, there is a section aiming at describing the role of the people taking care of the people challenged by spinal muscular atrophy from the early childhood to the middle adulthood. Furthermore, the diploma thesis points out the effort of these people to become independent and live their lives to the fullest with the help of care assistants, partners as well as start their families. For a better comprehension of the situation, there have been made semi-structured interviews with twelve families with a member of various age suffering from spinal muscular atrophy, inquiring not only the challenged persons, but also their parents and siblings.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are the commonest motor neuron diseases of adult- and childhood onset. Alterations of the RNA binding protein TDP-43 are associated with most cases of ALS, while SMA is caused by deletion of the Survival Motor Neuron (SMN1) gene. SMN has been well characterised in its role in the assembly of the cellular machinery that carries out splicing of pre-mRNA, but is thought to have other functions in RNA metabolism unrelated to pre-mRNA splicing. It is conceivable that specific aspects of RNA handling are disrupted in both SMA and ALS. A variety of genetic, molecular and neuropathological approaches were applied to investigate a potential common pathway in these diseases. The spectrum of genetic mutations underlying motor neuron disorders were explored by screening patient DNA. Cell culture and mouse models were used to test the hypothesis that altered pre-mRNA splicing causes motor neuron death. Human neuropathological specimens were examined for changes in proteins involved in RNA metabolism. The results indicate that altered pre-mRNA splicing is a late occurrence in disease and more likely to be a consequence rather than the cause of motor neuron degeneration. However, the notion that RNA metabolism is highly relevant to motor neuron diseases was strengthened by the discovery of mutations in another RNA binding protein, FUS, in cases of ALS without TDP-43 pathology. Overall the findings highlight the need to consider disruption of mRNA transport and regulation of mRNA translation in future motor neuron disease research.

Selective neuronal loss in response to loss or dysfunction of a ubiquitously expressed protein is a hallmark of neurodegenerative disease. Proximal spinal muscular atrophy (SMA) is caused by homozygous loss of the ubiquitously expressed survival motor neuron 1 (SMN1) gene, resulting in progressive neuromuscular weakness that eventually leads to flaccid paralysis and death from respiratory failure by two years of age in the most severely affected patients. Despite widespread motor neuron loss, certain motor pools are clinically spared. Type I SMA patients exhibit intercostal recession in conjunction with diaphragmatic sparing that produces a characteristic “bell-shaped chest.” Additionally, patients retain extraocular and external sphincter function, even in late disease stages. In order to fully define this differential vulnerability, I performed an extensive characterization of neuromuscular autopsies from Type I SMA patients and age-matched control patients. I found highly divergent degrees of motor unit degeneration, even within individual cranial nerves or a select anatomical region such as the neck. Remarkably, the diaphragm in a Type I SMA patient kept alive on life support for 17 years was still relatively preserved, despite virtually complete fibro-fatty infiltration in other muscles. Extraocular functions were also normal in this patient. These findings suggest that the molecular determinants of SMA-resistance provide indefinite protection against low SMN protein. Thus, identification and modulation of these genes and pathways represents a promising potential therapeutic strategy. Remarkably, this exquisite pattern of selectivity was preserved in the SMNΔ7 mouse, a widely used SMA mouse model. This suggests that the molecular determinants of differential vulnerability are conserved between mouse and human. Given the high degree of diversity between motor pools, I performed a comparative transcriptional microarray between multiple SMA-vulnerable and –resistant motor pools in healthy mice. This analysis revealed a small number of candidate therapeutic genes that segregate closely with vulnerability. I present a series of preliminary studies evaluating these targets in the SMNΔ7 mouse. Ongoing and future studies combine pharmacological, viral, and genetic approaches to modulate these candidate targets in the SMNΔ7 mouse and assess for improvements in neuromuscular pathology. Given the remarkable preservation of select motor pools in SMA patients, changing expression levels of the candidate targets I have identified may provide substantial clinical benefit.

Neuronal circuit perturbations are emerging as important determinants in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, and spinal muscular atrophy (SMA). SMA is a motor neuron disease caused by deficiency in the ubiquitously expressed survival motor neuron (SMN) protein. The hallmarks of SMA include loss of motor neurons, muscle atrophy, and abnormal postural reflexes. Although cell-autonomous mechanisms of motor neuron death have received much attention, recent studies in animal models of SMA revealed that motor circuit deficits resulting from early impairment of synaptic function and sensory-motor connectivity precede motor neuron death. It remains to be established whether motor circuit dysfunction is a consequence of SMN-deficiency in the motor neuron or SMN-dependent alterations in the activity of premotor neurons. Here I sought to address these outstanding issues through the development and characterization of a simplified in vitro model of the motor circuit based on the use of embryonic stem cell-derived motor neurons and interneurons. I found that SMN deficiency caused death of motor neurons in co-culture with other neurons as well as in isolation, demonstrating the cell autonomous origin of this defect. SMN requirement for motor neuron function was investigated using intracellular patch clamp recordings to measure both passive and active membrane properties. Remarkably, SMN deficiency induced hyperexcitability of motor neurons only when they are cultured in the presence of interneurons but not in isolation, providing initial evidence that SMN deficiency induces motor neuron hyperexcitability in a non-cell autonomous manner and that dysfunction and death of motor neurons are uncoupled. To determine the role of SMN-dependent interneuron dysfunction on motor neuron hyperexcitability, the effect of selective SMN depletion in either motor neurons or interneurons was investigated. Importantly, I found that SMN-deficient motor neurons cultured in the presence of wild type interneurons are not hyperexcitable, while the presence of SMN-deficient interneurons is necessary and sufficient to elicit hyperexcitability of wild type motor neurons. Therefore, in the context of SMN deficiency, increased excitability of motor neurons is a homeostatic response to interneuron dysfunction. Although the exact mechanism is currently unknown, reduced glutamatergic drive appears to play a role since glutamatergic receptor blockers phenocopied SMN deficiency in inducing motor neuron hyperexcitability but not neuronal death. Moreover, SMN deficiency caused reduction of excitatory VGluT2 synapses on motor neurons. In addition to changes in intrinsic membrane properties, SMN deficiency caused severe reduction in the spontaneous activity and firing pattern of motor neurons. However, in contrast to death and hyperexcitability, SMN-dependent deficits in both motor neurons and interneurons appear to underlie this complex phenotype. The findings presented in this study validate the use of in vitro models to study SMA disease mechanisms and shed new light on the cellular basis of motor circuit dysfunction induced by SMN deficiency that can have predictive value in vivo.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

Trichostatin A (TSA) is a histone deacetylase inhibitor with beneficial effects in spinal muscular atrophy mouse models that carry the human SMN2 transgene. Whether TSA specifically targets the upregulation of the SMN2 gene or whether other genes respond to TSA and in turn provide neuroprotection in SMA mice is unclear. We have taken advantage of the Smn2B/- mouse model that does not harbor the human SMN2 transgene, to test the hypothesis that TSA has its beneficial effects through a non-Smn mediated pathway. Daily intraperitoneal injection of TSA from postnatal day 12 to 25 was performed in the Smn2B/- mice and littermate controls. Previous work from our laboratory demonstrated that treatment with TSA increased the median lifespan of Smn2B/- mice from twenty days to eight weeks. As well, there was a significant attenuation of weight loss and improved motor behavior. Pen test and righting reflex both showed significant improvement, and motor neurons in the spinal cord of Smn2B/-mice were protected from degeneration. Both the size and maturity of neuromuscular junctions were significantly improved in TSA treated Smn2B/- mice. Here, we have shown that TSA treatment does not increase the levels of Smn protein in mouse embryonic fibroblasts or myoblasts obtained from the Smn2B/- mice. Further, qPCR analysis revealed no changes in the level of Smn transcripts in the brain or spinal cord of TSA-treated SMA mice. Similarly, western blot analysis revealed no significant increase in Smn protein levels in the brain, spinal cord, hind limb muscle, heart muscle, or the liver of TSA treated Smn2B/- mice. However, TSA has beneficial effects in the muscles of Smn2B/- mice and improves motor behavior and myofiber size. TSA improves muscle development by enhancing the activity of myogenic regulatory factors independent of the Smn gene. The beneficial effect of TSA is therefore likely through an Smn-independent manner. Identification of these protective pathways will be of therapeutic value for the treatment of SMA.