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Alström Syndrome (AS) is a rare autosomal recessive disease featuring early onset, severely insulin resistant diabetes, fatty liver and heart failure among other characteristics. AS is caused by biallelic loss-of-function mutations in the ALMS1 gene. Cardiometabolic complications in AS are the leading cause of mortality, occur despite only moderate obesity in many patients, and are the focus of this thesis. ALMS1 is a large protein found in the centrosome throughout the cell cycle, both in spindle poles in mitosis and in the basal body of primary cilia. Previous cellular studies suggested elongated cell cycle length in cells lacking ALMS1, despite no overt changes in morphology of primary cilia when viewed statically. One hypothesis to explain the changes in cell cycle length is that loss of ALMS1 in cells alters ciliary dynamics in ways that are not discernible in cross sectional imaging, and that this leads to cell cycle perturbation. To test this, CRISPR-Cas9 was used to induce biallelic loss of function mutations in Alms1 in NIH-3T3 cells. Stable expression of the cell cycle and ciliary fluorescent reporter Fucci2a-Arl13b was induced using the flp-in system. Live cell imaging of Alms1 knockout (KO)- and wild-type (WT)- Fucci2a-Arl13b+ cells was performed. No changes in time between cilia loss and cytokinesis were found, suggesting that in the period after disassembly of primary cilia, loss of Alms1 does not alter cell cycle length. This does not discount ciliary cycle dysfunction in other stages of the cell cycle or under different growth conditions. The metabolic profile of AS closely resembles that of lipodystrophy, including severe hepatosteatosis and dyslipidaemia, and in AS adipocyte hypertrophy is seen. This suggests that adipose tissue failure may be driving some or all cardiometabolic features in AS. To test this hypothesis, EUCOMM Tm1c Alms1 (flox/flox) mice were crossed with Pdgfrα-Cre mice to abrogate Alms1 function only in mesenchymal stem cells (MSCs) and their descendants including preadipocytes and adipocytes. For comparison, global Alms1 KO mice were generated by crossing EUCOMM Tm1c Alms1 with CAG-Cre. Metabolic phenotyping of global and MSC-specific Alms1 KO mice on a 45%-fat diet was performed over a 24 week period. MSC-specific Alms1 KO recapitulated key metabolic phenotypes of global Alms1 KO animals, including insulin resistance and hepatosteatosis in both male and female mice. Other phenotypes were found to differ by sex. Increased fat mass was only seen in female MSC-specific Alms1 KO mice. Furthermore, hyperphagia was not seen in male MSC-specific Alms1 KO mice, but was preserved in females, despite preservation of neuronal Alms1. These data show that MSC-derived lineages are critical in driving severe metabolic syndrome in AS, and support the theory of adipose tissue failure in AS. The global Alms1 KO mouse widely recapitulates the majority of the multisystemic characteristics of AS, including impaired auditory and visual perception, and metabolic aberration. However cardiac function has only been previously assessed in one ex vivo study of tissue from neonatal mice, despite heart failure presenting a leading cause of death in adult patients with AS. To comprehensively characterise cardiac function in global Alms1 KO mice, in vivo echocardiography was performed at three timepoints; 2, 8, and 23 weeks of age. Molecular analysis was also performed on hearts at 2 and 24 weeks of age. No changes in echocardiography were detected at 2 and 8 weeks of age. At 23 weeks of age, increased left atrial area and decreased isovolumic relaxation time was measured in female mice, indicative of restrictive cardiomyopathy. Reduced ejection fraction and fractional area change was also observed in female mice at this time, suggesting systolic cardiac dysfunction. There was no evidence of changes in males at 23 weeks of age. Quantification of transcript levels in heart of 24 week old female mice showed no transcriptional changes in genes typically associated with cardiomyopathy and fibrosis. In contrast with previously published findings, no changes in heart/body weight ratio were found in 2 week old mice. Together these data show that the Alms1 KO mouse does not mimic the human heart pathology well in the basal state. It remains possible that a clear cardiac pathology may become unmasked with pharmacological manipulation, age, or other stressors. In conclusion, this thesis presents data showing that loss of Alms1 does not change ciliary dynamics in relation to cytokinesis in vitro. Loss of Alms1 causes systolic and diastolic cardiac dysfunction indicative of restrictive cardiomyopathy in 23 week old female mice. However there is no evidence of cardiac dysfunction in male Alms1-deficient mice at this time, or in 2 week old mice of either sex. Loss of Alms1 in mice does model the human metabolic disease well, and the recapitulation of insulin resistance, obesity and hepatosteatosis with both global and MSC-specific Alms1 KO provide strong evidence that adipose tissue failure is a key driver of metabolic syndrome in AS. |
