SARS-CoV-2 infection alkalinizes the ERGIC and lysosomes through the viroporin activity of the viral envelope protein.

The coronavirus SARS-CoV-2, the agent of the deadly COVID-19 pandemic, is an enveloped virus propagating within the endocytic and secretory organelles of host mammalian cells. Enveloped viruses modify the ionic homeostasis of organelles to render their intra-luminal milieu permissive for viral entry, replication and egress. Here, we show that infection of Vero E6 cells with the delta variant of the SARS-CoV-2 alkalinizes the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as well as lysosomes, mimicking the effect of inhibitors of vacuolar proton ATPases. We further show the envelope protein of SARS-CoV-2 accumulates in the ERGIC when expressed in mammalian cells and selectively dissipates the ERGIC pH. This viroporin action is prevented by mutations of Val25 but not Asn15 within the channel pore of the envelope (E) protein. We conclude that the envelope protein acts as a proton channel in the ERGIC to mitigate the acidity of this intermediate compartment. The altered pH homeostasis of the ERGIC likely contributes to the virus fitness and pathogenicity, making the E channel an attractive drug target for the treatment of COVID-19.

TMBIM5 is the Ca2+ /H+ antiporter of mammalian mitochondria.

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+ /H+ exchanger (CHE). Originally identified as the mitochondrial K+ /H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+ -independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

Study on the Relationship Between ß2 Macroglobulin, Small Density, Low-density Lipoprotein and Carotid Plaque Instability after Acute Thrombolysis in Patients with Ischemic Stroke.

Objective: Study the relationship between ß2 microglobulin, small density, low-density lipoprotein and carotid plaque instability after acute thrombolysis in ischemic stroke patients (IS). Methods: 319 patients with acute cerebral infarction who were treated by thrombolysis in the Department of Neurology, Chongming Branch of Shanghai Xinhua Hospital from January 2017 to May 2022 were included retrospectively. All subjects have undergone a carotid artery ultrasound examination for plaque. According to the ultrasound results, the subjects were divided into plaque-free group (94 cases), a stable plaque group (38 cases) and an unstable plaque group (187 cases). Use an automatic blood biochemical analyzer to detect routine indicators. At the same time, compare the differences of risk factors and biochemical indicators among the groups according to the demographic data of the patient's previous hospitalization. To further evaluate the related risk factors of the instability of carotid plaque in patients through the multivariate logistic regression analysis, the odds ratio (OR) and 95% confidence interval (95% CI) were calculated. Analysis the predictive value of ß2 microglobulin and small density low density lipoprotein on the instability of carotid plaque in I.S. patients after acute thrombolysis through subject work characteristic curve (ROC). Results: Among 319 patients, 187 had unstable plaque accounting for 58.6% and 38 had stable plaque accounting for 11.9%, according to the comparison of general clinical data. Lymphocyte, neutrophil ratio, triglyceride, T3, Hcy, ß2 microglobulin has statistical significance in the presence or absence of plaque. Lymphocytes, small dense low-density lipoprotein, ß2 microglobulin have statistical significance in the stability of plaque (P < .05). Total cholesterol, hypertension, ß2 microglobulin and small density low-density lipoprotein may be independent risk factors of carotid plaque instability through multivariate logistic regression analysis (P < .05). The area under ROC curve showed that ß2 microglobulin AUC: 0.6388, P < .05, small density low-density lipoprotein AUC: 0.6086, P < .05, combined diagnosis AUC: 0.6924, P < .05. Conclusion: ß2 microglobulin and density low-density lipoprotein are independent risk factors of carotid artery plaque instability in I.S. patients after acute thrombolysis. Moreover, the sensibility and differential of combined diagnosis are higher, which has certain predictive value for the instability of carotid plaque in such patients.

The mammalian trafficking chaperone protein UNC93B1 maintains the ER calcium sensor STIM1 in a dimeric state primed for translocation to the ER cortex.

The stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that regulates the activity of Orai plasma membrane Ca2+ channels to mediate the store-operated Ca2+ entry pathway essential for immunity. Uncoordinated 93 homolog B1 (UNC93B1) is a multiple membrane-spanning ER protein that acts as a trafficking chaperone by guiding nucleic-acid sensing toll-like receptors to their respective endosomal signaling compartments. We previously showed that UNC93B1 interacts with STIM1 to promote antigen cross-presentation in dendritic cells, but the STIM1 binding site(s) and activation step(s) impacted by this interaction remained unknown. In this study, we show that UNC93B1 interacts with STIM1 in the ER lumen by binding to residues in close proximity to the transmembrane domain. Cysteine crosslinking in vivo showed that UNC93B1 binding promotes the zipping of transmembrane and proximal cytosolic helices within resting STIM1 dimers, priming STIM1 for translocation. In addition, we show that UNC93B1 deficiency reduces store-operated Ca2+ entry and STIM1-Orai1 interactions and targets STIM1 to lighter ER domains, whereas UNC93B1 expression accelerates the recruitment of STIM1 to cortical ER domains. We conclude that UNC93B1 therefore acts as a trafficking chaperone by maintaining the pool of resting STIM1 proteins in a state primed for activation, enabling their rapid translocation in an extended conformation to cortical ER signaling compartments.

Hyccin/FAM126A deficiency reduces glial enrichment and axonal sheath, which are rescued by overexpression of a plasma membrane-targeting PI4KIIIα in Drosophila.

Hyccin/FAM126A mutations are linked to hypomyelination and congenital cataract disease (HCC), but whether and how Hyccin/FAM126A deficiency causes hypomyelination remains undetermined. This study shows Hyccin/FAM126A expression was necessary for the expression of other components of the PI4KIIIα complex in Drosophila. Knockdown of Hyccin/FAM126A in glia reduced the enrichment of glial cells, disrupted axonal sheaths and visual ability in the visual system, and these defects could be fully rescued by overexpressing either human FAM126A or FAM126B, and partially rescued by overexpressing a plasma membrane-targeting recombinant mouse PI4KIIIα. Additionally, PI4KIIIα knockdown in glia phenocopied Hyccin/FAM126A knockdown, and this was partially rescued by overexpressing the recombinant PI4KIIIα, but not human FAM126A or FAM126B. This study establishes an animal model of HCC and indicates that Hyccin/FAM126A plays an essential role in glial enrichment and axonal sheath in a cell-autonomous manner in the visual system via controlling the expression and stabilization of the PI4KIIIα complex at the plasma membrane.

NPM1 is a diagnostic and prognostic biomarker associated with the clinicopathological characteristics of gastric cancer.

NPM1 plays an important role in the occurrence and development of leukemia and various solid tumors. This study aimed to investigate the expression of NPM1 in gastric cancer (GC) and adjacent normal tissues, study the relationship between NPM1 expression and clinicopathological characteristics in GC patients, and explore the impact of NPM1 expression on the diagnosis and prognosis of GC. We used tissue microarray immunohistochemical analysis to examine the expression level of NPM1 in GC and adjacent tissues and analyzed the relationship between NPM1 expression, clinicopathological factors, and GC prognosis. Prognostic values of NPM1 mRNA were also investigated using an online database. qRT-PCR was used to detect the expression of NPM1 mRNA in cancer and adjacent tissues. According to microarray immunohistochemical analysis and qRT-PCR results, NPM1 had a high expression in all adjacent normal tissues. Microarray immunohistochemical analyses demonstrated that the NPM1 was lowly expressed in 75.5% of GC tissues but highly expressed in 24.5% of GC tissues. qRT-PCR results showed NPM1 mRNA low expression in most GC tissues. NPM1 high expression group was associated with a better overall survival rate and disease-free survival rate than the NPM1 low expression group (p<0.01). This result is consistent with that of the online database. The receiver operating characteristics curve showed that NPM1 was valuable in the diagnosis of GC. The assessment of NPM1 expression in GC samples may represent a useful tool for GC diagnosis and prognosis assessment.

Proteins Interacting with STIM1 and Store-Operated Ca2+ Entry.

The endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) interacts with ORAI Ca2+ channels at the plasma membrane to regulate immune and muscle cell function. The conformational changes underlying STIM1 activation, translocation, and ORAI1 trapping and gating, are stringently regulated by post-translational modifications and accessory proteins. Here, we review the recent progress in the identification and characterization of ER and cytosolic proteins interacting with STIM1 to control its activation and deactivation during store-operated Ca2+ entry (SOCE).

Downregulation of RBO-PI4KIIIα Facilitates Aß42 Secretion and Ameliorates Neural Deficits in Aß42-Expressing Drosophila.

Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. In yeast and mammals, Eighty-five requiring 3 (EFR3), whose Drosophila homolog is Rolling Blackout (RBO), forms a plasma membrane-localized protein complex with phosphatidylinositol-4-kinase Type IIIα (PI4KIIIα) and a scaffold protein to tightly control the level of plasmalemmal phosphatidylinositol-4-phosphate (PI4P). Here, we report that RBO binds to Drosophila PI4KIIIα, and that in an Aß42-expressing Drosophila model, separate genetic reduction of PI4KIIIα and RBO, or pharmacological inhibition of PI4KIIIα ameliorated synaptic transmission deficit, climbing ability decline, premature death, and reduced neuronal accumulation of Aß42 Moreover, we found that RBO-PI4KIIIa downregulation increased neuronal Aß42 release and that PI4P facilitated the assembly or oligomerization of Aß42 in/on liposomes. These results indicate that RBO-PI4KIIIa downregulation facilitates neuronal Aß42 release and consequently reduces neuronal Aß42 accumulation likely via decreasing Aß42 assembly in/on plasma membrane. This study suggests the RBO-PI4KIIIα complex as a potential therapeutic target and PI4KIIIα inhibitors as drug candidates for Alzheimer's disease treatment.SIGNIFICANCE STATEMENT Phosphoinositides and their metabolizing enzymes are involved in Aß42 metabolism and Alzheimer's disease pathogenesis. Here, in an Aß42-expressing Drosophila model, we discovered and studied the beneficial role of downregulating RBO or its interacting protein PI4KIIIα-a protein that tightly controls the plasmalemmal level of PI4P-against the defects caused by Aß42 expression. Mechanistically, RBO-PI4KIIIα downregulation reduced neuronal Aß42 accumulation, and interestingly increased neuronal Aß42 release. This study suggests the RBO-PI4KIIIα complex as a novel therapeutic target, and PI4KIIIα inhibitors as new drug candidates.

The use of MMSE and MoCA in patients with acute ischemic stroke in clinical.

BACKGROUND: The Mini-Mental State Examination (MMSE) and the Montreal Cognitive Assessment (MoCA) are brief cognitive screening tools that have been developed for the screening of patients with Mild Cognitive Impairment. METHODS: A total of 105 patients were included in this study, aged 53-89 years, with acute ischemic stroke admitted to hospital and fell into two groups: stroke patients with cognitive impairment (SCI) and controls with no cognitive impairment (n-SCI). The patient's characteristics are collected and regression analyses were performed to predict cognitive impairments. We use MMSE and MoCA assessment as prognostic indices for cognitive impairments of patient's with stroke. OBJECTIVES: Our aim was to examine the effectiveness of the MMSE and MoCA in screening cognitive impairments. MAIN RESULTS: There were significant difference among the two groups in the prevalence of diabetes mellitus (p < 0.05) and intracranial atherosclerosis (p < 0.05). A linear regression determined that the age, diabetes, intracranial atherosclerosis predicted the cognitive impairments. The ROC results for MoCA with an AUC of 0.882 and the corresponding results for MMSE show a similar AUC of 0.839. CONCLUSION: Neuropsychological performance of stroke patients was influenced by biological and demographic variables: age, diabetes and intracranial atherosclerosis. The MoCA and MMSE are both reliable assessments for the diagnosis of cognitive impairment after stroke.

Endoplasmic reticulum stress associated responses in cancer.

The endoplasmic reticulum (ER) is responsible for many housekeeping functions within the cell and is an important site for pathways that regulates its state of homeostasis. When cellular states perturb ER functions, a phenomenon termed "ER stress" activates a number of pathways to counteract the associated damages; these pathways are together called the unfolded protein response (UPR). The UPR has a dualistic function; it exists to alleviate damage associated with ER stress, however, if this is not possible, then it signals for cell death through apoptosis. Cancer cells are shown to be very resilient under extreme environmental stress and an increasing number of studies have indicated that this may be largely due to an altered state of the UPR. The role of ER stress and the UPR in cancer is still not clear, however many components are involved and may prove to be promising targets in future anti-cancer therapy. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

High-level secretion of native recombinant human calreticulin in yeast.

BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

Treatment with recombinant tissue plasminogen activator alters the microRNA expression profiles in mouse brain after acute ischemic stroke.

Thrombolysis with recombinant tissue plasminogen activator (rtPA) is the only FDA approved treatment for the brain ischemic stroke. MicroRNAs, non-coding RNA sequences that regulate gene expression, might play important roles in regulating the rtPA thrombolysis process. The present study investigated changes in the microRNA profiles in a middle cerebral artery occlusion (MCAo) mouse model after rtPA treatment. Using microarrays containing 1179 microRNAs, we compared microRNAs expression profiles of brain tissues from C57 BL/6J mice subjected to focal cerebral ischemia with and without rtPA thrombolysis. We found that rtPA treatment upregulated 31 microRNAs and downregulated 11 microRNAs. Expression alterations of selected microRNAs mmu-miR-125a-3p, -208a-5p, -709, -721 were confirmed by real-time PCR. Differentially expressed microRNAs were analyzed using Targetscan v6.2 and David v6.7. 2200 predicted genes were subjected to GO analysis and pathway analysis, which identified mediators involved in multiple signaling pathways during proliferation. These data demonstrated that rtPA treatment alters microRNAs expression after stroke, and provided new insight into understanding the biological process of rtPA thrombolysis.

Efficacy of Intravitreal Injections Anti-Vascular Endothelial Growth Factor Treatment for Radiation Retinopathy: A Systematic Review and Meta-analysis.

PURPOSE: This study aims to appraise the therapeutic effectiveness of intravitreal injections anti-vascular endothelial growth factor (anti-VEGF) vs alternative therapies in managing radiation retinopathy (RR). DESIGN: Systematic review and meta-analysis. METHODS: We obtained comprehensive data retrieval using PubMed, Embase, Web of Science, Scopus, and the Cochrane Library from their inception until December 15, 2023. This review included randomized controlled trials (RCTs) and nonrandomized studies (NRSs) reporting on best-corrected visual acuity (BCVA) among RR patients treated with intravitreal anti-VEGF. Study selection and data extraction were meticulously performed by 2 independent reviewers. The Cochrane Risk of Bias Tool 2.0 (RoB 2.0) and Risk of Bias in Nonrandomized Studies of Interventions (ROBINS-I) scales were utilized for bias risk assessment. Quantification of heterogeneity was executed using Q, H, and I2 statistics. The primary endpoint was the BCVA at the final observation point of each study. Secondary endpoints included central retinal thickness (CRT), foveal avascular zone (FAZ) area, and capillary density (CD) at the level of superficial capillary plexus. Subgroup analyses were undertaken to explore potential heterogeneity sources possibly due to treatment duration and study design. Sensitivity analyses were conducted to ascertain result stability. RESULTS: This analysis incorporated 7 studies (including 3 RCTs) encompassing 922 patients afflicted with RR. Relative to other treatment modalities, intravitreal anti-VEGF therapy was associated with a statistically significant mean decrease in BCVA of -0.34 logMAR (95% CI, -0.39 to -0.30 logMAR; I2 = 87.70%; P < .001), and a substantial reduction in CRT of -34.65 µm (95% CI, -50.70 to -18.60 µm; I2 = 30.40%; P < .001). Additionally, a reduction in the FAZ area by -0.69 mm² (95% CI, -0.91 to -0.46 mm², I2 = 0%; P < .001) was observed. A positive tendency was noted in CD at the superficial capillary plexus between anti-VEGF and other therapeutic interventions. CONCLUSIONS: Intravitreal anti-VEGF injections, in comparison to other treatments, demonstrate superior efficacy in enhancing BCVA and reducing CRT, thereby underscoring the potential of anti-VEGF in ameliorating radiation retinopathy outcomes. However, the conclusions are constrained by the incorporation of data from some NRSs and the small sample sizes.

Modulation of epithelial-mesenchymal transition by gemcitabine: Targeting ionizing radiation-induced cellular senescence in lung cancer cell.

Ionizing radiation, a standard therapeutic approach for lung cancer, often leads to cellular senescence and the induction of epithelial-mesenchymal transition (EMT), posing significant challenges in treatment efficacy and cancer progression. Overcoming these obstacles is crucial for enhancing therapeutic outcomes in lung cancer management. This study investigates the effects of ionizing radiation and gemcitabine on lung cancer cells, with a focus on induced senescence, EMT, and apoptosis. Human-derived A549, PC-9, and mouse-derived Lewis lung carcinoma cells exposed to 10 Gy X-ray irradiation exhibited senescence, as indicated by morphological changes, ß-galactosidase staining, and cell cycle arrest through the p53-p21 pathway. Ionizing radiation also promoted EMT via TGFß/SMAD signaling, evidenced by increased TGFß1 levels, altered EMT marker expressions, and enhanced cell migration. Gemcitabine, a first-line lung cancer treatment, was shown to enhance apoptosis in senescent cells caused by radiation. It inhibited cell proliferation, induced mitochondrial damage, and triggered caspase-mediated apoptosis, thus mitigating EMT in vitro. Furthermore, in vivo studies using a lung cancer mouse model revealed that gemcitabine, combined with radiation, significantly reduced tumor volume and weight, extended survival, and suppressed malignancy indices in irradiated tumors. Collectively, these findings demonstrate that gemcitabine enhances the therapeutic efficacy against radiation-resistant lung cancer cells, both by inducing apoptosis in senescent cells and inhibiting EMT, offering potential improvements in lung cancer treatment strategies.

Experimental and Computational Analysis of Newly Identified Pathogenic Mutations in the Creatine Transporter SLC6A8.

Creatine is an essential metabolite for the storage and rapid supply of energy in muscle and nerve cells. In humans, impaired metabolism, transport, and distribution of creatine throughout tissues can cause varying forms of mental disability, also known as creatine deficiency syndrome (CDS). So far, 80 mutations in the creatine transporter (SLC6A8) have been associated to CDS. To better understand the effect of human genetic variants on the physiology of SLC6A8 and their possible impact on CDS, we studied 30 missense variants including 15 variants of unknown significance, two of which are reported here for the first time. We expressed these variants in HEK293 cells and explored their subcellular localization and transport activity. We also applied computational methods to predict variant effect and estimate site-specific changes in thermodynamic stability. To explore variants that might have a differential effect on the transporter's conformers along the transport cycle, we constructed homology models of the inward facing, and outward facing conformations. In addition, we used mass-spectrometry to study proteins that interact with wild type SLC6A8 and five selected variants in HEK293 cells. In silico models of the protein complexes revealed how two variants impact the interaction interface of SLC6A8 with other proteins and how pathogenic variants lead to an enrichment of ER protein partners. Overall, our integrated analysis disambiguates the pathogenicity of 15 variants of unknown significance revealing diverse mechanisms of pathogenicity, including two previously unreported variants obtained from patients suffering from the creatine deficiency syndrome.

Ionizing radiation-induced mitophagy promotes ferroptosis by increasing intracellular free fatty acids.

Ferroptosis is a type of cell death characterized by the accumulation of intracellular iron and an increase in hazardous lipid peroxides. Ferroptosis and autophagy are closely related. Ionizing radiation is a frequently used cancer therapy to kill malignancies. We found that ionizing radiation induces both ferroptosis and autophagy and that there is a form of mutualism between the two processes. Ionizing radiation also causes lipid droplets to form in proximity to damaged mitochondria, which, through the action of mitophagy, results in the degradation of the peridroplet mitochondria by lysosomes and the consequent release of free fatty acids and a significant increase in lipid peroxidation, thus promoting ferroptosis. Ionizing radiation has a stronger, fatal effect on cells with a high level of mitophagy, and this observation suggests a novel strategy for tumor treatment.

Ionizing radiation triggers mitophagy to enhance DNA damage in cancer cells.

Radiotherapy is an important cancer treatment strategy that causes DNA damage in tumor cells either directly or indirectly. Autophagy is a physiological process linked to DNA damage. Mitophagy is a form of autophagy, which specifically targets and eliminates impaired mitochondria, thereby upholding cellular homeostasis. However, the connection between DNA damage and mitophagy has yet to be fully elucidated. We found that mitophagy, as an upstream signal, increases ionizing radiation-induced DNA damage by downregulating or overexpressing key mitophagy proteins Parkin and BNIP3. Enhancing the basal level of mitophagy in conjunction with X-ray irradiation can potentially diminish cell cycle arrest at the G2/M phase, substantially elevate the accumulation of γ-H2AX, 53BP1, and PARP1 foci within the nucleus, augment DNA damage, and facilitate the demise of tumor cells. Consequently, this approach prolongs the survival of melanoma-bearing mice. The findings of this study are anticipated to offer a therapeutic approach for enhancing the therapeutic effectiveness of radiotherapy.

Calreticulin and the Heart.

Calreticulin is an endoplasmic Ca2+ binding protein and molecular chaperone. As a cardiac embryonic gene, calreticulin is essential for heart development. The protein supports Ca2+-dependent signaling events that are critical to cardiomyocyte differentiation and cardiogenesis. The increased expression of calreticulin and endoplasmic reticulum/sarcoplasmic reticulum Ca2+ capacity produces cardiomyocytes with enhanced efficiency, and detrimental mechanical stretching of cardiac fibroblasts, leading to cardiac pathology. Deletion of the calreticulin gene in adult cardiomyocytes results in left ventricle dilation, an impaired electrocardiogram, and heart failure. These observations indicate that a well-adjusted endoplasmic reticulum and calreticulin-dependent Ca2+ pool in cardiomyocytes are critical for the maintenance of proper cardiac function.

Expression of beta-amyloid induced age-dependent presynaptic and axonal changes in Drosophila.

Alzheimer's disease (AD) is attributable to synapse dysfunction and loss, but the nature and progression of the presynaptic structural and functional changes in AD are essentially unknown. We expressed wild-type or arctic form of beta amyloid(1-42) (Abeta) in a small group of neurons in the adult fly and performed extensive time course analysis of the function and structure of both axon and presynaptic terminals at the identified single-neuron level. Abeta accumulated intracellularly and induced a range of age-dependent changes, including depletion of presynaptic mitochondria, slowdown of bi-directional transports of axonal mitochondria, decreased synaptic vesicles, increased large vacuoles, and elevated synaptic fatigue. These structural and functional synaptic changes correlated with age-dependent deficit in motor behavior. All these alterations were accelerated in flies expressing the arctic form of Abeta. The depletion of presynaptic mitochondria was the earliest detected phenotype and was not caused by the change in axonal transport of mitochondria. Moreover, axonal mitochondria exhibited a dramatic reduction in number but a significant increase in size in aged Abeta-expressing flies, indicating a global depletion of mitochondria in the neuron and an impairment of mitochondria fission. These results suggest that Abeta accumulation depletes presynaptic and axonal mitochondria, leading to other presynaptic deficits.