During normal activity, tendons are subjected to dynamic tensile strains of approximately 1-10%, whilst mechanical overload can lead to damage and degradation and the development of tendinopathy. The tenocytes within tendon respond to this mechanical environment although the mechanisms are poorly understood. Primary cilia consist of a slender axoneme composed of acetylated α-tubulin and are known to regulate a variety of signalling pathways including mechanosignalling. In various cell types, mechanical loading also influences primary cilia length. However relatively little is known about tendon primary cilia structure and function. This thesis set out to examine the structure and organisation of primary cilia in tendon cells and the effect of mechanical loading, both in situ and in isolated cells cultured in monolayer. Studies analysed cilia expression using confocal immunofluorescence microscopy in tendon fascicles from rat tail and isolated human tenocytes. Results demonstrated that the prevalence and orientation of primary cilia was different in the fascicular matrix (FM) and interfascicular matrix (IFM) regions of the tendon. Stress deprivation caused differential cilia elongation between the FM and IFM, associated with disruption of the surrounding extracellular matrix and alterations in tissue biomechanics. In isolated tenocytes, primary cilia were significantly longer with a greater prevalence than in situ. Cyclic tensile loading applied using the Flexcell system resulted in cilia disassembly within 8 hours with a dramatic reduction in prevalence and length. This effect was completely reversible on removal of strain. A similar response was observed in situ within both FM and IFM regions of the tendon. This mechanically-induced cilia disassembly was shown to be mediated, at least in part, by the release of TGFβ and activation of HDAC6 which causes tubulin deacetylation. These results in this thesis suggest a novel feedback mechanism through which physiological and pathological mechanical loading may regulate primary cilia signalling.

oligodendrocytes migrate from the corpus callosum into the overlying cortex. The incidence of cilia did not change markedly across age groups, and did not vary consistently with the number of processes per cell, which was used as an indication of the maturation stage of OPCs and young OLs. The mean percent of Olig1 immunopositive (Olig1+) cells having cilia across ages was 33.1% + 16.5%, with all ages combined. In O4+ cells of these mice, 56.7 + 3.6% had primary cilia. If it is the case that adult OLs do not have cilia, the point in the lineage when primary cilia are lost is still unknown. Adult mice that had been injected with cyclopamine to block cilia-dependent Shh signaling were examined to determine whether the rate of generating new OPCs was influenced. In the CC of control mice, the numerical density of Olig1+/BrdU+ cells was 1.29 + 0.07/mm2 was reduced to 0.68 + 0.38/mm2 in the cyclopamine-injected group, and the numerical density of all BrdU+ cells (including both Olig1+ and Olig1- cells) of 4.55 + 1.50/mm2 in the control group was reduced to 3.14 + 1.27/mm2 in the cyclopamine-injected group. However, there were only 2 mice in each group and the differences were not statistically significant.

Indiana University-Purdue University Indianapolis (IUPUI) / Bone loss diseases, including osteoporosis affect millions of people worldwide. Understanding the underlying mechanisms behind bone homeostasis and adaptation is essential to uncovering new therapeutic targets for the prevention and treatment of bone loss diseases. Primary cilia have been implicated in the development and mechanosensation of various tissue types, including bone. The goal of the studies outlined in this thesis is to determine the mechanosensory role of primary cilia in bone cell function, bone growth, and adaptation. This goal was achieved by exploring two specific scenarios. In the first study, mice models with conditional knockouts of MKS5, a ciliary protein, in osteocytes were utilized to demonstrate that dysfunctional primary cilia in those cells result in impaired loading-induced bone formation. The hypothesis tested is that the existence of functioning primary cilia on osteocytes is crucial for proper bone adaptation following stress. The results of this study support the hypothesis, with the conditional knockout mice showing significantly lower loading-induced bone formation compared to controls. The second study highlighted the importance of the osteoblast primary cilia in bone growth by using mice models with osteoblast-specific deletion of the cilia. The hypothesis tested is that the presence of the primary cilia is crucial for proper bone growth. The results show that conditional knockout mice have lower body weights, decreased femur length, and a significantly lower rate of bone formation, confirming that the primary cilia play a great role in bone growth and development. This study has highlighted the role of primary cilia in bone health and this topic merits further investigation.

<p>The primary cilium is a singular signaling organelle found on most mammalian cell types. Dysfunction of the primary cilium or associated structures form a group of genetic disorders called ciliopathies. Recently, Huntington’s disease (HD), a monogenetic neurodegenerative disorder, was classified, at least in part, as a ciliopathy. How the primary cilium contributes to the pathogenesis of HD is the focus of this work. We demonstrate that huntingtin localization to the basal body or primary cilium is dependent on the phosphorylation status of serine residues 13 and 16. Furthermore, we demonstrate that, compared to controls, HD cell models have an increased number of cells with a primary cilium and that these cells have higher presence of huntingtin within the ciliary compartment. The primary cilia that form in HD cell lines demonstrate abnormal dynamics and morphology with bulging tips, characteristic of defective retrograde trafficking. We also demonstrate that alpha tubulin acetyltransferase 1 (αTAT1) expression and localization is increased in the primary cilium of HD cell lines. Subsequently, the primary cilium of HD cell lines are highly acetylated when compared to controls. These data support that primary cilia structure, ciliogenesis and ciliome are altered in HD.</p> / Master of Science (MSc)

No description available.

The developing brain requires an intricate network of signals to direct proliferation, differentiation and cell fate decisions. Primary cilia are vital organelles with an emerging role regulating several major signalling cascades, in particular the Hedgehog pathway. Talpid3 (Ta3) is 166.7 kD protein found at the distal tip of centrioles. It has been shown to interact with a number of key centriolar proteins and is essential for the formation of primary cilia. A recent mouse model has been designed to conditionally target the highly conserved coiled-coil domain of Ta3 using the Cre/loxP system. This project uncovers the role of Ta3 in the developing brain. It characterises in detail the phenotype of mice with conditional loss of Ta3 in the central nervous system using the Nestin-Cre deleter strain. Morphological and histological analyses demonstrate that significant defects occur postnatally with mice developing severe ataxia and hydrocephaly. Immunohistochemical techniques further characterise the distinct phenotypes of three key brain regions including the cerebellum, cortex and hippocampus. Ta3fl/fl;NesCre mutant mice exhibit defects in the proliferation, organisation, morphology and migration of both neuronal and glial cells. We have shown the mechanistic cause to be the result of widespread loss of primary cilia and a concomitant disruption in the transduction of the Hedgehog signalling pathway. The neural roles of Ta3 are explored further through the optimisation of an in vitro neurosphere system to culture postnatal hippocampal progenitors. The use of a tamoxifen inducible strain allows the timely recombination of Ta3 to study its role in a controlled environment. The cultured cells recapitulate many of the in vivo defects showing loss of primary cilia and reduced migration. Finally, characterisation of the phenotypes seen in the Ta3fl/fl;NesCre mice were shown to resemble neurological traits seen in human conditions with loss of Primary cilia, known as ‘human ciliopathies’. Through clinical collaboration this project demonstrated a human ciliopathy case of Joubert Syndrome with compound heterozygous mutations in TA3. This presents the Ta3fl/fl;NesCre mutant mice as a valuable model system to study a rare but clinically relevant condition.

No description available.

Lithium is given to millions of bipolar disorder or post-traumatic disorder patients. The recent studies also support a role for lithium in treating neurodegenerative disease such as Parkinson's disease and stroke. Lithium treatment leads to lithium nephropathy, which includes lithium-induced nephrogenic diabetic insipidus (NDI), lithium-induced renal cell proliferation leading to the formation of microcysts in the kidney, and lithium-induced renal fibrosis. However, there is still a gap in understanding the mechanisms and signaling pathways involved in regulating lithium-induced nephropathy. mTOR pathway activation and primary cilia are known to be associated with the abnormal renal cell proliferation and the formation of renal cysts in polycystic kidney disease, a renal disease model similar to our lithium model. The activation of hedgehog pathway is associated with the renal fibrosis observed in the unilateral ureteral obstruction and unilateral ischemia reperfusion injury models of chronic renal injury. Thus, I hypothesize that mTOR signaling pathway, primary cilia and hedgehog pathway may all contribute to lithium-induced nephropathy. To address the hypothesis that the mTOR signaling pathway may be responsible for lithium-induced renal collecting duct proliferation, mTOR pathway activation was assessed in lithium-treated mice and lithium-treated mouse inner medullary collecting duct (mIMCD3) cells. Lithium activated mTOR signaling pathway in renal collecting duct cells both in vivo and in vitro. Rapamycin, an inhibitor of mTOR, blocked lithium-induced renal cell proliferation in renal cortex and medulla in vivo and in renal collecting duct cells in vitro, supporting the hypothesis. However, rapamycin did not improve lithium-induced reduction of urine osmolality, suggesting mTOR signaling pathway may not contribute to lithium-induced NDI. To address the hypothesis that primary cilia may be necessary for lithium-induced mTOR activation and renal cell proliferation, primary cilia deficient cells were used to assess mTOR pathway activation and cell proliferation in response to lithium treatment. The absence of primary cilia abolished lithium-induced activation of mTOR pathway and cell proliferation, which supports the hypothesis. To address the hypothesis that lithium elongates primary cilia length, which is mediated by mTOR signaling pathway, primary cilia length alternation was assessed in the kidney and in mIMCD3 cells in response to lithium treatment. Lithium increased primary cilia length in renal collecting duct cells of cortex, outer medulla, and inner medulla kidney regions in vivo and in mIMCD3 cells in vitro. Rapamycin reversed lithium-induced elongation of primary cilia in renal cortical and outer medullary collecting duct cells in vivo, and blocked the increase of primary cilia length in mIMCD3 cells in vitro, which support the hypothesis. To address the hypothesis that lithium activates the hedgehog pathway in a Smoothened (smo, a key regulator of the hedgehog pathway)-dependent manner in renal collecting duct cells, mIMCD3 cells were treated with lithium or lithium/Smo inhibitor or lithium/Smo activator. Hedgehog signaling pathway is activated by lithium in mIMCD3 cells, which is partially Smo-dependent. However, the role of hedgehog signaling pathway in regulating lithium-induced fibrosis was not assessed in the study. Future studies are required to determine the role of the hedgehog pathway in the lithium model.

Bardet-Biedl syndrome (BBS) is a pleiotropic and genetically heterogeneous disorder, and a well-known ciliopathy. Nineteen different genes have been reported for BBS, mutations in which cause characteristic phenotypes including retinal degeneration, obesity, polydactyly, renal abnormalities, hypogenitalism and cognitive impairment. Protein products of eleven BBS genes are part of two major complexes: the BBSome complex and a CCT/CTRiC/BBS complex. The CCT/CTRiC/BBS complex assists in the formation of the BBSome complex, which in turn traffics numerous receptor proteins to the cilia. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. In fact, a complete picture of the cellular functions of BBS proteins is still missing, and gaps remain in our understanding of the pleiotropy and heterogeneity of the disease. With the aim of bridging those gaps, this thesis project was designed to identify tissue specific cargoes of the BBSome and to characterize their BBS-related functions. To this end, we generated a transgenic LAP-BBS4 mouse, which expresses the transgene in various tissues including brain, eye, testis, heart, kidney, and adipose tissue. We found that despite tissue specific variable expression, LAP-BBS4 was able to complement the deficiency of Bbs4 and rescue all the BBS phenotypes in the Bbs4 null mice. The finding provides an encouraging prospective for gene therapy for BBS related phenotypes and potentially for other ciliopathies. We also utilized the transgenic mice to search for tissue specific BBSome cargo proteins and identified CEP131 as a novel BBSome interacting protein. Using in vitro cell culture models we show that CEP131 interacts with the BBSome through BBS4. CEP131 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon CEP131 depletion. Our in vitro data implicate CEP131 as a negative regulator of ciliary BBSome trafficking. Finally, we show that cep131 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. This finding confirms the association of CEP131 with the BBS pathway. Overall, the work performed for this thesis provides further insight into the regulation of BBSome ciliary trafficking and suggests CEP131 as a BBS candidate gene.

Primary cilia are ubiquitous sensory organelles found on most cell types including cells of the dorsal root ganglia (DRG). The DRG are groups of peripheral neurons that relay sensory information from the periphery to the CNS. Other cell types in the DRG include a type of glial cell, the satellite glial cells (SGCs). The SGCs surround the DRG neurons and, with the neurons, form functional sensory units. Currently are no reports describing the numbers of DRG cells that have cilia. We found that 26% of the SGCs had primary cilia. The incidence of cilia on neurons varied with neuron size, a property that roughly correlates with physiological characteristics. We found that 29% of the small, 16% of the medium and 5% of the large neurons had primary cilia. Primary cilia have been shown to have a role in cell proliferation in a variety of cell types. In some of the cells the cilia mediate the proliferative effects of Sonic hedgehog (Shh). In the CNS, Shh signaling through primary cilia affects proliferation during development as well as following injury, but no studies have looked at this function in the PNS. The SGCs and neurons of the DRG undergo complex changes following peripheral nerve injury such as axotomy. One marked change seen after axotomy is SGC proliferation and at later stages, neuronal death. We found that following axotomy there is a significant increase in the percentage of SGCs with primary cilia. We also found a significant increase in the percentage of medium-sized neurons with primary cilia. In other experiments we tested the idea that Shh plays a role in SGC proliferation. When Shh signaling was blocked following axotomy we found decreased proliferation of SGCs. This is the first report of a change in the percentage of cells with cilia following injury in the PNS, and the first report of a role for Shh in SGC proliferation following axotomy.