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After traumatic injuries to the brachial plexus there is a risk that one or more of the spinal roots are torn from the spinal cord, known as avulsion injury. This often leads to paralysis and chronic pain, notoriously difficult to treat with current pharmacotherapy. Surgical treatment may improve motor function but sensory recovery is usually poor as sensory axons fail to establish functional connections inside the spinal cord. The aims of this thesis were to develop a model for dorsal root avulsion in rodents in order to investigate the potentials of stem cell therapy for enhancing sensory regeneration after spinal root avulsion. Two different types of stem cells, embryonic and neural crest stem cells, have been transplanted to the avulsion model and analysed using immunohistochemical methods. The results indicate that stem cells survive after transplantation to the avulsed dorsal root and associate with regenerating axons. Furthermore, the different stem cells display different phenotypes after transplantation where embryonic stem cells give rise to neurons located outside the spinal cord that could serve as projection neurons whereas the neural crest stem cells form elongated tubes outlining the avulsed dorsal root and are associated with regenerating neuronal fibers. We have also discovered that the neural crest stem cells migrate into the damaged spinal cord as single cells. The neural crest stem cells also display a diversity in generating both neuronal and glial cells that may have different beneficial effects in neural repair following dorsal root avulsion. To improve the survival of stem cell transplants, the potentials of co-transplanting embryonic stem cells together with nanoparticle delivered growth factor mimetics has been investigated. The results indicate that nanoparticle delivered growth factors improve both transplant survival and maturation in comparison to untreated controls and may be a promising strategy in stem cell transplantation.

Yukako Hattori, Tadao Usui, Daisuke Satoh, Sanefumi Moriyama, Kohei Shimono, Takehiko Itoh, Katsuhiko Shirahige, Tadashi Uemura, Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier, Developmental Cell, Volume 27, Issue 5, 9 December 2013, Pages 530-544, ISSN 1534-5807 / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18418号 / 生博第298号 / 新制||生||39(附属図書館) / 31276 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 西田 栄介, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

<p>Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.</p><p>In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.</p> / Dissertation

Luman (CREB3, LZIP) is a basic leucine zipper transcription factor involved in regulation of the unfolded protein response (UPR), dendritic cell maturation, and cell migration. But despite reported expression in primary sensory neurons, little is known about its role in the nervous system. Luman mRNA from rat sensory neurons was amplified and its coding sequence was determined. The rat Luman cDNA contains a full-length open reading frame encoding 387 amino acids, and the recombinant protein generated from this clone activated transcription from UPR elements. Quantitative RT-PCR revealed rat Luman transcripts in a variety of rat tissues with the highest levels in nervous system tissue. In situ hybridization confirmed the findings and demonstrated that the Luman mRNA hybridization signal localizes to neurons and satellite glial cells in dorsal root ganglia (DRG), the cytoplasm of hepatocytes in liver, and the hippocampal pyramidal cell layers in CA1 and CA3 and the granular cell layer of the dentate gyrus. Luman protein localizes with axonal endoplasmic reticulum (ER) components along the axon length within the sciatic nerve and is activated by sciatic nerve injury. Adult sensory axons also contain Luman mRNA which is translated within the axon and transported to the cell body via the importin-mediated retrograde transport system in response to nerve injury. Further, creation of an N-terminal, C-terminal dual fluorescence-tagged Luman adenoviral construct allowed visualization of the cleavage and retrograde translocation of the N-terminal portion of Luman to the nucleus in real time in vivo and in vitro. Neuronal or subcellular axonal knockdown of Luman significantly impaired the intrinsic ability of injury-conditioned, but not naïve, sensory neurons to extend the regeneration-associated elongating form of neurites. Sciatic nerve crush injury also induced activation of the UPR in axotomized DRGs, including genes linked to cholesterol biosynthesis. Knockdown of Luman decreased the activation of UPR and cholesterol biosynthesis, and axotomy-inducted increases in neurite outgrowth, which could be largely rescued with either mild UPR inducer treatment or cholesterol supplementation. Together these findings provide novel insights linking remote injury-associated axonal ER responses to the regenerative growth capacity of adult sensory neurons via axonal activation and synthesis of Luman and reveal a role for the UPR in regulation of axotomy-induced neurite outgrowth that is critically dependent on Luman.

Glutamate is the essential neurotransmitters in pain pathways. The discovery of the vesicular glutamate transporters (VGLUT1-3) has been a fundamental step on the way to describe glutamate-dependent pain pathways. We used the Cre-lox system to construct conditional knockouts with deficient Vglut2 transmission in specific neuronal populations. We generated a Vglut2f/f;Ht-Pa-Cre line to selectively delete Vglut2 from the peripheral nervous system. These Vglut2 deficient mice showed decreased acute nociceptive responses and were less prone to develop an inflammatory state. They did not develop cold allodynia, or heat hyperalgesia and were less hypersensitive to mechanical stimuli in the PSNL chronic pain model. Further analyses of genes with altered expression after nerve injury, revealed candidates for future studies of chronic pain biomarkers. Interestingly, the Vglut2f/f;Ht-Pa-Cre mice developed an elevated itch behavior. To investigate more specific neuronal populations, we analyzed mice lacking Vglut2 in the Nav1.8 population, as inflammatory hyperalgesia, cold pain, and noxious mechanosensation have been shown to depend upon Nav1.8Cre positive sensory neurons. We showed that deleting Vglut2 in Nav1.8Cre positive neurons abolished thermal hyperalgesia in persistent inflammatory models and responses to noxious mechanical stimuli. We also demonstrated that substance P and VGLUT2-dependent glutamatergic transmission are co-required for the development of formalin-induced inflammatory pain and heat hyperalgesia in persistent inflammatory states. Deletion of Vglut2 in a subpopulation of neurons overlapping with the vanilloid receptor (TRPV1) primary afferents in the dorsal root ganglia resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. Substance P signaling and VGLUT2-mediated glutamatergic transmission in TRPV1 neurons was co-required for the development of inflammatory pain states. Analyses of an itch phenotype uncovered the pathway within TRPV1 neurons, with VGLUT2 playing a regulatory role and GRPR neurons, which are to plausible converge the itch signal in the spinal cord. These studies confirmed the essential role of VGLUT2-dependent glutamatergic transmission in acute and persistent pain states and identified the roles of specific subpopulations of primary afferent neurons. Additionally, a novel pain and itch transmission pathway in TRPV1/VGLUT2 positive neurons was identified, which could be part of the gate control of pain.

In recent years it has become apparent that neuronal development and function relies not just on the regulation of transcription but also on post-transcriptional events. Two prevalent mRNA-based regulatory mechanisms in neurons are asymmetric mRNA localisation and the generation of different 3’UTR isoforms by alternative polyadenylation (APA). While experiments in mammalian systems indicate that subcellular mRNA localisation plays an important role in regulating local expression of proteins in neuronal processes, little is known about how mRNAs reach their destinations. It has been proposed that APA allows the production of mRNA isoforms with different roles. However, the importance of 3’UTR extensions has not been addressed in detail, particularly at the organismal level. In my PhD, I investigated the mechanisms of mRNA localisation and functional consequences of APA using the Drosophila embryonic nervous system as a genetically tractable model. I screened for mRNAs that localise in embryonic axons using an available transgenic library of 3’UTR sequences, as well as publically available in situ hybridisation data. I found that Ankyrin2 (Ank2) mRNA localises in Drosophila embryonic sensory neurons, and showed that this is dependent on the kinesin-1 motor and microtubules. These data reveal an active mRNA transport system in embryonic neurons. I also showed that the Ank2 mRNA has an extended 3’UTR that is found in axons, suggesting that APA could be relevant to axonal functions of Ank2. I demonstrated that while mRNA molecules could still localise to axons upon CRISPR-Cas9-mediated deletion of the Ank2 3’UTR extension, a fraction of the mutant embryos had a disrupted nervous system. Interestingly, embryos that lack the ability to make Ank2 protein have an overtly normal embryonic nervous system. This observation reveals that the extension does not simply promote Ank2 protein function. Further experiments revealed that the extended 3’UTR is required for efficient locomotion of adult flies. While the exact function of the Ank2 3’UTR extension requires future investigation, I show that it is unlikely to be associated with the trafficking of associated proteins into axons. RNA affinity purifications from embryonic extracts provide evidence that the 3’UTR extension selectively binds conserved RNA-binding proteins. I speculate that the extension plays a role in regulating axonal morphogenesis by regulating the relative expression level of different Ank2 protein isoforms.

Background Autonomic regulation therapy involving either vagus nerve stimulation (VNS) or spinal cord stimulation (SCS) represents emerging bioelectronic therapies for heart disease. The objective of this study was to determine if VNS and/or SCS modulate primary cardiac afferent sensory transduction of the ischemic myocardium. Methods Using extracellular recordings in 19 anesthetized canines, of 88 neurons evaluated, 36 ventricular-related nodose ganglia sensory neurons were identified by their functional activity responses to epicardial touch, chemical activation of their sensory neurites (epicardial veratridine) and great vessel (descending aorta or inferior vena cava) occlusion. Neural responses to 1 min left anterior descending (LAD) coronary artery occlusion (CAO) were then evaluated. These interventions were then studied following either: i) SCS [T1-T3 spinal level; 50 Hz, 90% motor threshold] or ii) cervical VNS [15–20 Hz; 1.2 × threshold]. Results LAD occlusion activated 66% of identified nodose ventricular sensory neurons (0.33 ± 0.08–0.79 ± 0.20 Hz; baseline to CAO; p < 0.002). Basal activity of cardiac-related nodose neurons was differentially reduced by VNS (0.31 ± 0.11 to 0.05 ± 0.02 Hz; p < 0.05) as compared to SCS (0.36 ± 0.12 to 0.28 ± 0.14, p = 0.59), with their activity response to transient LAD CAO being suppressed by either SCS (0.85 ± 0.39–0.11 ± 0.04 Hz; p < 0.03) or VNS (0.75 ± 0.27–0.12 ± 0.05 Hz; p < 0.04). VNS did not alter evoked neural responses of cardiac-related nodose neurons to great vessel occlusion. Conclusions Both VNS and SCS obtund ventricular ischemia induced enhancement of nodose afferent neuronal inputs to the medulla.

The olfactory system is a great model for studying regeneration due to the olfactory epithelium’s regenerative capability which makes it a potential a source of neural stem cells. The olfactory epithelium presents three types of cells: sustentacular cells which provide support and act as glial supporting cells; olfactory sensory neurons that are in charge of detecting odorant molecules in the air; and the stem cells that generated the aforementioned cell types. Olfactory sensory neurons are constantly dying and being replaced by new neurons originating from the stem cells that lie at the base of the olfactory epithelium. We have used an injury model that allows us to remove all the olfactory sensory neurons from the olfactory epithelium, via a single injection of methimazole. Then, at different timepoints after injury we measure the functional recovery of the olfactory epithelium by analyzing the expression of specific synaptic associated markers. Specifically, we analyzed the expression of synaptophysin, tyrosine hydroxylase, vesicular glutamate transporter 1, and vesicular glutamate transporter 2. Simultaneously, we measured glomerular size in order to serve as an indicator of anatomical recovery. Finally, we correlate these findings with previously generated data in the lab associated with functional recovery through behavior.

Pax3 has been used as a valuable marker in research aimed at understanding tissue interactions involved in trigeminal ophthalmic (opV) placode development. While Pax3 expression coincides with opV neuron specification, the function of Pax3 in these cells has not previously been investigated. Splotch mutant mice (which lack Pax3) have a reduced trigeminal ganglion; however it is not clear whether this reduction is due to neural crest or placode cells. We have used electroporation in the chick model system to block or ectopically express Pax3 at key times in opV placode development. Using several markers of placode cell differentiation, we have determined the experimental effects manipulating Pax3. Blocking placodal Pax3 with gene specific morpholinos resulted in a loss of migratory placode cells, and a downregulation of all opV placode markers in targeted cells. Ectopic expression of Pax3, either within the placode domain or in adjacent cranial ectoderm, resulted in the upregulation of some but not all placode markers. We conclude that opV placodal Pax3 expression is required for normal placode cell development, and hypothesize that its expression must be tightly regulated in order for placode cells to fully differentiate. The precise role of Pax3 and Pax7 in the restriction and differentiation of dorsal interneuron progenitors has been difficult to assess due to the many additional factors involved in specification and patterning of the neural tube. We have used electroporation in the chick model system to ectopically express Pax3 and Pax7 unilaterally in the neural tube. Using several markers for differentiation of ventral and dorsal neuronal progenitors, we have experimentally determined the effects of Pax3 and Pax7 ventrally and dorsally. Ectopic expression of these transcription factors in the ventral neural tube resulted in the loss of motorneurons. Though mis-expression did not qualitatively affect commissural neurons as assayed by neurofilament staining, ectopic expression of Pax3 and Pax7 in the dorsal neural tube stopped dorsal interneuron progenitors from differentiating. We conclude that Pax3 and Pax7 expression is sufficient to restrict ventral neuron identity. We also hypothesize that downregulation of these transcription factors in the dorsal neural tube is required for normal dorsal interneuron differentiation.