Ca2+signalling: A common language for organelles crosstalk in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disease caused by multifactorial pathogenic mechanisms. Familial PD is linked with genetic mutations in genes whose products are either associated with mitochondrial function or endo-lysosomal pathways. Of note, mitochondria are essential to sustain high energy demanding synaptic activity of neurons and alterations in mitochondrial Ca2+ signaling have been proposed as causal events for neurodegenerative process, although the mechanisms responsible for the selective loss of specific neuronal populations in the different neurodegenerative diseases is still not clear. Here, we specifically discuss the importance of a correct mitochondrial communication with the other organelles occurring at regions where their membranes become in close contact. We discuss the nature and the role of contact sites that mitochondria establish with ER, lysosomes, and peroxisomes, and how PD related proteins participate in the regulation/dysregulation of the tethering complexes. Unravelling molecular details of mitochondria tethering could contribute to identify specific therapeutic targets and develop new strategies to counteract the progression of the disease

Sorcin is an early marker of neurodegeneration, Ca2+ dysregulation and endoplasmic reticulum stress associated to neurodegenerative diseases

Dysregulation of calcium signaling is emerging as a key feature in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD), and targeting this process may be therapeutically beneficial. Under this perspective, it is important to study proteins that regulate calcium homeostasis in the cell. Sorcin is one of the most expressed calcium-binding proteins in the human brain; its overexpression increases endoplasmic reticulum (ER) calcium concentration and decreases ER stress in the heart and in other cellular types. Sorcin has been hypothesized to be involved in neurodegenerative diseases, since it may counteract the increased cytosolic calcium levels associated with neurodegeneration. In the present work, we show that Sorcin expression levels are strongly increased in cellular, animal, and human models of AD, PD, and HD, vs. normal cells. Sorcin partially colocalizes with RyRs in neurons and microglia cells; functional experiments with microsomes containing high amounts of RyR2 and RyR3, respectively, show that Sorcin is able to regulate these ER calcium channels. The molecular basis of the interaction of Sorcin with RyR2 and RyR3 is demonstrated by SPR. Sorcin also interacts with other ER proteins as SERCA2 and Sigma-1 receptor in a calcium-dependent fashion. We also show that Sorcin regulates ER calcium transients: Sorcin increases the velocity of ER calcium uptake (increasing SERCA activity). The data presented here demonstrate that Sorcin may represent both a novel early marker of neurodegenerative diseases and a response to cellular stress dependent on neurodegeneration

Utilizzo di zebrafish come organismo modello per lo studio in vivo delle funzioni di Presenilina2 e dei contatti tra organuli cellulari.

Alzheimer’s Disease (AD) is the most common form of dementia. It is mainly sporadic, however, autosomal dominant mutations on three different genes, coding for Amyloid Precursor Protein (APP), Presenilin 1 (PS1) and Presenilin 2 (PS2), are responsible for a little percentage of inherited cases (Familial AD, FAD). Presenilins (PS1 and PS2) form, alternately, the catalytic core of the ɣ- secretase complex, the enzyme that cleaves APP to produced Aβ peptides. Apart of their role in this enzymatic complex, PSs have also several ɣ-secretase-independent functions, such as those involved in the modulation of autophagy, mitochondrial axonal transport and Ca2+ homeostasis. Importantly, all of the above mentioned cellular processes have been found altered in AD. It is still unclear, however, how PSs and their FAD-linked mutants mechanistically influence these cellular processes. In our lab, our research specifically focuses on PS2. The aim of this study was to investigate in an in vivo model the possible effects of the protein absence in the cellular processes associated to its activity. To do so, we generated a psen2 knock out zebrafish line by CRISPR-Cas technology. On this model, by several techniques, we investigated the effect of Psen2 absence on mitochondrial axonal transport, mitochondrial respiration, ER-mitochondria communication and autophagy. In parallel, we used both Danio rerio and Drosophila melanogaster to test in vivo newly designed fluorescent probes that allows the visualization of interorganelle communications.La malattia di Alzheimer (AD) è la forma più comune di demenza. È principalmente sporadica; tuttavia, mutazioni autosomiche dominanti su tre diversi geni, codificanti per la Proteina Precursore dell'Amiloide (APP), Presenilina 1 (PS1) e Presenilina 2 (PS2), sono responsabili di una piccola percentuale di casi ereditari (AD familiare, FAD ). Le preseniline (PS1 e PS2) formano, alternativamente, il nucleo catalitico del complesso ɣ- secretasi, l'enzima che processa l'APP per produrre peptidi Aβ. Indipendentemente dal loro ruolo in questo complesso enzimatico, le PS sono coinvolte in altre funzioni cellulari, come quelle che riguardano la modulazione dell'autofagia, del trasporto assonale mitocondriale e dell'omeostasi del Ca2+. È importante sottolineare che tutti i processi cellulari sopra menzionati sono stati trovati alterati in AD. Non sono ancora chiari, tuttavia, i meccanismi attraverso cui le PS e i loro mutanti legati a FAD influenzano questi processi cellulari. Nel nostro laboratorio, la ricerca si concentra specificamente su PS2. Lo scopo di questa tesi è stato di indagare in un modello in vivo i possibili effetti dell'assenza di PS2 ​​nei processi cellulari associati alla sua attività. Per fare ciò, abbiamo generato una linea di zebrafish knock out per psen2 mediante la tecnologia CRISPR-Cas. Su questo modello, con diverse tecniche, abbiamo studiato l'effetto dell'assenza di Psen2 sul trasporto assonale mitocondriale, sulla respirazione mitocondriale, sulla comunicazione ER-mitocondri e sull'autofagia. Parallelamente, abbiamo utilizzato sia Danio rerio che Drosophila melanogaster per testare in vivo sonde fluorescenti di nuova generazione che consentono la visualizzazione delle comunicazioni tra organuli cellulari

Stable Integration of Inducible SPLICS Reporters Enables Spatio-Temporal Analysis of Multiple Organelle Contact Sites upon Modulation of Cholesterol Traffic

The study of organelle contact sites has received a great impulse due to increased interest in the understanding of their involvement in many disease conditions. Split-GFP-based contact sites (SPLICS) reporters emerged as essential tools to easily detect changes in a wide range of organelle contact sites in cultured cells and in vivo, e.g., in zebrafish larvae. We report here on the generation of a new vector library of SPLICS cloned into a piggyBac system for stable and inducible expression of the reporters in a cell line of interest to overcome any potential weakness due to variable protein expression in transient transfection studies. Stable HeLa cell lines expressing SPLICS between the endoplasmic reticulum (ER) and mitochondria (MT), the ER and plasma membrane (PM), peroxisomes (PO) and ER, and PO and MT, were generated and tested for their ability to express the reporters upon treatment with doxycycline. Moreover, to take advantage of these cellular models, we decided to follow the behavior of different membrane contact sites upon modulating cholesterol traffic. Interestingly, we found that the acute pharmacological inhibition of the intracellular cholesterol transporter 1 (NPC1) differently affects membrane contact sites, highlighting the importance of different interfaces for cholesterol sensing and distribution within the cell

PINK1/Parkin Mediated Mitophagy, Ca2+ Signalling, and ER–Mitochondria Contacts in Parkinson’s Disease

Endoplasmic reticulum (ER)–mitochondria contact sites are critical structures for cellular function. They are implicated in a plethora of cellular processes, including Ca2+ signalling and mitophagy, the selective degradation of damaged mitochondria. Phosphatase and tensin homolog (PTEN)-induced kinase (PINK) and Parkin proteins, whose mutations are associated with familial forms of Parkinson’s disease, are two of the best characterized mitophagy players. They accumulate at ER–mitochondria contact sites and modulate organelles crosstalk. Alterations in ER–mitochondria tethering are a common hallmark of many neurodegenerative diseases including Parkinson’s disease. Here, we summarize the current knowledge on the involvement of PINK1 and Parkin at the ER–mitochondria contact sites and their role in the modulation of Ca2+ signalling and mitophagy

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering

ER-Mitochondria Calcium Transfer, Organelle Contacts and Neurodegenerative Diseases

It is generally accepted that interorganellar contacts are central to the 7 control of cellular physiology. Virtually, any intracellular organelle can come into 8 proximity with each other and, by establishing physical protein-mediated contacts 9 within a selected fraction of the membrane surface, novel specific functions are 10 acquired. Endoplasmic reticulum (ER) contacts with mitochondria are among the 11 best studied with major roles in Ca2+ and lipid transfer, signaling, and membrane 12 dynamics. 13 This functional (and structural) diversity, their dynamic nature as well as the 14 growing number of new players concurred to make their monitoring difficult. 15 This review focuses on the most established examples of tethers/modulators of 16 the ER-mitochondria interface and on the roles of these contacts in health and 17 disease by specifically dissecting how Ca2+ transfer occur and how mishandling 18 eventually lead to disease. Additional functions of the ER-mitochondria interface 19 and an overview of the currently available methods to measure/quantify the ER- 20 mitochondria interface will also be considered

Unraveling Presenilin 2 Functions in a Knockout Zebrafish Line to Shed Light into Alzheimer’s Disease Pathogenesis

Mutations in presenilin 2 (PS2) have been causally linked to the development of inherited Alzheimer’s disease (AD). Besides its role as part of the γ-secretase complex, mammalian PS2 is also involved, as an individual protein, in a growing number of cell processes, which result altered in AD. To gain more insight into PS2 (dys)functions, we have generated a presenilin2 (psen2) knockout zebrafish line. We found that the absence of the protein does not markedly influence Notch signaling at early developmental stages, suggesting a Psen2 dispensable role in the γ-secretase-mediated Notch processing. Instead, loss of Psen2 induces an exaggerated locomotor response to stimulation in fish larvae, a reduced number of ER-mitochondria contacts in zebrafish neurons, and an increased basal autophagy. Moreover, the protein is involved in mitochondrial axonal transport, since its acute downregulation reduces in vivo organelle flux in zebrafish sensory neurons. Importantly, the expression of a human AD-linked mutant of the protein increases this vital process. Overall, our results confirm zebrafish as a good model organism for investigating PS2 functions in vivo, representing an alternative tool for the characterization of new AD-linked defective cell pathways and the testing of possible correcting drugs

A SPLICS reporter reveals α{{{{{\boldsymbol{\alpha }}}}}} α -synuclein regulation of lysosome-mitochondria contacts which affects TFEB nuclear translocation

Abstract Mitochondrial and lysosomal activities are crucial to maintain cellular homeostasis: optimal coordination is achieved at their membrane contact sites where distinct protein machineries regulate organelle network dynamics, ions and metabolites exchange. Here we describe a genetically encoded SPLICS reporter for short- and long- juxtapositions between mitochondria and lysosomes. We report the existence of narrow and wide lysosome-mitochondria contacts differently modulated by mitophagy, autophagy and genetic manipulation of tethering factors. The overexpression of α-synuclein (α-syn) reduces the apposition of mitochondria/lysosomes membranes and affects their privileged Ca2+ transfer, impinging on TFEB nuclear translocation. We observe enhanced TFEB nuclear translocation in α-syn-overexpressing cells. We propose that α-syn, by interfering with mitochondria/lysosomes tethering impacts on local Ca2+ regulated pathways, among which TFEB mediated signaling, and in turn mitochondrial and lysosomal function. Defects in mitochondria and lysosome represent a common hallmark of neurodegenerative diseases: targeting their communication could open therapeutic avenues

An expanded palette of improved SPLICS reporters detects multiple organelle contacts in vitro and in vivo

Membrane contact sites between virtually any known organelle have been documented and, in the last decades, their study received momentum due to their importance for fundamental activities of the cell and for the subtle comprehension of many human diseases. The lack of tools to finely image inter-organelle proximity hindered our understanding on how these subcellular communication hubs mediate and regulate cell homeostasis. We develop an improved and expanded palette of split-GFP-based contact site sensors (SPLICS) for the detection of single and multiple organelle contact sites within a scalable distance range. We demonstrate their flexibility under physiological conditions and in living organisms