α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.

α-Synuclein is an abundant nerve terminal protein and a primary component of the Lewy body pathology seen in Parkinson’s disease. While the precise biological and pathological role of α-synuclein remains unclear, its ability to bind to and dissociate from synaptic membranes may be linked to its function in these states. In this thesis, we characterized the role of the GTPase protein rab3a as a potential regulator of α-synuclein membrane binding and dissociation. We found evidence that GTP-bound rab3a sequesters α-synuclein on membranes during exocytosis, and that inhibition of rab3a dissociation from the membrane causes inhibition of α-synuclein dissociation as well. Furthermore, we found that the loss of rab3a in human neuroblastoma cells increases α-synuclein expression. This study identifies rab3a and proteins associated with its membrane dissociation as mediators of α-synuclein membrane binding and dissociation.

Alzheimer’s disease is a neurodegenerative disease characterised by progressive memory deterioration and cognitive impairment. Pathological hallmarks are extracellular senile plaques, neurofibrillary tangles and neuron loss. Senile plaques are produced through altered processing of the membrane-bound protein APP. Different neurotransmitter signal transduction pathways have been implicated in the formation or development of Alzheimer’s pathologies, but the molecular mechanisms behind these changes are not well known. The overall aims of this thesis were to identify novel genes with differential expression in Alzheimer’s disease and to investigate mechanisms initiating these changes and their relationship to the disease. A real-time RT-PCR strategy was developed to enable detection of small mRNA changes in human brain autopsy samples. This approach was first used to investigate levels of expression of a candidate gene (MAO), and later employed to verify gene expression differences detected by cDNA microarray analysis. Of several genes verified as differentially expressed in the patients, ITPKB (Inositol 1,4,5-trisphosphate 3-kinase B) and RGS4 (Regulator of G-protein signalling 4) presented the largest expression differences in Alzheimer’s cases compared to control samples. Several splice variants of RGS4 showed similar down-regulation levels and one rare haplotype was associated with decreased RGS4 expression. Functional studies in SH-SY5Y cell cultures overexpressing RGS4 showed that it is likely that RGS4 affects APP processing by regulating PRKC expression levels. The combined expression of RGS4 and ITPKB is for the first time presented in this thesis as genes with altered mRNA levels in Alzheimer’s disease. These two genes are implicated in the same signalling pathway that modifies calcium levels in the cell. Furthermore, the fact that RGS4 affects APP processing suggests that RGS4 is involved in the development of senile plaques. This motivates further functional studies of this gene and suggests that RGS4 may become a new potential drug target for Alzheimer’s disease.

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The function of GTP-binding proteins (G-proteins) in diverse intracellular pathways depends on their ability to switch between two forms, a GDP-bound (inactive) form and a GTP bound (active) form in a highly regulated GTPase cycle. The inactivation step of this cycle is regulated by GTPase-activating proteins (GAPs) which increase the intrinsic rate of hydrolysis of bound GTP; the activation step is regulated by a diverse family of GDP/GTP exchange factors (GEFs). A unique model system, which consists of the 13 kDa GEF Mss4 and the monomeric G protein Rab3A involved in presynaptic neurotransmission, was chosen to study the mechanism of G-protein regulation. Structure of Rab3A at high resolution The 2.0 Å crystal structure of Rab3A, bound to a non-hydrolyzable GTP-analog (GppNHp), enables a detailed description of the structural determinants that stabilize the active conformation and regulate GTPase activity within the Rab family. Although the overall structure is similar to that of GppNHp-bound Ras and other GTPases, localized but significant differences are observed in the vicinity of the conformational switch regions and the α3/β5 loop. The active conformation is stabilized primarily by extensive hydrophobic contacts between the switch I and II regions. Novel interactions with the γ phosphate, mediated by serine residues in the P-loop and switch I region, impose stereochemical constraints on the mechanism of GTP hydrolysis and provide a structural explanation for the broad range of GTPase activities within the Rab family. Residues implicated in interactions with effectors and regulatory factors map to a common face of the protein. The asymmetric distribution of charged and non-polar residues suggests a plausible orientation with respect to vesicle membranes that would position predominantly hydrophobic surfaces to interact with membrane-associated effectors and regulatory factors. Thus, the structure of Rab3A establishes a framework for understanding the molecular mechanisms underlying the function of Rab proteins in vesicle trafficking. High resolution structure of Mss4 and structure-based mutagenesis Activation of monomeric Rab GTPases, which function as ubiquitous regulators of intracellular membrane trafficking, requires the catalytic action of guanine nucleotide exchange factors. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic but not endocytic Rab GTPases. Chapter III describes the results of a high resolution crystallographic and mutational analysis of Mss4. The 1.65 Å crystal structure of Mss4 reveals a network of direct and water mediated interactions that stabilize a partially exposed structural sub-domain derived from four highly conserved but non-consecutive sequence elements. The conserved sub-domain contains the invariant cysteine residues required for Zn2+ binding as well as the residues implicated in the interaction with Rab GTPases. A strictly conserved DΦΦ motif, consisting of an invariant aspartic acid residue (Asp73) followed by two bulky hydrophobic residues (Met74 and Phe75), encodes a prominently exposed 310 helical turn in which the backbone is well ordered but the side chains of the conserved residues are highly exposed and do not engage in intramolecular interactions. Substitution of any of these residues with alariine dramatically impairs exchange activity towards Rab3A, indicating that the DΦΦ motif is a critical element of the exchange machinery. In particular, mutation of Phe75 results in a defect as severe as that observed for mutation of Asp96, which is located near the zinc binding site at the opposite end of Rab interaction epitope. Despite severe defects, however, none of the mutant proteins is catalytically dead. Taken together, the results suggest a concerted mechanism in which distal elements of the conserved Rab interaction epitope cooperatively facilitate GDP release. The basis for selective recognition of exocytic Rab family GTPases by Mss4 Rab3A is involved in Ca2+ -dependent exocytosis and neurotransmitter release. Mss4, an evolutionarily conserved Rab exchange factor, promotes nucleotide release from exocytic RabGTPase (Rab1, Rab3A, Rab8, and Rab10, Sec4 and Ypt1) but not endocytic Rab GTPases (Rab2, Rab4, Rab5, Rab6, Rab9 and Rab11). To understand the basis for selective recognition of exocytic Rab family GTPases by Mss4, a structure based mutagenesis study of Rab3A was conducted. Three residues in Rab3A (Phe51, Val61 and Thr89) were found to be critical for interaction with Mss4. Phe51 is located at the N- terminus of the switch region, adjacent to the Mg2+ and nucleotide binding site. Val61 in the β2 strand and Thr89 in the switch II region flank a triad of hydrophobic residues that is conserved in the Rab family. These residues comprise critical determinants underlying the broad specificity of Mss4 for exocytic Rab family proteins. In addition to determining the high resolution crystal structures of Rab3A and Mss4, the experiments described above identify critical structural determinants for the exchange activity of Mss4 and provide insight into the selective recognition of Mss4 by exocytic Rab GTPases.