ARF6: a mediator of dendritic filopodia maturation to spines and gamma-sescretase-independent presenilin functions

Korte inhoud:Mechanisms that regulate intracellular membrane/protein trafficking are of utmost physiological importance. By affecting the delivery of macromolecules to specific external and subcellular sites they impact majority, if not all relevant cellular processes. Not surprisingly, defects in intracellular membrane flow and its regulators are observed in many pathological conditions, including several neurodegenerative disorders (Howell et al., 2006; Wang et al., 2014). For instance, in Alzheimers’ disease (AD), the most frequent cause of age-related dementia, endo-lysosomal aberrancies followed by consequent defective turnover of autophagic vacuoles are one of the earliest neuropathological features to develop (Nixon, 2005; Nixon and Yang, 2011). Herein, AD-related and nbsp;associated presenilins (PSEN1/2) may have a two-fold exacerbating contribution. On the one hand, by cleaving amyloid precursor protein (APP) PSENs may facilitate the accumulation of toxic amyloid β (Aβ) species within these internal compartments, thereby compromising their functioning. On the other hand, PSENs (and in particular PSEN1) have a range of reported functions independent of their primary proteolytic activity. This e.g. includes the increasingly evident roles in endo-lysosomal trafficking of molecules and turnover of autophagic vacuoles, whereby in yet another (more direct) way PSEN dysfunction (as may occur in ageing and AD) could affect these internal compartments. The specific mechanism how this is mediated, however, in big part remains unsettled (reviewed in (Peric and Annaert, 2015)). One of the first accounts on this unconventional role of PSENs (later confirmed by independent sources) emerged from studies of the host laboratory, which demonstrated that PSEN1 interacts with neuron-specific cell surface localized adhesion protein Telencephalin (TLN), but does not proteolytically process it (Annaert et al., 2001; Esselens et al., 2004). Instead, in PSEN1-/- neurons TLN has a delayed turnover (prolonged half-life) and specifically and prominently deposits internally in neuronal soma and dendrites within autophagic-like vacuoles (devoid of APP). While similar TLN-specific accumulations are also noted in wild-type (WT) neurons, lack of PSEN1 clearly leads to their earlier and much more abundant occurrence. Notably, both human PSEN1 and its catalytically dead mutant successfully rescue this defect, underscoring that PSEN1 proteolytic activity does not play a role in this phenomenon (Esselens et al., 2004). Although disturbed turnover of TLN and its specific internal deposition strongly implied an underlying endo-lysosomal trafficking dysfunction, resulting from PSEN deficiency (as in PSEN1-/-) or its declining function (as in rdquo; in vitro WT) neurons, the molecular determinants of this deficit remained unknown. Under physiological conditions TLN is primarily found at the somato-dendritic plasma membrane of excitatory neurons, where it prominentlynbsp;to dendritic filopodia, thin (μm-long) protrusions that play a role in development of postsynaptic sites, and hence synaptogenesis. Previously, it has been established that during dendritic filopodia transition (maturation) to spines, TLN undergoes gradual exclusion from the postsynaptic cell surface, and that its selective removal is necessary in this process (Furutani et al., 2007; Matsuno et al., 2006). Specific contribution of endocytic trafficking regulators in this phenomenon, however, has not been evaluated. As regulators of TLN intracellular trafficking likely harbor molecules that may expand our understanding of both PSEN-related trafficking deficits as well as the negative regulatory role of TLN in development of postsynapses, the first objective of this thesis was to identify specific factors involved in endocytic TLN routing and to dissect the trafficking itinerary followed by this protein, starting from its internalization at the plasma membrane. Equipped with the knowledge gained through these studies, we also aimed to examine if and how the identified mediators of TLN endo-lysosomal trafficking functionally interact with this protein in context of spinogenesis. Finally, we also wanted to evaluate their functional relationship with PSENs. Towards the first major objective of this thesis, we identified a small GTPase ARF6 as an important mediator of TLN internalization and post-endocytic trafficking. Our experiments also revealed that once taken up from the cell surface, TLN follows a route through Rab5 positive early endosomes, to eventually become sorted towards intralumenal vesicles (ILVs) of the late endosomal multi-vesicular bodies (MVBs), the markers of which we found co-accumulating with TLN in previously reported intra-neuronal autophagic-like vacuoles. Similar colocalization with TLN in endosomes and neuronal accumulations was also shown for the lipid raft marker protein flotillin, which in addition co-distributed with TLN within distinct plasma membrane domains. Overall this suggested that after their internalization at the cell surface, both TLN and flotillin follow a common endocytic route, wherein ARF6 plays anbsp;function. Our experiments also revealed that ARF6-mediated internalization of TLN from dendritic filopodia surface, promotes their maturation to dendritic spines. This resolved the important outstanding question how during dendritic filopodia transition (maturation) to spines, TLN is excluded from the postsynaptic membranes. Thereby, we also addressed the second major objective of this thesis project. All these results were summarized and reported in the EMBO Journal, in a publication on which I share the first authorship (Raemaekers et al., 2012). This study comprises the Chapter III of the present work. Our novel findings that ARF6 mediates the intracellular routing of TLN, which previously has been show to also require PSEN1 (Esselens et al., 2004), suggested that ARF6 and PSEN1 may be functionally related. To study the cellular interplay between these two proteins, we next switched our focus to mouse embryonic fibroblasts (MEFs), in which PSEN deficiency, much like in neurons, results in endo-lysosomal transport deficits. We here first examined how ARF6 and PSENs relate to each other in context of cellular migration (as part of the third major objective of this thesis project). Our experiments revealed that both decreased levels of ARF6 as well as PSEN deficiency, severely compromise directional persistence of migration of MEF cells. We further show that expression of hPSEN1, catalytically inactive (hPSEN1 D257A/D385A) mutant or hARF6 (WT or its fast-cycling (T157A) mutant) in PSEN deficient MEFs, all successfully rescue this migratory deficit. Taken together, this implied that within the pathway that controls directional migration of MEF cells, PSEN1 acts independently of its proteolytic function, likely as an upstream modulator of ARF6. In line, we next demonstrated that this phenomenon (at least in part) may relate to a γ-secretase-independent influence of PSEN1 on ARF6 expression, as assessed on protein, mRNA and ARF6 promoter activity levels. These findings were further confirmed in two additional cell lines (namely N2a and HeLa cells), wherein upon genetically ablating PSEN1 and PSEN2, we observed congruent drops in ARF6 expression relative to WT cells in all tested assays. Finally, to identify additional trafficking regulators (affected in a similar way as ARF6 by the PSEN1) we performed comparative microarray mRNA profiling of PSEN double deficient MEFs, their hPSEN1, hPSEN1 D257A/D385A rescued and WT counterparts. Subsequent ranking of all genes according to the extent of rescue efficacy (up- or down-regulation in PSENdKOs and a successful expression recovery towards the levels observed in WT cells by both normal and catalytically inactive hPSEN1), confirmed our previous experimental findings by positioning ARF6 among the best rescued genes. In addition, this approach also revealed several additional interesting targets (of known involvement in membrane trafficking regulation) the expression of which seems to be prone to regulation by PSEN1 in a manner independent of its catalytic activity. This includes a few small GTPases of the RAB family, like e.g. Rab10, which interestingly enough, was recently identified as a novel functional partner of ARF6 in endocytic recycling (Shi and Grant, 2013). These findings are summarized in the Chapter IV of this thesis. In our two related studies, described in Chapters III and IV of the present work, we identified ARF6-regulated endocytic trafficking as annbsp;mediator of synaptogenesis (development of postsynaptic sites) and as well provided a strong support for its contributing role in PSEN-related, γ-secretase-independent endocytic trafficking phenomena. Regarding the well established role of synaptic deficits, endocytic abnormalities and aberrant PSEN-functioning to AD pathogenesis, taken together our findings are hence not only relevant to basic cell biology, but potentially also of importance to mechanisms governing AD progression. References Annaert, W.G., C. Esselens, V. Baert, C. Boeve, G. Snellings, P. Cupers, K. Craessaerts, and B. De Strooper. 2001. Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins. Neuron. 32:579-89. Esselens, C., V. Oorschot, V. Baert, T. Raemaekers, K. Spittaels, L. Serneels, H. Zheng, P. Saftig, B. De Strooper, J. Klumperman, and W. Annaert. 2004. Presenilin 1 mediates the turnover of telencephalin in hippocampal neurons via an autophagic degradative pathway. J Cell Biol. 166:1041-54. Furutani, Y., H. Matsuno, M. Kawasaki, T. Sasaki, K. Mori, and Y. Yoshihara. 2007. Interaction between telencephalin and ERM family proteins mediates dendritic filopodia formation. J Neurosci. 27:8866-76. Howell, G.J., Z.G.nbsp;C. Cobbold, A.P. Monaco, and S. Ponnambalam. 2006. Cell biology of membrane trafficking in human disease. Int Rev Cytol. 252:1-69. Matsuno, H., S. Okabe, M. Mishina, T. Yanagida, K. Mori, and Y. Yoshihara. 2006. Telencephalin slows spine maturation. J Neurosci. 26:1776-86. Nixon, R.A. 2005. Endosome functionnbsp;dysfunction in Alzheimer's disease and other neurodegenerative diseases. Neurobiol Aging. 26:373-82. Nixon, R.A., and D.S. Yang. 2011. Autophagy failure in Alzheimer's disease--locating the primary defect. Neurobiol Dis. 43:38-45. Peric, A., and W. Annaert. 2015. Early etiology of Alzheimer's disease: tipping the balance toward autophagy or endosomal dysfunction? Acta Neuropathol. Raemaekers, T., A. Peric, P. Baatsen, R. Sannerud, I. Declerck, V. Baert, C. Michiels, and W. Annaert. 2012. ARF6-mediated endosomal transport of Telencephalin affects dendritic filopodia-to-spine maturation. EMBO J. 31:3252-69. Shi, A., and B.D. Grant. 2013. Interactions between Rab and Arf GTPases regulate endosomal phosphatidylinositol-4,5-bisphosphate during endocytic recycling. Small GTPases. 4:106-9. Wang, X., T. Huang, G. Bu, and H. Xu. 2014. Dysregulation of protein trafficking in neurodegeneration. Mol Neurodegener. 9:31.

Jaar van publicatie:2015

Toegankelijkheid:Closed