Alzheimer's disease (AD) is a progressive neurodegenerative disease, clinically characterized by memory loss (amnesia), impaired linguistic ability (aphasia), inability to recognize objects or persons despite intact sensory organs (agnosia), and inability to perform complex motor tasks, without any physical limitations (apraxia). AD patients may also develop psychiatric symptoms such as personality changes, delusions, hallucinations and depression. The pathological hallmarks in the brains of AD patients are senile plaques, aggregated β-amyloid protein, and neurofibrillary tangles, entwined tau protein. The tau protein is involved in stability of the microtubules which are important for vesicle transport. The function of the β-amyloid protein is less well known, but may be involved in synaptic plasticity and neuroprotection.

There are roughly two types of AD, the familial and sporadic AD. Being a carrier for mutations in the amyloid precursor protein and presenilin -both proteins are involved in the formation of β-amyloid- leads to AD at an early age. The precise cause for sporadic AD is unknown; the disease has a multifactorial basis. Old age is the most obvious and major risk factor. Additional risk factors include, among others, genetic factors (APOε4 variant carriers), diabetes (type 2 diabetes mellitus), but also lack of exercise and a low level of education. To date, there is no medication that cures AD, and current available drugs (NMDA receptor agonists and acetylcholinesterase inhibitors) have a limited efficacy. Moreover, the current administration of medication is at a relatively late stage in the disease, when clear symptoms have arisen. By then underlying disease processes are already at an advanced stage. Ideally intervention occurs early in the disease, before the onset of symptoms. This requires understanding and insight into molecular processes occurring in AD.

One of these processes is the unfolded protein response (UPR) of the endoplasmic reticulum (ER). The endoplasmic reticulum is the organelle involved in the production of membrane proteins and proteins destined for exocytosis. The UPR is activated, when the homeostasis of the ER is disturbed; this situation is called ER stress. The UPR attempts to resolve ER stress but if this fails the UPR initiates apoptosis. Downstream of the UPR is the ER-associated degradation (ERAD), a process that targets ER proteins for degradation in the proteasome. Autophagy is the process in which cytosolic proteins and also whole organelles are engulfed by a double/multi membrane stucture which fuses with the lysosome to initiate degradation. All of these proteolytic mechanisms are integrated in order to maintain homeostasis of proteins (proteostasis). Vesicle transport is also important for these processes. The intracellular transport of is regulated by small GTPases called Rab proteins. For humans there are approximately 60 known Rab proteins which each act in a more or less specific domain. Rab6 features vesicle transportation within and in the surroundings of the Golgi complex, an organelle involved in packaging of vesicles and their associated content. In our laboratory, we have demonstrated that the UPR is activated early in AD and that it is accompanied by an increase of the Rab6. In this thesis we investigate the function of the protein Rab6 in proteostatic systems related to Alzheimer's disease.

In Chapter 1, the general introduction, the previous mentioned topics are discussed more elaborately. Chapter 2 addresses the question what the relationship between Rab6 and the UPR is. We show that increased Rab6 leads to a reduction of the UPR and that –vice versa- reduced Rab6 leads to increased UPR. We show that Rab6 does not interfere in the signaling of the UPR, but that Rab6 is involved in a yet to be defined feedback mechanism of the UPR. We conclude that Rab6 has an effect during recovery from the UPR and thus is a modulator of the UPR.

Chapter 3 describes the effect of ERAD inhibition on the UPR. We show that inhibition of ERAD reduces the UPR, similar to effect of increased Rab6. We hypothesized that there is a compensatory mechanism that reduces the UPR. We show that autophagy is probably not that compensatory mechanism, but that lysosomes are involved. ERAD inhibition creates a slightly increased number of lysosomes and altered positioning of lysosomes within the cell. We conclude that ERAD inhibition leads to activation of a compensatory mechanism, which involves lysosomes.

In Chapter 4 we discuss the strong correlation between the effect on the UPR of increased Rab6 and ERAD inhibition. In this chapter we show that inhibition of ERAD provides a concentration of Rab6 on membranes close to lysosomes. However, we show that Rab6 is not necessary for the effect of ERAD inhibition on the UPR. We conclude that ERAD inhibition and the effect of Rab6 independent mechanisms, most likely converging on the lysosome.

Chapter 5 discusses the effect of Rab6 on the lysosomes. We show that reduction in Rab6 has no effect on autophagy. However, a strong relocalisation of lysosomes can be observed at reduced Rab6 levels. In this condition, lysosomes show a strong clustering near the nucleus. In addition, the glycosylation of the lysosomal membrane protein LAMP1 and 2 are selectively changed. In addition, the proteolytic activity of the lysosomes is increased and the dynamic (re) positioning under stress conditions is disturbed. We conclude that Rab6, under normal conditions, has a function in the positioning of lysosomes and, in particular, in the dynamic repositioning during stress.

In Chapter 6 we study Rab6 in relation to the lysosomes in post-mortem Alzheimer brain material. We show that membrane bound Rab6 accumulates in cells, which do not perform autophagy. This indicates that membrane bound Rab6 is degraded by autophagy in this cell model. In the hippocampus of deceased AD patients, we find increased Rab6, as previously demonstrated. Moreover, we show that Rab6 is located both in and around autophagosomes and lysosomes. We hypothesize that the disturbed lysosomes and autophagy contribute to the ineffectiveness of Rab6 to resolve e.g. ER stress in AD.

In Chapter 7, we provide a brief overview of the findings, and we discuss the implications of our research for AD. We illustrate a model of the disease from our perspective. We discuss some technical limitations, including the cell models used. We speculate about the function of Rab6 related to lysosomes, discuss the role of Rab6 during ER stress, give an insight into the possible pharmacological therapeutic options and conclude with the proposal that Rab6 is a possible target for intervention to modulate lysosomal activity in the early stages of Alzheimer's disease.