Two sequential activation modules control the differentiation of protective T helper-1 (Th1) cells.

Interferon-γ (IFN-γ)-producing CD4+ T helper-1 (Th1) cells are critical for protection from microbes that infect the phagosomes of myeloid cells. Current understanding of Th1 cell differentiation is based largely on reductionist cell culture experiments. We assessed Th1 cell generation in vivo by studying antigen-specific CD4+ T cells during infection with the phagosomal pathogen Salmonella enterica (Se), or influenza A virus (IAV), for which CD4+ T cells are less important. Both microbes induced T follicular helper (Tfh) and interleukin-12 (IL-12)-independent Th1 cells. During Se infection, however, the Th1 cells subsequently outgrew the Tfh cells via an IL-12-dependent process and formed subsets with increased IFN-γ production, ZEB2-transcription factor-dependent cytotoxicity, and capacity to control Se infection. Our results indicate that many infections induce a module that generates Tfh and poorly differentiated Th1 cells, which is followed in phagosomal infections by an IL-12-dependent Th1 cell amplification module that is critical for pathogen control.

Immune tolerance of food is mediated by layers of CD4+ T cell dysfunction.

Gastrointestinal health depends on the adaptive immune system tolerating the foreign proteins in food1,2. This tolerance is paradoxical because the immune system normally attacks foreign substances by generating inflammation. Here we addressed this conundrum by using a sensitive cell enrichment method to show that polyclonal CD4+ T cells responded to food peptides, including a natural one from gliadin, by proliferating weakly in secondary lymphoid organs of the gut-liver axis owing to the action of regulatory T cells. A few food-specific T cells then differentiated into T follicular helper cells that promoted a weak antibody response. Most cells in the expanded population, however, lacked canonical T helper lineage markers and fell into five subsets dominated by naive-like or T follicular helper-like anergic cells with limited capacity to form inflammatory T helper 1 cells. Eventually, many of the T helper lineage-negative cells became regulatory T cells themselves through an interleukin-2-dependent mechanism. Our results indicate that exposure to food antigens causes cognate CD4+ naive T cells to form a complex set of noncanonical hyporesponsive T helper cell subsets that lack the inflammatory functions needed to cause gut pathology and yet have the potential to produce regulatory T cells that may suppress it.

Association between traditional Korean fermented vegetables (kimchi) intake and serum lipid profile: using the Korean Genome and Epidemiology Study (KoGES) cohort.

PURPOSE: Dyslipidemia is a major health issue worldwide. There is growing interest in understanding the potential role of kimchi consumption on serum lipid profiles. However, there are limited epidemiological studies available on this topic. Therefore, this study aims to investigate the association between kimchi intake and serum lipid profiles. METHODS: We conducted an epidemiological study on participants (aged 40-69 years old) selected from the Health Examinees (HEXA) cohort study (n = 61,761). Four types of kimchi, including Baechu kimchi (cabbage kimchi), Kkakdugi (radish kimchi), Nabak kimchi/Dongchimi (a type of water kimchi made with fermented vegetables), and other kimchi, were assessed by a 106-food item semi-quantitative validated food frequency questionnaire (FFQ). Each kimchi intake is the average value calculated from the FFQ of the baseline and follow-up surveys. Fasting blood data were obtained at baseline and follow-up visits. Linear regression was used to examine the relationship between the intake of kimchi and the change in serum lipid profiles. RESULTS: The mean years between the baseline survey and a follow-up survey was 4.97 years. In this study, compared to the lowest category (< 1 serving/day), Baechu kimchi intake (2- < 3 servings/day) had more negative correlations with the change in values of total cholesterol (ß: -1.600, 95% confidence interval [CI, -2.744, -0.456]), triglycerides (ß: -3.372, 95% CI [-5.414, -1.330]), and low-density lipoprotein cholesterol (ß: -1.155, 95% CI [-2.214, -0.095]) in women. In men, Baechu kimchi intake (2- < 3 servings/day) had a more positive correlation associated with the changes in values of high-density lipoprotein cholesterol (ß: 0.049, 95% CI [0.031, 0.907]) compared to the lowest intake category (< 1 serving/day). CONCLUSIONS: Among Korean adults, consumption of kimchi, particularly Baechu kimchi, was found to be associated with improvements in serum lipid profiles. Further studies are required to conduct additional interventions to confirm the association between kimchi and serum lipid profiles.

Novel virus-like nanoparticle vaccine effectively protects animal model from SARS-CoV-2 infection.

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.

Cutting Edge: Mouse SARS-CoV-2 Epitope Reveals Infection and Vaccine-Elicited CD8 T Cell Responses.

The magnitude of SARS-CoV-2-specific T cell responses correlates inversely with human disease severity, suggesting T cell involvement in primary control. Whereas many COVID-19 vaccines focus on establishing humoral immunity to viral spike protein, vaccine-elicited T cell immunity may bolster durable protection or cross-reactivity with viral variants. To better enable mechanistic and vaccination studies in mice, we identified a dominant CD8 T cell SARS-CoV-2 nucleoprotein epitope. Infection of human ACE2 transgenic mice with SARS-CoV-2 elicited robust responses to H2-Db/N219-227, and 40% of HLA-A*02+ COVID-19 PBMC samples isolated from hospitalized patients responded to this peptide in culture. In mice, i.m. prime-boost nucleoprotein vaccination with heterologous vectors favored systemic CD8 T cell responses, whereas intranasal boosting favored respiratory immunity. In contrast, a single i.v. immunization with recombinant adenovirus established robust CD8 T cell memory both systemically and in the respiratory mucosa.

Safety assessment of white colony-forming yeasts in kimchi.

White colony-forming yeasts (WCFYs) have been reported to form a white colony on the surface of kimchi, resulting in the deterioration of kimchi sensory quality. However, toxicity of WCFY has rarely been studied. Thus, to evaluate the safety of WCFY (i.e., Kazachstania servazzii, Candia sake, and Pichia kudriavzevii), we conducted cell and animal experiments as well as genomic analysis. In vitro studies indicated that WCFY did not induce cytotoxic responses such as lactate dehydrogenase release, excessive oxidative stress, and mitochondrial damage at concentrations of up to 2.5 × 105 CFU/mL in human intestinal and liver cells. In animal studies using rats (single-dose and 14-day repeated-dose oral toxicity studies), WCFY did not induce death, clinical signs of toxicity, histological alterations of the liver, or increases in the expression of pro-inflammatory cytokines nor cytochrome P450-2E1 in liver tissue at concentrations of up to 5 × 108 CFU/head/day. Genomic analysis revealed that P. kudriavzevii did not harbor genes related to toxicity and antimicrobial resistance. Taken together, our data suggest that exposure to WCFY through kimchi intake did not induce toxic response in the Caco-2, HepG2, and Sprague-Dawley rats. The current work provides evidence for the safety of accidental major WCFY ingestion via kimchi.

Unregulated antigen-presenting cell activation by T cells breaks self tolerance.

T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1-/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.

Cost-effective production of tag-less recombinant protein in Nicotiana benthamiana.

Plants have recently received a great deal of attention as a means of producing recombinant proteins. Despite this, a limited number of recombinant proteins are currently on the market and, if plants are to be more widely used, a cost-effective and efficient purification method is urgently needed. Although affinity tags are convenient tools for protein purification, the presence of a tag on the recombinant protein is undesirable for many applications. A cost-effective method of purification using an affinity tag and the removal of the tag after purification has been developed. The family 3 cellulose-binding domain (CBM3), which binds to microcrystalline cellulose, served as the affinity tag and the small ubiquitin-related modifier (SUMO) and SUMO-specific protease were used to remove it. This method, together with size-exclusion chromatography, enabled purification of human interleukin-6 (hIL6) with a yield of 18.49 mg/kg fresh weight from leaf extracts of Nicotiana benthamiana following Agrobacterium-mediated transient expression. Plant-produced hIL6 (P-hIL6) contained less than 0.2 EU/µg (0.02 ng/mL) endotoxin. P-hIL6 activated the Janus kinase-signal transducer and activator of transcriptional pathways in human LNCaP cells, and induced expression of IL-21 in activated mouse CD4+ T cells. This approach is thus a powerful method for producing recombinant proteins in plants.

Characterisation of the bacterial community in the gastrointestinal tracts of elk (Cervus canadensis).

The resident bacteria of the gastrointestinal tract (GIT) and the behaviour of these microbes have been poorly characterised in elk as compared to other ruminant animal species such as sheep and cattle. In addition, most microbial community studies of deer gut have focused on rumen or faeces, while other parts of the GIT such as the small and large intestine have received little attention. To address this issue, the present study investigated the diversity of the GIT bacterial community in elk (Cervus canadensis) by 16S rRNA pyrosequencing analysis. Eight distinct GIT segments including the stomach (rumen, omasum, and abomasum), small intestine (duodenum and jejunum), and large intestine (cecum, colon, and rectum) obtained from four elks were examined. We found that bacterial richness and diversity were higher in the stomach and large intestine than in the small intestine (P < 0.05). A total of 733 genera belonging to 26 phyla were distributed throughout elk GITs, with Firmicutes, Bacteroidetes, and Proteobacteria identified as the predominant phyla. In addition, there was spatial heterogeneity in the composition, diversity, and species abundance of microbiota in the GIT (P < 0.0001). To the best of our knowledge, this is the first study to characterise bacterial communities from eight GIT regions of elk by 16S rRNA pyrosequencing.

Dendritic cell expression of the signaling molecule TRAF6 is required for immune tolerance in the lung.

Immune tolerance in the lung is important for preventing hypersensitivity, such as allergic asthma. Maintenance of tolerance in the lung is established by coordinated activities of poorly understood cellular and molecular mechanisms, including participation of dendritic cells (DCs). We have previously identified DC expression of the signaling molecule TRAF6 as a non-redundant requirement for the maintenance of immune tolerance in the small intestine of mice. Because mucosal tissues share similarities in how they interact with exogenous antigens, we examined the role of DC-expressed TRAF6 in the lung. As with the intestine, we found that the absence TRAF6 expression by DCs led to spontaneous generation of Th2-associated immune responses and increased susceptibility to model antigen-induced asthma. To examine the role of commensal microbiota, mice deficient in TRAF6 in DCs were treated with broad-spectrum antibiotics and/or re-derived on a germ-free (GF) background. Interestingly, we found that antibiotics-treated specific pathogen-free, but not GF, mice showed restored immune tolerance in the absence of DC-expressed TRAF6. We further found that antibiotics mediate microbiota-independent effects on lung T cells to promote immune tolerance in the lung. This work provides both a novel tool for studying immune tolerance in the lung and an advance in our conceptual understanding of potentially common molecular mechanisms of immune tolerance in both the intestine and the lung.

Bacillus oryzaecorticis sp. nov., a moderately halophilic bacterium isolated from rice husks.

A Gram-stain-positive, aerobic, endospore-forming, moderately halophilic rod, designated strain R1(T), was isolated from rice husks and subjected to a taxonomic study using a polyphasic approach. Strain R1(T) produced spherical or ellipsoidal endospores at a subterminal position in swollen sporangia, and was catalase- and oxidase-positive. The isolate grew optimally at 37 °C and pH 6.0-7.0, and could grow in the presence of up to 9% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain R1(T) belongs to the genus Bacillus. The closest relatives of strain R1(T) were Bacillus subtilis subsp. subtilis NCIB 3610(T), Bacillus aquimaris TF-12(T), and Bacillus marisflavi TF-11(T), with 16S rRNA gene sequence similarities of 96.0%, 98.4%, and 98.7%, respectively. DNA-DNA relatedness values between the isolate and the reference strains were ≤42±3%. The predominant menaquinones were MK-5 (50%) and MK-7 (50%). The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and phosphatidylethanolamine. The major cellular fatty acids were iso-C(15 : 0) (48.6%) and anteiso-C(15 : 0) (20.6%), and the cell-wall diamino acid was meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analyses and chemotaxonomic and phenotypic characteristics, it is concluded that strain R1(T) represents a novel species of the genus Bacillus, for which we propose the name Bacillus oryzaecorticis sp. nov. The type strain is R1(T) ( = KACC 17217(T) = KCCM 90231(T) = JCM 19602(T)).

Exploring the anti-obesity effects of kimchi through enhanced thermogenesis in differentiated T37i brown adipocytes.

Background: Previous research has demonstrated the anti-obesity effects of kimchi in 3T3-L1 adipocytes and mice with diet-induced obesity by assessing the expression of obesity-associated genes. Additionally, recent studies have identified mechanisms involving thermogenesis that support these effects. Objective: This study aims to further investigate the anti-obesity properties of kimchi, focusing on its impact on thermogenic activity in differentiated T37i brown adipocytes. Design: The study first evaluated the antioxidant potential of kimchi using total antioxidant capacity (TAC) and ferric reducing antioxidant power (FRAP) assays. Optimal differentiation conditions for T37i adipocytes were established before proceeding with evaluations of cell viability, intracellular triglyceride (TG) content, lipid accumulation, and the expression of genes and proteins related to obesity and thermogenesis. Results: Kimchi maintained over 90% cell viability in T37i adipocytes at concentrations up to 1,000 µg/mL. Efficient differentiation of T37i preadipocytes was achieved using a medium containing 10% calf serum, 2 nM 3,3',5-triiodo-L-thyronin (T3), and 100 nM insulin. Kimchi significantly reduced intracellular TG levels and lipid accumulation, compared to the control group, and enhanced the expression of genes and proteins related to thermogenesis while reducing the expression of obesity-related genes. Discussion: The findings suggest that kimchi exerts its anti-obesity effects by modulating thermogenic and obesity-related pathways in brown adipocytes, which may be partially attributed to its antioxidant properties. Conclusions: Kimchi shows promise as a preventive measure against obesity by influencing metabolic pathways associated with both obesity and thermogenesis in T37i brown adipocytes.

Bacterial community of kimchi added with seafood based on culture-dependent investigations.

Previously, microbial communities of five commercial kimchi added with seafood and one kimchi without seafood were analyzed using a culture-independent (CI) method. In the current study, microbial communities of the same samples were analyzed using a culture-dependent (CD) method with two media: tryptic soy agar (TSA) and Lactobacilli de Man, Rogosa and Sharpe (MRS) agar. MRS agar showed a higher proportion of lactic acid bacteria, while TSA showed a higher proportion of Bacillus species. Leuconostoc mesenteroides became dominant over time except in kimchi added with hongeu (HBK, okamejei kenojei). In the case of HBK, Bacillus was dominant. The low pH of HBK was confirmed by cell size and heat treatment under pH 4-7 conditions that Bacillus could be present in the form of spores. With the CD method, only Lactococcus lactis, Leu. citreum, and Weissella cibaria were detected. With the CI method, only Pediococcus inopinatus was detected. A notable finding was that Leu. mesenteroides was more abundant than Latilactobacillus sakei with the CD method, whereas it was similar or lower with the CI method. This discrepancy was confirmed to be due to different rates of DNA recovered from the two strains. This shows that the assay method may influence the detection of these two strains.

Association between kimchi consumption and obesity based on BMI and abdominal obesity in Korean adults: a cross-sectional analysis of the Health Examinees study.

OBJECTIVE: Previous animal studies have shown the anti-obesity effect of kimchi-derived probiotic lactic acid bacteria. However, only a few epidemiological studies have investigated the association between kimchi consumption and obesity. Therefore, we aim to assess this relationship in Korean adults. DESIGN: Cross-sectional study. SETTING: The Health Examinees study was conducted from 2004 to 2013. PARTICIPANTS: This study analysed 115 726 participants aged 40-69 years enrolled in the Health Examinees study in Korea. PRIMARY AND SECONDARY OUTCOME MEASURES: Obesity was defined as body mass index ≥25 kg/m2, and abdominal obesity was defined as waist circumference ≥90 cm in men and ≥85 cm in women. Kimchi consumption was assessed by the validated food frequency questionnaire. RESULTS: In men, total kimchi consumption of 1-3 servings/day was related to a lower prevalence of obesity (OR: 0.875 in 1-2 servings/day and OR: 0.893 in 2-3 servings/day) compared with total kimchi consumption of <1 serving/day. Also, men with the highest baechu kimchi (cabbage kimchi) consumption had 10% lower odds of obesity and abdominal obesity. Participants who consumed kkakdugi (radish kimchi) ≥median were inversely associated with 8% in men and 11% in women with lower odds of abdominal obesity compared with non-consumers, respectively. CONCLUSIONS AND RELEVANCE: Consumption of 1-3 servings/day of total kimchi was associated with a lower risk of obesity in men. Baechu kimchi was associated with a lower prevalence of obesity in men, and kkakdugi was associated with a lower prevalence of abdominal obesity in both men and women. However, since all results showed a 'J-shaped' association, it is recommended to limit excessive kimchi intake.

Correction to: Bacterial diversity of baechu-kimchi with seafood based on culture­independent investigations.

[This corrects the article DOI: 10.1007/s10068-023-01471-2.].

Bacterial diversity of baechu-kimchi with seafood based on culture-independent investigations.

Baechu-kimchi is a traditional Korean dish of fermented vegetables, in which kimchi cabbage is the major ingredient. Seafood is added to baechu-kimchi in coastal areas, giving this dish regional diversity. However, little is known about how the addition of seafood affects the bacterial diversity of kimchi. Therefore, in this study, the bacterial diversity of five varieties of baechu-kimchi with seafood and one variety of baechu-kimchi without seafood was analyzed using culture-independent techniques. In 81.7% of all kimchi analyzed, the predominant species were members of the phylum Firmicutes and the lactic acid bacteria, Latilactobacillus sakei, Leuconostoc mesenteroides, Pediococcus inopinatus, and Weissella koreensis. These organisms were similar to those identified in baechu-kimchi without the addition of seafood, which was used as a control group, and bacterial community of previously reported kimchi. Therefore, the results of this study confirmed that the addition of seafood did not significantly affect the bacterial community in baechu-kimchi.

Anti-Obesity Effect of Kimchi with Starter Cultures in 3T3-L1 Cells.

Lactic acid bacteria (LAB) isolated from kimchi have various functions, including antioxidant, anti-inflammation, and anti-obesity activities, and are therefore widely used in the food, pharmaceutical, and medical fields. To date, the health functionalities of LAB have been widely reported; however, those of kimchi fermented with LAB as a starter have rarely been reported. Therefore, research on the selection of LAB with anti-obesity activity and the health functionality of kimchi fermented with LAB is needed. In the present study, LAB with anti-obesity activity were initially selected by measuring the Oil-Red O intensity. Among the four LAB strains, anti-obesity activity was confirmed by measuring cell viability, lipid levels, and lipid accumulation. Then, starter kimchi (SK) was prepared by inoculating selected LABs, and its pH, total acidity, and salinity were compared with those of naturally fermented kimchi (NK). Lastly, anti-obesity activity was also investigated in 3T3-L1 cells. Selected LAB showed no cytotoxicity up to 107 CFU/ml, with Lactobacillus brevis JC7 and Leuconostoc mesenteroides KCKM0828 having higher inhibitory effects on TG, TC content and lipid accumulation. Most SKs showed fermentation properties similar to those of the NK. SKs showed no cytotoxicity at concentrations of up to 1,000 µg/ml. SKs showed strong inhibitory effects on TG content, lipid accumulation, and obesity-related gene and protein expressions. Taken together, the utilization of LAB as a starter could improve the health benefits of kimchi.

Lactobacillus plantarum LPYC225 mixture partially modulates the vaginal bacterial community of Gardnerella vaginalis-infected bacterial vaginosis in mice.

Bacterial vaginosis (BV) is defined as dysbiosis of the vaginal microbiome associated with the depletion of Lactobacilli and excessive growth of commensal or pathogenic bacteria. This study investigated the effects of lactic acid bacteria (LAB) mixture (LM; InoRexyne™) on the vaginal bacterial community of Gardnerella vaginalis (G. vaginalis)-infected BV mice. Single LAB and LM exhibited antibacterial and anti-inflammatory effects by inhibiting G. vaginalis growth and pro-inflammatory markers in RAW 264.7 cells. Administering LM did not significantly alter the vaginal architecture or fecal short-chain fatty acids but did significantly inhibit the vaginal interleukin-6 levels in the high LM group compared to the GV group. LM administration decreased the relative abundances of Enterobacter, Escherichia coli, and Bacteroides vulgatus in vaginal flushing fluids compared to the GV group. LM partially alleviated BV by inhibiting G. vaginalis growth and modulating the vaginal bacterial community, providing new insights into its modulatory effects on the vaginal microbiome in BV. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-024-01641-w.

Effect of kimchi intake on body weight of general community dwellers: a prospective cohort study.

The impact of kimchi intake on weight management has been a topic of interest. We aimed to conduct an epidemiological study to investigate the association between kimchi intake and weight loss. Participants were selected from the Health Examinees cohort study. Kimchi intake was assessed by a 106-item semi-quantitative food frequency questionnaire, including four types of kimchi. Obesity was defined according to the Korean Society for the Study of Obesity guidelines. We performed a correlation analysis among all participants (N = 58 290) and conducted a prospective risk assessment analysis among participants with a baseline BMI value ≥25 kg m-2 (N = 20 066). In the correlation analysis, higher kimchi consumption was found to be associated with a lower increment in BMI change (men, ß 0.169, 95% CI (0.025, 0.313); women, ß 0.140, 95% CI (0.046, 0.236)) compared with the lower group. The risk assessment analysis indicated that moderate kimchi consumption is associated with normal weight development in men (Q3, hazard ratio, 1.28, 95% CI (1.06, 1.54)). Baechu [cabbage] kimchi intake also showed a significant association among men participants (all p for trend <0.05). In conclusion, moderate kimchi intake was associated with weight loss among middle-aged and older Koreans, especially in men.

A Novel Interaction between MFN2/Marf and MARK4/PAR-1 Is Implicated in Synaptic Defects and Mitochondrial Dysfunction.

As cellular energy powerhouses, mitochondria undergo constant fission and fusion to maintain functional homeostasis. The conserved dynamin-like GTPase, Mitofusin2 (MFN2)/mitochondrial assembly regulatory factor (Marf), plays a role in mitochondrial fusion, mutations of which are implicated in age-related human diseases, including several neurodegenerative disorders. However, the regulation of MFN2/Marf-mediated mitochondrial fusion, as well as the pathologic mechanism of neurodegeneration, is not clearly understood. Here, we identified a novel interaction between MFN2/Marf and microtubule affinity-regulating kinase 4 (MARK4)/PAR-1. In the Drosophila larval neuromuscular junction, muscle-specific overexpression of MFN2/Marf decreased the number of synaptic boutons, and the loss of MARK4/PAR-1 alleviated the synaptic defects of MFN2/Marf overexpression. Downregulation of MARK4/PAR-1 rescued the mitochondrial hyperfusion phenotype caused by MFN2/Marf overexpression in the Drosophila muscles as well as in the cultured cells. In addition, knockdown of MARK4/PAR-1 rescued the respiratory dysfunction of mitochondria induced by MFN2/Marf overexpression in mammalian cells. Together, our results indicate that the interaction between MFN2/Marf and MARK4/PAR-1 is fine-tuned to maintain synaptic integrity and mitochondrial homeostasis, and its dysregulation may be implicated in neurologic pathogenesis.