Neurocalcin Delta Research Articles

The role of DNA methylation in ovarian cancer chemoresistance: A narrative review.

Ovarian cancer (OC) is the most lethal gynecological cancer. In 2018, it was responsible for over 180,000 deaths worldwide. The high mortality rate is the culmination of a lack of early diagnosis and high rates of chemotherapy resistance, which is synonymous with disease recurrence. Over the last two decades, an increasingly significant role of epigenetic mechanisms, in particular DNA methylation, has emerged. This review will discuss several of the most significant genes whose hypo/hypermethylation profiles are associated with chemoresistance. Aside from functionally elucidating and evaluating these epimutations, this review will discuss recent trials of DNA methyltransferase inhibitors (DNMTi). Finally, we will propose future directions that could enhance the feasibility of utilizing these candidate epimutations as clinical biomarkers. To perform this review, a comprehensive literature search based on our keywords was conducted across the online databases PubMed and Google Scholar for identifying relevant studies published up until August 2022. Epimutations affecting MLH1, MSH2, and Ras-association domain family 1 isoform A (DNA damage repair and apoptosis);ATP-binding cassette subfamily B member 1 and methylation-controlled J (drug export); secreted frizzled-related proteins (Wnt/β-catenin signaling), neurocalcin delta (calcium and G protein-coupled receptor signaling), and zinc finger protein 671all have potential as biomarkers for chemoresistance. However, specific uncertainties relating to these epimutations include histotype-specific differences, intrinsic versus acquired chemoresistance, and the interplay with complete surgical debulking. DNMTi for chemoresistant OC patients has shown some promise; however, issues surrounding their efficacy and dose-limiting toxicities remain; a personalized approach is required to maximize their effectiveness. Establishing a panel of aberrantly methylated chemoresistance-related genes to predict chemoresponsiveness and patients' suitability to DNMTi could significantly reduce OC recurrence, while improving DNMTi therapy viability. To achieve this, a large-scale prospective genome-wide DNA methylation profile study that spans different histotypes, includes paired samples (before and after chemotherapy), and integrates transcriptomic and methylomic analysis, is warranted.

Long-Term SMN- and Ncald-ASO Combinatorial Therapy in SMA Mice and NCALD-ASO Treatment in hiPSC-Derived Motor Neurons Show Protective Effects

For SMA patients with only two SMN2 copies, available therapies might be insufficient to counteract lifelong motor neuron (MN) dysfunction. Therefore, additional SMN-independent compounds, supporting SMN-dependent therapies, might be beneficial. Neurocalcin delta (NCALD) reduction, an SMA protective genetic modifier, ameliorates SMA across species. In a low-dose SMN-ASO-treated severe SMA mouse model, presymptomatic intracerebroventricular (i.c.v.) injection of Ncald-ASO at postnatal day 2 (PND2) significantly ameliorates histological and electrophysiological SMA hallmarks at PND21. However, contrary to SMN-ASOs, Ncald-ASOs show a shorter duration of action limiting a long-term benefit. Here, we investigated the longer-term effect of Ncald-ASOs by additional i.c.v. bolus injection at PND28. Two weeks after injection of 500 µg Ncald-ASO in wild-type mice, NCALD was significantly reduced in the brain and spinal cord and well tolerated. Next, we performed a double-blinded preclinical study combining low-dose SMN-ASO (PND1) with 2× i.c.v. Ncald-ASO or CTRL-ASO (100 µg at PND2, 500 µg at PND28). Ncald-ASO re-injection significantly ameliorated electrophysiological defects and NMJ denervation at 2 months. Moreover, we developed and identified a non-toxic and highly efficient human NCALD-ASO that significantly reduced NCALD in hiPSC-derived MNs. This improved both neuronal activity and growth cone maturation of SMA MNs, emphasizing the additional protective effect of NCALD-ASO treatment.

Genome-Wide Association Study of Egg-Laying Traits and Egg Quality in LingKun Chickens.

Egg production is the most important trait of laying hens. To identify molecular markers and candidate genes associated with egg production and quality, such as body weight at first oviposition (BWF), the number of eggs produced in 500 days (EN500), egg weight (EW), egg shell thickness (EST), egg shell strength (ESS), and Haugh unit (HU), a genome-wide analysis was performed in 266 LingKun Chickens. The results showed that thirty-seven single nucleotide polymorphisms (SNPs) were associated with all traits (p < 9.47 × 10−8, Bonferroni correction). These SNPs were located in close proximity to or within the sequence of the thirteen candidate genes, such as Galanin And GMAP Prepropeptide (GAL), Centromere Protein (CENPF), Glypican 2 (GPC2), Phosphatidylethanolamine N-Methyltransferase (PEMT), Transcription Factor AP-2 Delta (TFAP2D), and Carboxypeptidase Q (CPQ) gene related to egg-laying and Solute Carrier Family 5 Member 7 (SLC5A7), Neurocalcin Delta (NCALD), Proteasome 20S Subunit Beta 2 (PSMB2), Slit Guidance Ligand 3 (SLIT3), and Tubulin Tyrosine Ligase Like 7 (TTLL7) genes related to egg quality. Interestingly, one of the genes involved in bone formation (SLIT3) was identified as a candidate gene for ESS. Our candidate genes and SNPs associated with egg-laying traits were significant for molecular breeding of egg-laying traits and egg quality in LingKun chickens.

Helicid Reverses the Effect of Overexpressing NCALD, Which Blocks the sGC/cGMP/PKG Signaling Pathway in the CUMS-Induced Rat Model.

Background Increasing evidence has shown that apoptosis in the hippocampus is closely related to depressive-like behavior. We previously reported that helicid had good antidepressant activities, which manifested as the alleviation of depression-like behaviors and the reversal of the high expression of neurocalcin delta (NCALD) in chronic unpredictable mild stress (CUMS) rats. The aim of this study was, therefore, to characterize the antidepressant-like effects and underlying mechanism of helicid on CUMS rats by silencing NCALD and using rescue experiments. Methods We developed the CUMS rat model using CUMS stimulation from week 0 to week 6. The rats were treated with helicid, or NCALD silenced, then we overexpressed NCALD using adeno-associated virus. We also measured the protein levels of sGCα1, sGCβ1, PKG1/2, and cleaved caspase-3 in hippocampal tissues using western blotting and measured cGMP using an ELISA. Results Treating CUMS rats by silencing NCALD or by the administration of helicid improved the depressive-like behavior. The levels of proteins, including sGC, PKG, cleaved caspase-3, and cGMP, in hippocampus all decreased. NCALD overexpression reversed these decreases and reversed the alleviation of depression-like behaviors in CUMS rats. Limitation. We only detected the antidepressant effects of helicid in the hippocampus; therefore, other parts of brain should also be studied. Conclusions Inhibition of NCALD, as well as helicid administration, alleviated antidepressant-like behavior by regulating the expressions of apoptotic cytokines and the sGC/cGMP/PKG signaling pathway. Overexpressing NCALD reversed the amelioration effects of silenced NCALD and helicid administration.

Antidepressant-like effects of helicid on a chronic unpredictable mild stress-induced depression rat model: Inhibiting the IKK/IκBα/NF-κB pathway through NCALD to reduce inflammation

We previously reported that helicid, an active plant monomer of Helicid nilgirica Bedd, had good antidepressant pharmacological activities. However, the potential mechanism of action remains unknown. Current investigation showed the antidepressant-like effects of helicid and its effects on the neurocalcin delta (NCALD) gene, and its mechanism of action through a depression model in rats exposed to chronic unpredictable mild stress (CUMS). We evaluated depression symptoms using the sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST). By silencing NCALD and using rescue experiments, the IL-6, iNOS, IL-1β, COX-2, and TNF-α levels in the hippocampus or peripheral blood were determined using western blotting and ELISAs. The expression of IKKβ, p-IкBα, p-IKKβ, NF-кB p65, and IкBα were tested using western blots of the cytoplasmic or nuclear samples. Helicid and silencing NCALD relieved the CUMS-irritated depressive-like actions of rats, which were shown by increased consumption of sucrose, numbers of rearings, total running distance, zone crossings, and reduced immobility times. Helicid or silencing NCALD reversed the CUMS-induced high levels of IL-1β, COX-2, IL-6, TNF-α, and iNOS in the hippocampus or peripheral blood. Helicid or silencing NCALD also reduced the expressions of p-IκBα and p-IKKβ in the cytoplasm and the expression of nuclear NF-κB p 65 in hippocampus, and simultaneously elevated cytoplasmic expressions of IκBα, IKKβ, and NF-κB p65 in the hippocampus. Notably, after NCALD overexpression, the biochemical indices of rat helicid administration were reversed. In conclusion, the antidepressant action of helicid was mediated through NCALD in rats of CUMS by repressing hippocampal neuro-inflammation and abating the activation of the IKK/IκBα/NF-κB pathway.

Calcium-Dependent Translocation of S100B Is Facilitated by Neurocalcin Delta.

S100B is a calcium-binding protein that governs calcium-mediated responses in a variety of cells—especially neuronal and glial cells. It is also extensively investigated as a potential biomarker for several disease conditions, especially neurodegenerative ones. In order to establish S100B as a viable pharmaceutical target, it is critical to understand its mechanistic role in signaling pathways and its interacting partners. In this report, we provide evidence to support a calcium-regulated interaction between S100B and the neuronal calcium sensor protein, neurocalcin delta both in vitro and in living cells. Membrane overlay assays were used to test the interaction between purified proteins in vitro and bimolecular fluorescence complementation assays, for interactions in living cells. Added calcium is essential for interaction in vitro; however, in living cells, calcium elevation causes translocation of the NCALD-S100B complex to the membrane-rich, perinuclear trans-Golgi network in COS7 cells, suggesting that the response is independent of specialized structures/molecules found in neuronal/glial cells. Similar results are also observed with hippocalcin, a closely related paralog; however, the interaction appears less robust in vitro. The N-terminal region of NCALD and HPCA appear to be critical for interaction with S100B based on in vitro experiments. The possible physiological significance of this interaction is discussed.

JAK/STAT signaling pathway-mediated microRNA-181b promoted blood-brain barrier impairment by targeting sphingosine-1-phosphate receptor 1 in septic rats.

BackgroundBlood-brain barrier (BBB) impairment plays a significant role in the pathogenesis of sepsis-associated encephalopathy (SAE). However, the molecular mechanisms are poorly understood. In the present study, we aimed to investigate the regulatory relationship between the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway, microRNA (miR)-181b and its target genes in sepsis in vivo and in vitro.MethodsFour rat models (sham, sepsis, sepsis plus STAT3 inhibitor (Stattic), and sepsis plus miR-181b inhibitor [sepsis + anta-miR-181b]) were established. For the in vitro experiments, rat brain microvascular endothelial cells (rBMECs) and rat brain astrocytes (rAstrocytes) were cultured with 10% serum harvested from sham, sepsis, and sepsis + anta-miR-181b rats. Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-QPCR) analysis was carried out to detect the binding and enrichment of the JAK/STAT3 signal core transcription complex in the miR-181b promoter region. Dual-luciferase reporter gene assay was conducted to test miR-181b and its target genes. The cell adhesion rate of rBMECs was also measured.ResultsDuring our investigations, the expression levels of miR-181b, p-JAK2, p-STAT3, and C/EBPβ were found to be significantly increased in the septic rats compared with the sham rats. STAT3 inhibitor halted BBB damage by downregulating the expression of miR-181b. In addition, miR-181b targeted sphingosine-1-phosphate receptor 1 (S1PR1) and neurocalcin delta (NCALD). The up-regulated miR-181b significantly decreased the cell adhesion rate of rBMECs. The administration of miR-181b inhibitor reduced damage to the BBB through increasing the expression of S1PR1 and NCALD, which again proved that miR-181b negatively regulates SIPR1 and NCALD to induce BBB damage.ConclusionsOur study demonstrated that JAK2/STAT3 signaling pathway induced expression of miR-181b, which promoted BBB impairment in rats with sepsis by downregulating S1PR1 and decreasing BBB cell adhesion. These findings strongly suggest JAK2/STAT3/miR-181b axis as therapeutic target in protecting against sepsis-induced BBB damage.

Low expression of NCALD is associated with chemotherapy resistance and poor prognosis in epithelial ovarian cancer

BackgroundLow expression of NCALD(neurocalcin delta) in peripheral blood of ovarian cancer patients predicts poor prognosis. However, the molecular mechanism of NCALD in ovarian cancer and its relationship with chemotherapy outcomes is unclear. The aim of this study was to investigate the potential signaling pathways of NCALD and to evaluate its ability to predict chemotherapy outcomes and prognosis.MethodsHigh-throughput RNA sequencing data were downloaded from TCGA. GSEA explored the potential signaling pathways of NCALD. The expression of NCALD in chemotherapy sensitive and chemotherapy resistant ovarian cancer patients was detected by TCGA data and clinical samples. ROC analysis confirmed the ability of NCALD to predict chemotherapy outcomes. The association between NCALD expression and prognosis in ovarian cancer patients was assessed using Kaplan-Meier plotter.ResultsIn patients with NCALD overexpression, genes expression related to ERK1 / 2 signaling pathway, NF-kappaB signaling pathway, TGF-β signaling pathway and immune response pathway was increased, especially ERK1 / 2 signaling pathway. The expression of NCALD in chemoresistant patients was significantly lower than chemosensitive patients. In TCGA data and immunohistochemical samples, the AUC of NCALD expression predicting chemotherapy outcome was 0.59 and 0.64, respectively. In clinical samples, low expression of NCALD was associated with poor OS and PFS.ConclusionsNCALD may activate the ERK1 / 2 signaling pathway in ovarian cancer. As a new biomarker of chemotherapy sensitivity, NCALD was significantly down-regulated in chemotherapy resistance ovarian cancer patients. Low expression of NCALD in ovarian cancer is associated with poor OS and PFS. In the future, further research will be needed on the potential mechanism and clinical application value of NCALD in ovarian cancer.

NCALD affects drug resistance and prognosis by acting as a ceRNA of CX3CL1 in ovarian cancer.

Drug resistance, an impenetrable barrier in the treatment of ovarian cancer (OC), is often associated with poor outcomes. Hence, it is urgent to discover new factors controlling drug resistance and survival. The association between neurocalcin delta (NCALD) and cancer drug resistance is poorly understood. Here, we reveal that NCALD messenger RNA expression, probably regulated by DNA methylation and microRNAs, was significantly downregulated in at least three independent microarrays covering 633 ovarian carcinomas and 16 normal controls, which includes the Cancer Genome Atlas (TCGA) ovarian cohort. In the sub-groups of the TCGA cohort, NCALD was suppressed in 90 platinum-resistant tissues vs in 197 sensitive tissues. It is consistent with the quantitative reverse transcription polymerase chain reaction results revealing gene downregulation in carboplatin-resistant SKOV3 and HeyA8 OC cells as compared with that in controls. Low expression of NCALD predicted poor overall survival (OS) in sub-groups of 1656 patients, progression-free survival (PFS) in 1435 patients, and post-progression survival (PPS) in 782 patients according to Kaplan-Meier plotter covering 1815 OC patients. Comprehensive bioinformatic analyses strongly implicated NCALD in the regulation of drug resistance, probably via competing for endogenous RNA (ceRNA) interactions with CX3CL1 and tumor immune-microenvironment. NCALD acted as a ceRNA for CX3CL1 in 21 different cancers includes OC according to Starbase. These two genes negatively correlated with tumor purity and positively correlated with infiltration levels of neutrophils and dendritic cells in OC. The combined low expression of NCALD and CX3CL1 showed better prognosis potential for OS, PFS, and PPS in the 1815 OC patients than any of the individually tested genes. In summary, NCALD acts as a ceRNA for CX3CL1, and its downregulation may affect drug resistance and prognosis in OC. Thus, NCALD could be a new therapeutic target for anticancer therapy and a new biomarker for survival prediction in OC.

NCALD Antisense Oligonucleotide Therapy in Addition to Nusinersen further Ameliorates Spinal Muscular Atrophy in Mice

Spinal muscular atrophy (SMA) is a neuromuscular disease causing the most frequent genetic childhood lethality. Recently, nusinersen, an antisense oligonucleotide (ASO) that corrects SMN2 splicing and thereby increases full-length SMN protein, has been approved by the FDA and EMA for SMA therapy. However, the administration of nusinersen in severe and/or post-symptomatic SMA-affected individuals is insufficient to counteract the disease. Therefore, additional SMN-independent therapies are needed to support the function of motoneurons and neuromuscular junctions. We recently identified asymptomatic SMN1-deleted individuals who were protected against SMA by reduced expression of neurocalcin delta (NCALD). NCALD reduction is proven to be a protective modifier of SMA across species, including worm, zebrafish, and mice. Here, we identified Ncald-ASO3-out of 450 developed Ncald ASOs-as the most efficient and non-toxic ASO for the CNS, by applying a stepwise screening strategy in cortical neurons and adult and neonatal mice. In a randomized-blinded preclinical study, a single subcutaneous low-dose SMN-ASO and a single intracerebroventricular Ncald-ASO3 or control-ASO injection were presymptomatically administered in a severe SMA mouse model. NCALD reduction of >70% persisted for about 1month. While low-dose SMN-ASO rescues multiorgan impairment, additional NCALD reduction significantly ameliorated SMA pathology including electrophysiological and histological properties of neuromuscular junctions and muscle at P21 and motoric deficits at 3months. The present study shows the additional benefit of a combinatorial SMN-dependent and SMN-independent ASO-based therapy for SMA. This work illustrates how a modifying gene, identified in some asymptomatic individuals, helps to develop a therapy for all SMA-affected individuals.

Clinicopathological and prognostic significance of NCALD protein expression in lung adenocarcinoma.

BackgroundAlthough there are great improvements in diagnostics and treatment of non-small cell lung cancer (NSCLC), it remains the leading cause of cancer-related death globally. NSCLC can be further categorized into two common subtypes, lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). The aims of this study were to evaluate the expression of neurocalcin delta (NCALD) in LUAD patients and to evaluate the relationship between NCALD expression and clinicopathological feature as well as overall survival (OS).MethodsThe expression level of NCALD protein was assessed by immunohistochemical analysis of tissue microarrays (TMAs) which contained 90 LUAD tissues and paired adjacent normal tissues. The relationship between NCALD expression level and clinicopathological characteristics as well as OS were investigated.ResultsThe results showed that the expression level of NCALD protein in LUAD tissues was significantly down-regulated (P<0.01) and that the NCALD level was correlated with the tumor size (P<0.001), lymph node metastasis (P=0.03) and TNM stage (P=0.015). Both Kaplan-Meier survival analysis and Cox regression univariate analysis identified that NCALD expression level, lymph node metastasis, and clinical TNM stages were prognostic factors for patients’ survival (P<0.05).ConclusionsOverexpression of NCALD is a significant marker for a good prognosis in patients with LUAD.

Clinicopathological and prognostic significance of NCALD protein expression in lung adenocarcinoma

Background: Although there are great improvements in diagnostics and treatment of non-small cell lung cancer (NSCLC), it remains the leading cause of cancer-related death globally. NSCLC can be further categorized into two common subtypes, lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD). The aims of this study were to evaluate the expression of neurocalcin delta (NCALD) in LUAD patients and to evaluate the relationship between NCALD expression and clinicopathological feature as well as overall survival (OS). Methods: The expression level of NCALD protein was assessed by immunohistochemical analysis of tissue microarrays (TMAs) which contained 90 LUAD tissues and paired adjacent normal tissues. The relationship between NCALD expression level and clinicopathological characteristics as well as OS were investigated. Results: The results showed that the expression level of NCALD protein in LUAD tissues was significantly down-regulated (P Conclusions: Overexpression of NCALD is a significant marker for a good prognosis in patients with LUAD.

Neurocalcin Delta Knockout Impairs Adult Neurogenesis Whereas Half Reduction Is Not Pathological.

Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor and its reduction acts protective against spinal muscular atrophy (SMA). However, the physiological function of NCALD and implications of NCALD reduction are still elusive. Here, we analyzed the ubiquitous Ncald knockout in homozygous (NcaldKO/KO) and heterozygous (NcaldKO/WT) mice to unravel the physiological role of NCALD in the brain and to study whether 50% NCALD reduction is a safe option for SMA therapy. We found that NcaldKO/KO but not NcaldKO/WT mice exhibit significant changes in the hippocampal morphology, likely due to impaired generation and migration of newborn neurons in the dentate gyrus (DG). To understand the mechanism behind, we studied the NCALD interactome and identified mitogen-activated protein kinase kinase kinase 10 (MAP3K10) as a novel NCALD interacting partner. MAP3K10 is an upstream activating kinase of c-Jun N-terminal kinase (JNK), which regulates adult neurogenesis. Strikingly, the JNK activation was significantly upregulated in the NcaldKO/KO brains. Contrary, neither adult neurogenesis nor JNK activation were altered by heterozygous Ncald deletion. Taken together, our study identifies a novel link between NCALD and adult neurogenesis in the hippocampus, possibly via a MAP3K10-JNK pathway and emphasizes the safety of using NCALD reduction as a therapeutic option for SMA.

Characterization of neurocalcin delta membrane binding by biophysical methods.

Neurocalcin delta (NCALD) is a member of the neuronal calcium sensors protein family. In the retina, NCALD is expressed by ganglion and amacrine cells. NCALD is composed of 4 EF-hand motifs but only 3 of them may bind calcium. The binding of calcium induces a conformational change of the protein which leads to the extrusion of its N-terminal myristoyl group as well as some hydrophilic residues. This mechanism, named calcium-myristoyl switch, is presumably involved in its membrane binding. The parameters responsible for the interaction of NCALD with membranes are only partially known. The purpose of this study was thus to gather more information on the membrane binding behavior of NCALD using lipid monolayers, including the influence of the lipid composition, the calcium and the myristoyl group. NCALD was injected underneath different lipid monolayers and this model membrane allowed the determination of the binding parameters as maximum insertion pressure (MIP) and synergy. The values of MIP are larger when monolayers were composed of a saturated phospholipid with phosphoethanolamine polar head. This trend is confirmed by polarization modulation infrared reflection absorption spectroscopy measurements. Moreover, the observations by fluorescence microscopy show that NCALD preferentially interacts with phospholipids which are in the liquid-condensed physical state, as found in membrane microdomains. This observation could explain the changes of NCALD expression level in the brains of patients suffering from Alzheimer's disease because of the alteration of lipid composition in microdomains structures.

Investigating the possible causal association of coffee consumption with osteoarthritis risk using a Mendelian randomization analysis.

This study examined whether coffee consumption is causally associated with osteoarthritis. A two-sample Mendelian randomization (MR) analysis using inverse-variance weighted (IVW) and weighted median estimates, and the MR-Egger regression method were performed. The publicly available summary statistical datasets of coffee consumption genome-wide association studies (GWASs) meta-analyses on coffee intake from eight Caucasian cohorts (n = 18,176), GWAS meta-analyses of predominately regular-type coffee consumers of European ancestry (n = up to 91,462), and a GWAS in 7410 patients with osteoarthritis in the arcOGEN study with 11,009 controls of European ancestry were evaluated. Four single-nucleotide polymorphisms (SNPs) from GWASs of coffee consumption as instrumental variables (IVs) to improve inference were selected. These SNPs were located at neurocalcin delta (NCALD) (rs16868941), cytochrome p450 oxidoreductase (POR) (rs17685), cytochrome p450 family 1 subfamily A member 1 (CYP1A1) (rs2470893), and neuronal cell adhesion molecule (NRCAM) (rs382140). The IVW method (beta = 0.381, SE = 0.170, p = 0.025) and the weighted median approach (beta = 0.419, SE = 0.206, p = 0.047) showed evidence to support a causal association between coffee consumption and osteoarthritis. MR-Egger regression revealed that directional pleiotropy was unlikely to be biasing the result (intercept = 0.064; p = 0.549), but showed no causal association between coffee consumption and osteoarthritis (beta = - 0.518, SE = 1.270, p = 0.723). Cochran's Q test and the funnel plot indicated no evidence of heterogeneity between IV estimates based on the individual variants. The results of MR analysis support the observation that coffee consumption is causally associated with an increased risk of osteoarthritis.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

Neurocalcin-delta: a potential memory-related factor in hippocampus of obese rats induced by high-fat diet.

Aberrant protein expression within the hippocampus has recently been implicated in the pathogenesis of obesity-induced memory impairment. The objective of the current study was to search for specific memory-related factors in the hippocampus in obese rats. Sprague-Dawley (SD) rats were fed either a high-fat (HF) diet or normal-fat (NF) diet for 10 weeks to obtain the control (CON), diet-induced obese rats (DIO) and diet-resistant (DR) rats. D-galactose was injected subcutaneously for 10 weeks to establish model (MOD) rats with learning and memory impairment. After the hippocampus of the rats sampling, the proteome analysis was conducted using two-dimensional get electrophoresis (2-DE) combined with peptide mass fingerprinting (PMF). We found 15 differential proteins that expressed in the hippocampus in rats induced by HF diet from the 2-DE map. In addition, Neurocalcin-delta (NCALD) was nearly down-regulated in the DR rats compared with CON rats and MOD rats, which was further confirmed by Western blot, real-time PCR and ELISA results. Our data demonstrates that the differential memory-related proteins were a reflection of the HF diet, but not potential factors in obesity proneness or obesity resistance. Furthermore, NCALD is proved to be a potential hippocampus-memory related factor related to obesity.

Neurocalcin Delta Suppression Protects against Spinal Muscular Atrophy in Humans and across Species by Restoring Impaired Endocytosis

Homozygous SMN1 loss causes spinal muscular atrophy (SMA), the most common lethal genetic childhood motor neuron disease. SMN1 encodes SMN, a ubiquitous housekeeping protein, which makes the primarily motor neuron-specific phenotype rather unexpected. SMA-affected individuals harbor low SMN expression from one to six SMN2 copies, which is insufficient to functionally compensate for SMN1 loss. However, rarely individuals with homozygous absence of SMN1 and only three to four SMN2 copies are fully asymptomatic, suggesting protection through genetic modifier(s). Previously, we identified plastin 3 (PLS3) overexpression as an SMA protective modifier in humans and showed that SMN deficit impairs endocytosis, which is rescued by elevated PLS3 levels. Here, we identify reduction of the neuronal calcium sensor Neurocalcin delta (NCALD) as a protective SMA modifier in five asymptomatic SMN1-deleted individuals carrying only four SMN2 copies. We demonstrate that NCALD is a Ca2+-dependent negative regulator of endocytosis, as NCALD knockdown improves endocytosis in SMA models and ameliorates pharmacologically induced endocytosis defects in zebrafish. Importantly, NCALD knockdown effectively ameliorates SMA-associated pathological defects across species, including worm, zebrafish, and mouse. In conclusion, our study identifies a previously unknown protective SMA modifier in humans, demonstrates modifier impact in three different SMA animal models, and suggests a potential combinatorial therapeutic strategy to efficiently treat SMA. Since both protective modifiers restore endocytosis, our results confirm that endocytosis is a major cellular mechanism perturbed in SMA and emphasize the power of protective modifiers for understanding disease mechanism and developing therapies.

Datasets depicting mobility retardation of NCS proteins observed upon incubation with calcium, but not with magnesium, barium or strontium.

In this data article we show the specificity of the Ca2+-induced mobility shift in three proteins that belong to the neuronal calcium sensor (NCS) protein family: Hippocalcin, GCAP1 and GCAP2. These proteins did not display a shift in mobility in native gels when incubated with divalent cations other than Ca2+ – such as Mg2+, Ba2+, and Sr2+, even at 10× concentrations. The data is similar to that obtained with another NCS protein, neurocalcin delta (Viviano et al., 2016, “Electrophoretic Mobility Shift in Native Gels Indicates Calcium-dependent Structural Changes of Neuronal Calcium Sensor Proteins”, [1]).

Data on the calcium-induced mobility shift of myristoylated and non-myristoylated forms of neurocalcin delta.

This data article presents the differences observed between the myristoylated and non-myristoylated forms of the neuronal calcium sensor protein, neurocalcin delta (NCALD). Analysis of the myristoylated and non-myristoylated versions of the protein by mass spectrometry provided difference in mass values consistent with addition of myristoyl group. In the presence of calcium, mobility retardation was observed upon electrophoresis of the protein in native gels. The retardation was dose-dependent and was exhibited by both the myristoylated and non-myristoylated forms of the protein.