Analysis of the Cellular Functions of TRAPPC9 and Its Role in Brain Development and Body Weight Regulation.

Abstract

The nervous system development is a complex and tightly controlled process, and disturbance of this process leads to neurological disorders. Intellectual disability is characterised by a deficit in cognitive functions, which is usually accompanied by microcephaly. Intellectual disability affects around 1.39% of the population globally, and it has a significant impact on both patients and their families. Understanding the mechanism behind the genetic causes of intellectual disability will provide the right therapy or prevent the occurrence of this condition, for example, through genetic counselling. Trafficking Protein Particle Complex Subunit 9 (TRAPPC9) is the main subunit of the TRAPPII complex, which acts as a tethering factor in intracellular trafficking. Mutations of TRAPPC9 in humans have been identified as one of the causes of non-syndromic intellectual disability. Lack of TRAPPC9 led to microcephaly, intellectual disability, speech impairment, developmental delay and, in some cases, obesity. The main aim of this thesis is to understand how TRAPPC9 is involved in brain development and body weight regulation using Trappc9 knock-out (KO) mice as a model for this purpose. In Chapter 3, I have analysed the microcephaly phenotype of Trappc9 KO mice. The results showed that these mice do not have any difference in brain weight at birth but develop microcephaly postnatally with significant differences detectable by weaning age. Further analysis of adult brain weight showed a progressive reduction of brain weight in KO mice compared to wild-type (WT) by the age of 9 months (9% and 11% reduction of the brain weight in males and females, respectively). Next, I have analysed the neural stem cells (neurospheres) in KO mice to investigate the role of Trappc9 in neurogenesis. Investigation of the hippocampal neurospheres in vitro showed no difference in cell viability and proliferation between WT and KO. However, the characterisation of neurospheres using a differentiation assay showed a reduction of neuronal differentiation in KO cells, which may contribute to the microcephaly phenotype. Chapter 4 focuses on the role of lipid droplets (LDs) and LD related protein analysis in Trappc9 KO primary neuron cultures. TRAPPC9 works as guanine nucleotide exchange factor (GEF) for Rab18, which is involved in LD homeostasis in different cell lines. I analysed LD accumulation in the hippocampal and cortical neuron cultures and showed abnormal accumulations of LDs in Trappc9 KO neurons, which were more significant in hippocampal neurons. These abnormal accumulations were associated with Plin2 mis-localisation around LDs. Analysis of the cell viability revealed a reduction in Trappc9 KO neuron viability regardless of LD accumulation at two tested time points. Chapter 5 describes the characterisation of body weight and obesity phenotype in Trappc9 KO mice. The results showed no significant body weight difference at the newborn stage and 1 month of age in both genders. A cohort study of body weight was performed starting from age 2 months to observe any change. The results showed an increased body weight in Trappc9 KO mice, which was more severe in females. Analysis of white and brown adipose tissues using RT-PCR revealed the expression of Trappc9 in these tissues. In addition, abnormal LD accumulations were observed in white and brown adipose tissues of KO mice. Further characterisation using blood plasma analysis showed elevated leptin levels in KO samples, which were correlated positively with increased body weight. In conclusion, Trappc9 is essential for postnatal development and maintenance of the brain and body weight regulation, which involves the regulation of LDs in neurons. However, further investigation is needed to understand the molecular role of Trappc9 in neuronal LD homeostasis and how the defect in LD metabolism might contribute to microcephaly in Trappc9 deficient mice.