Gut microbiota composition modulates inflammation and structure of the vagal afferent pathway.

Vagal afferent neurons (VAN), located in the nodose ganglion (NG) innervate the gut and terminate in the nucleus of solitary tract (NTS) in the brainstem. They are the primary sensory neurons integrating gut-derived signals to regulate meal size. Chronic high-fat diet (HFD) consumption impairs vagally mediated satiety, resulting in overfeeding. There is evidence that HFD consumption leads to alterations in both vagal nerve function and structural integrity. HFD also leads to marked gut microbiota dysbiosis; in rodent models, dysbiosis is sufficient to induce weight gain. In this study, we investigated the effect of microbiota dysbiosis on gut-brain vagal innervation independently of diet. To do so, we recolonized microbiota-depleted rats with gastrointestinal (GI) contents isolated from donor animals fed either a HFD (45 or 60% fat) or a low fat diet (LFD, 13% fat). We used two different depletion models while maintaining the animals on LFD: 1) conventionally raised Fischer and Wistar rats that underwent a depletion paradigm using an antibiotic cocktail and 2) germ free (GF) raised Fischer rats. Following recolonization, receiver animals were designated as ConvLF and ConvHF. Fecal samples were collected throughout these studies and analyzed via 16S Illumina sequencing. In both models, bacteria that were identified as characteristic of HFD were successfully transferred to recipient animals. Three weeks post-colonization, ConvHF rats showed significant increases in ionized calcium-binding adapter molecule-1 (Iba1) positive immune cells in the NG compared to ConvLF animals. Additionally, using isolectin B4 (IB4) staining to identify c-fibers, we found that, compared to ConvLF animals, ConvHF rats displayed decreased innervation at the level of the medial NTS; c-fibers at this level are believed to be primarily of vagal origin. This alteration in vagal structure was associated with a loss in satiety induced by the gut peptide cholecystokinin (CCK). Increased presence of immunocompetent Iba1+ cells along the gut-brain axis and alterations in NTS innervation were still evident in ConvHF rats compared to ConvLF animals 12 weeks post-colonization and were associated with increases in food intake and body weight (BW). We conclude from these data that microbiota dysbiosis can alter gut-brain vagal innervation, potentially via recruitment and/or activation of immune cells.

Evidence for redox regulation of cytochrome C release during programmed neuronal death: antioxidant effects of protein synthesis and caspase inhibition.

Sympathetic neurons die by apoptosis when they are deprived of nerve growth factor (NGF). Activation of caspases by cytochrome c released from mitochondria is central to this death. In this report we present evidence that cellular redox state regulates cytochrome c redistribution in these neurons. An increase of mitochondrial-produced reactive oxygen species (ROS) occurred in rat sympathetic neurons in cell culture within 3 hr of NGF withdrawal. Caspase inhibitors blocked this ROS burst. By 6 hr after NGF deprivation, glutathione (GSH) levels had increased, neutralizing elevated hydrogen peroxide levels and returning cellular redox state to basal levels. By 12 hr after deprivation, ROS levels had again increased and remained elevated during the rest of the apoptotic process. The later ROS burst appeared to have both caspase-dependent and caspase-independent components and was coincident with the period of cytochrome c release. Inhibition of protein synthesis with cycloheximide (CHX) and treatment with the antioxidant compound, N-acetyl-l-cysteine (l-NAC), blocked both the early and late ROS bursts by increasing cellular GSH levels (Ratan et al., 1994; Tan et al., 1998). Both compounds, and a membrane-permeant form of GSH, also inhibited cytochrome c release and death. Treatment of NGF, CHX-, l-NAC-, and GSH-saved cells with hydrogen peroxide caused rapid cytochrome c release. These data suggest a role for cellular redox state in regulating cytochrome c release during apoptosis induced by NGF withdrawal.

Loss of cardiolipin and mitochondria during programmed neuronal death: evidence of a role for lipid peroxidation and autophagy.

Cardiolipin, a lipid of the mitochondrial inner membrane, is lost from many types of cells during apoptotic death. Here we show that the cardiolipin content of nerve growth factor (NGF)-deprived rat sympathetic neurons undergoing apoptotic death in cell culture decreased before extensive loss of mitochondria from the cells. By 18-24 h after NGF deprivation, many neurons did not stain with the cardiolipin-specific dye, Nonyl Acridine Orange, suggesting complete loss of cardiolipin. Gas chromatography confirmed the decline of cardiolipin content in NGF-deprived neurons. Electron microscopy and immunoblots for the mitochondrial-specific protein, heat shock protein 60 (HSP60), revealed that there was only a slight decrease in mitochondrial mass at this time. Cardiolipin loss after NGF deprivation was concurrent with increased production of mitochondrial-derived reactive oxygen species [Kirkland, R.A., Franklin, J.L., 2001. J. Neurosci. 21, 1949-1963] and increased lipid peroxidation. Compounds having antioxidant effects blocked peroxidation, loss of cardiolipin, and the decrease of mitochondrial mass in NGF-deprived neurons. These compounds also blocked an increase in the number of lysosomes and autophagosomes in NGF-deprived cells. The findings reported here show that the important mitochondrial inner membrane lipid, cardiolipin, is lost from mitochondria during neuronal apoptosis and that this loss occurs before significant loss of mitochondria from cells. They suggest that the loss of cardiolipin is mediated by free radical oxygen.