Dissertations / Theses on the topic 'Spinal muscular atrophy; Neurodegenerative'

Spinal bulbar muscular atrophy (SBMA) is a member of the polyglutamine (polyQ) expansion diseases family; the most famous of which is Huntington disease (HD). SBMA is caused by the expansion of the coding region for the polyQ tract in the exon 1 of androgen receptor (AR), which represents the N-terminal intrinsically disordered transactivation domain (NTD). AR is the nuclear receptor sensible to testosterone and aggregates of this polyQ-expanded protein are observed in the motor neurons of SBMA patients. The aggregation mechanism of polyQ proteins depends both on the length of the tract and on the chemical properties of the regions flanking it, that can increase or decrease the rate of aggregation depending on their secondary structure. In order to study the structure of the polyQ tract in AR and the mechanism by which this protein forms aggregates, we developed recombinant proteins designed over the N-terminal fragment of cleavage of a caspase (Caspase 3) associated to the onset of the toxicity in SBMA. We also developed a set of biophysical tools for rendering these aggregation-prone proteins monomeric and to monitor their evolution from the monomer level to the fibril. These methodologically challenging endeavor allowed us to study the secondary structure of this intrinsically disordered protein as a monomer and then to monitor what regions are important in its oligomerization and aggregation. Bulk biophysical experiments and NMR indicated that the polyQ tract of AR is in α-helical conformation, unlike other polyQ tracts described in literature, and we demonstrated that this conformation is caused by the nucleating effect of an N-terminal flanking sequence of four Leu residues (54LLLL58). We also showed that the helical conformation of this tract prevents the polyQ to acquire the ß-sheet conformation and to progress as a fibril, as a deletion mutant of the 54LLLL58 motif aggregates and forms fibrils faster than the wild type. By measuring the aggregation rates of three different AR recombinant proteins with progressively higher polyQ length (4Q, 25Q and 51Q) emerged that the polyQ is not the only region responsible for oligomerization and we identified by NMR that a second region, N- terminal and far apart from the polyQ is responsible of the early oligomerization. By analysis of the chemical shifts in different NMR experiments we obtained that this region (23FQNLF27) however not entirely helical, is prone to interact and acquire secondary structure. Furthermore, this sequence is known to bind to the ligand binding domain (LBD) of AR in an interaction critical for its dimerization and subsequent translocation into the nucleus, which is called N/C interaction. The crystal structure of this complex shows 23FQNLF27 in α-helical conformation when bound to LBD. We then investigated what amino-acids were important in the interaction stabilizing the intereaction of 23FQNLF27. By mutational analysis and measurements of aggregation rates we demonstrated that the helicity of this region is important for the aggregation and mutations that increase the helicity also an increase the aggregation propensity of the protein. We also identified that the residues responsible for the contact are the Gln in position 2, 28 and 36 which form a ‘spine’ of polar residues in register along the α-helix. This polar side of the helix is not the one in contact with LBD during the N/C interaction and it is possible that the two events occur in parallel. In the complex, we characterized the early oligomerization of AR in the aggregation process associated to SBMA with the perspective to provide valuable information for the development of drugs for this diseases that has currently no treatment.
Les malalties neurodegeneratives són una de les malediccions de la civilització moderna i es troben estretament lligades a l’augment de l’esperança de vida de la població mundial. La majoria d’aquestes malalties estan associades a la deposició de material proteic, altrament conegut com a fibres amiloides, a les neurones i el cervell en general. Les fibres amiloides són conjunts supramoleculars lineals, composats per proteïnes disposades en fulla beta, que mostren una alta rigidesa i estabilitat termodinàmica. Exemples famosos de proteïnes amiloides són la beta amiloide (Aβ), associada a la malaltia d’Alzheimer, i l’α-­‐sinucleïna i la proteïna tau, més estretament lligades a la malaltia de Parkinson. Una altra família de desordres neurodegeneratius associats a la deposició de proteïnes és la de les malalties poliglutamines (poliQ). Aquesta família està formada per nou patologies, entre les que es troben sis atàxies espinocerebrals diferents (de les sigles en anglès, SCA 1, 2, 3, 6, 7, 17), la atròfia dentatorubral-­‐pallidoluysian (de les sigles en anglès, DRPLA) i la atròfia muscular espinal bulbar (de les sigles en anglès, SBMA), històricament la primera en ser descrita. Totes elles són hereditàries, dominants i es manifesten en edat avançada. D’altra banda, totes elles estan associades a l’adquisició de neurotoxicitat degut a l’agregació de la proteïna causant de la malaltia, que s’acumula progressivament a les neurones amb el temps. La mutació responsable de la malaltia és una expansió genètica a la regió polimòrfica de l’ADN que és comuna a totes le proteïnes associades en aquests enfermetats. Aquesta regió polimòrfica és un conjunt de repeticions CAG que codifiquen l’aminoàcid glutamina a nivell d’expressió de proteïna, és per això que es coneix com a tram de poliglutamines. Aquest tram pot tenir diverses longituds, però l’efecte tòxic només té lloc quan es supera un determinat límit d’allargada. Aquest límit fluctua entre 30 i 40 repeticions i varia de malaltia a malaltia, però en tots els casos el número de repeticions influencia la severitat i l’edat en la que s’inicia la malaltia. La raó que explica aquesta inestabilitat genètica resulta de la propensitat de les seqüències d’ADN altament repetitives (com ara els hairpins) que en determina el slippage de la cadena principal durant la replicació de l’ADN. Les expansions més llargues són causades per la reiteració d’aquesta petita mutació i s’ha observat una reducció progressiva de l’estabilitat genètica amb l’increment del número de repeticions, que en última instància determina un avançament temporal i empitjorament dels símptomes. Considerant l’estreta relació entre la presència d’agregats en els teixits dels pacients malalts i l’estadiatge de la malaltia, és fonamental entendre les propietats biofísiques dels trams de poliQ, com aquestes seqüències determinen l’agregació de la proteïna i el tipus d’estructura que presenten els agregats.

Spinal Muscular Atrophy (SMA) is a neuronal degenerative disease caused by the mutation or loss of the Survival Motor Neuron (SMN) gene. The cause for the specific motor neuron susceptibility in SMA has not been identified. The high axonal transport/localization demand on motor neurons may be one potentially disrupted function, more specific to these cells. We therefore used a large-scale immunoprecipitation (IP) experiment, to identify potential interactors of SMN involved in neuronal transport and localization of mRNA targets. We identified KH-type splicing regulatory protein (KSRP), a multifunctional RNA-binding protein that has been implicated in transcriptional regulation, neuro-specific alternative splicing, and mRNA decay. KSRP is closely related to chick zipcode-binding protein 2 and rat MARTA1, proteins involved in neuronal transport/localization of beta-actin and microtubule-associated protein 2 mRNAs, respectively. We demonstrated that KSRP is arginine methylated, a novel SMN interactor (specifically with the SMN Tudor domain; and not with SMA causing mutants). We also found this protein to be misregulated in the absence of SMN, resulting in increased mRNA stability of KSRP mRNA target, p21cip/waf1. A role for SMN as an axonal chaperone of methylated RBPs could thus be key in SMA pathophysiology.

Mounting evidence suggests that synaptic connections are early pathological targets in many neurodegenerative diseases, including motor neuron disease. A better understanding of synaptic pathology is therefore likely to be critical in order to develop effective therapeutic strategies. Spinal muscular atrophy (SMA) is a common autosomal recessive childhood form of motor neuron disease. Previous studies have highlighted nerve- and muscle-specific events in SMA, including atrophy of muscle fibres and postsynaptic motor endplates, loss of lower motor neuron cell bodies and denervation of neuromuscular junctions caused by loss of pre-synaptic inputs. Here I have undertaken a detailed morphological investigation of neuromuscular synaptic pathology in the Smn-/- ;SMN2 and Smn-/-;SMN2;Δ7 mouse models of SMA. Results imply that synaptic degeneration is an early and significant event in SMA, with progressive denervation and neurofilament accumulation being present at early symptomatic time points. I have identified selectively vulnerable motor units, which appear to conform to a distinct developmental subtype compared to more stable motor units. I have also identified significant postsynaptic atrophy which does no correlate with pre-synaptic denervation, suggesting that there is a requirement for Smn in both muscle and nerve and pathological events can occur in both tissues independently. Rigorous investigation of lower motor neuron development, connectivity and gene expression at pre-symptomatic time points revealed developmental abnormalities do not underlie neuromuscular vulnerability in SMA. Equivalent gene expression analysis at end-stage time points has implicated growth factor signalling and extracellular matrix integrity in SMA pathology. Using an alternative model of early onset neurodegeneration, I provide evidence that the processes regulating morphologically distinct types of synaptic degeneration are also mechanistically distinct. In summary, in this work I highlight the importance and incidence of synaptic pathology in mouse models of spinal muscular atrophy and provide mechanistic insight into the processes regulating neurodegeneration.

Spinal Muscular Atrophy (SMA) is an autosomal, recessive form of childhood motor neuron disease and the most common genetic cause of infant mortality in the western world. SMA displays the characteristic hallmarks of a motor neuron disease, including loss of motor neurons in the spinal cord and atrophy of skeletal muscles. However, mounting evidence suggests that multiple tissues and body systems, beyond the neuromuscular system, are affected in SMA. Previous studies have highlighted alterations in the vascular system in both SMA patients and in a variety of mouse models of the disease, reporting alterations in vessel structure and perfusion abnormalities in peripheral tissues. In this project a detailed morphological investigation of the capillary beds of skeletal muscle and the spinal cord, two of the key pathological tissues in SMA was undertaken. This work was conducted in the Smn-/-;SMN2, Smn-/-;SMN2tg/+ and Smn-/-;SMN2;Δ7 mouse models of SMA. Significant alterations in the form and extent of the skeletal muscle and spinal cord capillary bed in SMA mice were identified, the most striking of which being a reduction in capillary density in SMA tissue when compared to control littermate tissue. In skeletal muscle, this reduction in capillary density was found to be a postnatal phenomenon, which occurred independently of denervation, in a variety of phenotypically distinct muscles and in all three SMA mouse models investigated. In the spinal cord, the capillary defect was seen to develop in a similar postnatal pattern to that observed in skeletal muscle. Importantly, a reduction in capillary density was observed in the ventral horn of the spinal cord, which houses motor neuron cell bodies, a known pathological target in SMA. These motor neurons were seen to be surrounded by fewer capillaries than their control counterparts. Using an injectable marker of hypoxia, it was determined that the cells of the ventral horn of SMA spinal cords are hypoxic. This suggests that the capillary defect identified has a functional impact on the tissues it is observed in. Having established the presence of capillary defect in SMA tissue, the effect of potential SMA therapeutics on the capillary defect was then investigated. The effect of HDAC inhibitors, which have been successfully shown to increase the levels of the disease causing Smn protein, was investigated. Treatment with the HDAC inhibitor SAHA was found to ameliorate the capillary defect, significantly improving capillary density in SMA skeletal muscle. This implies that the capillary defect is related to Smn levels in tissue and is amenable to therapeutics which increase Smn levels. Having characterised the capillary defect in SMA tissues in detail, a selection of tools were then used to investigate the underlying mechanisms resulting in the defect. First, using primary cell cultures, the growth and morphology of the key cellular component of capillaries, the endothelial cell, was examined. While displaying reduced levels of the Smn protein, endothelial cells isolated from SMA tissues showed no difference in growth rate, morphology or endothelial cell marker expression when compared to endothelial cells isolated from control tissue. This suggests that the defects seen in SMA capillary beds are not the result of defects in the structure and growth of endothelial cells. Second, retinas from SMA mice were found to exhibit similar capillary defects to those observed in SMA skeletal muscle and spinal cord. Given the entirely postnatal development of the retinal capillary network, the retina was identified as a useful experimental preparation for the further investigation of the mechanisms underlying the capillary defect in SMA. In summary, this work highlights the incidence and importance of capillary defects in mouse models of spinal muscular atrophy.

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 25, 2010). Vita. Thesis advisor: Christian Lorson. "July 2009" Includes bibliographical references.

The childhood neuromuscular disease spinal muscular atrophy (SMA) is caused by low levels of survival motor neuron (SMN) protein. Historically, SMA has been characterised as a disease primarily affecting lower motor neurons. However, recent breakthroughs have revealed defects in other non-neuronal cells and tissues. In vivo analysis of peripheral nerve showed defects in Schwann cells, manifesting as abnormal myelination and delayed maturation of axo-glia interactions. The experiments in this thesis were designed to build on these observations and examine whether Schwann cell defects are intrinsic and occur as a primary result of low levels of SMN in that cell type, or rather represent a secondary consequence of pathology in neighbouring motor neurons. I initially developed a protocol to allow isolation of high-yields of purified, myelination-competent Schwann cells from ‘Taiwanese’ SMA mice. SMA-derived Schwann cells had significantly reduced SMN levels and failed to respond normally to differentiation cues. Increasing SMN levels restored myelin protein expression in Schwann cells from SMA mice. Perturbations in expression of key myelin proteins were likely due to failure of protein translation and/or stability rather than transcriptional defects. Co-cultures of healthy neurons with SMA Schwann cells revealed a significant reduction in myelination compared to cultures where wild-type Schwann cells were used. The presence of SMA Schwann cells also disrupted neurite stability. Perturbations in the expression of key extracellular matrix proteins, such as laminin α2, in SMA-derived Schwann cells suggests that Schwann cells were influencing neurite stability by modulating the composition of the extracellular matrix. Previous studies have demonstrated that low levels of SMN lead to disruption of ubiquitin homeostasis and decreased expression of ubiquitin-like modifier activating enzyme (UBA1) in the neuromuscular system, driving neuromuscular pathology via a beta-catenin dependent pathway. Label-free proteomics analysis of SMA and control Schwann cells identified 195 proteins with modified expression profiles. Bioinformatic analysis of these proteins using Ingenuity Pathway Analysis (IPA) software confirmed that major disruption of protein ubiquitination pathways was also present in Schwann cells from SMA mice. Immunolabeling and proteomics data both revealed that UBA1 levels were significantly reduced in SMA-derived Schwann cells. However, loss of UBA1 in Schwann cells did not lead to downstream modifications in beta-catenin pathways. Pharmacological inhibition of UBA1 in healthy Schwann cells was sufficient to induce defects in myelin protein expression, suggesting that UBA1 defects contribute directly to Schwann cell disruption in SMA. I conclude that low levels of SMN induce intrinsic defects in Schwann cells, mediated at least in part through disruption to ubiquitination pathways.

Low levels of survival motor neuron (SMN) protein cause the autosomal recessive neurodegenerative disease spinal muscular atrophy (SMA), through mechanisms that are poorly defined. SMN protein is ubiquitously expressed, however the major pathological hallmarks of SMA are focused on the neuromuscular system, including a loss of lower motor neurons in the ventral horn of the spinal cord and atrophy of skeletal muscle. At present there is no cure for SMA. Most research to date has focused on examining how low levels of SMN lead to pathological changes in motor neurons, therefore the contribution of other tissues, for example muscle, remains unclear. In this thesis I have used proteomic techniques to identify intrinsic molecular changes in muscle of SMA mice that contribute to neuromuscular pathology in SMA. I demonstrate significant disruption to the molecular composition of skeletal muscle in pre-symptomatic SMA mice, in the absence of any detectable degenerative changes in lower motor neurons and with a molecular profile distinct from that of denervated muscle. Functional cluster analysis of proteomics data and phospho-histone H2AX labelling of DNA damage revealed increased activity of cell death pathways in SMA muscle. In addition robust up-regulation of VDAC2 and down-regulation of parvalbumin was confirmed in two mouse models of SMA as well as in patient muscle biopsies. Thus intrinsic pathology of skeletal muscle is an important event in SMA. I then used proteomics to identify individual proteins in skeletal muscle of SMA that report directly on disease status. Two proteins, GRP75 and calreticulin, showed increased expression levels over time in different muscles as well as in skin samples, a more accessible tissue for biopsies in patients. Preliminary results suggest that GRP75 and calreticulin can be detected and measured in SMA patient muscle biopsies. These results show that proteomics provides a powerful platform for biomarker identification in SMA, revealing GRP75 and calreticulin as peripherally accessible potential protein biomarkers capable of reporting on disease progression in muscle as well as in skin samples. Finally I identified a role for ubiquitin-dependent pathways in regulating neuromuscular pathology in SMA. Levels of ubiquitin-like modifier activating enzyme 1 (UBA1) were reduced in spinal cord and skeletal muscle tissue of SMA mice. Dysregulation of UBA1 and subsequently the ubiquitination pathways led to the accumulation of β-catenin. I show here that pharmacological inhibition of β-catenin robustly ameliorates neuromuscular pathology in animal models of SMA. Interestingly, downstream disruption of β-catenin was restricted to the neuromuscular system in SMA mice. Pharmacological inhibition of β-catenin failed to prevent systemic pathology in organs. Thus disruption of ubiquitin homeostasis, with downstream consequences for β-catenin signalling, contributes to the pathogenesis of SMA, thereby highlighting novel therapeutic targets for this disease.

L'Atròfia Muscular Espinal (AME) és una malaltia neurodegenerativa greu i la primera causa genètica de mort infantil. S'origina per la pèrdua o mutació del gen Survival Motor Neuron 1 (SMN1) que causa una deficiència de la proteïna de Survival Motor Neuron (SMN). La reducció d'aquesta proteïna condueix principalment a la degeneració de les motoneurones (MNs) de la medul·la espinal i, en conseqüència, produeix atròfia i feblesa del múscul esquelètic. Actualment, només es coneix parcialment quins mecanismes cel·lulars i moleculars exactes són els responsables de la pèrdua de funció de les MNs. La reducció de SMN causa degeneració de les neurites i mort cel·lular sense característiques apoptótiques clàssiques. L'autofàgia és un procés important i altament regulat, essencial per a l'eliminació d'orgànuls danyats i substàncies o proteïnes tòxiques a través de la degradació amb els lisosomes. L'autofàgia és especialment important en cèl·lules post-mitòtiques, com les MNs, on l'acumulació d’autofagosomes provoca la interrupció del transport axonal, la interferència del trànsit intracel·lular i la degeneració de les neurites. El que és ben sabut en l'AME és que el nivell intracel·lular de proteïna SMN defineix l'inici i la gravetat de la malaltia i això està parcialment determinat pel nombre de còpies del gen SMN2, la duplicació centromérica de SMN i el principal modificador de l'AME. Per aquesta raó, comprendre els processos que regulen la degradació de SMN amb la finalitat d'identificar compostos que augmentin els nivells de proteïnes és el principal objectiu en el desenvolupament terapèutic per a l’AME. Les calpaínes són una família de proteases dependents de calci que s'han relacionat amb trastorns musculars i malalties neurodegeneratives. Específicament, s'ha descrit en el múscul que SMN pot ser proteolizada per calpaína. L'activitat de la calpaína també està involucrada en la regulació de l'autofàgia mitjançant la modulació de múltiples de les proteïnes involucrades en el procés. L'objectiu en el present treball ha estat analitzar la desregulació de l'autofàgia i determinar la participació de la calpaína en la regulació de la proteïna SMN en les MNs per a aprofundir en l'origen de la neurodegeneración i desenvolupar un nou enfocament terapèutic per a l'AME. Per aquesta finalitat, hem analitzat marcadors autofágics en diferents models in vitro d’AME, tant de ratolí com d'humà. Els resultats van mostrar que, tant els autofagosomes com els nivells de LC3 es troben augmentats en les mostres d’AME en comparació amb els controls, la qual cosa suggereix una desregulació del procés d'autofàgia al llarg de la progressió de la malaltia. A més, la reducció dels nivells endògens de calpaína utilitzant un shRNA van mostrar un augment dels nivells de Smn i LC3, alhora que prevenia la degeneració neurítica que es produeix en les MNs de ratolí afectats per AME. Es van obtenir resultats similars en experiments in vitro utilitzant un inhibidor farmacològic de calpaína, la calpeptina. Tanmateix, l'activació de la calpaína produïda per condicions despolarizants induïa la proteólisis de l’α-fodrina i de SMN, la qual cosa confirma que calpain regula directament els nivells de proteïna SMN en les MNs. A més, el tractament amb calpeptina in vivo va millorar significativament l'esperança de vida i la funció motora de dos models de ratolins amb AME, la qual cosa demostra la utilitat potencial dels inhibidors de la calpaína en la teràpia per a la malaltia. Finalment, l'anàlisi de la via de la calpaína en ratolins i models cel·lulars humans d’AME va indicar un augment de l'activitat de la calpaína en les MNs amb nivells reduïts de SMN. Per tant, els nostres resultats demostren que l'activitat de la calpaína es troba sobreactivada en les MNs d’AME i que la seva inhibició pot tenir un efecte beneficiós sobre el fenotip de la malaltia a través de l'augment de SMN i la regulació del procés d'autofàgia en les MNs de la medul·la espinal.
La atrofia muscular espinal (AME) es una enfermedad neurodegenerativa grave y la primera causa genética de muerte infantil. Se origina por la pérdida o mutación del gen Survival Motor Neuron 1 (SMN1) que causa una deficiencia de la proteína de Survival Motor Neuron (SMN). La reducción de esta proteína conduce predominantemente a la degeneración de las motoneuronas (MNs) de la médula espinal y, en consecuencia, produce atrofia y debilidad del músculo esquelético. Actualmente, solo se conoce parcialmente que mecanismos celulares y moleculares exactos son los responsables de la pérdida de función de las MNs. La reducción de SMN causa degeneración de neuritas y muerte celular sin características apoptóticas clásicas. La autofagia es un proceso importante y altamente regulado, esencial para la eliminación de orgánulos dañados y sustancias o proteínas tóxicas a través de la degradación con los lisosomas. La autofagia es especialmente importante en células post-mitóticas, como las MNs, donde la acumulación de autofagosomas provoca la interrupción del transporte axonal, la interferencia del tráfico intracelular y la degeneración de las neuritas. Lo que es bien sabido en la AME es que el nivel intracelular de proteína SMN define el inicio y la gravedad de la enfermedad y esto está parcialmente determinado por el número de copias del gen SMN2, la duplicación centromérica de SMN y el principal modificador de la AME. Por esa razón, comprender los procesos que regulan la degradación de SMN con la finalidad de identificar compuestos que aumentan los niveles de proteínas es el principal objetivo en el desarrollo terapéutico de AME. Las calpaínas son una familia de proteasas dependientes de calcio que se han relacionado con trastornos musculares y enfermedades neurodegenerativas. Específicamente, se ha descrito en el músculo que SMN puede ser proteolizada por calpaína. La actividad de la calpaína también está involucrada en la regulación de la autofagia mediante la modulación de múltiples de las proteínas involucradas en el proceso. El objetivo en el presente trabajo ha sido analizar la desregulación de la autofagia y determinar la participación de la calpaína en la regulación de la proteína SMN en las MNs para profundizar en el origen de la neurodegeneración y desarrollar un nuevo enfoque terapéutico para la AME. Con este fin, hemos analizado marcadores autofágicos en diferentes modelos in vitro de AME, tanto de ratón como de humano. Los resultados mostraron que los autofagosomas y los niveles de LC3 se encuentran aumentados en las muestras de AME en comparación con los controles, lo que sugiere una desregulación del proceso de autofagia a lo largo de la progresión de la enfermedad. Además, la reducción de los niveles endógenos de calpaína utilizando un shRNA muestraron un aumento de los niveles de Smn y LC3, a la vez que previene la degeneración neuritica que se produce en las MNs de ratón afectados por AME. Se obtuvieron resultados similares en experimentos in vitro utilizando un inhibidor farmacológico de calpaína, la calpeptina. Asimismo, la activación de calpaína producida por condiciones despolarizantes inducia la proteólisis de α-fodrina y de SMN, lo que confirma que calpain regula directamente los niveles de proteína SMN en las MNs. Además, el tratamiento con calpeptina in vivo mejoró significativamente la esperanza de vida y la función motora de dos modelos de ratones con AME, lo que demuestra la utilidad potencial de los inhibidores de la calpaína en la terapia para la enfermedad. Finalmente, el análisis de la vía de la calpaína en ratones y modelos celulares humanos de AME indicó un aumento de la actividad de la calpaína en las MNs con niveles reducidos de SMN. Por lo tanto, nuestros resultados demuestran que la actividad de la calpaína se encuentra sobreactivada en las MNs de AME y su inhibición puede tener un efecto beneficioso sobre el fenotipo de la enfermedad a través del aumento de SMN y la regulación del proceso de autofagia en las MNs de la médula espinal.
Spinal Muscular Atrophy (SMA) is a severe neurodegenerative disease and the first genetic cause of infant death. It is originated by the deletion or mutation of Survival Motor Neuron 1 (SMN1) gene causing a Survival Motor Neuron (SMN) protein deficiency. The reduction of this protein predominantly leads to the degeneration of spinal cord motoneurons (MNs) and consequently produces skeletal muscle atrophy and weakness. The exact cellular and molecular mechanisms responsible for MN loss of function are only partially known. SMN reduction causes neurite degeneration and cell death without classical apoptotic features. Autophagy is an important and highly regulated process, essential for the removal of damaged organelles and toxic substances or proteins through lysosome degradation. This mechanism is specifically important in post-mitotic cells like MNs where autophagosome accumulation causes axonal transport disruption, interference of intracellular space trafficking, and neurite degeneration. What is well known in SMA is that intracellular SMN protein levels are critical to define the disease onset and severity, and this is partially determined by the number of copies of SMN2, the centromeric duplication of the SMN gene and the main modifier of SMA. For that reason, understanding the processes of SMN stability and degradation to identify compounds that increase protein levels is a major goal in SMA therapeutics development. Calpains are a family of calcium-dependent proteases that have been related to muscle disorders and neurodegenerative diseases. Specifically, it has been described in muscle that SMN can be a proteolytic target of calpain. Calpain activity is also involved in autophagy regulation by modulation of multiple proteins involved in the process. The objectives in the present work have been to analyze the autophagy deregulation and determine the involvement of calpain in SMN protein regulation on MNs, in order to deepen in the origin of neurodegeneration and to develop a new therapeutic approach for SMA disease. To this end, we have analyzed autophagic markers in different mouse and human SMA in vitro models. The results showed that autophagosomes and LC3 levels were increased in SMA samples compared to controls, suggesting a deregulation of the autophagy process throughout the disease progression. Moreover, calpain knockdown using an shRNA approach showed an increase of both, Smn and LC3 levels and prevented neurite degeneration occurred in SMA affected mouse MNs. Similar results were obtained in in vitro experiments using a pharmacological calpain inhibitor, calpeptin. Likewise, calpain activation produced by depolarized conditions induced α-fodrin and SMN proteolysis, confirming that calpain directly regulates the SMN protein level in MNs. Additionally, calpeptin in vivo treatment significantly improved the lifespan and motor function of two severe SMA mouse models, demonstrating the potential utility of calpain inhibitors in SMA therapeutics. Finally, the analysis of calpain pathway members in mice and human cellular SMA models indicated an increase of calpain activity in SMN-reduced MNs. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on the SMA phenotype through the increase of SMN and the regulation of the autophagy process in spinal cord MNs.

Spinal muscular atrophy (SMA) is the most commonly inherited neurodegenerative disease that leads to infant mortality worldwide. There are no known cures for SMA, but small increase in protein levels of SMN can be beneficial. We have developed adenoviral (Ad) vectors that express a human transgene of SMN and have tested their safety in vitro. We have demonstrated that these viruses can effectively express the transgene following cell entry and that the levels are relative to the virus dose. The viruses do not appear to impact the health and function of the cells, and are capable of increasing the number of Gems. We also attempted to change the tropism of the viruses through fiber protein modifications in order to target muscles and motor neurons. Our results suggest that a therapy based on an Ad-SMN fiber-modified vector may ultimately be successful in treating patients of SMA.

The disruption of the survival motor neuron (SMN1) gene leads to the children’s genetic disease spinal muscular atrophy (SMA). SMA is characterized by the degeneration of α-motor neurons and skeletal muscle atrophy. Although SMA is primarily considered a motor neuron disease, the involvement of muscle in its pathophysiology has not been ruled out. To gain a better understanding of the involvement of skeletal muscle pathophysiology in SMA, we have developed three aims: to identify cell-specific Smn-interacting proteins, to characterize postnatal skeletal muscle development in mouse models of SMA, and to assess the functional capacity of muscles from SMA model mice. We have used tandem affinity purification to discover Smn interacting partners in disease relevant cell types. We have identified novel cell-specific Smn interacting proteins of which we have validated myosin regulatory light chain as a muscle-specific Smn associated protein in vivo. We have taken advantage of two different mouse models of SMA, the severe Smn-/-;SMN2 mouse and the less severe Smn2B/- mouse, to study the postnatal development of skeletal muscle. Primary myoblasts from Smn2B/- mice demonstrate delayed myotube fusion and aberrant expression of the myogenic program. In addition, the expression of myogenic proteins was delayed in muscles from severe Smn-/-;SMN2 and less severe Smn2B/- SMA model mice. Muscle denervation and degeneration, however, are not the cause of the aberrant myogenic program. At the functional level, we demonstrate a significant decrease in force production in pre-symptomatic Smn-/-;SMN2 and Smn2B/- mice indicating that muscle weakness is an early event in these mice. Immunoblot analyses from hindlimb skeletal muscle samples revealed aberrant levels of developmentally regulated proteins important for muscle function, which may impact muscle force production in skeletal muscle of SMA model mice. The present study demonstrates early and profound intrinsic muscle weakness and aberrant expression of muscle proteins in mouse models of SMA, thus demonstrating how muscle defects can contribute to the disease phenotype independently of and in addition to that caused by motor neuron pathology.

Spinal Muscular Atrophy (SMA), traditionally described as a predominantly childhood form of motor neuron disease, is a leading genetic cause of infant mortality. Although motor neurons are undoubtedly the primary affected cell type, SMA is now widely recognised as a multisystem disorder, where a variety of organs and systems in the body are also affected. Vascular perfusion abnormalities have previously been reported in both patients and mouse models of SMA, however it remains unclear whether these defects are secondary to the motor neuron pathology for which this disease is known. Through analysis of the 'Taiwanese' murine model of severe SMA (Smn-/-;SMN2tg/0, Smn-/+) we report significant vascular defects in the retinas of SMA mice, a tissue devoid of motor neurons, thus providing strong evidence that these vascular defects are independent of motor neuron pathologies. We show that restoration of Smn levels by antisense oligonucleotide treatment at birth significantly ameliorates retinal vascular defects. Next, we report defects in the neural retina, with a significant decrease in key neural cells in SMA mice. A similar vascular pathology was expected in the spleen of SMA mice given that the spleen is small and pale in appearance; however, the density of the intrinsic vasculature remained unchanged. We report that the spleen is disproportionately small in SMA mice, correlated to low levels of cell proliferation, increased cell death, and multiple lacunae. The SMA spleen lacks its distinctive red appearance and presents with a degenerated capsule and a disorganized fibrotic architecture. Histologically distinct white pulp fails to form and this is reflected in an almost complete absence of B lymphocytes necessary for normal immune function. Taken together, these results highlight both the vascular and immune systems as key targets of SMA pathology that should be considered during treatment of this disease.

Spinal muscular atrophy (SMA) is a devastating recessive neurological disorder thought to be affecting primarily the motor neurons. As such, paralysis, motor weakness and death ensue. While SMA is most commonly seen in infants and children, it can span all ages. Its genetic etiology revolves around the homozygous deletion or mutation of the SMN1 gene, whose product (SMN protein) has critical and ubiquitous roles in mRNA splicing, amongst various other functions in mRNA metabolism. As such, SMN depletion in other non-neuronal cells type is likely to have physiological repercussions, and perhaps modulate the SMA phenotype. Herein, we identify the molecular pathways of atrophy in skeletal and cardiac muscle of two mouse models of SMA and their therapeutic modulation via the histone deacetylase inhibitor trichostatin A. We also identify dramatic changes in immune organs in mouse models of SMA, which could impact susceptibility to infections. Furthermore, we establish the presence of important defects in fatty acid homeostasis in the liver and plasma seen in both mouse models and SMA patients. Finally, we provide the first mild mouse model of SMA that reliably reproduces canonical features of SMA, permitting aging studies. This model presents with a prominent myopathic phenotype prior to motor neuron death, without extra-neuronal involvement during the course of its lifespan. Overall, our work shows multiple potentially clinically relevant defects in extra-neuronal organs, provides ways to abrogate them and provides a framework to study them over the course of aging.

Spinal Muscular Atrophy (SMA) is a devastating autosomal-recessive pediatric neurodegenerative disease characterized by loss of spinal motor neurons. It is caused by mutation in the survival of motor neuron 1, SMN1, gene and leads to loss of function of the full-length SMN protein. SMN has a number of functions related to RNA processing in neurons, including RNA trafficking in neurites, and RNA splicing and snRNP biogenesis in the nucleus. While previous work has focused on the alternative splicing and expression of traditional mRNAs, our lab has focused on the contribution of another RNA species, microRNAs (miRNAs), to the SMA phenotype. miRNAs are ~22 nucleotide small RNAs that are involved in post-transcriptional regulation of gene expression. They function by translational repression or mRNA decay of target RNAs. Interestingly, dysregulation of RNA processing and miRNA expression has been identified in motor neuron diseases including SMA and Amyotrophic Lateral Sclerosis. Our lab previously discovered a miRNA, miR-183, that is dysregulated in SMA and impacts its targets in cortical neurons and SMA mouse spinal cords. However, spinal motor neurons are the cell type most affected by SMN loss. We hypothesized that motor neuron specific miRNA changes are involved selective vulnerability in SMA. Therefore, we sought to determine the effect of loss of SMN on spinal motor neurons. To accomplish this, I used microarray and RNAseq to profile both miRNA and mRNA expression in primary spinal motor neurons after acute SMN knockdown. By integrating the miRNA:mRNA profiles we identified dysregulated miRNAs with enrichment in differentially expressed putative targets. miR-431 was the most substantially increased miRNA and a number of its putative targets were downregulated after SMN loss. Further, I confirm that miR-431 directly regulates its target chondrolectin and impacts neurite length. This work is critical to understanding the cell-type specific effect of aberrant miRNA expression in SMN knockdown motor neurons. It demonstrates the contribution of dysregulated RNA processing in motor neurons to neurodegeneration. Furthermore, this work highlights the impact of non-coding RNAs in human disease and points to specific miRNA whose dysregulation potentially impacts motor neuron health.
Medical Sciences

Spinal muscular atrophy (SMA) is a devastating childhood motor neuron disease caused by mutations in the survival motor neuron 1 gene (SMN1). SMN1 and SMN2 are nearly identical genes producing the survival of motor neuron (SMN) protein. SMN protein plays a crucial role in mRNA splicing and β-actin mRNA transport along the axons. In SMA the mutation leads to the loss of SMN1, which cannot be fully compensated by the SMN2 gene, which predominantly produces a truncated protein. The loss or reduction of SMN protein leads to motor axonal defects and motor neuron cell death. There are currently no treatments available but therapies have focused on increasing SMN through replacing SMN1 or increasing full length SMN from SMN2. The actin-binding protein Plastin 3 (PLS3) has been reported as a modifier for SMA, making it a potential therapeutic target. Recently, it was shown that the overexpression of the PLS3 gene improved axonal outgrowth in SMN-deficient motor neurons of SMA Zebrafish and cultured motor neurons from mouse embryos. Gene therapy using viral vectors was carried out in vitro and in vivo to assess whether the overexpression of PLS3 could rescue neuronal loss in SMA and be developed as a therapy. The SMNΔ7 mouse model produces low levels of SMN, modelling severe SMA disease with an average lifespan of 12 days and loss of motor neurons. This study has established that the SMNΔ7 mice have little or no detectable PLS3 from birth, making it a good model for developing PLS3 gene therapy. Lentiviral vectors were able to upregulate PLS3 expression in different cell lines. Transduction of NSC34 cells with LV-PLS3 vector led to a five-fold increase in expression of PLS3 compared to controls. In smn-deficient MNs, expression of PLS3 restored axonal length and showed a strong neuroprotective effect. Pre-clinical in vivo proof-of-concept studies using adeno-associated virus serotype 9 (AAV9) encoding PLS3 in SMNΔ7 mice showed high transduction efficiency and overexpression of PLS3 specifically targeted to neurons in the central nervous system (CNS). This led to a small but significant increase of lifespan by 54%. However, PLS3 was not able to prevent disease onset. Although there was no improvement of phenotype, this study has demonstrated the potential use of PLS3 as a target for gene therapy, possibly in conjunction with other modulators of disease.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by reduced levels of the survival motor neuron (SMN) protein, which results in degeneration of motor neurons, leading to muscle weakness, and, ultimately, death. To provide an effective therapy for SMA, SMN expression must be restored in motor neurons. The goal of our research is to develop protein-based therapeutics for SMA. Protein transduction domain (PTD), from the human immunodeficiency virus-transactivator of transcription (HIV-TAT), mediates the transduction of any polypeptide to which they are fused. We produced bacterial expression cassettes of PTD-SMN. The PTD-SMN is able to transduce cells in vitro, reaching the nucleus and forming punctate structures similar to that of endogenous SMN. Internalization of PTD-SMN results in an increased nuclear-staining foci (gems) number. Importantly, this PTD-SMN co-localizes with coiled bodies, indicating PTD-SMN is able to localize to the correct region of the nucleus. However, we were unable to detect interaction of PTD-SMN with SMN known binding partners, suggesting that majority of PTD-SMN has low activity. Our results suggest that PTD-SMN produced from a prokaryotic source may be useful for SMA therapy, but optimization of protein production is required before this therapy can reach its full potential.

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is a neurodegenerative disease characterised by the loss of motor neurones in the anterior horn of the spinal cord with concomitant muscle weakness. However, a growing body of evidence shows that different motor pools in the anterior horn have a very different vulnerability to SMA. For example, previous studies on mouse have suggested motor neurones innervating three different muscles in the lower leg, tibialis anterior (TA), extensor digitorium longus (EDL) and gastrocnemius (GS) show different levels of axonal degeneration upon Smn loss. It indicated that there were undiscovered factors endeavouring motor neurones with distinct disease resistance. As such, we attempted to discover these disease-modifying factors mainly by investigating the transcriptomes of different motor neurones with differing vulnerability. Further functionality experiments were carried out on both in vivo and in vitro systems to validate the microarray result. Furthermore, various new techniques were also tested to advance research in the future. In general, we identified that higher bioenergetics could be the potential disease modulator, which was further illustrated by the manipulation of this pathway in zebrafish. However, bioenergetics status of the motor neurone was independent of SMN levels and likely to be a result of complex interaction of motor neurone and the surrounding environment in which astrocyte might play an important role. Moreover, the use of DEPArray (dielectrophoresis array) might offer an opportunity to explore this subject ex vivo. Lastly, because axon terminal tends to degenerate prior to the cell body, we extended the concept of selective vulnerability to motor neurone itself and attempted to specifically profile axonal transcriptome using RNAseq combined with the microfluidic device.

Spinal Bulbar Muscular Atrophy (SBMA), also known as Kennedy's disease, is an X-linked, late-onset progressive neurodegenerative disease. SBMA is characterised by the selective loss of spinal and bulbar motor neurons and progressive muscle weakness. The disease is caused by an expansion in the CAG repeat in the androgen receptor (AR) gene which encodes a polyglutamine tract in the protein. The underlying pathophysiology of the disease is thought to be related to abnormal accumulation of the pathogenic AR protein within the nucleus. The AR100 transgenic mouse model of SBMA has a progressive neuromuscular phenotype accompanied by motor neuron degeneration, thereby mirroring the human disease. Since ER stress has been suggested to play a role in motor neuron death in SBMA, I investigated the underlying mechanism by which ER stress may result in cell death. In particular, I tested whether the motor neuron-specific Fas/NO cell death pathway plays a role in SBMA. In addition, I examined whether the ER chaperone, Calreticulin forms a link between the Fas/NO MN-specific death pathway and ER stress. I found that Fas/NO induced cell death is not observed in AR100 MNs. However, an increase in Calreticulin is observed in the spinal cord of AR100 mice, suggesting it may contribute to SBMA pathology. Although SBMA is considered to be a neurodegenerative disease affecting motor neurons, emerging evidence suggests that SBMA may also involve a primary muscle deficit. I examined this possibility by characterising muscle histopathology longitudinally, at different stages of disease progression, in AR100 mice. My results show that muscle atrophy is evident during early stages of disease, prior to any loss of motor neurons. Physiological deficits were accompanied by a change in the properties of the muscle fibres such as increase in oxidative capacity and signs of myogenic and neurogenic induced muscle atrophy. Furthermore, RNA-sequencing and pathway enrichment analysis of hindlimb muscles of AR100 mice identified some of the molecular signalling pathways which may underlie the changes within the muscle. These findings indicate that muscle deficits are an early and primary manifestation of disease in SBMA.

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xvi, 137 p.; also includes graphics (some col.). Includes bibliographical references (p. 127-137).

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by widespread loss of lower motor neurons from the spinal cord. Lower motor neuron degeneration leads to a progressive decline in motor development, manifesting as muscle atrophy and weakness. It is now well characterised that ubiquitin homeostasis is altered in SMA and that reduction of the ubiquitin-like modifier-activating enzyme 1 (UBA1) is central to this disruption. UBA1 is responsible for activating ubiquitin as the first step in the ubiquitin conjugation process, marking unwanted proteins for degradation by the proteasome. While it is known that therapies targeting UBA1 rescue neuromuscular phenotypes in SMA models, the mechanism by which UBA1 mediates neurodegeneration is unclear. In fact, very little is known about the function of UBA1 beyond its canonical role in the ubiquitin proteasome system. To better understand the role of UBA1 in motor neuron degeneration, a robust set of antibodies for both in vivo and in vitro work to study UBA1 have been identified. This enabled a novel characterisation of UBA1 distribution throughout disease progression in SMA spinal motor neurons to be performed, revealing that UBA1 reduction is an important pre-symptomatic molecular feature of SMA. To identify downstream targets of UBA1 critical for UBA1-mediated degeneration in SMA, label-free proteomics was performed on HEK293 cells after overexpression or knockdown of UBA1. The proteomics data was analysed across multiple platforms, including Biolayout, IPA and DAVID to identify UBA1-dependent pathways and demonstrated that modulation of UBA1 levels lead to disruption of key cellular pathways including translation elongation, nuclear transport, and tRNA synthetases. Validation of target proteins from these UBA1-dependent pathways identified that the tRNA synthetease GARS behaves in a UBA1-dependent manner across a range of model systems in vitro and in vivo. It was then identified that GARS expression is significantly dysregulated across a range of neuronal tissues in a mouse model of SMA. Interestingly, mutations in GARS cause Charcot-Marie-Tooth disease type 2D (CMT2D), an axonal neuropathy, in which a disruption to sensory neuron fate in dorsal root ganglia has recently been identified. In a mouse model of SMA we identified a phenotype consistent with that in the CMT2D mouse model and showed that disruption to sensory neuron fate is reversible and dependent on changes in UBA1 and GARS expression in SMA. In conclusion, modulation of UBA1 levels leads to disruption of key cellular pathways, with dysregulation of tRNA synthetases a prominent feature that is likely to play a role in the pathogenesis of SMA.

Spinal muscular atrophy (SMA) is a genetic disease which is characterized by muscle weakness and atrophy. The disease arises from mutations in the survival motor neuron 1 (SMN1) gene causing degeneration of spinal cord motor neurons. No effective treatment is currently available for SMA however it may be possible to treat the disease using gene therapy. The aim of this project is to develop potential therapies for SMA by investigating different viral and non-viral gene therapy vectors and assessing the effect of potential disease modifying genes. The data collected are described under four chapters as follows: 1: The aim here was to develop a novel approach based on polymer nanoparticles (polymersomes) for gene delivery. Encapsulation of DNA by polymersomes was optimised and polymersomes were used to restore SMN levels into a fibroblast cell line isolated from a child with severe SMA. 2: The ability of adeno-associated virus serotype 5 (AAV5) vectors expressing GFP to transduce the central nervous system (CNS) following intravenous injection was tested in neonatal wild-type mice. Overall transduction efficiency of AAV5-GFP in the brain was low and very few lumbar spinal cord neurons were found to be transduced, suggesting that AAV5 is not an appropriate vector to treat diseases such as SMA. 3: AAV6 was used to overexpress hnRNP R in an in vivo model of SMA. hnRNP R is a candidate disease modifying gene for SMA due to its interaction with SMN and β-actin. However this strategy had only a very marginal effect on the phenotype and life-span of this SMA mouse model. 4: Finally AAV9 was used to silence phosphatase and tensin homolog (PTEN) in an in vivo model of SMA. PTEN is a negative regulator of growth which acts on the PI3K/Akt pathway. AAV9-mediated PTEN silencing resulted in a significant increase in the lifespan of a SMA mouse model coupled with an improved phenotype. In conclusion this work highlights two major findings: i) polymersomes can be used to deliver SMN plasmid DNA to restore SMN mRNA and protein levels in an in vitro model of SMA; ii) AAV9-mediated silencing of PTEN can improve the phenotype and increase lifespan of a SMA mouse model.

The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.

Thesis (Ph. D.)--University of Missouri-Columbia, 2009.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2009" Includes bibliographical references.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced levels of the survival motor neuron (SMN) protein. SMA results in degeneration of motor neurons, progressive muscle atrophy, and death in severe forms of the disease. Currently, there is a lack of inexpensive, readily accessible, accurate biomarkers to study the disease. Furthermore, the current FDA approved therapeutic is neither 100 % effective nor accessible for all patients, thus more research is required. Tiny cell derived vesicles known as exosomes have been evaluated in an attempt to identify novel biomarkers for many disease states and have also shown therapeutic promise through their ability to deliver protein and nucleic acid to recipient cells. The research presented herein investigates whether (1) the level of SMN protein in exosomes isolated from the medium of cells, and serum from animal models and patients of SMA is indicative of disease, to serve as a biomarker for monitoring disease progression and therapeutic efficacy; (2) SMN-protein loaded exosomes can be utilized to deliver SMN protein to SMN-deficient cells; (3) adenoviral vectors are effective at creating SMN protein-loaded exosomes in situ for body wide distribution of SMN protein. This research has shown SMN protein is naturally released in extracellular vesicles, and the level of exosomal SMN protein is reflective of the disease state. Exosomes can also be modified to hold enhanced levels of SMN protein and deliver them to both the cytoplasm and nucleus of SMN-deficient cells. Furthermore, adenoviral vectors expressing luciferase-tagged SMN1 cDNA, targeted to the liver, results in SMN protein-loaded exosomes and detectable luciferase activity, body-wide. Thus, exosomes present as an effective biomarker and potentially a novel approach to treat SMA.