published_or_final_version / Paediatrics / Master / Master of Philosophy

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine (polyGln) tract in the human androgen receptor (hAR). One mechanism by which polyGln-expanded proteins are believed to cause neuronotoxicity is through aberrant interaction(s) with, and possible sequestration of, critical cellular protein(s). / Our goal was to confirm and further characterize the interaction between hAR and cytochrome c oxidase subunit Vb (COXVb), a nuclear-encoded mitochondrial protein. We had previously isolated COXVb as an AR-interacting protein in a yeast two-hybrid search to identify candidates that interact with normal and polyGln-expanded AR. Using the mammalian two-hybrid system, we confirm that COXVb interacts with normal and mutant AR and demonstrate that the COXVb-normal AR interaction is stimulated by heat shock protein 70 (Hsp70). Also, BFP-tagged AR specifically co-localizes with cytoplasmic aggregates formed by GFP-labelled polyGln-expanded AR in androgen-treated cells. / Mitochondrial dysfunction may precede neuropathological findings in polyGln-expanded disorders and may thus represent an early event in neuronotoxicity. Interaction of COXVb and hAR, with subsequent sequestration of COXVb, may provide a mechanism for putative mitochondrial dysfunction in SBMA.

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 is caused by mutation of the SMN1 gene resulting in the selective loss of spinal cord motor neurons. HuD has been shown to interact with SMN and to localize to RNA granules along axons. In conditions where SMN is decreased, like in SMA, HuD’s localization to RNA granules affected. Overexpression of HuD in an SMA cell culture model was shown to rescue SMA-like axonal defects. Here, existence of a signaling pathway downstream of PKC leading to the activation of HuD was investigated in MN-1 cells. Stimulation of this pathway using a pharmacological agonist of PKC increased HuD levels and enhanced its binding to GAP-43 and Tau mRNAs. An scAAV9 viral expression system to overexpress HuD in vivo was established, laying the foundation for the next phase of the study. Overall, modulating HuD expression and activity would be beneficial and could constitute an attractive therapeutic approach for SMA.

Disruption of RNA homeostasis is a shared feature across multiple neurodegenerative diseases that are associated with mutations in RNA binding proteins or factors involved in RNA processing. One prime example is the neurodegenerative disease spinal muscular atrophy (SMA), which is characterized by the degeneration of spinal motor neurons and atrophy of skeletal muscle through poorly defined mechanisms. SMA is the consequence of ubiquitous deficiency in the survival motor neuron (SMN) protein, which has a well-characterized role in the assembly of small nuclear ribonucleoproteins (snRNPs). SMN-dependent dysfunction of major (U2) and minor (U12) spliceosomal snRNPs as well as U7 snRNP – which functions in histone mRNA processing – along with consequent RNA misprocessing events have been characterized in SMA. Additionally, SMN has been implicated in additional RNA pathways that may also be involved in SMA etiology. With the broad implications of multifaced roles of SMN in RNA regulation, an outstanding challenge in the SMA field has been the identification of key downstream RNA-dependent events and their contributions to pathogenesis. While the selective loss of spinal motor neurons is a key hallmark of SMA pathology, the molecular mechanisms remain incompletely understood. Through my dissertation work, I aimed to characterize the RNA mediated pathways that underlie neurodegeneration in SMA. We previously demonstrated that SMA motor neuron death is driven by converging mechanisms of p53 activation that include upregulation and phosphorylation – the latter of which establishes the vulnerability of specific motor neuron pools – however, the upstream triggers remained unknown. Here, I show that the function of SMN in the assembly of Sm-class snRNPs of the splicing machinery regulates alternative splicing of Mdm2 and Mdm4 – two non-redundant repressors of p53 – and increased skipping of critical exons in these genes is associated with p53 stabilization. Further investigation uncovered that dysfunction of Stasimon – a U12 intron-containing gene regulated by SMN – converges on p53 upregulation to induce phosphorylation of p53 through the activation of p38α MAPK and contributes to the demise of SMA motor neurons. Thus, this work elucidated the upstream RNA mediated mechanisms underlying multiple modes of p53 activation, implicating impairments in both the U2 and U12 snRNP pathways in SMA motor neuron death. It further established Mdm2, Mdm4, and Stasimon as effector genes that are regulated by SMN’s role in snRNP assembly and play key roles in the degeneration of SMA motor neurons. Studies in mouse models of SMA have revealed broader deficits in the motor circuit beyond motor neuron death, which include reduced excitatory drive on motor neurons brought on by a loss of proprioceptive synapses. Restoration in SMA mice revealed that Stasimon dysfunction also contributes to the deafferentation of motor neurons – a cellular defect originating in proprioceptive neurons – revealing Stasimon’s dual contribution to motor circuit pathology in SMA. However, the Stasimon-dependent molecular mechanisms that mediate synaptic loss in proprioceptive neurons, along with the pathway through which Stasimon impairment induces p38α MAPK activation in motor neurons are not well established. Stasimon was initially identified as a novel contributor to motor circuit dysfunction in Drosophila and motor axon outgrowth deficits in zebrafish models of SMN deficiency. However its cellular roles remain poorly understood. In an effort to address this, I identify Stasimon as an endoplasmic reticulum (ER) resident protein that localizes at mitochondria-associated ER membranes (MAM) – specialized contact sites between ER and mitochondria membranes. Additionally, through characterization of novel knockout mice, I show that Stasimon is an essential gene for mouse embryonic development. These findings provide key insight into Stasimon function and set the stage for further investigation of the p53-dependent mechanisms of motor neuron degeneration as well as cellular pathways driving proprioceptive synaptic loss in SMA. This dissertation also expands beyond RNA mediated mechanisms of SMA – which occur as a consequence of SMN-deficiency – to translational efforts aimed to treat the disease by describing an unexpected gain of toxic function associated with SMN overexpression that is accompanied by RNA dysregulation and sensory-motor circuit pathology. I further explore these surprising findings of neuronal toxicity induced by AAV9-mediated SMN overexpression that paradoxically affects sensory-motor function and reveal that they parallel features of SMA pathogenesis. Accordingly, I find that the functional basis of long-term motor toxicity of AAV9-SMN involves motor neuron deafferentation and proprioceptive neurodegeneration. At the cellular level, toxicity is associated with the accumulation of large cytoplasmic aggregates of SMN in motor circuit neurons that sequester Sm proteins, disrupt snRNP biogenesis, and induce widespread transcriptome alterations and splicing deficits. These findings identify a novel deleterious role for SMN when expressed at supraphysiological levels that acts through inhibition of SMN’s normal function in snRNP biogenesis, akin to disease mechanisms of SMA. These observations have important implications regarding the approved use of AAV9-SMN for gene therapy in humans and suggest a need for further, careful consideration of potential detrimental effects when SMN is expressed at supraphysiological levels. Collectively, this dissertation identifies the direct involvement of key SMN-dependent splicing events in select aspects of SMA pathology in a mouse model, in particular those converging on the p53-mediated mechanisms of motor neuron death and the loss of proprioceptive synapses. This work also establishes causal links between impairments in snRNP biology and neuronal dysfunction in SMA, providing mechanistic insight into the process of motor neuron death. Lastly, it uncovers a new, clinically relevant aspect of SMN biology associated with its long-term overexpression which has shared features with the RNA mediated mechanisms of neurodegeneration in SMA.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by muscle weakness and atrophy and death of motor neurons in humans. Although almost all cases of SMA occur due to mutations in a gene called survival motor neuron 1 (SMN1), SMA patients present with a wide range of severities of the symptoms. The most severe cases never achieve any developmental motor milestone and die within a few years after birth. On the other hand, mild cases of SMA have a normal life span and show trivial motor deficits. This suggests the role of other factors (rather than the function of SMN1) in the outcome of the disease. Indeed, the copy number of an almost identical gene, called SMN2, is the main determining factor for the severity of SMA. In addition, a few other genes (e.g. Plastin 3) are proposed as disease modifiers in SMA. SMN1 is a housekeeping gene, but due to unknown reasons, the most prominent pathologies in SMA are atrophy of myofibers and death of motor neurons. However, recent studies showed that some other cell types are also affected in the course of SMA disease. We investigated the alterations of central synapses in Smn2B/- mice, a model of SMA. We did not observe any degeneration of central synapses in these mice until a post symptomatic stage. However, mass spectrometry (MS) analysis on isolated synaptosomes from spinal cords of these animals revealed widespread alterations in the proteome of their central synapses at a presymptomatic stage. Functional cluster analysis on MS results suggested that several molecular pathways are affected within synapses of spinal cords of Smn2B/- mice prior to the onset of any obvious pathology in their motor units. The affected molecular pathways are involved in basic cell biological functions including energy production, protein synthesis, cytoskeleton regulation and intracellular trafficking. We showed that the levels of several proteins involved in actin cytoskeleton regulation are altered in synaptosomes isolated from spinal cords of Smn2B/- mice. More investigations are required to determine the exact functional abnormalities of affected pathways in central synapses of these mice. We also generated congenic Smn2B/- mice in two different mouse genetic backgrounds; FVB and BL6. Using a systematic approach, we showed that congenic Smn2B/- mice in the FVB background show a more severe SMA phenotype than Smn2B/- mice in a BL6 background. Smn2B/- mice in the FVB background had a shorter survival, higher rate of weight loss, earlier and more severe pathologic changes compared to Smn2B/- mice in the BL6 background. We investigated the levels of several actin binding proteins in spinal cords of these animals and found higher induction of plastin 3 in Smn2B/- mice in the BL6 background. More investigations are underway to determine the role of plastin 3 in the severity of the phenotype of Smn2B/- mice, and to find other possible SMA modifier genes in these animals.

Die spinale Muskelatrophie (SMA) ist eine monogenetische Erkrankung, bei der es durch den Verlust des SMN Proteins zur Degeneration der α-Motoneurone im Rückenmark kommt. Abhängig vom Schweregrad zeigen die Patienten bereits innerhalb der ersten Lebensmonate ausgeprägte Lähmungen der Skelettmuskulatur und eine Zwerchfellparese einhergehend mit einer reduzierten Lebenserwartung. Mithilfe von Mausmodellen für die SMA konnte gezeigt werden, dass der Motoneuronenverlust bei Smn-defizienten Mäusen mit Störungen der Neurotransmission an der motorischen Endplatte und mit Differenzierungsstörungen der Motoneurone einhergeht. Die Differenzierungs-störungen primärer Smn-defizienter Motoneurone sind eng gekoppelt mit einer verminderten Clusterbildung spannungsabhängiger Kalziumkanäle im distalen axonalen Bereich. Dies wiederum führt zu einer verminderten Frequenz spontaner Kalziumeinströme am Axonterminus und hat eine veränderte axonale Elongation zur Folge. Es wurden folgende Aspekte in Bezug auf die Verstärkung und die Induktion spontaner Kalziumeinströme in Mausmodellen für spinale Muskelatrophien in dieser Arbeit adressiert: 1) Lassen sich spontane Kalziumeinströme in Smn-defizienten Motoneuronen durch die externe Applikation von Kalziumkanalagonisten verstärken? 2) Sind spontane Kalziumeinströme in primären Motoneuronen durch den Brain-derived-neurotrophic-factor (BDNF) induzierbar? 3) Zeigen primäre Motoneurone eines Mausmodells für spinale Muskelatrophie mit Ateminsuffizienz Typ 1 (SMARD1) ebenfalls veränderte Kalziumtransienten? Die Ergebnisse meiner Arbeit zeigen, dass durch den Kalziumkanalagonisten R-Roscovitine die Frequenz der spontanen Kalziumeinströme im distalen Axon von Smn-defizienten Motoneuronen signifikant erhöht wird. Dies hat wiederum einen regulierenden Effekt auf die Differenzierung der SMA Motoneurone zur Folge. Smn-defiziente Motoneurone zeigen somit keine Unterschiede mehr in Bezug auf Axonlängen und Wachstumskegelflächen im Vergleich zu Kontrollzellen. Für R- 10 Roscovitine ist neben der agonistischen Wirkung am Kalziumkanal auch ein inhibitorischer Effekt auf die Cyclin-abhängige Kinase 5 beschrieben. Es konnte jedoch gezeigt werden, dass die erhöhten Kalziumtransienten unter der Behandlung mit R-Roscovitine durch eine direkte Bindung an die Cav2 Kalziumkanäle verursacht werden und nicht durch eine Cdk5 Blockade. Dafür spricht die schnelle und reversible Wirkung von R-Roscovitine, sowie die Aufhebung des R-Roscovitines Effekts bei gleichzeitiger Gabe des Cav2.2 Antagonisten ω-Conotoxin MVIIC. Der zweite Aspekt dieser Arbeit behandelt den Einfluss der neurotrophen Faktoren BDNF, CNTF und GDNF auf die Kalziumtransienten am Wachstumskegel wildtypischer Motoneurone. Der Vergleich der neurotrophen Faktoren zeigt, dass nur BDNF eine induzierende Wirkung auf spontane Kalziumtransienten am Wachstumskegel hat. Der letzte Abschnitt dieser Arbeit beschäftigt sich mit den Kalziumtransienten bei Motoneuronen aus dem Nmd2J (SMARD1) Mausmodell. Die SMARD1 gilt als eigenständige Form der spinalen Muskelatrophien mit unterschiedlicher Genetik und unterschiedlichen klinischen Merkmalen. Die Motoneurone weisen in Bezug auf die Kalziumtransienten keine Unterschiede zwischen Wildtyp und Nmd2J Mutante auf. Es ergibt sich somit kein Hinweis darauf, dass die Degeneration der Motoneurone bei der SMARD1 von einer Störung der Kalziumhomöostase im distalen axonalen Bereich ausgeht. / Spinal muscular atrophy (SMA) is a monogenetic disorder which is caused by the loss of the SMN Protein and leads to the degeneration of α-motoneurons. Within the first few months of life most patients are clinically affected with severe motor deficits of skeletal muscles and a diaphragm paralysis, going along with a reduced life expectancy depening on the degree of severity. With the aid of SMA mouse models it was shown that the loss of motoneurons with Smn deficiancy lies in an impaired neurotransmission of the motoneuron endplat leading to a differentiation disorder of the motoneurons. This differentiation disorder is strongly connected to a reduced cluster formation of voltage-dependent calcium channels in the distal axonal area. The impaired cluster formation in turn leads to a reduced frequency of spontanous calcium transients at the axon terminus, followed by an altered axonal elongation. In this work the following aspects concerning the enhancement and induction of spontanous calcium transients in mouse models of spinal muscular atrophy were adressed: 1) Does the external application of calcium channel agonists increase spontanous calcium transients in Smn-deficient motoneurons? 2) Is the neurotrophic factor Brain-derived neurotrophic factor (BDNF) able to induce spontanous calcium transients in primary motoneurons? 3) Do primary motoneurons of a mouse model for spinal muscular atrophy with respiratory distress (SMARD1) show altered calcium transients as well? The results of my work show that the calcium channel agonist R-Roscovitine significantly increases the frequency of spontanous calcium transients in growth cones of Smn-deficient motoneurons which in turn has a regulatory effect on the differentiation of SMA motoneurons. Smn-deficient motoneurons treated with R-Roscovitine do not show any differences concerning axon length and growth cone size compared to control cells. Apart from the agonist effect on the calcium channels, R-Roscovitine also has an inhibitory impact on the cyclin-dependant kinase 5. The results of this work show that the positive effect on the calcium 12 transients under R-Roscovitine treatment is because R-Roscovitine binds directly to the calcium channel rather than due to an inhibition of cdk5. Arguments supporting this idea are the rapid and reversible channel kinetics of R-Roscovitine. Plus, the effect of R-Roscovitine can be repealed when the Cav2 channal antagonist ω-conotoxin is given simultaneously. In the second part of this work the influence of the neurotrophic factors BDNF, CNTF and GDNF on the calcium transients of wildtype motoneurons is investigated. Comparing these neurotrophic factors show that only BDNF has an impact on local calcium channel kinetics in growth cones of motoneurons. The last part of this work deals with the investigation of calcium transients in motoneurons from the Nmd2J (SMARD1) mouse model. SMARD1 is an independent form of spinal muscular atrophies with different genetical and clinical aspects compared to proximal SMA. The results of this work show that Nmd2J motoneurons do not show any difference in growth cone calcium influx between wildtype and mutant. Thus, there is no indication that the degeneration of SMARD1 motoneurons has any pathophysiological similarities with motoneurons from the proximal SMA mouse model. Hence, there are also no indications that the reason for motoneuron degeneration in SMARD1 lies in an impaired calcium homeostasis in the distal axonal area.

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Ever since the advent of polymer science, polyurethanes have played a huge role in the industrial world. They have been used in endless applications from furniture padding to aircraft coatings, to binders for insensitive munitions. It is therefore important that the chemistry of polyurethanes is well understood as well as the ability to draw relationships between the raw materials selected and the end-use properties of the polymer. Because of the multitude of practical applications, the development of an undergraduate polymer chemistry laboratory focused on polyurethane elastomers is developed and described herein. Polymer chemistry students are exposed to hydroxyterminated polybutadiene (HTPB) polyols as well as di- and multifunctional isocyanates for use in a tin-catalyzed reaction. The effect of catalyst concentration and crosslinking agent on cure time, prepolymer structure on end-use properties, and the effect of crosslink density on physical properties are explored. Students also receive a very important introduction to statistical experimental design. They learn when using statistical experimental design is necessary, and they learn how to manipulate, analyze, and interpret data using two-way ANOVA in Minitab. The development of the lab experiment also led to extrapolating the use of polyurethane elastomers into a new application, the development of a polyurethane spinal muscular atrophy (SMA) brace. SMA is a neurodegenerative disorder that results in the mutation or deletion of the spinal motor neuron gene, resulting in the atrophy of a subject’s spine muscles throughout the continuation of their life. These patients are therefore forced to wear a brace for the entirety of their lives. The current brace technology in use by SMA patients is limited by the fact that SMA affects a very small amount of the population and therefore it is not cost-effective for industry to develop a brace technology designed for these patients. Scoliosis braces such as thoracolumbrosacral orthoses (TLSOs) are too hard and too uncomfortable for patients with SMA; therefore, the polyurethane elastomer was extrapolated to develop a brace with more flexibility and more durability. Two generations of polyurethane elastomeric brace were developed and evaluated by a subject and family with an SMA background. The brace is a much improved technology to the TLSO braces and provides more flexibility, more mobility, greater comfort, and superior modularity to the old technology. An instruction manual is also included with a step-by-step process of how to reproduce the brace.