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Exploring Orai2 Function in Alzheimer's Disease Models

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Exploring Orai2 Function in Alzheimer's Disease Models Sede Amministrativa: Università degli Studi di Padova Dipartimento di Biologia ___________________________________________________________________ CORSO DI DOTTORATO DI RICERCA IN: BIOSCIENZE e BIOTECNOLOGIE CURRICOLO: NEUROBIOLOGIA CICLO XXIX EXPLORING ORAI2 FUNCTION IN ALZHEIMER’S DISEASE MODELS BASED ON PRESENILIN 2 AND AMYLOID PRECURSOR PROTEIN MUTANTS Coordinatore: Ch.mo Prof. Paolo Bernardi Supervisore: Dott.ssa Cristina Fasolato Dottorando: Mario Agostini INDEX SUMMARY ................................................................................................ 1 RIASSUNTO ............................................................................................... 3 ABBREVIATIONS ........................................................................................ 5 INTRODUCTION ......................................................................................... 8 γ-SECRETASE ................................................................................................................... 11 PRESENILINS ................................................................................................................... 12 APP ................................................................................................................................. 14 THE AMYLOID CASCADE HYPOTHESIS ............................................................................ 16 THE Ca2+ HYPOTHESIS ..................................................................................................... 17 Ca2+ homeostasis ........................................................................................................ 18 PLASMA MEMBRANE Ca2+ CHANNELS IN PHYSIOLOGY ................................................. 19 Ligand-gated channels ................................................................................................ 19 Voltage-operated Ca2+ channels (VOCCs) ................................................................... 20 Store-operated Ca2+ channels (SOCCs) ....................................................................... 21 PLASMA MEMBRANE Ca2+ CHANNELS IN AD ................................................................. 25 VOCCs ......................................................................................................................... 26 nAChRs ........................................................................................................................ 27 Ionotropic glutamate receptors ................................................................................. 28 SOCCs .......................................................................................................................... 30 INTRACELLULAR Ca2+ CHANNELS IN PHYSIOLOGY ......................................................... 33 Endoplasmic Reticulum/Golgi apparatus ................................................................... 33 Mitochondria .............................................................................................................. 36 Acidic compartments .................................................................................................. 37 INTRACELLULAR Ca2+ CHANNELS IN AD ......................................................................... 38 III Endoplasmic reticulum/Golgi apparatus .................................................................... 38 Mitochondria .............................................................................................................. 39 Lysosomes ................................................................................................................... 41 Ca2+ IMAGING ................................................................................................................. 42 Ca2+ measurements in living cells ............................................................................... 42 Synthetic Ca2+ indicators ............................................................................................ 42 Genetically-Encoded Ca2+ Indicators (GECIs) .............................................................. 44 RESULTS ........................................................................................................................... 50 Ca2+ stores, CCE and Aβ42 production: a close interplay .............................................. 50 The SOCE subunit Orai2 is overexpressed in mutant PS2-based AD mouse models .... 52 The SOCE subunit Orai2 is mainly expressed in cortical and hippocampal neurons in situ. ........................................................................................................................................ 54 Orai2 overexpression impairs IP3-induced ER Ca2+ release ........................................... 56 Orai2 overexpression decreases the ER Ca2+ content .................................................... 58 Orai2 overexpression affects the Store-Operated Ca2+ Entry ........................................ 59 Orai2 is a less efficient SOCE mediator than Orai1 ........................................................ 60 Orai2 down-regulation does not alter the internal store Ca2+ handling ........................ 64 Orai2 downregulation boosts the Store-Operated Calcium Entry ................................. 66 Orai2 minimally localizes to the ER ................................................................................ 67 Orai2 localizes to the early endosome compartment ................................................... 68 Orai2 localizes to the early-endosome compartment of wild-type and PS2-N141I cortical neurons .......................................................................................................................... 70 Orai2 and Orai1 localization to early endosomes changes upon cell stimulation ......... 72 DISCUSSION ............................................................................................ 79 MATERIALS AND METHODS .................................................................... 87 REFERENCES ............................................................................................ 94 IV V VI SUMMARY Alzheimer’s disease (AD) is the most common form of dementia among elderly population. More than twenty years ago the so-called amyloid hypothesis was formulated based on the major histopathological hallmarks of AD, among which the amyloid plaques are the most known and studied. This hypothesis was prompted by the discovery of three genes that, whereas mutated, are associated with the familial forms of the disease (FAD). One of these genes encodes for the amyloid precursor protein (APP), a single-pass type I transmembrane protein that undergoes sequential cleavages operated by the secretase family of enzymes. The last and key secretase, called γ-secretase, is composed of four proteins, among which we found either presenilin 1 (PS1) or presenilin 2 (PS2), encoded by other two genes (PSEN1/PSEN2) that are responsible for FAD pathogenesis. Autosomic dominant mutations in either APP, PSEN1 or PSEN2 cause accelerated Aβ deposition due to an increased Aβ42/Aβ40 ratio. While the vast majority of AD cases are sporadic, FAD patients bearing PS2 mutations show a clinical course much similar to that of sporadic patients. By many groups it was found that PSs are capable of perturbing cellular Ca2+ homeostasis, and, particularly, our group demonstrated that PS2, either bearing FAD-linked mutations or wild-type (WT), lowers endoplasmic reticulum (ER) and Golgi apparatus Ca2+ content, interacts with SERCA pump, dampening its function, and tethers ER and mitochondria; all of these pleiotropic effects are independent of its γ-secretase activity. Recently another group identified PS2 as a regulator of the ER Ca2+ content, together with Orai2, a plasma membrane channel implicated in the Store- Operated Ca2+ Entry (SOCE). This latter phenomenon is impaired in AD, and specifically it is down- tuned in mutant PS-bearing cells. Taken together this body of information offered an interesting background to study the interplay between ER Ca2+ levels, SOCE defects and APP processing/Aβ production. Taking advantage of the PS2-based AD mouse models available in our laboratory, namely the homozygous single transgenic (TG) line expressing the FAD-linked mutant PS2-N141I (line PS2.30H) and the homozygous double transgenic (2TG) line expressing PS2-N141I together with the Swedish double mutant APP-K670M/N671L (line B6.152H), we could investigate the expression pattern of Orai2 in the nervous tissue. Western blot analyses on cortices and hippocampi revealed that Orai2 was overexpressed in cortices from TG and 2TG mice, when compared to C57BL/6 (WT) mice. This overexpression was mainly due to the neuronal contribution since it was even higher in cortical neuronal cultures and in situ Orai2 was found only in neurons, as assayed by immunohistochemical analysis of brain 1 slices. Orai2 up-regulation, that is the condition found in TG and 2TG neurons, is capable of perturbing cellular Ca2+ homeostasis. Particularly, when overexpressed it caused a significant decrease in IP3-induced ER Ca2+ release in both H4-APPswe and HEK29T cells; these results are consistent with a decreased ER Ca2+ level, as measured with the ER-targeted probe G-CEPIA1er. In addition to this, Orai2 revealed to be a less efficient mediator of SOCE than Orai1, since it dampened SOCE when overexpressed alone and it produced a much smaller SOCE when overexpressed with STIM1 as compared with Orai1 plus STIM1 overexpression. Conversely to our
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