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.

The retromer is a protein complex whose function is to mediate the recycling of proteins from the endosome to either the plasma membrane or the trans-Golgi network. A deficit in retromer function has been associated with multiple neurodegenerative disorders, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). In both AD and PD, deficiencies have been found in retromer expression both in patient tissues and in animal models of disease. Furthermore, mutations in the retromer and in retromer-associated genes have been strongly linked with both diseases. Despite ample evidence of the link between the retromer and neurodegeneration, little is known about the retromer in the context of amyotrophic lateral sclerosis (ALS), another common neurodegenerative disorder. ALS is an adult-onset neurodegenerative disorder of the upper and lower motor neurons (MNs) characterized by muscle wasting and weakness leading to death within 3-5 years after diagnosis. To date, the most commonly used model of ALS is a transgenic (Tg) mouse that overexpresses an ALS-causing G93A mutation in the human superoxide dismutase 1 (SOD1) gene. In this study, I first establish a link between the retromer and ALS by showing that cells from ALS patients as well as tissues and cells from SOD1G93A-Tg mice express lower protein levels of the retromer core components—vacuolar protein sorting 35 (Vps35), Vps26a, and Vps29. I then establish that deficiencies in retromer core proteins have functional consequences in an in vitro model of ALS. Having found significant deficiencies in the retromer in SOD1G93A-Tg mice, I then followed the model of studies performed in mouse models of other neurodegenerative disorders by investigating whether repletion of retromer levels, either virally or pharmacologically, in SOD1G93A-Tg mice confers a therapeutic benefit. Surprisingly, I find that rather than ameliorating disease, repletion of retromer levels in SOD1G93A-Tg mice exacerbates it, resulting in a faster decline in motor performance, earlier mortality, and a decrease in MNs in the spinal cord. Finally, since retromer repletion causes deleterious effects on SOD1G93A-Tg mouse disease progression, I study the effect of a single allele deletion of Vps35 in SOD1G93A-Tg mice and find that this depletion of the retromer results in amelioration of disease, including delayed onset of symptomatology, slower decline of motor deficits, delayed mortality, and an increase in MNs in the spinal cord. Altogether, the findings reported herein, support the notion that a mild defect in retromer develops over the course of the disease, which, rather than being deleterious may be therapeutic in mutant SOD1-induced MN degeneration. Perhaps this unexpected outcome may be explained by the fact that the observed mild nature of the defect is not sufficient to kill MNs but enough to alter the trafficking of specific cargos such as AMPA receptors, allowing MNs to better withstand the neurodegenerative process.

The exceptional abilities of top-tier athletes – from Simone Biles’ dizzying gymnastics to LeBron James’ gravity-defying bounds – can easily lead one to forget to marvel at the exceptional breadth of everyday movements. Whether holding a cup of coffee, reaching out to grab a falling object, or cycling at a quick clip, every motor action requires activating multiple muscles with the appropriate intensity and timing to move each limb or counteract the weight of an object. These actions are planned and executed by the motor cortex, which transmits its intentions to motoneurons in the spinal cord, which ultimately drive muscle contractions. A central problem in neuroscience is precisely how neural activity in cortex and the spinal cord gives rise to this diverse range of behaviors. At the level of spinal cord, this problem is considered to be well understood. A foundational tenet in motor control asserts that motoneurons are controlled by a single input to which they respond in a reliable and predictable manner to drive muscle activity, akin to the way that depressing a gas pedal by the same degree accelerates a car to a predictable speed. Theories of how motor cortex flexibly generates different behaviors are less firmly developed, but the available evidence indicates that cortical neurons are coordinated in a similarly simplistic, well-preserved manner. Yet a potential complication for both these old and new theories are the relative paucity of diverse behaviors during which motor cortex and spinal motoneurons have been studied. In this dissertation, I present results from studying these two neuronal populations during a broader range of behaviors than previously considered. These results indicate, in essence, that diverse behaviors involve greater complexity and flexibility in cortical and spinal neural activity than indicated by current theories.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20609号 / 医博第4258号 / 新制||医||1023(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 伊佐 正, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The stretch reflex circuit is one of the most primitive circuits of mammalian system and serves mainly to control the length of the muscle. It consists of four elements: the stretch sensor (muscle spindle/ intrafusal fiber lie parallel between extrafusal, contractile musculature), extrafusal muscle fiber, sensory neuron and motoneuron. The basic principle of the stretch reflex arc circuit is as follows: whenever there is a sudden stretch in a muscle, it needs to compensate back to its original length so as to prevent any kind of injury. It performs this compensation process using a simple negative feed back circuit called the stretch reflex arc. Any form of stretch in a muscle activates the stretch sensors (muscle spindle/ intrafusal fiber) lying deep in each muscle. After the stretch sensors get activated, it sends a train of signals to the spinal cord through the sensory neurons. The sensory neurons relay this information to the motoneuron. The motoneuron performs the necessary information processing and sends the message to the extrafusal fibers so as to compensate for the sudden stretch action. The motoneuron conveys this message to the extrafusal fibers by communicating through the special synaptic junctions called neuromuscular junctions. Based on this information, the extrafusal fibers act accordingly so as to counter the effect of sudden stretch. This is also called the monosynaptic stretch reflex that involves a single synapse between a sensory neuron and a motoneuron. To date studying these stretch reflex circuits is only feasible in animal models. Almost no effort has been made to tissue engineer such circuits for a better understanding of the complex development and repair processes of the stretch reflex circuit formation. The long-term goal of this research is to tissue engineer a cellular prototype of the entire iii stretch reflex circuit. The specific theme of this dissertation research was to tissue engineer the motoneuron to muscle segment of the stretch reflex arc circuit utilizing micro-fabrication, interface design and defined medium formulations. In order to address this central theme, the following hypothesis has been proposed. The first part of the hypothesis is that microfabrication technology, interface design and defined medium formulations can be effectively combined to tissue engineer the motoneuron to muscle segment of the stretch reflex arc. The second part of the hypothesis is that different growth factors, hormones, nanoparticles, neurotransmitters and synthetic substrate can be optimally utilized to regenerate the adult mammalian spinal cord neurons so as to replace the embryonic motoneurons in the stretch reflex tissue engineered construct with adult motoneurons. In this body of work, the different tissue engineering strategies and technologies have been addressed to enable the recreation of a in vitro cellular prototype of the stretch reflex circuit with special emphasis on building the motoneuron to muscle segment of the circuit. In order to recreate the motoneuron to muscle segment of the stretch reflex arc, a successful methodology to tissue engineer skeletal muscle and motoneuron was essential. Hence the recreation of the motoneuron to muscle segment of the stretch reflex circuit was achieved in two parts. In the part 1 (Chapters 2-5), the challenges in skeletal muscle tissue engineering were examined. In part 2 (Chapters 6-7), apart from tissue engineering the motoneuron to muscle segment, the real time synaptic activity between motoneuron and muscle segment were studied using extensive video recordings. In part 3 (Chapters 8-10), an innovative attempt had been made to tissue engineer the adult mammalian spinal cord neurons so that in future this technology could utilized to replace the iv embryonic neurons used in the stretch reflex circuit with adult neurons. The advantage of using adult neurons is that it provides a powerful tool to study older neurons since these neurons are more prone to age related changes, neurodegenerative disorders and injuries. This study has successfully demonstrated the recreation of the motoneuron to muscle segment of the stretch reflex arc and further demonstrated the successful tissue engineering strategies to grow adult mammalian spinal cord neurons. The different cell culture technologies developed in these studies could be used as powerful tools in nerve-muscle tissue engineering, neuro-prosthetic devices and in regenerative medicine.

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Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

No description available.