Stress and Rab35 modulate Alzheimer’s disease-related protein trafficking

Chronic stress and elevated glucocorticoids (GCs), the major stress hormones, are risk factors for Alzheimer’s disease (AD) and promote AD pathomechanisms in animal models. These include overproduction of synaptotoxic amyloid-β (Aβ) peptides and intraneuronal accumulation of microtubule-associated protein Tau. Tau accumulation is linked to downregulation of the small GTPase Rab35, which mediates Tau degradation via the endolysosomal pathway. Whether Rab35 is also involved in stress/GC-induced Aβ overproduction remains an open question. Here, I find that hippocampal Rab35 levels are decreased not only by stress/GCs, but also by aging, another AD risk factor. Moreover, I show that Rab35 negatively regulates Aβ production by sorting amyloid precursor protein (APP) and β-secretase (BACE1) out of the endosomal network, where they interact to produce Aβ. Interestingly, Rab35 coordinates distinct intracellular trafficking events for BACE1 and APP, mediated by its effectors OCRL and ACAP2, respectively. Additionally, I show that Rab35 overexpression prevents the amyloidogenic trafficking of APP and BACE1 induced by GCs. Finally, I begin to investigate how GCs and/or Rab35 affect the intercellular spread of Aβ and Tau through exosomes. I describe methods for purifying exosomes and measuring their secretion from neurons, astrocytes, and microglial cells in order to determine the effects of stress/GCs and Rab35 on this process. These studies identify Rab35 as a key regulator of Alzheimer’s disease-related protein trafficking, and suggest that its downregulation contributes to stress- and AD-related pathomechanisms

Nerve growth factor and chemokine receptors regulate macrophage phenotype via activation of transient receptor potential and inositol triphosphate receptor channels

Human Immunodeficiency Virus (HIV) activates macrophages and microglia cells in the central nervous system (CNS) and triggers the secretion of neurotoxins, causing neuronal damage. Therapeutic approaches to restore cognitive function by suppressing macrophage and microglial activation have not been successful, partially due to limited knowledge of cellular mechanisms that control toxin secretion. In this investigation, we study the pathway to HIV-induced neurotoxin release, focusing on how calcium influx and phosphorylation of the HIV co-receptor CXCR4 lead to macrophage activation, including alteration of the actin cytoskeleton resulting in the formation of ruffles and podosomes. We found that Nerve Growth Factor (NGF) increases CXCR4 phosphorylation, increases calcium signaling, and reverses the HIV-induced increase in podosome formation. To further elucidate the pathway that mediates this effect, we challenged macrophages with specific inhibitors for CXCR4 and CCR5 receptors, as well as for PI3K and Pyk2. To investigate the source of calcium leading to macrophage activation, we inhibited the P2X7 receptor, CRAC channels, TrpC channels, and IP3 receptor channels. Our data indicated that calcium entry through the P2X7 receptor contributed to podosome formation while the IP3 receptor channel favored expression of the less toxic ruffled phenotype, Pyk2 signaling and calcium entry through the TrpC channel were necessary for both ruffling and podosomes. Because podosome expression is correlated with toxin secretion from macrophages, determining the pathway to morphological modifications enables a better overall understanding of macrophage activation. This knowledge can lead to better regulation of macrophages and control of inflammation in the CNS.Bachelor of Scienc

Endolysosomal degradation of Tau and its role in glucocorticoid-driven hippocampal malfunction

Emerging studies implicate Tau as an essential mediator of neuronal atrophy and cognitive impairment in Alzheimer's disease (AD), yet the factors that precipitate Tau dysfunction in AD are poorly understood. Chronic environmental stress and elevated glucocorticoids (GC), the major stress hormones, are associated with increased risk of AD and have been shown to trigger intracellular Tau accumulation and downstream Tau-dependent neuronal dysfunction. However, the mechanisms through which stress and GC disrupt Tau clearance and degradation in neurons remain unclear. Here, we demonstrate that Tau undergoes degradation via endolysosomal sorting in a pathway requiring the small GTPase Rab35 and the endosomal sorting complex required for transport (ESCRT) machinery. Furthermore, we find that GC impair Tau degradation by decreasing Rab35 levels, and that AAV-mediated expression of Rab35 in the hippocampus rescues GC-induced Tau accumulation and related neurostructural deficits. These studies indicate that the Rab35/ESCRT pathway is essential for Tau clearance and part of the mechanism through which GC precipitate brain pathology.work was supported by NIH grants R01NS080967and R21MH 104803 to C.L.W., Portuguese Foundation for Science & Technology (FCT) PhD fellowships to J. Vaz-Silva and T. Meira (PD/BD/105938/2014; PD/BD/113700/2015, respectively), and the following grants to I.S.: FCT Investigator grant IF/01799/2013, the Portuguese North Regional Operational Program (ON.2) under the National Strategic Reference Framework (QREN), through the European Regional Development Fund (FEDER), the Project Estratégico co-funded by FCT (PEst-C/SAU/LA 0026/2013) and the European Regional Development Fund COMPETE (FCOMP-01 -0124-FEDER-037298) as well as the project NORTE- 01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER)info:eu-repo/semantics/publishedVersio