Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury.

Spinal cord injury (SCI) results in numerous systemic dysfunctions, including intestinal dysmotility and enteric nervous system (ENS) atrophy. The ENS has capacity to recover following perturbation, yet intestinal pathologies persist. With emerging evidence demonstrating SCI-induced alterations to gut microbiome composition, we hypothesized that microbiome modulation contributes to post-injury enteric recovery. Here, we show that intervention with the dietary fiber, inulin, prevents SCI-induced ENS atrophy and dysmotility in mice. While SCI-associated microbiomes and specific injury-sensitive gut microbes are not sufficient to modulate intestinal dysmotility after injury, intervention with microbially-derived short-chain fatty acid (SCFA) metabolites prevents ENS dysfunctions in injured mice. Notably, inulin-mediated resilience is dependent on IL-10 signaling, highlighting a critical diet-microbiome-immune axis that promotes ENS resilience post-injury. Overall, we demonstrate that diet and microbially-derived signals distinctly impact ENS survival after traumatic spinal injury and represent a foundation to uncover etiological mechanisms and future therapeutics for SCI-induced neurogenic bowel.

Modulation of Ceramide-Induced Apoptosis in Enteric Neurons by Aryl Hydrocarbon Receptor Signaling: Unveiling a New Pathway beyond ER Stress.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a persistent organic pollutant and a potent aryl hydrocarbon receptor (AHR) ligand, causes delayed intestinal motility and affects the survival of enteric neurons. In this study, we investigated the specific signaling pathways and molecular targets involved in TCDD-induced enteric neurotoxicity. Immortalized fetal enteric neuronal (IM-FEN) cells treated with 10 nM TCDD exhibited cytotoxicity and caspase 3/7 activation, indicating apoptosis. Increased cleaved caspase-3 expression with TCDD treatment, as assessed by immunostaining in enteric neuronal cells isolated from WT mice but not in neural crest cell-specific Ahr deletion mutant mice (Wnt1Cre+/-/Ahrb(fl/fl)), emphasized the pivotal role of AHR in this process. Importantly, the apoptosis in IM-FEN cells treated with TCDD was mediated through a ceramide-dependent pathway, independent of endoplasmic reticulum stress, as evidenced by increased ceramide synthesis and the reversal of cytotoxic effects with myriocin, a potent inhibitor of ceramide biosynthesis. We identified Sptlc2 and Smpd2 as potential gene targets of AHR in ceramide regulation by a chromatin immunoprecipitation (ChIP) assay in IM-FEN cells. Additionally, TCDD downregulated phosphorylated Akt and phosphorylated Ser9-GSK-3ß levels, implicating the PI3 kinase/AKT pathway in TCDD-induced neurotoxicity. Overall, this study provides important insights into the mechanisms underlying TCDD-induced enteric neurotoxicity and identifies potential targets for the development of therapeutic interventions.

Molecular mechanisms of enteric neuropathies in high-fat diet feeding and diabetes.

BACKGROUND: Obesity and diabetes are associated with altered gastrointestinal function and with the development of abdominal pain, nausea, diarrhea, and constipation among other symptoms. The enteric nervous system (ENS) regulates gastrointestinal motility. Enteric neuropathies defined as damage or loss of enteric neurons can lead to motility disorders. PURPOSE: Here, we review the molecular mechanisms that drive enteric neurodegeneration in diabetes and obesity, including signaling pathways leading to neuronal cell death, oxidative stress, and microbiota alteration. We also highlight potential approaches to treat enteric neuropathies including antioxidant therapy to prevent oxidative stress-induced damage and the use of stem cells.

Diet-microbiome interactions promote enteric nervous system resilience following spinal cord injury.

Spinal cord injury (SCI) results in a plethora of physiological dysfunctions across all body systems, including intestinal dysmotility and atrophy of the enteric nervous system (ENS). Typically, the ENS has capacity to recover from perturbation, so it is unclear why intestinal pathophysiologies persist after traumatic spinal injury. With emerging evidence demonstrating SCI-induced alterations to the gut microbiome composition, we hypothesized that modulation of the gut microbiome could contribute to enteric nervous system recovery after injury. Here, we show that intervention with the dietary fiber, inulin prevents ENS atrophy and limits SCI-induced intestinal dysmotility in mice. However, SCI-associated microbiomes and exposure to specific SCI-sensitive gut microbes are not sufficient to modulate injury-induced intestinal dysmotility. Intervention with microbially-derived short-chain fatty acid (SCFA) metabolites prevents ENS dysfunctions and phenocopies inulin treatment in injured mice, implicating these microbiome metabolites in protection of the ENS. Notably, inulin-mediated resilience is dependent on signaling by the cytokine IL-10, highlighting a critical diet-microbiome-immune axis that promotes ENS resilience following SCI. Overall, we demonstrate that diet and microbially-derived signals distinctly impact recovery of the ENS after traumatic spinal injury. This protective diet-microbiome-immune axis may represent a foundation to uncover etiological mechanisms and future therapeutics for SCI-induced neurogenic bowel.

Targeting PKCα alleviates iron overload in diabetes and hemochromatosis through the inhibition of ferroportin

Ferroportin (Fpn) is the only iron exporter, playing a crucial role in systemic iron homeostasis. Fpn is negatively regulated by its ligand hepcidin, but other potential regulators in physiological and disease conditions remain poorly understood. Diabetes is a metabolic disorder that develops body iron loading with unknown mechanisms. By utilizing diabetic mouse models and human duodenal specimens, we demonstrated that intestinal Fpn expression was increased in diabetes in a hepcidin-independent manner. Protein kinase C (PKC) is hyperactivated in diabetes. We showed that PKC was required to sustain baseline Fpn expression and diabetes induced Fpn upregulation in the enterocytes and macrophages. Knockout of PKC abolished diabetes associated iron overload. Mechanistically, activation of PKC increased the exocytotic while decreased the endocytic trafficking of Fpn in the resting state. Hyperactive PKC also suppressed hepcidin-induced ubiquitination, internalization, and degradation of Fpn. We further observed that iron loading in the enterocytes and macrophages activated PKC, acting as a novel mechanism to enhance Fpn-dependent iron efflux. Finally, we demonstrated that the loss-of-function of PKC and pharmacological inhibition of PKC significantly alleviated hereditary hemochromatosis associated iron overload. Our study has highlighted, for the first time, that PKC is an important positive regulator of Fpn and a new target in the control of iron homeostasis.

Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

Treatment of Obesity Through Glial Cell-Derived Neurotrophic Factor Lipid Nanoparticle Delivery in Mice.

BACKGROUND AND AIMS: The overexpression of glial cell-derived neurotrophic factor (GDNF) in the liver and adipose tissues offers strong protection against high-fat diet (HFD)-induced obesity in mice. We hypothesize that sustainably enhancing GDNF expression in the liver may provide a therapeutic effect that can prevent the progression of HFD-induced obesity in mice. METHODS: Expression lentivector encoding mouse GDNF (GDNF(pDNA) or empty vector (pDNA, control) were encapsulated in lipid nanoparticles (LNPs) using the thin-film hydration method. Mice were fed with regular diet (RD) or HFD for 20 weeks prior to injection and the GDNF and control vector-loaded LNPs were administered by intravenous (IV) injection to mice once weekly for 5 weeks. Changes in body weight were monitored and mice tissues were collected and imaged for fluorescence using an IVIS in vivo imaging system. Post-treatment abdominal fat weight, colon length, and spleen weight were obtained. GDNF protein levels in the liver and serum were quantified by enzyme-linked immunosorbent assay, while liver AKT serine/threonine kinase and AMP-activated protein kinase phosphorylation levels were evaluated by Western blotting. RESULTS: IV-injected GDNF(pDNA)-loaded LNPs targeted the liver and remained in there for up to 15 days postinjection. A single injection of GDNF(pDNA)-loaded LNPs significantly increased GDNF expression for 7 days and consequently increased the levels of phosphorylated AKT serine/threonine kinase and AMP-activated protein kinase. Once weekly injections of GDNF(pDNA)-loaded LNPs for 5 weeks slowed increase in body weight, reduced abdominal fat, and modulated the gut microbiota toward a healthier composition in HFD-fed mice. CONCLUSION: GDNF(pDNA)-loaded LNPs could potentially be developed as a therapeutic strategy to reverse weight gain in obese patients.

Targeting PKC alleviates iron overload in diabetes and hemochromatosis.

Diabetes is one of the most prevalent chronic diseases worldwide. Iron overload increases the incidence of diabetes and aggravates diabetic complications that cause mortality. Reciprocally, diabetes potentially promotes body iron loading, but the mechanism remains not well understood. In this study, we demonstrated systemic iron excess and the upregulation of iron exporter ferroportin (Fpn) in the enterocytes and macrophages of multiple diabetic mouse models. Increased Fpn expression and iron efflux was also seen in the enterocytes of type 2 diabetic human patients. We further showed that protein kinase C (PKC), which is activated in hyperglycemia, was responsible for the sustained membrane expression of Fpn in physiological and in diabetic settings. For the first time, we identified that PKCs were novel binding proteins and positive regulators of Fpn. Mechanistically, hyperactive PKC promoted exocytotic membrane insertion while inhibited the endocytic trafficking of Fpn in the resting state. PKC also protected Fpn from internalization and degradation by its ligand hepcidin dependent on decreased ubiquitination and increased phosphorylation of Fpn. Importantly, the loss-of-function and pharmacological inhibition of PKC alleviated systemic iron overload in diabetes and hemochromatosis. Our study thus highlights PKC as a novel target in the control of systemic iron homeostasis.

Microbiome differences related to metformin intolerance among Black individuals with diabetes, a pilot cross-sectional study.

Aims: Metformin is the broadly accepted the first-line medication for diabetes. Its use, however, is limited by gastrointestinal side effects present in approximately 25% of patients. This study aimed to better understand the interplay between metformin intolerance and gut microbiota among Black individuals with diabetes. Methods: We performed a cross-sectional study among 29 Black individuals living with diabetes with or without metformin intolerance. Participants with mean age 59±11, 58% female, were stratified into three groups: 1)intolerant: metformin intolerance in the past, not on metformin; 2)partially intolerant: mild to moderate gastrointestinal symptoms, currently taking metformin 3)tolerant: using metformin without symptoms. We collected and analyzed rectal swabs and analyzed microbiota composition using V3-V4 regions of the 16s rRNA. Results: Metformin intolerant subjects trended towards having greatest alpha diversity, followed by tolerant and partially tolerant (Intolerant:4.9; Tolerant:4.2; Partially tolerant:3.9). Mean difference in alpha diversity for intolerant versus partially tolerant was 1.0 (95% CI-0.1,2.1) and intolerant versus tolerant were 0.7 (95% CI -0.4,1.8). Conclusion: This was the first study to evaluate the role of microbiota and metformin intolerance among Black individuals. We report on differences in alpha diversity as well as microbiota composition.

Differences in hepatocellular iron metabolism underlie sexual dimorphism in hepatocyte ferroptosis.

Males show higher incidence and severity than females in hepatic injury and many liver diseases, but the mechanisms are not well understood. Ferroptosis, an iron-mediated lipid peroxidation-dependent death, plays an important role in the pathogenesis of liver diseases. We determined whether hepatocyte ferroptosis displays gender difference, accounting for sexual dimorphism in liver diseases. Compared to female hepatocytes, male hepatocytes were much more vulnerable to ferroptosis by iron and pharmacological inducers including RSL3 and iFSP1. Male but not female hepatocytes exhibited significant increases in mitochondrial Fe2+ and mitochondrial ROS (mtROS) contents. Female hepatocytes showed a lower expression of iron importer transferrin receptor 1 (TfR1) and mitochondrial iron importer mitoferrin 1 (Mfrn1), but a higher expression of iron storage protein ferritin heavy chain 1 (FTH1). It is well known that TfR1 expression is positively correlated with ferroptosis. Herein, we showed that silencing FTH1 enhanced while knockdown of Mfrn1 decreased ferroptosis in HepG2 cells. Removing female hormones by ovariectomy (OVX) did not dampen but rather enhanced hepatocyte resistance to ferroptosis. Mechanistically, OVX potentiated the decrease in TfR1 and increase in FTH1 expression. OVX also increased FSP1 expression in ERK-dependent manner. Elevation in FSP1 suppressed mitochondrial Fe2+ accumulation and mtROS production, constituting a novel mechanism of FSP1-mediated inhibition of ferroptosis. In conclusion, differences in hepatocellular iron handling between male and female account, at least in part, for sexual dimorphism in induced ferroptosis of the hepatocytes.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.

Defective neurite elongation and branching in Nibp/Trappc9 deficient zebrafish and mice.

Loss of function in transport protein particles (TRAPP) links a new set of emerging genetic disorders called "TRAPPopathies". One such disorder is NIBP syndrome, characterized by microcephaly and intellectual disability, and caused by mutations of NIBP/TRAPPC9, a crucial and unique member of TRAPPII. To investigate the neural cellular/molecular mechanisms underlying microcephaly, we developed Nibp/Trappc9-deficient animal models using different techniques, including morpholino knockdown and CRISPR/Cas mutation in zebrafish and Cre/LoxP-mediated gene targeting in mice. Nibp/Trappc9 deficiency impaired the stability of the TRAPPII complex at actin filaments and microtubules of neurites and growth cones. This deficiency also impaired elongation and branching of neuronal dendrites and axons, without significant effects on neurite initiation or neural cell number/types in embryonic and adult brains. The positive correlation of TRAPPII stability and neurite elongation/branching suggests a potential role for TRAPPII in regulating neurite morphology. These results provide novel genetic/molecular evidence to define patients with a type of non-syndromic autosomal recessive intellectual disability and highlight the importance of developing therapeutic approaches targeting the TRAPPII complex to cure TRAPPopathies.

Differences in Hepatocellular Iron Metabolism Underlie Sexual Dimorphism in Hepatocyte Ferroptosis.

Males show higher incidence and severity than females in hepatic injury and many liver diseases, but the mechanisms are not well understood. Ferroptosis, an iron-mediated lipid peroxidation-dependent death, plays an important role in the pathogenesis of liver diseases. We determined whether hepatocyte ferroptosis displays gender difference, accounting for sexual dimorphism in liver diseases. Compared to female hepatocytes, male hepatocytes were much more vulnerable to ferroptosis by iron and pharmacological inducers including RSL3 and iFSP1. Male but not female hepatocytes exhibited significant increases in mitochondrial Fe 2+ and mitochondrial ROS (mtROS) contents. Female hepatocytes showed a lower expression of iron importer transferrin receptor 1 (TfR1) and mitochondrial iron importer mitoferrin 1 (Mfrn1), but a higher expression of iron storage protein ferritin heavy chain 1 (FTH1). It is well known that TfR1 expression is positively correlated with ferroptosis. Herein, we showed that silencing FTH1 enhanced while knockdown of Mfrn1 decreased ferroptosis in HepG2 cells. Removing female hormones by ovariectomy (OVX) did not dampen but rather enhanced hepatocyte resistance to ferroptosis. Mechanistically, OVX potentiated the decrease in TfR1 and increase in FTH1 expression. OVX also increased FSP1 expression in ERK-dependent manner. Elevation in FSP1 suppressed mitochondrial Fe 2+ accumulation and mtROS production, constituting a novel mechanism of FSP1-mediated inhibition of ferroptosis. In conclusion, differences in hepatocellular iron handling between male and female account, at least in part, for sexual dimorphism in induced ferroptosis of the hepatocytes.

Role of stimulator of interferon genes (STING) in the enteric nervous system in health and disease.

Stimulator of Interferon Genes (STING) is a crucial protein that controls the immune system's reaction to bacterial and viral infections. As a pattern-recognition receptor, STING is found in immune cells as well as in neurons and glia in the enteric nervous system (ENS). Recent studies have linked STING to the pathogenesis of several neurological disorders like multiple sclerosis (MS), Alzheimer's disease (AD), and gastrointestinal disorders, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), which are characterized by chronic inflammation and dysregulation of the enteric nervous system (ENS) in the digestive tract. STING plays a crucial role in the pathway that induces the production of interferon in response to viral infection in the central nervous system (CNS). A new study by Dharshika et al. in the current issue of Neurogastroenterology and Motility has demonstrated distinct roles for STING in enteric neurons and glia, namely activation of STING leads to IFN-ß production in enteric neurons but not in glia and reducing STING activation in enteric glia does not modulate the severity of Dextran sulfate sodium (DSS) colitis or subsequent loss of enteric neurons. Rather, the role of STING in enteric glia is related to enhancing autophagy. STING can influence gastrointestinal motility and barrier function and therefore be involved in the pathophysiology of IBS and IBD. This mini review highlights the current knowledge of STING in the pathophysiology of CNS and gastrointestinal diseases as well as these newly uncovered roles STING in enteric neurons and glia.

SARS-CoV-2 Induces Epithelial-Enteric Neuronal Crosstalk Stimulating VIP Release.

BACKGROUND: Diarrhea is present in up to 30-50% of patients with COVID-19. The mechanism of SARS-CoV-2-induced diarrhea remains unclear. We hypothesized that enterocyte-enteric neuron interactions were important in SARS-CoV-2-induced diarrhea. SARS-CoV-2 induces endoplasmic reticulum (ER) stress in enterocytes causing the release of damage associated molecular patterns (DAMPs). The DAMPs then stimulate the release of enteric neurotransmitters that disrupt gut electrolyte homeostasis. METHODS: Primary mouse enteric neurons (EN) were exposed to a conditioned medium from ACE2-expressing Caco-2 colonic epithelial cells infected with SARS-CoV-2 or treated with tunicamycin (ER stress inducer). Vasoactive intestinal peptides (VIP) expression and secretion by EN were assessed by RT-PCR and ELISA, respectively. Membrane expression of NHE3 was determined by surface biotinylation. RESULTS: SARS-CoV-2 infection led to increased expression of BiP/GRP78, a marker and key regulator for ER stress in Caco-2 cells. Infected cells secreted the DAMP protein, heat shock protein 70 (HSP70), into the culture media, as revealed by proteomic and Western analyses. The expression of VIP mRNA in EN was up-regulated after treatment with a conditioned medium of SARS-CoV-2-infected Caco-2 cells. CD91, a receptor for HSP70, is abundantly expressed in the cultured mouse EN. Tunicamycin, an inducer of ER stress, also induced the release of HSP70 and Xbp1s, mimicking SARS-CoV-2 infection. Co-treatment of Caco-2 with tunicamycin (apical) and VIP (basolateral) induced a synergistic decrease in membrane expression of Na+/H+ exchanger (NHE3), an important transporter that mediates intestinal Na+/fluid absorption. CONCLUSIONS: Our findings demonstrate that SARS-CoV-2 enterocyte infection leads to ER stress and the release of DAMPs that up-regulates the expression and release of VIP by EN. VIP in turn inhibits fluid absorption through the downregulation of brush-border membrane expression of NHE3 in enterocytes. These data highlight the role of epithelial-enteric neuronal crosstalk in COVID-19-related diarrhea.

Aryl hydrocarbon receptor activation affects nitrergic neuronal survival and delays intestinal motility in mice.

Despite progress describing the effects of persistent organic pollutants (POPs) on the central nervous system, the effect of POPs on enteric nervous system (ENS) function remains underexplored. We studied the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a POP, and a potent aryl hydrocarbon receptor (AHR) ligand, on the ENS and intestinal motility in mice. C57Bl/6J mice treated with TCDD (2.4 µg/kg body weight) for 8 weeks (once per week) exhibited significant delay in intestinal motility as shown by reduced stool frequency, prolonged intestinal transit time, and a persistence of dye in the jejunum compared to control mice with maximal dye retention in the ileum. TCDD significantly increased Cyp1a1 expression, an AHR target gene, and reduced the total number of neurons and affected nitrergic neurons in cells isolated from WT mice, but not Ahr-/- mice. In immortalized fetal enteric neuronal cells, TCDD-induced nuclear translocation of AHR as well as increased Cyp1a1 expression. AHR activation did not affect neuronal proliferation. However, AHR activation resulted in enteric neuronal toxicity, specifically, nitrergic neurons. Our results demonstrate that TCDD adversely affects nitrergic neurons and thereby contributes to delayed intestinal motility. These findings suggest that AHR signaling in the ENS may play a role in modulating TCDD-induced gastrointestinal pathophysiology.

SIRT3 activation promotes enteric neurons survival and differentiation.

Enteric neuron degeneration has been observed during aging, and in individuals with metabolic dysfunction including obesity and diabetes. Honokiol, a naturally occurring compound, is an activator of Sirtuin-3 (SIRT3) that has antioxidant activity. Its role in modulating enteric neuron-specific neurodegeneration is unknown. We studied the effects of honokiol and its fluorinated analog, hexafluoro-honokiol, on enteric neuronal differentiation and survival. We used a previously established model of mouse primary enteric neuronal cells and an enteric neuronal cell line treated with palmitate (PA) and lipopolysaccharide (LPS) to induce mitochondrial dysfunction and enteric neuronal cell death. The effect of honokiol and hexafluoro-honokiol was assessed on neuronal phenotype, fiber density, differentiation, and pyroptosis. Honokiol and hexafluoro-honokiol significantly increased neuronal networks and fiber density in enteric neurons and increased levels of neuronal nitric oxide synthase and Choline acetyltransferase mRNA. Hexafluoro-honokiol and honokiol also significantly increased SIRT3 mRNA levels and suppressed palmitate and LPS-induced neuronal pyroptosis. SIRT3 knock-down prevented the hexafluoro-honokiol mediated suppression of mitochondrial superoxide release. Our data supports a neuroprotective effect of honokiol and its derivative and these could be used as prophylactic or therapeutic agents for treating enteric neurodegeneration and associated motility disorders.

Glial cell derived neurotrophic factor prevents western diet and palmitate-induced hepatocyte oxidative damage and death through SIRT3.

Nonalcoholic fatty liver disease (NAFLD) is associated with increased oxidative stress that leads to hepatocyte and mitochondrial damage. In this study we investigated the mechanisms involved in the induction of oxidative stress and impairment of mitochondrial quality control and mitophagy in hepatocytes by the saturated fatty acid palmitate and Western diet feeding in mice and if their harmful effects could be reversed by the neurotrophic factor glial cell derived neurotrophic factor (GDNF). Western diet (WD)-feeding increased hepatic lipid peroxidation in control mice and, in vitro palmitate induced oxidative stress and impaired the mitophagic clearance of damaged mitochondria in hepatocytes. This was accompanied by reductions in hepatocyte sirtuin 3 (SIRT3) deacetylase activity, gene expression and protein levels as well as in superoxide dismutase enzyme activity. These reductions were reversed in the liver of Western diet fed GDNF transgenic mice and in hepatocytes exposed to palmitate in the presence of GDNF. We demonstrate an important role for Western diet and palmitate in inducing oxidative stress and impairing mitophagy in hepatocytes and an ability of GDNF to prevent this. These findings suggest that GDNF or its agonists may be a potential therapy for the prevention or treatment of NAFLD.