Back pain is a highly prevalent condition whose pathogenesis is associated with intervertebral disc (IVD) degeneration. Degeneration is driven by abnormal cell biology, particularly within the IVD’s inner core, the nucleus pulposus (NP). In recent years, there has been an ever-increasing search for cell-based therapies aimed at correcting the cell biology and thus repairing/regenerating the degenerate IVD. The success of these novel therapies, however, requires a thorough understanding of IVD development and of the phenotype of its cells. The embryonic, foetal and juvenile NP is populated by large vacuolated notochordal cells that with skeletal maturity are replaced by smaller NP cells. Since notochordal cells have been shown to display protective and anabolic roles in the IVD their loss in humans has often been suggested to initiate the degenerative process. As such, a detailed understanding of notochordal cells and their regulatory pathways may help identify factors involved in IVD homeostasis and aid the development of novel cell-based therapies targeting IVD degeneration. The study of human notochordal cells has, however, been hindered by ethical, logistical and technical difficulties in obtaining suitable samples and, as such, the human notochordal cell phenotype is, to date, unknown, constituting a major limitation in the field. The work presented here was conducted with the objective of developing a methodology to isolate human developing notochordal cells (NP progenitors) from adjacent sclerotomal cells (annulus fibrosus and vertebral body progenitors), to characterise the notochordal cell phenotype and identify potential factors involved in notochordal cell biology. Initially, human embryonic and foetal spines were characterised to assess their suitability as a source of notochordal cells and to identify a notochord-specific marker that could be used to isolate notochordal cells for microarray studies. The human developing spine contained large vacuolated notochordal cells in all stages analysed (3.5-18 weeks post-conception (WPC)) that specifically expressed KRT8, KRT18 and KRT19 at all stages and CD24 between 5.5-18 WPC. KRT18 and CD24 were independently used to label notochordal cells (7.5-14 weeks post-conception) and separate them from sclerotomal cells. Methodologies were developed to allow extraction of RNA of sufficient quality for microarray analysis from fixed, permeabilised (in the case of KRT18) and/or, labelled and sorted cells (CD24). Microarray analysis identified and real-time qPCR and, for some markers, immunohistochemistry, validated GRB14, SLC19A1, FGF10, ADORA3, TBXA2R, CDH6, ANPEP, CD69, CD24, RTN1, PRPH, MAP1B, ISL1 and CLDN1 as human notochordal cell markers. Ingenuity pathway analysis was performed to investigate the pathways/networks and upstream regulators and downstream effectors of notochordal cells. Inhibition of inflammation and angiogenesis were identified as relevant to notochordal cell biology, function and, possibly, to the known protective and anabolic role notochordal cells display in the IVD. Notochordal marker gene expression was identified in adult NP tissue, and negatively correlated with degeneration. Proteins encoded by ADORA3 and MAP1B were expressed by a proportion of adult NP cells, suggesting the presence of notochord-derived cells in the adult NP.Importantly, this is the first study to detail a methodology and successfully isolate human notochordal cells. Such methodology has the potential to be used to culture and investigate the biology of viable human notochordal cells (CD24+ve). Future studies aimed at developing cell-based therapies for IVD degeneration could also use these identified markers to assess appropriate stem cell differentiation to notochordal cells.

Intervertebral disc (IVD) degeneration is associated with low back pain (LBP). It has been suggested that changes in the IVD physio-chemical microenvironment (i.e. hypoxia, reduced nutrient and acidic conditions) may lead to disc degeneration. Studying the response of human nucleus pulposus (NP) cells to these conditions could establish the causal relationship between IVD microenvironment and aberrant cellular behaviour, characteristic of disc degeneration. Human bone marrow mesenchymal stem cells (BM-MSCs) are a promising cell population for disc regeneration. However, knowledge of their survival and functioning in the microenvironment of the IVD is still lacking. Moreover, in vitro co-culture model studies that are used to study MSC/disc cell interaction, also need to consider the effect of the microenvironment on cellular responses. BM-MSCs and degenerate NP cells were cultured alone or co-cultured in monolayer under hypoxia (2%O2), reduced nutritional (2% serum or/and 5mM glucose) and acidic (moderate pH 6.8 or severe pH 6.5) conditions alone or in combination for 7 days. Cell viability, proliferation, gene and protein expression was assessed. Degenerate NP cells and BM-MSCs maintained good cell viability under all conditions. Both cell types demonstrated overall similar proliferation and gene and protein responses under the majority of the conditions and combinations studied. Hypoxia promoted aggrecan and versican matrix biosynthesis in both cell types. Nutrient deprived and moderate acidic conditions (pH 6.8) inhibited proliferation of both cell types. Interestingly the combination of hypoxia with these conditions showed a protective effect in modulating cell proliferation. These results imply that hypoxia may be beneficial in some instances. Nutrient deprived conditions had a relatively minor effect on degenerate NP cell gene and protein expression but these conditions specifically inhibited VCAN expression in BM-MSCs. The combination of hypoxia with these conditions increased or restored VCAN expression. Interestingly the combination of hypoxia with reduced glucose conditions increased aggrecan and versican matrix biosynthesis in both NP cells and BM-MSCs. The combination of hypoxia and complete nutrient deprived conditions (both reduced serum and reduced glucose) impaired ACAN, VCAN and PAX-1 gene and aggrecan and versican protein expression in degenerate NP cells implicating disc hypoxia and complete nutrient deprived combined microenvironment in accelerating degenerate changes in NP cells. In contrast, these conditions showed no such detrimental effects on BM-MSC gene and protein expression. pH 6.5 was critical for both cell types proliferation and ACAN and VCAN gene expression suggesting that severe acidic conditions may exacerbate degenerative changes and be inhibitory for implanted MSCs. Finally, a combination of hypoxia, complete nutrient deprived and moderate acidic conditions, reduced cell proliferation without affecting the gene expression profile of both cell types. IVD-like physio-chemical microenvironmental conditions also appeared to influence differentiation of BM-MSC and modulation of degenerate NP cell phenotype observed during co-culture. Noticeably hypoxia, reduced serum or reduced glucose conditions stimulated BM-MSC differentiation and modulation of degenerate NP cell phenotype. Hypoxia also increased or recovered changes at gene expression level in both BM-MSCs and degenerate NP cells under nutrient deprived (reduced serum or/and reduced glucose) conditions during co-culture. Degenerate NP cell and BM-MSC co-culture also showed noticeable increase in aggrecan and versican biosynthesis under hypoxia and reduced glucose combine conditions, implicating these in improving the co-culture responses. Severe pH condition alone, pH 6.8 in combination with hypoxia and finally all IVD-like physio-chemical conditions together compromised co-culture responses. Such results imply that IVD-like physio-chemical microenvironmental conditions may influence MSC based regenerative outcomes. This work has increased our understanding about the influence of disc harsh microenvironment on degeneration and regeneration processes.

Retinose Pigmentar (RP) é uma doença hereditária que conduz progressivamente à cegueira. Mais de 150 mutações da rodopsina associadas à RP foram descritas, e causam a alteração da sua conformação. Esta tese testou a hipótese de que pequenas moléculas auxiliam na formação da rodopsina e/ou reduzem a morte dos fotorreceptores. As mutações da RP, N15S e P23H, revelaram diferenças quanto às características e gravidade devido à má-formação das proteínas mutantes. Ligação de pequenas moléculas (retinóides, íons metálicos, clorofilas e antocianinas) à rodopsina foi demonstrada in vitro. O derivado da clorofila, Ce6, mostrou-se mais efetivo, conferindo maior estabilidade e foi então testado em ratos submetidos à degeneração por luz ou em modelos de RP (P23H e S334ter). Observou-se uma proteção contra a degeneração por luz e uma significante diminuição da degeneração no P23H. Em contraste, Ce6 causou um aumento na degeneração dos fotorreceptores do S334ter. Finalmente, resultados clínicos, bioquímicos e in vivo foram comparados e mostraram estar altamente relacionados. / Retinitis Pigmentosa (RP) is an inherited disease that progressively leads to blindness. More than 150 mutations associated with RP are known in rhodopsin, causing its misfolding. This thesis tested the hypothesis that small molecules can rescue folded rhodopsin and/or reduce photoreceptor cell death. RP mutations, N15S and P23H, revealed differences in characteristics and severity of misfolding of the mutant proteins. Binding of small molecule classes (retinals, metal ions, chlorophylls and anthocyanins) to rhodopsin was demonstrated in vitro. The chlorophyll derivative, Ce6, was most effective in conferring stability and therefore tested in rats subjected to light-damage and RP rat models, P23H and S334ter. Protection against the light-induced retinal degeneration and more importantly a significant slowing of the photoreceptor degeneration rate in the P23H rat were observed. In contrast, Ce6 increased photoreceptor degeneration in the S334ter rat. Finally, clinical, biochemical and in vivo rat data were compared and it was found to be highly correlated.

After dendrite transection, two primary injury current pathways may acount for cell death: (1) the lesion current at the site of injury and (2) the voltage sensitive calcium channels along the dendrite. Lesions were made with a laser microbeam in mouse spinal monolayer cell cultures. Polylysine was tried as a positively charged "molecular bandage" to block the lesion current. The calcium channel blockers, verapamil and nifedipine, were used to reduce the calcium channel current. Control toxicity curves were obtained for all three compounds. The results show that neither verapamil, nifedipine, nor polylysine (MW: 3,300) protect nerve cells after dendrite amputation 100 ptm from the soma. The data also indicate that these compounds do not slow the process of cell death after such physical trauma.

abstract: Patients suffering from Retinitis Pigmentosa (RP), the most common type of inherited retinal degeneration, experience irreversible vision loss due to photoreceptor degeneration. The preservation of cone photoreceptors has been deemed medically relevant as a therapy aimed at preventing blindness in patients with RP. Cones rely on aerobic glycolysis to supply the metabolites necessary for outer segment (OS) renewal and maintenance. The rod-derived cone viability factor (RdCVF), a protein secreted by the rod photoreceptors that preserves the cones, accelerates the flow of glucose into the cone cell stimulating aerobic glycolysis. This dissertation presents and analyzes ordinary differential equation (ODE) models of cellular and molecular level photoreceptor interactions in health and disease to examine mechanisms leading to blindness in patients with RP. First, a mathematical model composed of four ODEs is formulated to investigate the progression of RP, accounting for the new understanding of RdCVF’s role in enhancing cone survival. A mathematical analysis is performed, and stability and bifurcation analyses are used to explore various pathways to blindness. Experimental data are used for parameter estimation and model validation. The numerical results are framed in terms of four stages in the progression of RP. Sensitivity analysis is used to determine mechanisms that have a significant affect on the cones at each stage of RP. Utilizing a non-dimensional form of the RP model, a numerical bifurcation analysis via MATCONT revealed the existence of stable limit cycles at two stages of RP. Next, a novel eleven dimensional ODE model of molecular and cellular level interactions is described. The subsequent analysis is used to uncover mechanisms that affect cone photoreceptor functionality and vitality. Preliminary simulations show the existence of oscillatory behavior which is anticipated when all processes are functioning properly. Additional simulations are carried out to explore the impact of a reduction in the concentration of RdCVF coupled with disruption in the metabolism associated with cone OS shedding, and confirms cone-on-rod reliance. The simulation results are compared with experimental data. Finally, four cases are considered, and a sensitivity analysis is performed to reveal mechanisms that significantly impact the cones in each case. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2020

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22357号 / 医博第4598号 / 新制||医||1042(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 井上 治久, 教授 伊佐 正, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Temporal lobe epilepsy (TLE) is among the most common forms of epilepsy in adults. A significant proportion of patients experience drug-resistant seizures associated with hippocampal sclerosis (HS), in which there is extensive cell loss in the hippocampal cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) subfields. The dentate gyrus (DG) and cornu ammonis 2 (CA2) subfield are more resilient to neurodegeneration, and a prior report found that CA2 neurons in tissue from TLE patients show interictal-like firing and receive aberrant perisomatic excitatory synapses from DG granule cell (GC) mossy fibers (Wittner et al. Brain. 2009;132:3032–3046). Furthermore, findings from a collaborative study in the laboratory of Dr. Helen Scharfman demonstrated that chronic chemogenetic inhibition of CA2 pyramidal neurons (PNs) in vivo significantly reduced the frequency of spontaneous recurring convulsive seizures in epileptic mice. I therefore explored the hypothesis that pathophysiological changes to CA2 PN excitability or synaptic connectivity may be associated with chronic epilepsy by examining CA2 properties in a mouse model of TLE.Pilocarpine-induced status epilepticus in mice leads to a pattern of hippocampal sclerosis-like neurodegeneration and recurring spontaneous seizures, and thus recapitulates key features of TLE. I performed whole-cell electrophysiological recordings from PNs in acute hippocampal slices from pilocarpine (PILO)-treated mice in the chronic phase of epilepsy as well as age-matched controls. In some experiments I used Cre-expressing mouse lines to selectively express a light-activated excitatory channel in CA2 PNs or DG GCs. I also performed immunohistochemistry to examine CA2 interneuron (IN) populations following PILO-induced status epilepticus. I found that in healthy tissue CA2 PNs, like those in CA3, both directly excited other CA2 PNs via a recurrent CA2-CA2 PN circuit and indirectly inhibited other CA2 PNs by recruiting local INs. The CA2 and CA3 subfields also form reciprocal excitatory and feedforward inhibitory circuits. These recurrent and reciprocal circuits constitute an auto-associative network in which INs crucially control CA2/CA3 population excitability. DG GC mossy fibers made direct but relatively weak excitatory synapses onto CA2 PNs. Following PILO-induced status epilepticus, feedforward inhibition is diminished in the DG GC mossy fiber circuit to CA2, in the CA2/CA3 recurrent network, and in the forward-projecting circuit from CA2 PNs to CA1. I found a modest decrease in the density of parvalbumin-immunopositive INs and a profound decrease of cholecystokinin-immunopositive IN density, combined with degradation of the pyramidal neuron-associated perisomatic perineuronal net, which together may contribute to this inhibitory disruption. DG GC mossy fiber excitatory input to CA2 PNs is strengthened, along with CA2 PN excitatory input to CA1 PNs. Finally, in hippocampal slices from PILO-treated mice I found an increase in CA2 PN input resistance and thus elevated intrinsic excitability, leading to a higher firing rate upon direct current injection. The combined effect of these changes may drive the emergence of epileptiform synchronization in the CA2 network and facilitate the propagation of seizure activity from the DG and entorhinal cortex directly to CA1 via the CA2-centered disynaptic (EC LII --> CA2 --> CA1) and alternate trisynaptic circuits (EC LII --> DG --> CA2 --> CA1).

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23087号 / 医博第4714号 / 新制||医||1050(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 木村 剛, 教授 YOUSSEFIAN Shohab, 教授 大森 孝一 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Glia, whose name derives from the original Greek word, meaning “glue,” have long been understood to be cells that play an important functional role in the nutritive and structural support of the central nervous system, yet their full involvement has been historically undervalued. Despite the strong evidence that glial reactions to cellular debris govern the health of the nervous system, the specific properties of damaged axonal debris and the mechanisms by which glia sense them, morphologically adapt to their presence, and initiate phagocytosis for clearance, have remained poorly understood. The work presented in this thesis was aimed at addressing this fundamental gap in our understanding of the role for glia in neurodegenerative processes. I demonstrate that the cellular machinery responsible for the phagocytosis of apoptotic cell corpses is well conserved from worms to mammals. Draper is a key component of the glial response machinery and I am able to show here, for the first time, that it signals through Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70. Shark binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. I show that Shark activity is essential for Draper-mediated signaling events in vivo, including the recruitment of glial membranes to axons undergoing Wallerian degeneration. I further show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. Therefore I propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the subsequent downstream activation of the Draper pathway. I observed that these Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signaling events in myeloid and lymphoid cells. Thus, Draper appears to be an ancient immunoreceptor with an extracellular domain tuned to modified-self antigens and an intracellular domain that promotes phagocytosis through an ITAM domain-SFK-Syk-mediated signaling cascade. I have further identified the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. I am able to demonstrate that Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway to promote a distinct step in the clearance of axonal debris. Whereas Draper signaling is required early during glial responses, promoting glial activation and extension of glial membranes to degenerating axons, the Crk/Mbc/dCed-12 complex functions at later stages of glial response, promoting the actual phagocytosis of axonal debris. Finally, many interesting mutants have been identified in primary screens for genes active in neurons that are required for axon fragmentation or clearance by glia, and genes potentially active in glia that orchestrate clearance of fragmented axons. The further characterization of these genes will likely unlock the mystery surrounding “eat me” and “find me” cues hypothesized to be released or exposed by neurons undergoing degeneration. Illuminating these important glial pathways could lead to a novel therapeutic approach to brain trauma or other neurodegenerative conditions by providing a druggable means of inducing early attenuation of the glial response to injury down to levels less damaging to the brain. Taken together, my combined work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and the termination of glial responses to injury are genetically separable events mediated by distinct signaling pathways.

No description available.