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Increasing genetic evidence supports the hypothesis that variants in the annexin A11 gene (ANXA11) contribute to amyotrophic lateral sclerosis pathogenesis. Therefore, we studied the clinical aspects of sporadic amyotrophic lateral sclerosis patients carrying ANXA11 variants. We also implemented functional experiments to verify the pathogenicity of the hotspot variants associated with amyotrophic lateral sclerosis-frontotemporal dementia. Korean patients diagnosed with amyotrophic lateral sclerosis (n = 882) underwent genetic evaluations through next-generation sequencing, which identified 16 ANXA11 variants in 26 patients. We analysed their clinical features, such as the age of onset, progression rate, initial symptoms and cognitive status. To evaluate the functional significance of the ANXA11 variants in amyotrophic lateral sclerosis-frontotemporal dementia pathology, we additionally utilized patient fibroblasts carrying frontotemporal dementia-linked ANXA11 variants (p.P36R and p.D40G) to perform a series of in vitro studies, including calcium imaging, stress granule dynamics and protein translation. The frequency of the pathogenic or likely pathogenic variants of ANXA11 was 0.3% and the frequency of variants classified as variants of unknown significance was 2.6%. The patients with variants in the low-complexity domain presented unique clinical features, including late-onset, a high prevalence of amyotrophic lateral sclerosis-frontotemporal dementia, a fast initial progression rate and a high tendency for bulbar-onset compared with patients carrying variants in the C-terminal repeated annexin homology domains. In addition, functional studies using amyotrophic lateral sclerosis-frontotemporal dementia patient fibroblasts revealed that the ANXA11 variants p.P36R and p.D40G impaired intracellular calcium homeostasis, stress granule disassembly and protein translation. This study suggests that the clinical manifestations of amyotrophic lateral sclerosis and amyotrophic lateral sclerosis-frontotemporal dementia spectrum patients with ANXA11 variants could be distinctively characterized depending upon the location of the variant.

Background Ankle sprains are one of the most common musculoskeletal injuries. To guide management decisions, a clear insight into the relevant subgroups of patients with a potentially better or worse prognosis is important. This study aimed to evaluate injury severity, using ultrasonography (US), as a prognostic factor of acute ankle sprain and other possible factors including age, sex, body mass index (BMI), level of job activity, and level of sports activity. Materials and methods We retrospectively reviewed 28 patients with acute ankle sprain who reported at initial examination with an acutely twisted ankle. All patients had received a standard physical examination, radiography and standard ultrasound, to diagnose specific ligament injuries and their ankle sprain had been treated using standard conservative management. Various data including age, sex, BMI, level of sports activity, level of daily job activity, and final functional score (Foot and Ankle Outcome Scores, FAOS) were obtained. Mean comparison and correlations were used to assess risk factors. Risk factors associated with functional outcomes were evaluated using a multiple linear regression test. Results At final follow-up as 1 year after injury, FAOS differed significantly for injury severity, age, and BMI. There were no significant differences in sex, job activity, and exercise levels. The factor most affecting FAOS for both pain (FAOS-Pain) and symptoms (FAOS-Sx) was the number of completely torn ligaments. Age was the most important factor affecting the FAOS-Daily Living Activity (ADL). BMI was the most important factor for sports activity level (FAOS-Sports). Age and the number of completely torn ligaments were both important to FAOS-Sports and quality of life (FAOS-QOL). Conclusion The severity of injury, defined using US, was a prognostic factor for long-term outcome following acute ankle sprain. Therefore, US imaging of acute ankle ligament injury may be important to predict prognosis of acute ankle sprain.

In this study, a numerical simulation of edge-defined film-fed growth (EFG) was performed to determine the appropriate capillary conditions for Ga2O3 melt. Meniscus and capillary rise were significantly influenced by the design of the die in the EFG system. The ratio of the seed crystal and die width was >0.73 for a die width of 4.4 mm. Narrower slit width resulted in higher capillary rise with longer process time compared with wider slit width. Under conditions consistent with the simulation results, highly crystalline (100) β-Ga2O3 single crystals were successfully achieved.

Background Previous quantitative proteomic study from our laboratory had identified leucine-rich-alpha-2-glycoprotein 1 (LRG1) as the most prominent marker for colorectal cancer (CRC). This marker was significantly up-regulated in the serum of late-stage CRC patients and present in a stage-dependent manner in the CRC cells. Many studies have linked autophagy to cancer, but none had explored the relationship between LRG1 and autophagy in CRC. This study (FRGS/1/2018/SKK08/UKM/03/3) is aimed to investigate the effect of LRG1 modulation on the autophagy mechanism in colorectal cancer. Methods Colorectal adenocarcinoma cells, HT29 (low-grade CRC) with the lowest endogenous level of LRG1 were chosen for the over-expression of human LRG1 gene, whereas colorectal carcinoma cells, HCT116 (high-grade CRC) with the highest endogenous level of LRG1 were used for the LRG1 knockdown experiment. The cells were stably transduced with lentivirus containing LRG1 open reading frame (ORFs) and short hairpin RNA (shRNA), respectively. The selection was carried out with Blasticidin S and Puromycin, respectively prior to RNA extraction and gene expression assessment via RT-qPCR. Statistical analysis of Independent T-test was performed using GraphPad Prism 8.0.1. Results Over-expression of LRG1 in HT29 increased cell proliferation by activation of Ki67 mRNA (p 0.05), but several autophagy markers (Beclin-1, ATG4D, ATG5, LC3 and GABARAP, except ATG3) were in a decreasing trend. Conclusions In summary, LRG1 enhanced the survival of low-grade colorectal cancer and tumour progression by suppression of autophagy but failed to reverse the mechanism in the knockdown model of high-grade cancer. More related works are currently on-going.

Autophagy is a conserved cellular process required to maintain homeostasis. The hallmark of autophagy is the formation of a phagophore that engulfs cytosolic materials for degradation and recycling to synthesize essential components. Basal autophagy is constitutively active under normal conditions and it could be further induced by physiological stimuli such as hypoxia, nutrient starvation, endoplasmic reticulum stress,energy depletion, hormonal stimulation and pharmacological treatment. In cancer, autophagy is highly context-specific depending on the cell type, tumour microenvironment, disease stage and external stimuli. Recently, the emerging role of autophagy as a double-edged sword in cancer has gained much attention. On one hand, autophagy suppresses malignant transformation by limiting the production of reactive oxygen species and DNA damage during tumour development. Subsequently, autophagy evolved to support the survival of cancer cells and promotes the tumourigenicity of cancer stem cells at established sites. Hence, autophagy is an attractive target for cancer therapeutics and researchers have been exploiting the use of autophagy modulators as adjuvant therapy. In this review, we present a summary of autophagy mechanism and controlling pathways, with emphasis on the dual-role of autophagy (double-edged sword) in cancer. This is followed by an overview of the autophagy modulation for cancer treatment and is concluded by a discussion on the current perspectives and future outlook of autophagy exploitation for precision medicine.

Dysregulation of calcium ion homeostasis and abnormal protein aggregation have been proposed as major pathogenic hallmarks underpinning selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS). Recently, mutations in annexin A11 (ANXA11), a gene encoding a Ca2+-dependent phospholipid-binding protein, have been identified in familial and sporadic ALS. However, the physiological and pathophysiological roles of ANXA11 remain unknown. Here, we report functions of ANXA11 related to intracellular Ca2+ homeostasis and stress granule dynamics. We analyzed the exome sequences of 500 Korean patients with sALS and identified nine ANXA11 variants in 13 patients. The amino-terminal variants p.G38R and p.D40G within the low-complexity domain of ANXA11 enhanced aggregation propensity, whereas the carboxyl-terminal ANX domain variants p.H390P and p.R456H altered Ca2+ responses. Furthermore, all four variants in ANXA11 underwent abnormal phase separation to form droplets with aggregates and led to the alteration of the biophysical properties of ANXA11. These functional defects caused by ALS-linked variants induced alterations in both intracellular Ca2+ homeostasis and stress granule disassembly. We also revealed that p.G228Lfs*29 reduced ANXA11 expression and impaired Ca2+ homeostasis, as caused by missense variants. Ca2+-dependent interaction and coaggregation between ANXA11 and ALS-causative RNA-binding proteins, FUS and hnRNPA1, were observed in motor neuron cells and brain from a patient with ALS-FUS. The expression of ALS-linked ANXA11 variants in motor neuron cells caused cytoplasmic sequestration of endogenous FUS and triggered neuronal apoptosis. Together, our findings suggest that disease-associated ANXA11 mutations can contribute to ALS pathogenesis through toxic gain-of-function mechanisms involving abnormal protein aggregation.

Eight carbon (C8) compounds are the key characteristic flavors of mushrooms. The quantitative analysis of the volatiles in mushrooms is challenging especially with the unattended HS-SPME-GC-MS. An unattended HS-SPME-GC-MS/MS in combination with novel sample preparation of the complete control of volatile emissions was developed for the quantitation of the C8 volatiles in mushrooms. The sample preparation strategy was composed of freeze-drying, rehydration, and the addition of a 15% citric acid solution. With this strategy, the volatile emission from mushroom was fully controlled at a certain time point. This method was found to be highly reliable, sensitive, precise, and accurate. This method was successfully applied to measure the contents of the C8 volatiles in the beech, button, and shiitake mushrooms. 1-Octene-3-ol was the most predominant compound in the mushrooms, representing 62.4, 69.0, and 89.2% of the total C8 volatiles in the beech, button, and shiitake mushrooms, respectively.

Aronia melamocarpa (AM) is a rich source of anthocyanins, which are known to help prevent obesity. The cyanidine-3-O-galactoside enriched AM extract (AM-Ex) containing more cyanidine-3-O-galactoside than conventional AM extract was recently developed. The objective of this study was to examine the effect of AM-Ex on adipogenesis and its action mechanisms in vitro using 3T3-L1 adipocytes. To examine the anti-obesity effect of AM-Ex, 3T3-L1 cells were induced adipocyte differentiation and incubated with various concentration of AM-Ex. Lipid accumulation, cellular triglyceride content, mRNA expression of transcription factors and adipogenic genes were analyzed. Treatment with 100 - 400 μg/mL of AM-Ex resulted in a dose-dependent decrease in adipocyte differentiation and triglyceride accumulation. mRNA expression of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein α, sterol regulatory element-binding protein 1 were decreased. The level of gene expression of adipogenesis and lipogenesis-related genes, such as adipocyte protein 2, lipoprotein lipase, acetyl-CoA carboxylase, ATP-citrate lyase and fatty acid synthase were decreased. These results suggest that AM-Ex alleviated risk factors related to obesity by modulating multiple pathways associated with adipogenesis.

Fused in sarcoma (FUS) is a DNA/RNA-binding protein that is involved in DNA repair and RNA processing. FUS is associated with neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the molecular mechanisms underlying FUS-mediated neurodegeneration are largely unknown. Here, using a Drosophila model, we showed that the overexpression of glutathione transferase omega 2 (GstO2) reduces cytoplasmic FUS aggregates and prevents neurodegenerative phenotypes, including neurotoxicity and mitochondrial dysfunction. We found a FUS glutathionylation site at the 447

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation.

In the present study, we examined whether Lactobacillus johnsonii CJLJ103 (LJ) could alleviate cholinergic memory impairment in mice. Oral administration of LJ alleviated scopolamine-induced memory impairment in passive avoidance and Y-maze tasks. Furthermore, LJ treatment increased scopolamine-suppressed BDNF expression and CREB phosphorylation in the hippocampi of the brain, as well as suppressed TNF-α expression and NF-κB activation. LJ also increased BDNF expression in corticosterone-stimulated SH-SY5Y cells and inhibited NF-κB activation in LPS-stimulated microglial BV2 cells. However, LJ did not inhibit acetylcholinesterase activity. These findings suggest that LJ, a member of human gut microbiota, may mitigate cholinergic memory impairment by increasing BDNF expression and inhibiting NF-κB activation.

BACKGROUND/OBJECTIVE There is intense interest in soy isoflavone as a hormone replacement therapy for the prevention of postmenopausal osteoporosis. A new kind of isoflavone-enriched whole soy milk powder (I-WSM) containing more isoflavones than conventional whole soy milk powder was recently developed. The aim of this study was to investigate the effects of I-WSM on bone metabolism in ovariectomized mice. MATERIALS/METHODS Sixty female ICR mice individually underwent ovariectomy (OVX) or a sham operation, and were randomized into six groups of 10 animals each as follows: Sham, OVX, OVX with 2% I-WSM diet, OVX with 10% I-WSM diet, OVX with 20% I-WSM diet, and OVX with 20% WSM diet. After an 8-week treatment period, bone mineral density (BMD), calcium, alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) 5b, osteocalcin (OC), procollagen 1 N-terminal propeptide (P1NP), and osteoprotegenin (OPG) were analyzed. RESULTS BMD was significantly lower in the OVX group compared to the Sham group but was significantly higher in OVX + 10% I-WSM and OVX + 20% I-WSM groups compared to the OVX group (P < 0.05). Serum calcium concentration significantly increased in the OVX + 10% and 20% I-WSM groups. Serum ALP levels were significantly lower in the OVX + 10% and 20% I-WSM groups compared to the other experimental groups (P < 0.05). OC was significantly reduced in the OVX group compared to the Sham group (P < 0.05), but a dose-dependent increase was observed in the OVX groups supplemented with I-WSM. P1NP and OPG levels were significantly reduced, while TRAP 5b level was significantly elevated in the OVX group compared with the Sham group, which was not affected by I-WSM (P < 0.05). CONCLUSIONS This study suggests that I-WSM supplementation in OVX mice has the effect of preventing BMD reduction and promoting bone formation. Therefore, I-WSM can be used as an effective alternative to postmenopausal osteoporosis prevention.

The aim of the present study was to evaluate the physiochemical properties and resorption progress of two cross-linked, porcine skin-derived collagen membranes and compare their features with those of a membrane without cross-linking (Bio-Gide® [BG], Geistlich Biomaterials, Wolhusen, Switzerland). Three porcine skin-derived collagen membranes, dehydrothermally (DHT) cross-linked (experimental), DHT and 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (DHT/EDC) cross-linked (experimental) and BG were investigated for their morphology, enzyme resistance, and tensile strength in vitro and biodegradation in vivo. DHT and DHT/EDC membranes exhibited irregular, interconnected macro- and micropores that formed a 3D mesh, whereas BG exhibited individual collagen fibrils interlaced to form coarse collagen strands. In enzyme resistance and tensile strength tests, DHT and DHT/EDC membranes demonstrated good resistance and mechanical properties compared with BG. In vivo, all three membranes were well integrated into the surrounding connective tissue. Thus, the DHT membrane exhibited its potential as a barrier membrane for guided bone and tissue regeneration.

Lactobacillus plantarum C29-fermented defatted soybean (FDS), which contains soyasaponins such as soyasaponin I (SI) and soyasapogenol B (SB) and isoflavones such as genistin (GE) and genistein (GT), attenuated memory impairment in mice. Moreover, in the preliminary study, FDS and its soyasaponins and isoflavones significantly inhibited NF-κB activation in LPS-stimulated microglial BV2 cells. Therefore, we examined the effects of FDS and its constituents SI, SB, GT, and GE on LPS-induced memory impairment in mice. Oral administration of FDS (80 mg/kg), which has higher concentrations of SB and GE than DS, recovered LPS-impaired cognitive function in Y-maze (55.1 ± 3.5%) and passive avoidance tasks (50.9 ± 19.2 s) to 129.2% (74.1 ± 3.5%) and 114.2% (290.0 ± 22.4 s) of normal mice, respectively (P0.05). SB and GE (10 μM) also more potently attenuated LPS-impaired cognitive behavior than SI and GT, respectively. SB (10 mg/kg) was the most effective: treatment recovered LPS-impaired spontaneous alternation and latency time to 105.7% and 126.8% of normal control mice, respectively (P0.05). SB and GE significantly increased BDNF expression and CREB phosphorylation in LPS-treated mice and corticosterone-stimulated SH-SY5Y cells. Furthermore, SB and GE (10 μM) also significantly inhibited NF-κB activation in LPS-treated mice. These findings suggested that FDS and its constituent soyasaponins and isoflavones may attenuate memory impairment by the regulation of NF-κB-mediated BDNF expression.

Exposure to excessive lipopolysaccharide (LPS) by the gut microbiota disturbance causes ulcerative colitis and memory impairment. Therefore, we isolated anti-inflammatory Lactobacillus johnsonii CJLJ103 (LJ) from human fecal microbiota as an inhibitor for Escherichia coli growth and LPS production and investigated anti-colitic and memory impairment-ameliorating effects in mice. Oral administration of LJ inhibited 2,3,6-trinitrobenzenesulfonic acid (TNBS)-induced colon shortening, colonic myeloperoxidase activity, and NF-κB activation in mice. Furthermore, LJ inhibited TNBS-induced TNF and IL-1β expression in the colon but increased TNBS-suppressed expression of IL-10 and tight-junction proteins. Treatment with LJ inhibited fecal LPS levels and proteobacteria/bacteroidetes ratio in mice with TNBS-induced colitis. Furthermore, LJ ameliorated LPS-induced memory impairment in mice. LJ also suppressed LPS-induced NF-κB activation in the hippocampus and increased LPS-suppressed brain-derived neurotrophic factor expression. These findings suggest that LJ, a member of human gut microbiota, may ameliorate colitis and memory impairment, thereby acting as a useful probiotic.

Gut microbiota play essential roles in the regulation of human metabolism via symbiotic interactions with the host. Prolonged consumption of high-fat diet (HFD) elevates the Firmicutes to Bacteroidetes ratio and lipopolysaccharide (LPS) production by gut microbiota, thereby increasing the probability of developing metabolic and immune disorders such as obesity and colitis. The use of probiotics with anti-inflammatory properties has been suggested to counteract this effect. Here, we tested whether Bifidobacterium adolescentis IM38, which inhibited nuclear factor-kappa B (NF-κB) activation in Caco-2 cells and peritoneal macrophages and inhibited Escherichia coli LPS production, exerted an anticolitic effect in mice with HFD-induced obesity. Oral administration of IM38 (2×109CFU/mouse per day) for 6 weeks in mice with HFD-induced obesity inhibited whole-body and epididymal fat weight gain. IM38 also increased HFD-suppressed expression of interleukin (IL)-10 and tight junction proteins but significantly downregulated HFD-induced NF-κB activation and tumor necrosis factor expression in the colon. IM38 inhibited differentiation into helper T17 cells and reduced IL-17 levels in the colon of mice with HFD-induced obesity but increased HFD-suppressed differentiation into regulatory T cells and IL-10 levels. Furthermore, treatment with IM38 lowered the HFD-induced LPS levels in blood and colonic fluid, as well as the Proteobacteria to Bacteroidetes ratio in gut microbiota. Therefore, we suggest that IM38 can inhibit HFD-induced LPS production in gut microbiota through the regulation of Proteobacteria to Bacteroidetes ratio and NF-κB activation in the colon, which ultimately attenuates colitis. Thus, IM38 may be a suitable ingredient of functional foods designed for treating or preventing colitis.

The roots of Abrus precatorius (AP, Fabaceae) have traditionally been used in Vietnam and China for the treatment of inflammatory diseases such as stomatitis, asthma, bronchitis, and hepatitis. Therefore, in this study, we isolated 4-methoxylonchocarpin (ML), an anti-inflammatory compound present in AP, and studied its anti-inflammatory effects in mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. In lipopolysaccharide (LPS)-stimulated macrophages, ML was found to inhibit nuclear factor (NF)-κB activation and tumor necrosis factor (TNF) and interleukin (IL)-6 expression by inhibiting LPS binding to Toll-like receptor 4 (TLR4) in vitro. Oral administration of ML in mice with TNBS-induced colitis suppressed colon shortening and colonic myeloperoxidase activity. ML treatment significantly inhibited the activation of nuclear factor (NF)-κB and phosphorylation of transforming growth factor β-activated kinase 1 in the colon. Treatment with ML also inhibited TNBS-induced expression of IL-1β, IL-17A, and TNF. While ML reduced the TNBS-induced expression of M1 macrophage markers such as arginase-2 and TNF, it was found to increase the expression of M2 macrophage markers such as arginase-1 and IL-10. In conclusion, oral administration of ML attenuated colitis in mice by inhibiting the binding of LPS to TLR4 on immune cells and increasing the polarization of M1 macrophages to M2 macrophages.

Thymoma is associated with a wide spectrum of autoimmune paraneoplastic syndromes, though it is uncommon for multiple paraneoplastic syndromes to be present in a single individual. We report a rare case of an elderly gentleman who was found to have thymoma-associated myasthenia gravis and LGI1-encephalitis with myokymia, who presented with nephrotic syndrome (minimal change glomerulopathy) after thymectomy. The latter two paraneoplastic syndromes had manifested when prednisolone was tapered down to low dose. This case serves to remind neurologists that apart from paraneoplastic neurological manifestations, thymoma may also be associated with renal disease. Nephropathy in myasthenia patients with thymoma should be properly evaluated, as it is treatable with immunotherapy, and it may even occur post-thymectomy.

Cholesterol metabolism operates autonomously within the central nervous system (CNS), where the majority of cholesterol resides in myelin. We demonstrate that TDP-43, the pathological signature protein for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), influences cholesterol metabolism in oligodendrocytes. TDP-43 binds directly to mRNA of SREBF2, the master transcription regulator for cholesterol metabolism, and multiple mRNAs encoding proteins responsible for cholesterol biosynthesis and uptake, including HMGCR, HMGCS1, and LDLR. TDP-43 depletion leads to reduced SREBF2 and LDLR expression, and cholesterol levels in vitro and in vivo. TDP-43–mediated changes in cholesterol levels can be restored by reintroducing SREBF2 or LDLR. Additionally, cholesterol supplementation rescues demyelination caused by TDP-43 deletion. Furthermore, oligodendrocytes harboring TDP-43 pathology from FTD patients show reduced HMGCR and HMGCS1, and coaggregation of LDLR and TDP-43. Collectively, our results indicate that TDP-43 plays a role in cholesterol homeostasis in oligodendrocytes, and cholesterol dysmetabolism may be implicated in TDP-43 proteinopathies–related diseases.

Gynostemma pentaphyllum is widely used in Asia as a herbal medicine to treat type 2 diabetes, dyslipidemia, and inflammation. Here, we investigated the anti-obesity effect and underlying mechanism of G. pentaphyllum extract (GPE) enriched in gypenoside L, gypenoside LI, and ginsenoside Rg3 and obtained using a novel extraction method. Five-week-old male C57BL/6N mice were fed a control diet (CD), high-fat diet (HFD), HFD + 100 mg/kg body weight (BW)/day GPE (GPE 100), HFD + 300 mg/kg BW/day GPE (GPE 300), or HFD + 30 mg/kg BW/day Orlistat (Orlistat 30) for 8 weeks. The HFD-fed mice showed significant increases in body weight, fat mass, white adipose tissue, and adipocyte hypertrophy compared to the CD group, but GPE inhibited those increases. GPE reduced serum levels of triglyceride, total cholesterol, and LDL-cholesterol, without affecting HDL-cholesterol. GPE significantly increased AMPK activation and suppressed adipogenesis by decreasing the mRNA expression of CCAAT/enhancer binding protein-&alpha, (C/EBP&alpha, ), peroxisome proliferator-activated receptor-&gamma, (PPAR&gamma, ), sterol regulatory element-binding protein-1c (SREBP1c), PPAR&gamma, coactivator-1&alpha, fatty acid synthase (FAS), adipocyte protein 2 (AP2), and sirtuin 1 (SIRT1) and by increasing that of carnitine palmitoyltransferase (CPT1) and hormone- sensitive lipase (HSL). This study demonstrated the ameliorative effect of GPE on obesity and elucidated the underlying molecular mechanism.

Aronia melanocarpa are a rich source of anthocyanins that have received considerable interest for their relations to human health. In this study, the anti-adipogenic effect of cyanidin-3-O-galactoside-enriched Aronia melanocarpa extract (AM-Ex) and its underlying mechanisms were investigated in an in vivo system. Five-week-old male C57BL/6N mice were randomly divided into five groups for 8-week feeding with a control diet (CD), a high-fat diet (HFD), or a HFD with 50 (AM-Ex 50), 100 (AM-Ex 100), or 200 AM-Ex (AM-Ex 200) mg/kg body weight/day. HFD-fed mice showed a significant increase in body weight compared to the CD group, and AM-Ex dose-dependently inhibited this weight gain. AM-Ex significantly reduced the food intake and the weight of white fat tissue, including epididymal fat, retroperitoneal fat, mesenteric fat, and inguinal fat. Treatment with AM-Ex (50 to 200 mg/kg) reduced serum levels of leptin, insulin, triglyceride, total cholesterol, and low density lipoprotein (LDL)-cholesterol. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AM-Ex suppressed adipogenesis by decreasing CCAAT/enhancer binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor gamma coactivator-1α, acetyl-CoA carboxylase 1, ATP-citrate lyase, fatty acid synthase, and adipocyte protein 2 messenger RNA (mRNA) expressions. These results suggest that AM-Ex is potentially beneficial for the suppression of HFD-induced obesity by modulating multiple pathways associated with adipogenesis and food intake.

Anemarrhena asphodeloides (AA, family Liliaceae) inhibits macrophage activation by inhibiting IRAK1 phosphorylation and helper T (Th)17 differentiation. Coptis chinensis (CC, family Ranunculaceae), which inhibits macrophage activation by inhibiting the binding of lipopolysaccharide (LPS) on toll-like receptor 4 and inducing regulatory T (Treg) cell differentiation. The mixture of AA and CC (AC-mix) synergistically attenuates 2,4,6-trinitrobenzenesulfonic acid or dextran sulfate sodium-induced colitis in mice by inhibiting NF-[Formula: see text]B activation and regulating Th17/Treg balance. In the present study, we examined the effect of AC-mix on high-fat diet (HFD)-induced colitis in mice, which induced NF-[Formula: see text]B activation and disturbed Th17/Treg balance. Long-term feeding of HFD in mice caused colitis, including increased macroscopic score and myeloperoxidase activity. Oral administration of AC-mix (20[Formula: see text]mg/kg) suppressed HFD-induced myeloperoxidase activity by 68% ([Formula: see text]). Furthermore, treatment with the AC-mix (20[Formula: see text]mg/kg) inhibited HFD-induced activation of NF-[Formula: see text]B and expression of cyclooxygenase-2, inducible NO synthase, interleukin (IL)-17, and tumor necrosis factor-alpha but increased HFD- suppressed expression of IL-10. AC-mix suppressed HFD-induced differentiation into Th17 cells by 46% ([Formula: see text]) and increased HFD-induced differentiation into regulatory T cells 2.2-fold ([Formula: see text]). AC-mix also suppressed the HFD-induced Proteobacteria/Bacteroidetes ratio on the gut microbiota by 48% ([Formula: see text]). These findings suggest that AC-mix can ameliorate HFD-induced colitis by regulating innate and adaptive immunities and correcting the disturbance of gut microbiota.

In the present study, we isolated Bifidobacterium longum CH57, which suppresses macrophage activation, from human gut microbiota and Lactobacillus brevis CH23, which inhibit Th17 cell differentiation, from kimchi, and investigated anti-inflammatory effects of CH23, CH57, and their probiotic mixture (PM) in mice with TNBS-induced colitis. CH57 inhibited NF-κB activation in LPS-stimulated macrophages. Oral administration of CH57 in mice attenuated TNBS-induced colitis and TNF-α expression. CH23 inhibited IL-17 and RORγt expression in splenic T cells. Oral administration of CH23 in mice inhibited TNBS-induced colon shortening, myeloperoxidase activity, and Th17 cell differentiation and induced TNBS-suppressed Treg differentiation. PM increased TNBS-suppressed claudin, occludin, and ZO-1 expression and Treg differentiation, while CH57 inhibited Th17 cell differentiation and RORγt and IL-17 expression. PM inhibited TNBS-induced macrophage activation. Moreover, CH23 and CH57 synergistically inhibited macrophage activation and Th17 cell differentiation in vitro. The PM may synergistically ameliorate colitis by inhibiting macrophage activation and restoring Th17/Treg balance.

In this study, we investigated the effects of antibiotics on the pharmacological effects of aspirin. The antithrombotic activity of aspirin was evaluated after antibiotic treatment using tail bleeding assay. The pyrosequencing analysis and selective medium culture assay were performed to investigate the alterations in gut microbiota. In addition, the in vitro metabolism assay with fecal suspension and in vivo pharmacokinetic experiments with antibiotic treatment were conducted. Ampicillin treatment significantly prolonged the bleeding time in aspirin-dosed rats. Oral administration of ampicillin significantly reduced gut microbial aspirin-metabolizing activity by 67.0% in rats. Furthermore, systemic exposure to aspirin and its primary metabolite (M1) was significantly increased in ampicillin-treated rats. The results from the pyrosequencing and selective medium culture with rat fecal samples revealed that ampicillin treatment led to the changes of the amounts and composition profile of gut microbiota. These findings suggest that co-administration of antibiotics can modulate the metabolism and pharmacokinetics of aspirin via suppression of metabolic activity of gut microbiota, which could potentiate the therapeutic potency of aspirin.

The type II C-type lectin CLEC4C is a transmembrane protein selectively expressed on plasmacytoid dendritic cells (PDCs). Although its mechanism of action remains unclear, triggering of the extracellular C-terminal C-type carbohydrate recognition region of CLEC4C regulates the secretion of proinflammatory cytokines and type I IFNs in PDCs. Applying whole-exome sequencing in a patient with juvenile amyotrophic lateral sclerosis (ALS) and both healthy parents, we identified a de novo CLEC4C variant (c.629_631delAGA; p.Lys210del). In this study, we report that the deletion of a lysine residue at the extracellular region of CLEC4C yields a C-terminal dilysine motif that results in endoplasmic reticulum (ER) retention of the protein in transfected HeLa and Jurkat T lymphoma cell models. As a consequence, a decrease in the surface expression of CLEC4C and the ER localization of the mutant construct were observed. Furthermore, depletion of the cell surface CLEC4C level was also observed in the patient PDCs, further suggesting that the variant p.Lys210del CLEC4C may contribute to juvenile ALS susceptibility.

Background Anemarrhena asphodeloides (Liliaceae family) and Mangifera indica L. (Anacardiaceae family) contain neomangiferin as the main active constituent and have been used to treat inflammation, asthma, and pain. Purpose A preliminary study found that neomangiferin inhibited splenic T cell differentiation into Th17 cells and promoted Treg cell production in vitro. Therefore, we examined its anti-colitic effects in vitro and in vivo. Methods Splenocytes isolated from C57BL/6 J mice were treated with neomangiferin. Colitis was either induced in vivo by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) to C57BL/6 J mice or occurred spontaneously in colitis caused by interleukin (IL)-10 knockout at age of 13 weeks. Mice were treated daily with neomangiferin or sulfasalazine. Inflammatory markers, cytokines, enzymes and transcription factors were measured by enzyme-linked immunosorbent assay, immunoblot, and flow cytometry. Results Neomangiferin suppressed retinoic acid receptor-related orphan receptor gamma t (RORγt) and IL-17 expression in IL-6/transforming growth factor β-stimulated Th17 splenocytes and increased IL-10 expression in vitro. Mouse TNBS-induced colon shortening, macroscopic score, and myeloperoxidase activity were inhibited by neomangiferin, which also reduced TNBS-induced activation of nuclear factor-κB and extracellular signal-regulated kinases, as well as expression of inducible nitric oxide synthase and cyclooxygenase-2. In addition, neomangiferin inhibited TNBS-induced expression of tumor necrosis factor-α, IL-17, IL-6, and IL-1β, and increased IL-10 expression. Neomangiferin inhibited TNBS-induced differentiation to Th17 cells and promoted the development of Treg cells. Moreover, in IL-10−/− mice, neomangiferin inhibited colonic myeloperoxidase activity, suppressed Th17 cell differentiation, and reduced levels of TNF-α and IL-17. Conclusion Neomangiferin may restore the balance between Th17/Treg cells by suppressing IL-17 and RORγt expression and inducing IL-10 and forkhead box P3 expression, thus ameliorating colitis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is frequently linked to microtubule abnormalities and mitochondrial trafficking defects. Whole exome sequencing (WES) of patient-parent trios has proven to be an efficient strategy for identifying rare de novo genetic variants responsible for sporadic ALS (sALS). Using a trio-WES approach, we identified a de novo RAPGEF2 variant (c.4069G>A, p.E1357K) in a patient with early-onset sALS. To assess the pathogenic effects of this variant, we have used patient-derived skin fibroblasts and motor neuron-specific overexpression of the RAPGEF2-E1357K mutant protein in Drosophila. Patient fibroblasts display reduced microtubule stability and defective microtubule network morphology. The intracellular distribution, ultrastructure, and function of mitochondria are also impaired in patient cells. Overexpression of the RAPGEF2 mutant in Drosophila motor neurons reduces the stability of axonal microtubules and disrupts the distribution of mitochondria to distal axons and neuromuscular junction (NMJ) synapses. We also show that the recruitment of the pro-apoptotic protein BCL2-associated X (BAX) to mitochondria is significantly increased in patient fibroblasts compared with control cells. Finally, increasing microtubule stability through pharmacological inhibition of histone deacetylase 6 (HDAC6) rescues defects in the intracellular distribution of mitochondria and BAX. Overall, our data suggest that the RAPGEF2 variant identified in this study can drive ALS-related pathogenic effects through microtubule dysregulation., Graphical Abstract

Recently, studies on the relationship between gut dysbiosis and Parkinson’s disease (PD) have increased, but whether a specific gut bacterium may cause PD remains unexplored. Here, we report, for the first time, that a specific gut bacterium directly induces PD symptoms and dopaminergic neuronal damage in the mouse brain. We found that the number of Enterobacteriaceae, particularly Proteus mirabilis, markedly and commonly increased in PD mouse models. Administration of P. mirabilis isolated from PD mice significantly induced motor deficits, selectively caused dopaminergic neuronal damage and inflammation in substantia nigra and striatum, and stimulated α-synuclein aggregation in the brain as well as in the colon. We found that lipopolysaccharides, a virulence factor of P. mirabilis, may be associated in these pathological changes via gut leakage and inflammatory actions. Our results suggest a role of P. mirabilis on PD pathogenesis in the brain.

The aim of the present study was to evaluate the physiochemical properties and resorption progress of two cross-linked, porcine skin-derived collagen membranes and compare their features with those of a membrane without cross-linking (Bio-Gide

Orally administered probiotics change gut microbiota composition and enzyme activities. Thus, coadministration of probiotics with drugs may lead to changes in the pharmacokinetic parameters of the drugs. In this study, we investigated the pharmacokinetics of acetaminophen in mice treated with probiotics. Oral administration of probiotics changed the gut microbiota composition in the mice. Of these probiotics, Lactobacillus reuteri K8 increased the numbers of clostridia, bifidobacteria, and enterococci, and Lactobacillus rhamnosus K9 decreased the number of bifidobacteria, determined by culturing in selective media. Next, we performed a pharmacokinetic study of acetaminophen in mice orally treated with K8 and K9 for 3 days. Treatment with K8 reduced the area under the curve (AUC) of orally administered acetaminophen to 68.4% compared with normal control mice, whereas K9 did not affect the AUC of acetaminophen. Oral administration to mice of K8, which degraded acetaminophen, increased the degradation of acetaminophen by gut microbiota, whereas K9 treatment did not affect it. Treatment with K8 increased the number of L. reuteri adhered in the upper small intestine, whereas the number of L. rhamnosus was not affected by treatment with K8 or K9. K8 increased the number of cyanobacteria, whereas K9 increased the number of deferribacteres. These results suggest that the intake of probiotics may make the absorption of orally administered drugs fluctuate through the disturbance of gut microbiota-mediated drug metabolism and that the subsequent impact on microbiota metabolism could result in altered systemic concentrations of the intact drug.

Vaccination is the most efficient method for infectious disease prevention. Parenteral injections such as intramuscular, intradermal, and subcutaneous injections have several advantages in vaccine delivery, but there are many drawbacks. Thus, the development of a new vaccine delivery system has long been required. Recently, microneedles have been attracting attention as new vaccination tools. Microneedle is a highly effective transdermal vaccine delivery method due to its mechanism of action, painlessness, and ease of use. Here, we summarized the characteristics of microneedles and the possibilities as a new vaccine delivery route.

Alpha-melanocyte stimulating hormone (α-MSH) is known as a regulator of the blood glucose homeostasis and food intake. In the present study, the possible roles of α-MSH located in the spinal cord in the regulation of the blood glucose level were investigated in d-glucose-fed and immobilization stress (IMO) mouse models. We found in the present study that intrathecal (i.t.) injection with α-MSH alone did not affect the blood glucose level. However, i.t. administration with α-MSH reduced the blood glucose level in d-glucose-fed model. The plasma insulin level was increased in d-glucose-fed model and was further increased by α-MSH, whereas α-MSH did not affect plasma corticosterone level in d-glucose-fed model. In addition, i.t. administration with glucagon alone enhanced blood glucose level and, i.t. injection with glucagon also increased the blood glucose level in d-glucose-fed model. In contrasted to results observed in d-glucose-fed model, i.t. treatment with α-MSH caused enhancement of the blood glucose level in IMO model. The plasma insulin level was increased in IMO model. The increased plasma insulin level by IMO was reduced by i.t. treatment with α-MSH, whereas i.t. pretreatment with α-MSH did not affect plasma corticosterone level in IMO model. Taken together, although spinally located α-MSH itself does not alter the blood glucose level, our results suggest that the activation of α-MSH system located in the spinal cord play important modulatory roles for the reduction of the blood glucose level in d-glucose fed model whereas α-MSH is responsible for the up-regulation of the blood glucose level in IMO model. The enhancement of insulin release may be responsible for modulatory action of α-MSH in down-regulation of the blood glucose in d-glucose fed model whereas reduction of insulin release may be responsible for modulatory action of α-MSH in up-regulation of the blood glucose in IMO model.

Meglitinides (nateglinide and repaglinide) are widely used oral drugs for the treatment of type II diabetes mellitus. In the present study, the effects of meglinitides administered supraspinally on kainic acid (KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30 μg of nateglinide and repaglinide for 10 min and then, mice were administered i.c.v. with KA (0.1 μg). The neuronal cell death in the CA3 region in the hippocampus was assessed 24h after KA administration and the blood glucose level was measured 30, 60, and 120 min after KA administration. We found that i.c.v. pretreatment with repaglinide attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and hyperglycemia. However, nateglinide pretreated i.c.v. did not affect the KA-induced neuronal cell death and hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corticosterone level and a reduction of the plasma insulin level. Furthermore, i.c.v. pretreatment with repaglinide attenuated KA-induced up-regulation of plasma corticosterone level. Furthermore, i.c.v. administration of repaglinide alone increased plasma insulin level and repaglinide pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered repaglinide, but not nateglinide, exerts a protective effect against the KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of repaglinide appears to be mediated by lowering the blood glucose level induced by KA.

Sulfonylureas are widely used oral drugs for the treatment of type II diabetes mellitus. In the present study, the effects of sulfonylureas administered supraspinally on kainic acid (KA)-induced hippocampal neuronal cell death and hyperglycemia were studied in ICR mice. Mice were pretreated intracerebroventricularly (i.c.v.) with 30μg of tolbutamide, glyburide or glipizide for 10min and then, mice were administered i.c.v. with KA (0.1μg). The neuronal cell death in the CA3 region in the hippocampus was assessed 24h after KA administration and the blood glucose level was measured 30, 60, and 120min after KA administration. We found that i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated the KA-induced neuronal cell death in CA3 region of the hippocampus and hyperglycemia. In addition, KA administered i.c.v. caused an elevation of plasma corticosterone level and a reduction of the plasma insulin level. The i.c.v. pretreatment with tolbutamide, glyburide or glipizide attenuated KA-induced increase of plasma corticosterone level. Furthermore, i.c.v. pretreatment with tolbutamide, glyburide or glipizide causes an elevation of plasma insulin level. Glipizide, but not tolbutamide or glyburide, pretreated i.c.v. caused a reversal of KA-induced hypoinsulinemic effect. Our results suggest that supraspinally administered tolbutamide, glyburide and glipizide exert a protective effect against KA-induced neuronal cells death in CA3 region of the hippocampus. The neuroprotective effect of tolbutamide, glyburide and glipizide appears to be mediated by lowering the blood glucose level induced by KA.

Background/Aims: To show whether intrathecal (i.t.) treatment with pertussis toxin (PTX) produces a hypoglycemic effect in ICR, db/db and streptozotocin-treated mice. Methods: The blood glucose level (BGL) was measured after i.t. treatment with PTX, AB5 toxins and PTX subunits. Insulin or leptin levels were measured after PTX injection. The effect of PTX on the BGL was examined in adrenalectomized (ADX) mice. Glucose transporter (GLUT) levels were determined by Western blotting. Results: PTX attenuated the elevated BGL in the D-glucose-fed model in a long-term manner. Heat-labile toxin (HLT), HLT subunit B or Shiga toxin, which belong to the AB5 toxins, administered i.t. did not affect the BGL. PTX A protomer (PTX-A) or PTX B oligomers (PTX-B) injected i.t. did not have an effect on the BGL as well. However, combined treatment with PTX-A and PTX-B subunits caused a hypoglycemic effect. The leptin level was gradually reduced by PTX for up to 6 days, without affecting the insulin level. PTX administered i.t. significantly decreased the BGL further in ADX mice. Moreover, GLUT-2 (hypothalamus and pituitary gland), GLUT-4 (muscle) and GLUT-3 (adrenal gland) expression levels were increased, whereas GLUT-1 (brain cortex, liver, muscle and spinal cord), GLUT-2 (liver) and GLUT-3 (brain cortex and pituitary gland) expression levels were decreased. Discussion: Our data suggest that PTX administered spinally produces a hypoglycemic effect in a long-term manner, and PTX-induced hypoglycemia appears to be mediated by the reduction in activity of the glucocorticoid system. Furthermore, PTX may modulate the insulin level during hypoglycemia. Among GLUTs, GLUT-4 in muscle, GLUT-2 in the liver, hypothalamus and pituitary gland as well as GLUT-1 in the adrenal gland may be responsible for PTX-induced hypoglycemia.

Proinflammatory cytokines have been implicated in the production of neuropathic pain. We have previously reported that several ginsenosides produce antinoception. Especially, we have previously demonstrated that ginsenosides administered supraspinally reduce pain behaviors induced by proinflmmatory cytokines administered spinally. However, spinal action of ginsenosides in the regulation of proinflammtory cytokine-induced pain behavior has not been characterized yet. In the present study, we investigated the effects of ginsenosides following intrathecal treatment on pain behaviors induced by pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β), and interferon-γ (IFN-γ) injected intrathecally. The ginsenosides such as Rb1, Rb2, Rc, Rf, Rg1, and Rg3 pretreated intrathecally reduced the pain behavior induced by TNF-α, IL-1β, and IFN-γ injection. However, ginsenoside Rd and Re treated intrathecally did not affect the pain behavior induced by proinflammatory cytokines i...

We examined the role of spinally located β-adrenergic receptors in the regulation of the blood glucose level. The intrathecal (i.t.) injections with dobutamine (β1-adrenergic receptor agonist) or terbutaline (β2-adrenergic receptor agonist) caused an elevation of the blood glucose level, whereas metoprolol (β1-adrenergic receptor antagonist) or butoxamine (β2-adrenergic receptor antagonist) did not. In addition, i.t. pretreatment with pertussis toxin (PTX) attenuated the hyperglycemic effect induced by dobutamine or terbutaline. Moreover, plasma insulin level was increased by dobutamine but not by terbutaline, and PTX reduced dobutamine-induced up-regulation of the plasma insulin level. Terbutaline significantly increased plasma corticosterone level, and PTX further enhanced terbutaline-induced corticosterone level. Furthermore, intraperitoneal (i.p.) pretreatment with hexamethonium- (a preganglionic blocker) attenuated dobutamine- and terbutaline-induced hyperglycemic effects. Our results suggest that activation of spinal β1- and β2-adrenergic receptors produces hyperglycemic effects in a different manner. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by terbutaline. Furthermore, dobutamine- or terbutaline-induced hyperglycemia appears to be mediated through the spinal nerves.

In the present study, the antinociceptive profiles of coumarin were examined in ICR mice. Coumarin administered orally (from 1 to 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Duration of antinociceptive action of coumarin maintained at least for 60 min. But, the cumulative response time of nociceptive behaviors induced by a subcutaneous (s.c.) formalin injection, intrathecal (i.t.) substance P (0.7 µg) or glutamate (20 µg) injection was not affected by coumarin. In addition, intracerebroventricular (i.c.v.) or intrathecal (i.t.) administration with coumarin (10-40 µg) attenuated acetic acid-induced writhing response in a dose dependent manner. Intraperitoneal (i.p.) pretreatment with naloxone (an opioid receptor antagonist) attenuated antinociceptive effect induced by coumarin in the writhing test. Furthermore, i.c.v. or i.t. pretreatment with naloxone (5 µg) reversed the decreased acetic acid-induced writhing response. However, methysergide (a 5-HT serotonergic receptor antagonist) or yohimbine (an α2-adrenergic receptor antagonist) did not affect antinociception induced by coumarin in the writhing test. Our results suggest that coumarin exerts a selective antinociceptive property in the acetic acid-induced visceral-derived pain model. Furthermore, the antinociceptive effect of coumarin may be mediated by activation of central opioid receptors, but not serotonergic and adrenergic receptors.

// Su Min Lim 1,2,* , Byung-Ok Choi 3,* , Seong-il Oh 4,* , Won Jun Choi 5 , Ki-Wook Oh 2,6 , Minyeop Nahm 2 , Yuanchao Xue 7 , Jae Hyeok Choi 2 , Ji Young Choi 2 , Young-Eun Kim 8 , Ki Wha Chung 9 , Xiang-Dong Fu 10 , Chang-Seok Ki 11 and Seung Hyun Kim 2,6 1 Biomedical Research Institute, Hanyang University, Seoul, Republic of Korea 2 Cell Therapy Center, Hanyang University Hospital, Seoul, Republic of Korea 3 Department of Neurology and Neuroscience Center, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea 4 Department of Neurology, Busan Paik Hospital, Inje University College of Medicine, Busan, Republic of Korea 5 Department of Neurology, Sheikh Khalifa Specialty Hospital, Ras Al Khaimah, United Arab Emirates 6 Department of Neurology, College of Medicine, Hanyang University, Seoul, Republic of Korea 7 Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China 8 Green Cross Genome, Yongin, Republic of Korea 9 Department of Biological Sciences, Gongju National University, Gongju, Republic of Korea 10 Department of Cellular Molecular Medicine, University of California, San Diego, La Jolla, CA, USA 11 Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea * These authors have contributed equally to this work Correspondence: Chang-Seok Ki, email: // Seung Hyun Kim, email: // Keywords : krabbe disease; globoid cell leukodystrophy; β-galactosylceramidase; psychosine; induced neuron; Gerotarget Received : October 17, 2015 Accepted : October 14, 2016 Published : October 21, 2016 Abstract Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by defective β-galactosylceramidase (GALC), a lysosomal enzyme responsible for cleavage of several key substrates including psychosine. Accumulation of psychosine to the cytotoxic levels in KD patients is thought to cause dysfunctions in myelinating glial cells based on a comprehensive study of demyelination in KD. However, recent evidence suggests myelin-independent neuronal death in the murine model of KD, thus indicating defective GALC in neurons as an autonomous mechanism for neuronal cell death in KD. These observations prompted us to generate induced neurons (iNeurons) from two adult-onset KD patients carrying compound heterozygous mutations (p.[K563*];[L634S]) and (p.[N228_S232delinsTP];[G286D]) to determine the direct contribution of autonomous neuronal toxicity to KD. Here we report that directly converted KD iNeurons showed not only diminished GALC activity and increased psychosine levels, as expected, but also neurite fragmentation and abnormal neuritic branching. The lysosomal-associated membrane proteins 1 (LAMP1) was expressed at higher levels than controls, LAMP1-positive vesicles were significantly enlarged and fragmented, and mitochondrial morphology and its function were altered in KD iNeurons. Strikingly, we demonstrated that psychosine was sufficient to induce neurite defects, mitochondrial fragmentation, and lysosomal alterations in iNeurons derived in healthy individuals, thus establishing the causal effect of the cytotoxic GALC substrate in KD and the autonomous neuronal toxicity in KD pathology.