Calpain Research Articles

CAPN2 acts as an indicator of hepatitis B virus to induce hepatic fibrosis.

This study is aimed to investigate whether calpain 2 (CAPN2) serves as an indicator of the hepatitis B virus (HBV) to induce hepatic fibrosis. Differentially-expressed genes (DEGs) in HBV-induced hepatic fibrosis and normal liver tissues were analyzed, and signal pathway which was analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis using DEGs. Next, the gene-related network map was constructed using the Search Tool for the Retrieval of Interacting Genes. Moreover, CAPN2 protein expression, level of hepatic fibrosis, CAPN2 messenger RNA level, and protein levels of CAPN2, a-SAM, COL3A1, COL1A1, and MAPK1 were determined using Immunohistochemistry (IHC), hematoxylin and eosin, RT-qPCR, and western blot (WB), respectively. There were 420 DEGs screened in HBV-induced hepatic fibrosis and normal liver tissues, among which, 373 were significantly upregulated and 47 were obviously downregulated. KEGG analysis showed that the upregulated DEGs were mainly concentrated in extracellular matrix-receptor interaction, protein digestion, and absorption signaling pathways. The network diagram analysis showed that the DEGs, such as CAPN2, ITGAV, and CCR2, play the key role in the DEG network map, and CAPN2 related to hepatic fibrosis via MAPK1. The increased CAPN2 expression and obvious hepatic fibrosis was displayed in the HBV-induced hepatic fibrosis tissues. In addition, HBV could induce CAPN2 expression, and the interference of CAPN2 could inhibit the expression of hepatic fibrosis markers, including a-SAM, COL3A1, COL1A1, and MAPK1. CAPN2 is regarded as a biomarker of hepatic fibrosis induced by HBV.

Regulation of calpain 2 expression by miR-223 and miR-145

Calpain 2 (CAPN2) is a Ca2+-dependent cysteine-protease that is involved in different cellular processes. Despite its important role, little is known about how CAPN2 expression is regulated. This study addressed the potential regulation of CAPN2 by microRNAs (miRNAs) in human endothelial cells. Two miRNAs were found to regulate CAPN2 expression by two distinct mechanisms, one direct and the other indirect. MiR-223 directly targeted CAPN2 by binding to the CAPN2 3′-untranslated region. Mir-223 overexpression decreased CAPN2 protein levels in cultured cells and in mice miR-223 antagonism led to an increase in CAPN2 protein in lung tissue. MiR-145 overexpression also decreased CAPN2 expression but did not affect a CAPN2 luciferase construct, indicating that the effect was indirect. MiR-145 targets histone deacetylase (HDAC) 2, and HDAC inhibition transcriptionally regulated CAPN2 expression by hyperacetylation of the promoter of CAPN2 gene and a subsequent decrease in polymerase 2 binding. Indeed, down regulation of HDAC2 by miR-145 not only decreased CAPN2 protein expression and calpain activity, but also protected paxillin against calpain-dependent degradation. Thus, protein levels of CAPN2 are regulated by miR-223, acting directly on the 3′-untranslated region as well as by miR-145, which acts via an increase in HDAC2. EnzymesCalpain 2 (EC 3.4.22.53), histone deacetylase 2 (EC 3.5.1.98).

Effects of the MC4R, CAPN1, and ADSL genes on body weight and purine content in slow-growing chickens

Consumer preference for slow-growing broiler chickens is rising because of increased demand for high-quality poultry products. Korat chicken (KRC) is a slow-growing chicken generated in Thailand. A goal of the KRC breeding program is to produce meat with a low purine content to benefit an aging population, without interfering with growth performance. Thus, this study aimed to investigate the effects of genes encoding melanocortin 4 receptor (MC4R), calpain 1 (CAPN1), and adenylosuccinate lyase (ADSL) on body weight, muscle fiber, and content of purine and its derivatives (i.e., adenine, guanine, hypoxanthine, and xanthine), to develop molecular markers for breeding programs. Genotypes of MC4R, CAPN1, and ADSL were obtained from 583 KRCs by PCR-single-strand conformation polymorphism. The body weight and purine contents of the KRCs were measured every 2 wk until the KRCs reached market weight at 10 wk of age. A significant association between the MC4R genotype and body weight at 2, 4, and 10 wk of age was detected. KRC possessing the BB genotype of CAPN1 showed significantly heavier body weight at 6 wk of age and guanine content at 4 wk of age, and a smaller muscle fiber diameter in the breast muscle at 10 wk of age, compared with those of the other genotypes. In addition, high expression levels of the CAPN1 and ADSL genes were detected in the breast muscle at 2 wk of age. Although higher purine contents were detected at a young age, no significant associations with the MC4R, CAPN1, and ADSL genes were detected. Our results indicate that MC4R and CAPN1 could be used as genetic markers for growth and meat quality in the slow-growing chicken breeding program.

P.394Coalition to cure calpain 3: a patient organization committed to treating and ultimately curing limb girdle muscular dystrophy type 2A

Coalition to cure calpain 3 (C3) was founded in 2010 with a mission to fund research and clinical trials relevant to limb girdle muscular dystrophy type 2A (LGMD2A, also called LGMDR1), a form of calpainopathy. The mission of C3 is to support a network of affected families, to raise awareness of LGMD2A in the global community and to support promising research focused on understanding pathomechanisms of or finding treatments for LGMD2A. We are governed by a Board of Directors including two LGMD2A patients, a Scientific Advisory Board composed of academic scientists, and a Scientific Director. Patients are usually diagnosed before their second decade. LGMD2A is initially characterized by proximal muscle weakness that progressively spreads to all muscles, leading to loss of ambulation approximately 10 years after diagnosis. LGMD2A is caused by mutations in CAPN3, the gene which encodes the proteolytic enzyme calpain 3. Despite being the most common subtype of limb girdle muscular dystrophy, the pathophysiology of LGMD2A is still not well understood. Furthermore, the biological function of the calpain 3 enzyme is not entirely clear. C3 has funded research on the basic biology of calpain 3, has supported preclinical studies in LGMD2A models, and has sponsored the generation of tools for use in studying LGMD2A. Additionally, C3 has created the first global patient registry for LGMD2A and is working in coordination with peer organizations to ensure that we reach a broad audience. C3 has also organized three workshops (in the US in 2011 and 2018, and in the Netherlands in 2013) that have been instrumental in bringing together experts in relevant fields to discuss basic science and clinical information pertaining to LGMD2A. A travel grant program, aimed at early career researchers, provides travel support for investigators to present on LGMD2A at major scientific conferences. Our current priorities are to fund research efforts and support collaborations focused on finding a treatment for LGMD2A.

P.138C-terminal titin fragments in mouse muscles

Mutations in the extreme C-terminal (M-band) part of titin cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J, aka LGMD R10 titin-related). The titin region affected by the TMD/LGMD2J mutations contains a binding site of the muscle-specific protease calpain 3 (CAPN3), mutations in which underlie LGMD2A (aka LGMD R1 calpain 3 -related). The biological function of this interaction is not well understood.

Loss of TRIM29 suppresses cancer stem cell-like characteristics of PDACs via accelerating ISG15 degradation.

TRIM family proteins are defined as E3 ubiquitin ligases because of their RING-finger domains. The ubiquitin-like protein interferon-stimulated gene 15 (ISG15) encodes a 15-kDa protein, that is implicated in the posttranslational modification of diverse proteins. Both TRIM29 and ISG15 play both pro-tumoral and anti-tumoral functions in cancer cells derived from different histology. In the current study, we demonstrated that correlation expression of TRIM29 and ISG15 in pancreatic ductal adenocarcinomas (PDACs). The current study demonstrated that TRIM29 knockdown destabilized ISG15 protein via promoting its processing by calpain 3 (CAPN3). Importantly, the current study found that TRIM29 knockdown suppressed cancer stem cell-like features of PDACs, which can be rescued by ISG15 independent of its conjugation function. In addition, the current study demonstrated that extracellular free ISG15 played an important role in maintenance of cancer stem cell-like features of PDACs. Thereby, the current study displayed a novel mechanism by which TRIM29 modulates ISG15 stability via CAPN3-mediated processing, and subsequently extracellular ISG15 maintains the cancer stem cell-like features of PDAC via autocrine mode of action.

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.

Gene Correction of LGMD2A Patient-Specific iPSCs for the Development of Targeted Autologous Cell Therapy.

Limb girdle muscular dystrophy type 2A (LGMD2A), caused by mutations in the Calpain 3 (CAPN3) gene, is an incurable autosomal recessive disorder that results in muscle wasting and loss of ambulation. To test the feasibility of an autologous induced pluripotent stem cell (iPSC)-based therapy for LGMD2A, here we applied CRISPR-Cas9-mediated genome editing to iPSCs from three LGMD2A patients to enable correction of mutations in the CAPN3 gene. Using a gene knockin approach, we genome edited iPSCs carrying three different CAPN3 mutations, and we demonstrated the rescue of CAPN3 protein in myotube derivatives invitro. Transplantation of gene-corrected LGMD2A myogenic progenitors in a novel mouse model combining immunodeficiency and a lack of CAPN3 resulted in muscle engraftment and rescue of the CAPN3 mRNA. Thus, we provide here proof of concept for the integration of genome editing and iPSC technologies to develop a novel autologous cell therapy for LGMD2A.

Expression of calpain small subunit 1 in breast cancer and its clinical significance

Objective To investigate the expression of calpain small subunit 1 (Capn4) in breast cancer and its clinical significance. Methods The cancer tissues and the paraffin-embeded specimens of the corresponding adjacent normal tissues from 12 patients with breast cancer between May 2015 and July 2015 in Shanghai General Hospital were collected. The expression of Capn4 was detected by using immunohistochemistry. The paraffin-embeded specimens of cancer tissues from 70 breast cancer patients in Shanghai General Hospital between January 2009 and September 2011 were also collected. And the expression of Capn4 was detected. The correlation between Capn4 expression level and TNM stage, pathological grade, molecular markers as well as the prognosis of breast cancer was also analyzed. Results The expression of Capn4 in breast cancer tissues was higher than that in the corresponding adjacent normal tissues. The expression level of Capn4 was correlated with TNM stage (P = 0.002), T stage (P = 0.004), N stage (P = 0.004), M stage (P = 0.025), estrogen receptor (ER) status (χ 2 = 4.787, P = 0.029) and survival status (χ 2 = 5.826, P = 0.016). Kaplan-Meier survival analysis revealed that the median survival time in the higher expression of Capn4 group 90 months was shorter than that in the lower expression of Capn4 group (not reaching the median survival time), and there were statistical differences (χ 2 = 4.351, P = 0.037). Conclusions The expression of Capn4 in breast cancer tissues is upregulated. The expression difference is correlated with TNM stage, pathological grade, ER status and the prognosis of breast cancer, suggesting that it may become a new target and molecular marker for breast cancer treatment. Key words: Breast neoplasms; Calcium-activated neutral protease; Calpain small subunit 1; Prognosis; Immunohistochemistry

Inhibition of the ubiquitous calpains protects complex I activity and enables improved mitophagy in the heart following ischemia-reperfusion.

Activation of calpain 1 (CPN1) and calpain 2 (CPN2) contributes to cardiac injury during ischemia (ISC) and reperfusion (REP). Complex I activity is decreased in heart mitochondria following ISC-REP. CPN1 and CPN2 are ubiquitous calpains that exist in both cytosol (cs)-CPN1 and 2 and mitochondria (mit)-CPN1 and 2. Recent work shows that the complex I subunit (NDUFS7) is a potential substrate of the mit-CPN1. We asked whether ISC-REP led to decreased complex I activity via proteolysis of the NDUFS7 subunit via activation of mit-CPN1 and -2. Activation of cs-CPN1 and -2 decreases mitophagy in hepatocytes following ISC-REP. We asked whether activation of cs-CPN1 and -2 impaired mitophagy in the heart following ISC-REP. Buffer-perfused rat hearts underwent 25 min of global ISC and 30 min of REP. MDL-28170 (MDL; 10 µM) was used to inhibit CPN1 and -2. Cytosol, subsarcolemmal mitochondria (SSM), and interfibrillar mitochondria (IFM) were isolated at the end of heart perfusion. Cardiac ISC-REP led to decreased complex I activity with a decrease in the content of NDUFS7 in both SSM and IFM. ISC-REP also resulted in a decrease in cytosolic beclin-1 content, a key component of the autophagy pathway required to form autophagosomes. MDL treatment protected the contents of cytosolic beclin-1 and mitochondrial NDUFS7 in hearts following ISC-REP. These results support that activation of both cytosolic and mitochondrial calpains impairs mitochondria during cardiac ISC-REP. Mitochondria-localized calpains impair complex I via cleavage of a key subunit. Activation of cytosolic calpains contributes to mitochondrial dysfunction by impairing removal of the impaired mitochondria through depletion of a key component of the mitophagy process.

Absolute expressions of hypoxia-inducible factor-1 alpha (HIF1A) transcript and the associated genes in chicken skeletal muscle with white striping and wooden breast myopathies

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as “non-defective” (n = 4), “medium-WS” (n = 6), “heavy-WS” (n = 7) and “heavy-WS+WB” (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm2. Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated.

Arsenic impairs GLUT1 trafficking through the inhibition of the calpain system in lymphocytes

Exposure to arsenic is associated with increased risk of developing insulin resistance and type 2 diabetes. The proteases calpain-1 (CAPN1), calpain-2 (CAPN2) and calpain-10 (CAPN10) and their endogenous inhibitor calpastatin (CAST) regulate glucose uptake in skeletal muscle and adipocytes. We investigated whether arsenic disrupts GLUT1 trafficking and function through calpain inhibition, using lymphocytes as a cell model. Lymphocytes from healthy subjects were treated with 0.1 or 1 μM of sodium arsenite for 72 h and challenged with 3.9 or 11.1 mM of glucose. Our results showed that arsenite inhibited GLUT1 trafficking, glucose uptake, and calpain activity in the presence of 11.1 mM of glucose. These correlated with a decrease in the autolytical fragment of 50 kDa of CAPN1 and increased levels of CAST, but there were no changes in CAPN2 and CAPN10. We used a cell-free system to evaluate the effect of arsenite over CAPN1, finding that arsenite induced CAPN1 autolysis. To confirm that calpains are involved in GLUT1 trafficking and glucose uptake in lymphocytes, we generated stable CAPN1 or CAPN10 knockdowns in Jurkat cells using short hairpin RNA (shRNA). CAPN1 knockdown induced glucose uptake, while CAPN10 knockdown diminished glucose uptake, which correlated with a significant reduction of calpain activity after the pulse with 11.1 mM of glucose. These data showed that CAPN10 was responsible for the induction of calpain activity after the challenge with 11.1 mM of glucose and that CAPN1 and CAPN10 regulate glucose uptake in lymphocytes. Altogether, our results suggest that arsenite impairs GLUT1 trafficking and function through calpain dysregulation.

Use of a Mouse Model and Human Umbilical Vein Endothelial Cells to Investigate the Effect of Arsenic Exposure on Vascular Endothelial Function and the Associated Role of Calpains.

Background:Arsenic (As) is a well-known environmental contaminant. Chronic exposure to As is known to increase the risk of cardiovascular diseases, including atherosclerosis, hypertension, diabetes, and stroke. However, the detailed mechanisms by which As causes vascular dysfunction involving endothelial integrity and permeability is unclear.Objectives:Our goal was to investigate how exposure to As leads to endothelial dysfunction.Methods:Arsenic trioxide (ATO) was used to investigate the effects and mechanisms by which exposure to As leads to endothelial dysfunction using a mouse model and cultured endothelial cell monolayers.Results:Compared with the controls, mice exposed chronically to As (10 ppb in drinking water supplied by ATO) exhibited greater vascular permeability to Evans blue dye and fluorescein isothiocyanate–labeled bovine serum albumin (FITC-BSA). In addition, endothelial monolayers treated with ATO ( As) exhibited greater intracellular gaps and permeability to low-density lipoprotein or transmigrating THP-1 cells. Furthermore, activity and protein levels of calpain-1 (CAPN-1) were significantly higher in aortas and human umbilical vein endothelial cells (HUVECs) treated with ATO. These results were consistent with effects of ATO treatment and included a rapid increase of intracellular calcium () and higher levels of CAPN-1 in the plasma membrane. Endothelial cell dysfunction and the proteolytic disorganization of vascular endothelial cadherin (VE-cadherin) in HUVECs in response to ATO were partially mitigated by treatment with a CAPN-1 inhibitor (ALLM) but not a CAPN-2 inhibitor (Z-LLY-FMK).Conclusions:This study found that in mice and HUVEC models, exposure to ATO led to CAPN-1 activation by increasing and CAPN-1 translocation to the plasma membrane. The study also suggested that inhibitor treatment may have a role in preventing the vascular endothelial dysfunction associated with As exposure. The findings presented herein suggest that As-induced endothelial dysfunction involves the hyperactivation of the CAPN proteolytic system. https://doi.org/10.1289/EHP4538

The Identification of Novel Single Nucleotide Polymorphisms in Calpain 1 (CAPN1) Gene of Japanese Quail (Coturnix coturnix japonica)

Objective: Calpains, in particular μ-calpain, are responsible for the post mortem proteolysis processes in muscle tissue and have main influences on meat quality. The CAPN1 gene that codes for large subunit of μ-calpain is revealed as a candidate gene related with meat quality and tenderization traits for livestock. For this reason, in this study it is aimed to investigate the genetic variation of CAPN1 gene in Japanese quails. Material and Methods: In this study, the genetic variation of CAPN1 gene was analyzed via DNA sequencing of 35 (13 males and 12 females) Japanese quails which were reared in Tekirdağ Namık Kemal University, Animal Research Unit. Results: Some genetic variants which are found in the 4th and 5th exons are as g.103969C>T in the 4th exon region and eight novel SNPs as g.104116A>T, g.104118T>G, g.104148G>C, g.104169G>C, g.104172A>G, g.104179C>G, g.104181G>A, g.104184T>C in the 5th exon of CAPN1 gene. The novel DNA polymorphisms of CAPN1 gene in Japanese quails are reported for the first time in this study and these sequences were deposited to NCBI GenBank Database, with the accession numbers MK496828-MK496837, respectively. g.103969C>T transversion which is localized in the 4th exon region and g.104148G>C, g.104169G>C transversions and g.104172A>G, g.104181G>A, g.104184T>C transitions which are localized in the 5th exon region have not caused an amino acid change. Instead, g.104116A>T, g.104118T>G tranversions caused the change from Threonine to Serine amino acid. Similarly, C→G transversion which was observed on the 104179th position caused the amino acid change from Proline to Alanine. Conclusion: These observed SNPs may have an effect on meat yield and tenderness in quails, so further researches are needed to demonstrate this hypothesis and these SNPs may be candidate SNPs for quails breeding.

Effects of okadaic acid and hematein on human lung adenocarcinoma A549 cells and responses of mitochondria and endoplasmic reticulum apoptosis pathways

Background: Okadaic acid (OA) and hematein are both able to inhibit the proliferation of human lung adenocarcinoma A549 cells. However, it is largely unknown about their combined effects on proliferation and apoptosis of A549 cells. Methods: The combined effects of the two drugs on proliferation of A549 cells and the responses of mitochondria and endoplasmic reticulum (ER) apoptosis pathways were investigated. Results: MTT assay showed that both OA and hematein significantly inhibited proliferation of A549 cells, and the combined drugs could further increase the inhibition ratio with lower dosages. Inverted phase contrast microscope and scanning electron microscope (SEM) analysis indicated that the combined drugs aggravated the appearance of apoptotic bodies and damaged cells. Compared to the single drug treated groups, reactive oxygen species (ROS) contents were significantly higher in the groups treated by the combined drugs, and mitochondrial membrane potential was significant lower. Western blot indicated that pro-apoptotic Bax that involved in mitochondrial apoptosis pathway increased, the anti-apoptotic protein Bcl-2 decreased, and the ER apoptosis-related proteins CHOP, Calpain2, JNK1 and IRE1 increased. Conclusions: This work demonstrates that OA combined hematein can more effectively inhibit proliferation of A549 cells, and induce apoptosis of A549 cells via mitochondria and ER dependent pathways.

Genome-Wide Scan Identifies Selection Signatures in Chinese Wagyu Cattle Using a High-Density SNP Array.

Simple SummaryAnalysis of the genomic regions under consideration can provide important insights into the genetic basis of complex traits. In this study, we utilized a high-density SNP (Single nucleotide polymorphism) array for analysis of genome selection signatures in Chinese Wagyu cattle. In total, 239 selected regions responding to 162 candidate genes were identified, which related to economic traits, including fatty acid, meat quality, growth and developmental traits. Moreover, we found that these selected genes were enriched for the four Gene Ontology (GO) terms of biological regulation and metabolic process. These results also contribute to the understanding of genetic basis of these traits during the formation of this population.Selective breeding can lead to genetic diversity and diverse phenotypes in farm animals. Analysis of the genomic regions under selection can provide important insights into the genetic basis of complex traits. In this study, a high-density SNP array was used for analysis of genome selection signatures in Chinese Wagyu cattle. In total, we obtained 478,903 SNPs and 24,820 no-overlap regions for |iHS| (integrated haplotype score) estimations. Under the threshold of the top 1%, 239 regions were finally identified as candidate selected regions and 162 candidate genes were found based on the UMD3.1 genome assembly. These genes were reported to be associated with fatty acids, such as Bos taurus nitric oxide synthase 1 adaptor protein (NOS1AP), Bos taurus hydroxysteroid 17-beta dehydrogenase 7 (HSD17B7), Bos taurus WD repeat domain 7 (WDR7), Bos taurus ELOVL fatty acid elongase 2 (ELOVL2), Bos taurus calpain 1 (CAPN1), Bos taurus parkin RBR E3 ubiquitin protein ligase (PRKN, also known as PARK2), Bos taurus mitogen-activated protein kinase kinase 6 (MAP2K6), meat quality, including Bos taurus ADAM metallopeptidase domain 12 (ADAM12), Bos taurus 5′-aminolevulinate synthase 1 (ALAS1), Bos taurus small integral membrane protein 13 (SMIM13) and Bos taurus potassium two pore domain channel subfamily K member 2 (KCNK2), growth, and developmental traits, such as Bos taurus insulin like growth factor 2 receptor (IGF2R), Bos taurus RAR related orphan receptor A (RORA), Bos taurus fibroblast growth factor 14 (FGF14), Bos taurus paired box 6 (PAX6) and Bos taurus LIM homeobox 6 (LHX6). In addition, we identified several genes that are associated with body size and weight, including Bos taurus sorting nexin 29 (SNX29), Bos taurus zinc finger imprinted 2 (ZIM2), Bos taurus family with sequence similarity 110 member A (FAM110A), immune system, including Bos taurus toll like receptor 9 (TLR9), Bos taurus TAFA chemokine like family member 1 (TAFA1), Bos taurus glutathione peroxidase 8 (putative) (GPX8), Bos taurus interleukin 5 (IL5), Bos taurus PR domain containing 9 (PRDM9), Bos taurus glutamate ionotropic receptor kainate type subunit 2 (GRIK2) and feed intake efficiency, Bos taurus sodium voltage-gated channel alpha subunit 9 (SCN9A), Bos taurus relaxin family peptide/INSL5 receptor 4 (RXFP4), Bos taurus RNA polymerase II associated protein 3 (RPAP3). Moreover, four GO terms of biological regulation (GO:0009987, GO:0008152) and metabolic process (GO:0003824, GO:0005488) were found based on these genes. In addition, we found that 232 candidate regions (~18 Mb) overlapped with the Quantitative trait loci (QTL)regions extracted from cattle QTLdb. Our findings imply that many genes were selected for important traits in Chinese Wagyu cattle. Moreover, these results can contribute to the understanding of the genetic basis of the studied traits during the formation of this population.

Downregulation of Calpain 1 Causes Neurotoxicity and Apoptosis in Primary Neurons Induced by Aβ25-35

Amyloid-peptides (Aβ), which can aggregate oligomers or fibrils into neurons, play a critical role in Alzheimer's disease (AD). Calpain 1 (CAPN1) is one of the Ca2+ regulated proteases, involved in cell death, apoptosis, and motility. As an important signal transduction pathway, it has been reported to be closely related to muscle atrophy, myoblast fusion, diabetes mellitus, and neurode-generative diseases, including Alzheimer's disease. Our previous study investigated the effect of CAPN1 RNAi on neurotoxicity and apoptosis in primary neurons induced by Aβ25-35. The primary neurons were cultured and separated into different groups pretreated with Aβ25-35 (10 μM). Three groups were treated with CAPN1 shRNA, while one group was not. Along with CAPN1 shRNA, P25 (2 μg/mL) and rapamycin (20 ng/mL) were added to the culture. The results showed that CAPN1 RNAi enhanced the formation of autophagosomes in cells, as observed by confocal microscopy. Parallelly, CAPN1 RNAi enhanced the cell viability, as observed by Cell Counting Kit-8 (CCK8) assay, and decreased the apoptotic rate, as observed by flow cytometry. In addition, CAPN1 RNAi decreased the mRNA level of CAPN1 and downregulated the expression levels of CDK5, GSK3β, and p-tau in primary neurons induced by Aβ25-35. All these results indicated the effect of CAPN1 RNAi against Aβ-induced neurotoxicity. Our results suggested that CAPN1 RNAi exhibits a neuroprotective effect on the treatment of AD and provided a pharmacological basis for its clinical treatment.

Calpastatin Mediates Development of Alzheimer's Disease in Diabetes.

Aged people have a high chance to develop two prevalent diseases, diabetes and Alzheimer's disease (AD), which are characterized with hyperglycemia and neurodegeneration, respectively. Interestingly, recent evidence suggest that diabetes is a predisposing factor for AD. Nevertheless, the mechanisms underlying the association of diabetes with AD remain poorly defined. Here, we studied the effects of diabetes on AD in mice. The APP-PS1 mouse, an AD-prone strain, was administrated with streptozotocin (STZ) to destroy 75% beta cell mass to induce sustained hyperglycemia. We found that STZ-treated APP-PS1 mice exhibited poorer performance in the social recognition test, Morris water maze, and plus-maze discriminative avoidance task, compared to saline-treated normoglycemic APP-PS1 mice, likely resulting from increases in brain deposition of amyloid-β peptide aggregates (Aβ). Since formation of Aβ is known to be induced by protein hyperphosphorylation mediated by calpain (CAPN)-induced cleavage of p35 into p25, we examined levels of these proteins in mouse brain. We detected not only increased p35-to-p25 conversion, but also enhanced CAPN1 activity via increased protein but not mRNA levels. The internal CAPN1 inhibitor, calpastatin (CAST), was downregulated in STZ-treated APP-PS1 mouse brain, as a basis for the increase in CAPN1. In vitro, a human neuronal cell line, HCN-2, increased CAPN1 activity and downregulated CAST levels when incubated for 8 days in high glucose level, resulting in increased cell apoptosis. Together, these data suggest that chronic hyperglycemia may promote AD development through downregulating CAST.

Activation of Mitochondrial Calpains Contributes to the Selective Degradation of Specific Mitochondrial Proteins

INTRODUCTIONCalpain 1 (CPN1) and 2 (CPN2) are ubiquitous calpains that exist in both cytosol and mitochondria. Calpains are calcium‐activated proteases. Pharmacologic inhibition of CPN1 and CPN2 decreases cardiac injury by improving mitochondrial function during ischemia‐reperfusion. Since both CPN1 and CPN2 include one large subunit and a small regulatory subunit (calpain 4, CPN4), genetic removal of CPN4 eliminates the activities of CPN1 and CPN2. CPN4 knockout mice were used in this study to test if activation of mitochondrial calpains leads to mitochondrial damage by proteolysis of mitochondrial proteins.HYPOTHESISWe hypothesize that exogenous calcium‐mediated activation of mitochondrial CPN1 and CPN2 impairs mitochondrial function through degradation of mitochondrial proteins.METHODSPurified cardiac mitochondria were isolated from wild type and CPN4 knockout mice. Exogenous calcium (25 uM) was used to activate mitochondrial CPN1 and CPN2. Mitochondria were subjected to proteomic analysis using tryptic digestion, liquid chromatography, and mass spectrometry with iTRAQ quantification.RESULTSWe focused on the proteins that were decreased by exogenous calcium exposure in mitochondria from wild type but not in CPN4 knockout mice as specific targets of mitochondrial CPN1 and CPN2. In wild type mice, calcium treatment significantly decreased the contents of multiple metabolic enzymes involving fatty acid oxidation and oxidative phosphorylation (electron transport chain components and ATP synthase) compared to non‐calcium treated mitochondria. Knockout of CPN4 preserved these enzyme contents in calcium‐treated mitochondria (Table). Decreases in ATPase factor 6 and very long chain acyl‐coA dehydrogenase were confirmed by Western blot.CONCLUSIONActivation of mitochondrial calpains leads to decreased mitochondrial function by impairing metabolic enzyme activity. Prevention of CPN1 and CPN2 activation provides an attractive approach to protect mitochondria during pathologic conditions including ischemia‐reperfusion and heart failure.Support or Funding InformationThis work was supported by the Office of Research and Development, Medical Research Service Merit Review Award (2IO1BX001355‐01A2) and Department of Veterans Affairs (EJL); National Institutes of Health NIH R21AG054975‐01 (QC).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of Post-Transcriptional Control of Calpain by miR-124-3p in the Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disease prevalent in aged people, clinically characterized by progressive memory loss, behavioral and learning dysfunction, and cognitive deficits. The pathogenesis of AD is hallmarked by formation of amyloid-β peptide aggregates (Aβ) and intraneuronal neurofibrillary tangles (NFTs), which are induced by hyperphosphorylation of amyloid-β protein precursor and tau protein, respectively. The hyperphosphorylation is controlled by cyclin-dependent kinase-5 (CDK5), the aberrant activation of which is mediated by calpain (CAPN)-induced cleavage of p35 into p25. However, the regulation of CAPN in AD remains largely unknown. Here, we studied the post-transcriptional control of CAPN1 by microRNAs (miRNAs) in the setting of AD. We found that miR-124-3p, previously reported as a miRNA that was downregulated in AD, was a CAPN1-targeting miRNA that functionally inhibited the protein translation of CAPN1 in a human neural cell line, HCN-2. In vitro, transfection with miR-124-3p reduced the levels of CAPN1 protein, the cleavage of p35 into p25, and cell apoptosis dose-dependently in HCN-2 cells. Moreover, a significant inverse correlation was detected between the levels of miR-124-3p and CAPN1 in AD specimens. Furthermore, intracranial injection of adeno-associated virus expressing miR-124-3p into APP/PS1-AD mice significantly reduced Aβ deposition and significantly improved the AD-mouse behavior in the social recognition test and plus-maze discriminative avoidance task. Together, our data suggest that post-transcriptional control of calpain by miR-124-3p plays an essential role in the development of AD.