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.

Licentiatavhandling (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 2 uppsatser.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / 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. "December 2006" Includes bibliographical references.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

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.

Abstract The complete exon-intron organization of the gene coding for the mouse α1(XIII) collagen chain, Col13a1, was characterized from genomic clones and multiple transcription initiation points were determined. Detailed comparison of the human and mouse genes showed that the exon-intron structures are completely conserved between the species, and both genes have their 5' untranslated region preceded by a highly conserved putative promoter region. The chromosomal location of the mouse gene was determined to be at chromosome 10, band B4, between markers D10Mit5 – (2.3 ± 1.6 cM) – Col13a1 – (3.4 ± 1.9 cM) – D10Mit15. The location of the genes for both the catalytically important α-subunit of prolyl 4-hydroxylase (P4HA) and human type XIII collagen (COL13A1) were previously mapped to 10q21.3-23.1. Prolyl-4-hydroxylase catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of peptide-bound proline and plays a crucial role in the synthesis of these proteins. The order and transcriptional orientation of the COL13A1 and P4HA was determined. These two genes were found to lie at tail to tail orientation on chromosome 10 and the distance between these genes was determined to be about 550 kbp. To study the function of type XIII collagen we used gene targeting in ES cells to generate a mouse line that carries a mutated type XIII collagen gene. Instead of normal protein, mutant mice express type XIII collagen with an altered amino-terminus in which the cytosolic and the transmembrane domains have been replaced with an unrelated sequence. The homozygous mice are fertile and viable but they show alterations in skeletal muscles, mainly wavy sarcolemma and increased variation in muscle fiber diameter. Ultrastructural studies revealed additional abnormalities such as streaming of z-disks, accumulation and enlargement of mitochondria, and disorganized myofilaments. The basement membranes of the muscle cells showed areas of detachment from the plasma membrane and the fibrillar matrix of the cells was less compact than in control animals. Fibroblasts cultured from mutant mice had normal levels of type XIII collagen but exhibited decreased adhesion to substratum which might be explained by a reduced anchoring strength of the altered protein.

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.

No description available.

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.