Nucleolin: a cell portal for viruses, bacteria, and toxins.

The main localization of nucleolin is the nucleolus, but this protein is present in multiple subcellular sites, and it is unconventionally secreted. On the cell surface, nucleolin acts as a receptor for various viruses, some bacteria, and some toxins. Aim of this review is to discuss the characteristics that make nucleolin able to act as receptor or co-receptor of so many and different pathogens. The important features that emerge are its multivalence, and its role as a bridge between the cell surface and the nucleus. Multiple domains, short linear motifs and post-translational modifications confer and modulate nucleolin ability to interact with nucleic acids, with proteins, but also with carbohydrates and lipids. This modular multivalence allows nucleolin to participate in different types of biomolecular condensates and to move to various subcellular locations, where it can act as a kind of molecular glue. It moves from the nucleus to the cell surface and can accompany particles in the reverse direction, from the cell surface into the nucleus, which is the destination of several pathogens to manipulate the cell in their favour.

Structural Integrity of Nucleolin Is Required to Suppress TDP-43-Mediated Cytotoxicity in Yeast and Human Cell Models.

The Transactivating response (TAR) element DNA-binding of 43 kDa (TDP-43) is mainly implicated in the regulation of gene expression, playing multiple roles in RNA metabolism. Pathologically, it is implicated in amyotrophic lateral sclerosis and in a class of neurodegenerative diseases broadly going under the name of frontotemporal lobar degeneration (FTLD). A common hallmark of most forms of such diseases is the presence of TDP-43 insoluble inclusions in the cell cytosol. The molecular mechanisms of TDP-43-related cell toxicity are still unclear, and the contribution to cell damage from either loss of normal TDP-43 function or acquired toxic properties of protein aggregates is yet to be established. Here, we investigate the effects on cell viability of FTLD-related TDP-43 mutations in both yeast and mammalian cell models. Moreover, we focus on nucleolin (NCL) gene, recently identified as a genetic suppressor of TDP-43 toxicity, through a thorough structure/function characterization aimed at understanding the role of NCL domains in rescuing TDP-43-induced cytotoxicity. Using functional and biochemical assays, our data demonstrate that the N-terminus of NCL is necessary, but not sufficient, to exert its antagonizing effects on TDP-43, and further support the relevance of the DNA/RNA binding central region of the protein. Concurrently, data suggest the importance of the NCL nuclear localization for TDP-43 trafficking, possibly related to both TDP-43 physiology and toxicity.

Single cell analysis reveals the involvement of the long non-coding RNA Pvt1 in the modulation of muscle atrophy and mitochondrial network.

Long non-coding RNAs (lncRNAs) are emerging as important players in the regulation of several aspects of cellular biology. For a better comprehension of their function, it is fundamental to determine their tissue or cell specificity and to identify their subcellular localization. In fact, the activity of lncRNAs may vary according to cell and tissue specificity and subcellular compartmentalization. Myofibers are the smallest complete contractile system of skeletal muscle influencing its contraction velocity and metabolism. How lncRNAs are expressed in different myofibers, participate in metabolism regulation and muscle atrophy or how they are compartmentalized within a single myofiber is still unknown. We compiled a comprehensive catalog of lncRNAs expressed in skeletal muscle, associating the fiber-type specificity and subcellular location to each of them, and demonstrating that many lncRNAs can be involved in the biological processes de-regulated during muscle atrophy. We demonstrated that the lncRNA Pvt1, activated early during muscle atrophy, impacts mitochondrial respiration and morphology and affects mito/autophagy, apoptosis and myofiber size in vivo. This work corroborates the importance of lncRNAs in the regulation of metabolism and neuromuscular pathologies and offers a valuable resource to study the metabolism in single cells characterized by pronounced plasticity.

Perturbations of the Proteome and of Secreted Metabolites in Primary Astrocytes from the hSOD1(G93A) ALS Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose pathophysiology is largely unknown. Despite the fact that motor neuron (MN) death is recognized as the key event in ALS, astrocytes dysfunctionalities and neuroinflammation were demonstrated to accompany and probably even drive MN loss. Nevertheless, the mechanisms priming astrocyte failure and hyperactivation are still obscure. In this work, altered pathways and molecules in ALS astrocytes were unveiled by investigating the proteomic profile and the secreted metabolome of primary spinal cord astrocytes derived from transgenic ALS mouse model overexpressing the human (h)SOD1(G93A) protein in comparison with the transgenic counterpart expressing hSOD1(WT) protein. Here we show that ALS primary astrocytes are depleted of proteins-and of secreted metabolites-involved in glutathione metabolism and signaling. The observed increased activation of Nf-kB, Ebf1, and Plag1 transcription factors may account for the augmented expression of proteins involved in the proteolytic routes mediated by proteasome or endosome-lysosome systems. Moreover, hSOD1(G93A) primary astrocytes also display altered lipid metabolism. Our results provide novel insights into the altered molecular pathways that may underlie astrocyte dysfunctionalities and altered astrocyte-MN crosstalk in ALS, representing potential therapeutic targets to abrogate or slow down MN demise in disease pathogenesis.

Regulation of Endoplasmic Reticulum-Mitochondria Tethering and Ca2+ Fluxes by TDP-43 via GSK3ß.

Mitochondria-ER contacts (MERCs), tightly regulated by numerous tethering proteins that act as molecular and functional connections between the two organelles, are essential to maintain a variety of cellular functions. Such contacts are often compromised in the early stages of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). TDP-43, a nuclear protein mainly involved in RNA metabolism, has been repeatedly associated with ALS pathogenesis and other neurodegenerative diseases. Although TDP-43 neuropathological mechanisms are still unclear, the accumulation of the protein in cytoplasmic inclusions may underlie a protein loss-of-function effect. Accordingly, we investigated the impact of siRNA-mediated TDP-43 silencing on MERCs and the related cellular parameters in HeLa cells using GFP-based probes for MERCs quantification and aequorin-based probes for local Ca2+ measurements, combined with targeted protein and mRNA profiling. Our results demonstrated that TDP-43 down-regulation decreases MERCs density, thereby remarkably reducing mitochondria Ca2+ uptake after ER Ca2+ release. Thorough mRNA and protein analyses did not highlight altered expression of proteins involved in MERCs assembly or Ca2+-mediated ER-mitochondria cross-talk, nor alterations of mitochondrial density and morphology were observed by confocal microscopy. Further mechanistic inspections, however, suggested that the observed cellular alterations are correlated to increased expression/activity of GSK3ß, previously associated with MERCs disruption.

Microglia in Prion Diseases: Angels or Demons?

Prion diseases are rare transmissible neurodegenerative disorders caused by the accumulation of a misfolded isoform (PrPSc) of the cellular prion protein (PrPC) in the central nervous system (CNS). Neuropathological hallmarks of prion diseases are neuronal loss, astrogliosis, and enhanced microglial proliferation and activation. As immune cells of the CNS, microglia participate both in the maintenance of the normal brain physiology and in driving the neuroinflammatory response to acute or chronic (e.g., neurodegenerative disorders) insults. Microglia involvement in prion diseases, however, is far from being clearly understood. During this review, we summarize and discuss controversial findings, both in patient and animal models, suggesting a neuroprotective role of microglia in prion disease pathogenesis and progression, or-conversely-a microglia-mediated exacerbation of neurotoxicity in later stages of disease. We also will consider the active participation of PrPC in microglial functions, by discussing previous reports, but also by presenting unpublished results that support a role for PrPC in cytokine secretion by activated primary microglia.

The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons.

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aß Toxicity.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

ALS-Associated SOD1(G93A) Decreases SERCA Pump Levels and Increases Store-Operated Ca2+ Entry in Primary Spinal Cord Astrocytes from a Transgenic Mouse Model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective death of motor neurons (MNs), probably by a combination of cell- and non-cell-autonomous processes. The past decades have brought many important insights into the role of astrocytes in nervous system function and disease, including the implication in ALS pathogenesis possibly through the impairment of Ca2+-dependent astrocyte-MN cross-talk. In this respect, it has been recently proposed that altered astrocytic store-operated Ca2+ entry (SOCE) may underlie aberrant gliotransmitter release and astrocyte-mediated neurotoxicity in ALS. These observations prompted us to a thorough investigation of SOCE in primary astrocytes from the spinal cord of the SOD1(G93A) ALS mouse model in comparison with the SOD1(WT)-expressing controls. To this purpose, we employed, for the first time in the field, genetically-encoded Ca2+ indicators, allowing the direct assessment of Ca2+ fluctuations in different cell domains. We found increased SOCE, associated with decreased expression of the sarco-endoplasmic reticulum Ca2+-ATPase and lower ER resting Ca2+ concentration in SOD1(G93A) astrocytes compared to control cells. Such findings add novel insights into the involvement of astrocytes in ALS MN damage.

Regulation of ER-mitochondria contacts by Parkin via Mfn2.

Parkin, an E3 ubiquitin ligase and a Parkinson's disease (PD) related gene, translocates to impaired mitochondria and drives their elimination via autophagy, a process known as mitophagy. Mitochondrial pro-fusion protein Mitofusins (Mfn1 and Mfn2) were found to be a target for Parkin mediated ubiquitination. Mfns are transmembrane GTPase embedded in the outer membrane of mitochondria, which are required on adjacent mitochondria to mediate fusion. In mammals, Mfn2 also forms complexes that are capable of tethering mitochondria to endoplasmic reticulum (ER), a structural feature essential for mitochondrial energy metabolism, calcium (Ca2+) transfer between the organelles and Ca2+ dependent cell death. Despite its fundamental physiological role, the molecular mechanisms that control ER-mitochondria cross talk are obscure. Ubiquitination has recently emerged as a powerful tool to modulate protein function, via regulation of protein subcellular localization and protein ability to interact with other proteins. Ubiquitination is also a reversible mechanism, which can be actively controlled by opposing ubiquitination-deubiquitination events. In this work we found that in Parkin deficient cells and parkin mutant human fibroblasts, the tether between ER and mitochondria is decreased. We identified the site of Parkin dependent ubiquitination and showed that the non-ubiquitinatable Mfn2 mutant fails to restore ER-mitochondria physical and functional interaction. Finally, we took advantage of an established in vivo model of PD to demonstrate that manipulation of ER-mitochondria tethering by expressing an ER-mitochondria synthetic linker is sufficient to rescue the locomotor deficit associated to an in vivo Drosophila model of PD.

Almost a century of prion protein(s): From pathology to physiology, and back to pathology.

Prions are one of the few pathogens whose name is renowned at all population levels, after the dramatic years pervaded by the fear of eating prion-infected food. If now this, somehow irrational, scare of bovine meat inexorably transmitting devastating brain disorders is largely subdued, several prion-related issues are still unsolved, precluding the design of therapeutic approaches that could slow, if not halt, prion diseases. One unsolved issue is, for example, the role of the prion protein (PrPC), whole conformational misfolding originates the prion but whose physiologic reason d'etre in neurons, and in cells at large, remains enigmatic. Preceded by a historical outline, the present review will discuss the functional pleiotropicity ascribed to PrPC, and whether this aspect could fall, at least in part, into a more concise framework. It will also be devoted to radically different perspectives for PrPC, which have been recently brought to the attention of the scientific world with unexpected force. Finally, it will discuss the possible reasons allowing an evolutionary conserved and benign protein, as PrPC is, to turn into a high affinity receptor for pathologic misfolded oligomers, and to transmit their toxic message into neurons.

Absolute quantification of myosin heavy chain isoforms by selected reaction monitoring can underscore skeletal muscle changes in a mouse model of amyotrophic lateral sclerosis.

Skeletal muscle fibers contain different isoforms of myosin heavy chain (MyHC) that define distinctive contractile properties. In light of the muscle capacity to adapt MyHC expression to pathophysiological conditions, a rapid and quantitative assessment of MyHC isoforms in small muscle tissue quantities would represent a valuable diagnostic tool for (neuro)muscular diseases. As past protocols did not meet these requirements, in the present study we applied a targeted proteomic approach based on selected reaction monitoring that allowed the absolute quantification of slow and fast MyHC isoforms in different mouse skeletal muscles with high reproducibility. This mass-spectrometry-based method was validated also in a pathological specimen, by comparison of the MyHC expression profiles in different muscles from healthy mice and a genetic mouse model of amyotrophic lateral sclerosis (ALS) expressing the SOD1(G93A) mutant. This analysis showed that terminally ill ALS mice have a fast-to-slow shift in the fiber type composition of the tibialis anterior and gastrocnemius muscles, as previously reported. These results will likely open the way to accurate and rapid diagnoses of human (neuro)muscular diseases by the proposed method. Graphical Abstract Methods for myosin heavy chain (MyHC) quantification: a comparison of classical methods and selected reaction monitoring (SRM)-based mass spectrometry approaches.

Age-dependent neuromuscular impairment in prion protein knockout mice.

INTRODUCTION: The cellular prion protein (PrP(C) ) is commonly recognized as the precursor of prions, the infectious agents of the fatal transmissible spongiform encephalopathies, or prion diseases. Despite extensive effort, the physiological role of PrP(C) is still ambiguous. Evidence has suggested that PrP(C) is involved in different cellular functions, including peripheral nerve integrity and skeletal muscle physiology. METHODS: We analyzed the age-dependent influence of PrP(C) on treadmill test-based aerobic exercise capacity and on a series of morphological and metabolic parameters using wild-type and genetically modified mice of different ages expressing, or knockout (KO) for, PrP(C) . RESULTS: We found that aged PrP-KO mice displayed a reduction in treadmill performance compared with PrP-expressing animals, which was associated with peripheral nerve demyelination and alterations of skeletal muscle fiber type. CONCLUSION: PrP-KO mice have an age-dependent impairment of aerobic performance as a consequence of specific peripheral nerve and muscle alterations.

Mitochondria Dysfunction and Neuroinflammation in Neurodegeneration: Who Comes First?

Neurodegenerative diseases (NDs) encompass an assorted array of disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, each characterised by distinct clinical manifestations and underlying pathological mechanisms. While some cases have a genetic basis, many NDs occur sporadically. Despite their differences, these diseases commonly feature chronic neuroinflammation as a hallmark. Consensus has recently been reached on the possibility that mitochondria dysfunction and protein aggregation can mutually contribute to the activation of neuroinflammatory response and thus to the onset and progression of these disorders. In the present review, we discuss the contribution of mitochondria dysfunction and neuroinflammation to the aetiology and progression of NDs, highlighting the possibility that new potential therapeutic targets can be identified to tackle neurodegenerative processes and alleviate the progression of these pathologies.

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.

Secreted Metabolome of ALS-Related hSOD1(G93A) Primary Cultures of Myocytes and Implications for Myogenesis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease associated with progressive muscle atrophy, paralysis, and eventually death. Growing evidence demonstrates that the pathological process leading to ALS is the result of multiple altered mechanisms occurring not only in MNs but also in other cell types inside and outside the central nervous system. In this context, the involvement of skeletal muscle has been the subject of a few studies on patients and ALS animal models. In this work, by using primary myocytes derived from the ALS transgenic hSOD1(G93A) mouse model, we observed that the myogenic capability of such cells was defective compared to cells derived from control mice expressing the nonpathogenic hSOD1(WT) isoform. The correct in vitro myogenesis of hSOD1(G93A) primary skeletal muscle cells was rescued by the addition of a conditioned medium from healthy hSOD1(WT) myocytes, suggesting the existence of an in trans activity of secreted factors. To define a dataset of molecules participating in such safeguard action, we conducted comparative metabolomic profiling of a culture medium collected from hSOD1(G93A) and hSOD1(WT) primary myocytes and report here an altered secretion of amino acids and lipid-based signaling molecules. These findings support the urgency of better understanding the role of the skeletal muscle secretome in the regulation of the myogenic program and mechanisms of ALS pathogenesis and progression.

Relative quantification of membrane proteins in wild-type and prion protein (PrP)-knockout cerebellar granule neurons.

Approximately 25% of eukaryotic proteins possessing homology to at least two transmembrane domains are predicted to be embedded in biological membranes. Nevertheless, this group of proteins is not usually well represented in proteome-wide experiments due to their refractory nature. Here we present a quantitative mass spectrometry-based comparison of membrane protein expression in cerebellar granule neurons grown in primary culture that were isolated from wild-type mice and mice lacking the cellular prion protein. This protein is a cell-surface glycoprotein that is mainly expressed in the central nervous system and is involved in several neurodegenerative disorders, though its physiological role is unclear. We used a low specificity enzyme α-chymotrypsin to digest membrane proteins preparations that had been separated by SDS-PAGE. The resulting peptides were labeled with tandem mass tags and analyzed by MS. The differentially expressed proteins identified using this approach were further analyzed by multiple reaction monitoring to confirm the expression level changes.

SOD1 in ALS: Taking Stock in Pathogenic Mechanisms and the Role of Glial and Muscle Cells.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of motor neurons in the brain and spinal cord. While the exact causes of ALS are still unclear, the discovery that familial cases of ALS are related to mutations in the Cu/Zn superoxide dismutase (SOD1), a key antioxidant enzyme protecting cells from the deleterious effects of superoxide radicals, suggested that alterations in SOD1 functionality and/or aberrant SOD1 aggregation strongly contribute to ALS pathogenesis. A new scenario was opened in which, thanks to the generation of SOD1 related models, different mechanisms crucial for ALS progression were identified. These include excitotoxicity, oxidative stress, mitochondrial dysfunctions, and non-cell autonomous toxicity, also implicating altered Ca2+ metabolism. While most of the literature considers motor neurons as primary target of SOD1-mediated effects, here we mainly discuss the effects of SOD1 mutations in non-neuronal cells, such as glial and skeletal muscle cells, in ALS. Attention is given to the altered redox balance and Ca2+ homeostasis, two processes that are strictly related with each other. We also provide original data obtained in primary myocytes derived from hSOD1(G93A) transgenic mice, showing perturbed expression of Ca2+ transporters that may be responsible for altered mitochondrial Ca2+ fluxes. ALS-related SOD1 mutants are also responsible for early alterations of fundamental biological processes in skeletal myocytes that may impinge on skeletal muscle functions and the cross-talk between muscle cells and motor neurons during disease progression.

Serum Metabolomics and Proteomics to Study the Antihypertensive Effect of Protein Extracts from Tenebrio molitor.

Hypertension is the leading risk factor for premature death worldwide and significantly contributes to the development of all major cardiovascular disease events. The management of high blood pressure includes lifestyle changes and treatment with antihypertensive drugs. Recently, it was demonstrated that a diet supplemented with Tenebrio molitor (TM) extracts is useful in the management of numerous pathologies, including hypertension. This study is aimed at unveiling the underlying mechanism and the molecular targets of intervention of TM dietary supplementation in hypertension treatment by means of proteomics and metabolomics techniques based on liquid chromatography coupled with high-resolution mass spectrometry. We demonstrate that serum proteome and metabolome of spontaneously hypertensive rats are severely altered with respect to their normotensive counterparts. Additionally, our results reveal that a diet enriched with TM extracts restores the expression of 15 metabolites and 17 proteins mainly involved in biological pathways associated with blood pressure maintenance, such as the renin-angiotensin and kallikrein-kinin systems, serin protease inhibitors, reactive oxygen scavenging, and lipid peroxidation. This study provides novel insights into the molecular pathways that may underlie the beneficial effects of TM, thus corroborating that TM could be proposed as a helpful functional food supplement in the treatment of hypertension.

The ataxia-linked E1081Q mutation affects the sub-plasma membrane Ca2+-microdomains by tuning PMCA3 activity.

Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.