Membrane-bound phosphoinositides are signalling molecules that have a key role in vesicle trafficking in eukaryotic cells 1 . Proteins that bind specific phosphoinositides mediate interactions between membrane-bounded compartments whose identity is partially encoded by cytoplasmic phospholipid tags. Little is known about the localization and regulation of mammalian phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P 2 ), a phospholipid present in small quantities that regulates membrane trafficking in the endosome-lysosome axis in yeast 2 . Here we describe a multi-organ disorder with neuronal degeneration in the central nervous system, peripheral neuronopathy and diluted pigmentation in the 'pale tremor' mouse. Positional cloning identified insertion of ETn2β (early transposon 2β) 3 into intron 18 of Fig4 (A530089I17Rik), the homologue of a yeast SAC (suppressor of actin) domain PtdIns(3,5)P 2 5-phosphatase located in the vacuolar membrane. The abnormal concentration of PtdIns(3,5)P 2 in cultured fibroblasts from pale tremor mice demonstrates the conserved biochemical function of mammalian Fig4. The cytoplasm of fibroblasts from pale tremor mice is filled with large vacuoles that are immunoreactive for LAMP-2 (lysosomal-associated membrane protein 2), consistent with dysfunction of the late endosomelysosome axis. Neonatal neurodegeneration in sensory and autonomic ganglia is followed by loss of neurons from layers four and five of the cortex, deep cerebellar nuclei and other localized brain regions. The sciatic nerve exhibits reduced numbers of large-diameter myelinated axons, slowed nerve conduction velocity and reduced amplitude of compound muscle action potentials. We identified pathogenic mutations of human FIG4 (KIAA0274) on chromosome 6q21 in four unrelated

CREBH is a liver-specific transcription factor that is localized in the endoplasmic reticulum (ER) membrane. Our previous work demonstrated that CREBH is activated by ER stress or inflammatory stimuli to induce an acute-phase hepatic inflammation. Here we demonstrate that CREBH is a key metabolic regulator of hepatic lipogenesis, fatty acid (FA) oxidation, and lipolysis under metabolic stress. Saturated FA, insulin signals, or an atherogenic high-fat diet can induce CREBH activation in the liver. Under the normal chow diet, CrebH knockout mice display a modest decrease in hepatic lipid contents but an increase in plasma triglycerides (TG). After feeding an atherogenic high-fat diet, massive accumulation of hepatic lipid metabolites and significant increase in plasma TG levels were observed in the CrebH knockout mice. Along with the hypertriglyceridemia phenotype, the CrebH null mice displayed significantly reduced body weight gain, diminished abdominal fat, and increased non-alcoholic steatohepatitis (NASH) activities under the atherogenic high-fat diet. Gene expression analysis and chromatin-immunoprecipitation (ChIP) assay indicated that CREBH is required to activate expression of the genes encoding functions involved in de novo lipogenesis, TG and cholesterol biosynthesis, FA elongation and oxidation, lipolysis, and lipid transport. Supporting the role of CREBH in lipogenesis and lipolysis, forced expression of an activated form of CREBH protein in the liver significantly increases accumulation of hepatic lipids but reduces plasma TG levels in mice. All together our study shows that CREBH plays a key role in maintaining lipid homeostasis by regulating expression of the genes involved in hepatic lipogenesis, FA oxidation, and lipolysis under metabolic stress. The identification of CREBH as a stress-inducible metabolic regulator has important implications in the understanding and treatment of metabolic disease.

Background Air pollution is a global challenge to public health. Epidemiological studies have linked exposure to ambient particulate matter with aerodynamic diameters < 2.5 μm (PM2.5) to the development of metabolic diseases. In this study, we investigated the effect of PM2.5 exposure on liver pathogenesis and the mechanism by which ambient PM2.5 modulates hepatic pathways and glucose homeostasis. Methods Using “Ohio’s Air Pollution Exposure System for the Interrogation of Systemic Effects (OASIS)-1”, we performed whole-body exposure of mice to concentrated ambient PM2.5 for 3 or 10 weeks. Histological analyses, metabolic studies, as well as gene expression and molecular signal transduction analyses were performed to determine the effects and mechanisms by which PM2.5 exposure promotes liver pathogenesis. Results Mice exposed to PM2.5 for 10 weeks developed a non-alcoholic steatohepatitis (NASH)-like phenotype, characterized by hepatic steatosis, inflammation, and fibrosis. Mice after PM2.5 exposure displayed impaired hepatic glycogen storage, glucose intolerance, and insulin resistance. Further investigation revealed that exposure to PM2.5 led to activation of inflammatory response pathways mediated through c-Jun N-terminal kinase (JNK), nuclear factor kappa B (NF-κB), and Toll-like receptor 4 (TLR4) but suppression of the insulin receptor substrate 1 (IRS1)-mediated signaling. Moreover, PM2.5 exposure repressed expression of the peroxisome proliferator-activated receptor (PPAR) γ and PPARα in the liver. Conclusions Our study suggests that PM2.5 exposure represents a significant “hit” that triggers a NASH-like phenotype and impairs hepatic glucose metabolism. The information from this work has important implications in our understanding of air pollution-associated metabolic disorders.

Recessive Charcot-Marie-Tooth disease type-4J (CMT4J) and its animal model, the pale tremor mouse (plt), are caused by mutations of the FIG4 gene encoding a PI(3,5)P(2) 5-phosphatase. We describe the 9-year clinical course of CMT4J, including asymmetric, rapidly progressive paralysis, in two siblings. Sensory symptoms were absent despite reduced numbers of sensory axons. Thus, the phenotypic presentation of CMT4J clinically resembles motor neuron disease. Time-lapse imaging of fibroblasts from CMT4J patients demonstrates impaired trafficking of intracellular organelles because of obstruction by vacuoles. Further characterization of plt mice identified axonal degeneration in motor and sensory neurons, limited segmental demyelination, lack of TUNEL staining and lack of accumulation of ubiquitinated protein in vacuoles of motor and sensory neurons. This study represents the first documentation of the natural history of CMT4J. Physical obstruction of organelle trafficking by vacuoles is a potential novel cellular mechanism of neurodegeneration.

Patients with PMP22 deficiency present with focal sensory and motor deficits when peripheral nerves are stressed by mechanical force. It has been hypothesized that these focal deficits are due to mechanically induced conduction block (CB). To test this hypothesis, we induced 60-70% CB (defined by electrophysiological criteria) by nerve compression in an authentic mouse model of HNPP with an inactivation of one of the two pmp22 alleles (pmp22+/−). Induction time for the CB was significantly shorter in pmp22+/− mice than that in pmp22+/+ mice. This shortened induction was not found in the mice with deficiency of myelin protein zero (MPZ), a major structural protein of compact myelin. Pmp22+/− nerves showed intact tomacula with no segmental demyelination in both non-compressed and compressed conditions, normal molecular architecture, and normal concentration of voltage-gated sodium channels by H3-saxitoxin binding assay. However, focal constrictions were observed in the axonal segments enclosed by tomacula, a pathological hallmark of HNPP. The constricted axons increase axial-resistance to action potential propagation, which should hasten the induction of CB in pmp22 deficiency. Taken together, these results demonstrate that a function of Pmp22 is to protect the nerve from mechanical injury.

The circadian clock orchestrates diverse physiological processes critical for health and disease. CREB, hepatocyte specific (CREBH) is a liver-enriched, endoplasmic reticulum (ER)–tethered transcription factor known to regulate the hepatic acute phase response and energy homeostasis under stress conditions. We demonstrate that CREBH is regulated by the circadian clock and functions as a circadian regulator of hepatic lipid metabolism. Proteolytic activation of CREBH in the liver exhibits typical circadian rhythmicity controlled by the core clock oscillator BMAL1 and AKT/glycogen synthase kinase 3β (GSK3β) signaling pathway. GSK3β-mediated phosphorylation of CREBH modulates the association between CREBH and the coat protein complex II transport vesicle and thus controls the ER-to-Golgi transport and subsequent proteolytic cleavage of CREBH in a circadian manner. Functionally, CREBH regulates circadian expression of the key genes involved in triglyceride (TG) and fatty acid (FA) metabolism and is required to maintain circadian amplitudes of blood TG and FA in mice. During the circadian cycle, CREBH rhythmically regulates and interacts with the hepatic nuclear receptors peroxisome proliferator–activated receptor α and liver X receptor α as well as with the circadian oscillation activator DBP and the repressor E4BP4 to modulate CREBH transcriptional activities. In conclusion, these studies reveal that CREBH functions as a circadian-regulated liver transcriptional regulator that integrates energy metabolism with circadian rhythm.

Background Hepatic fibrosis, featured by accumulation of excessive extracellular matrix in liver tissues, is associated with metabolic disease and cancer. Inhalation exposure to airborne particulate matter in fine ranges (PM2.5) correlates with pulmonary dysfunction, cardiovascular disease, and metabolic syndrome. In this study, we investigated the effect and mechanism of PM2.5 exposure on hepatic fibrogenesis. Methods Both inhalation exposure of mice and in vitro exposure of specialized cells to PM2.5 were performed to elucidate the effect of PM2.5 exposure on hepatic fibrosis. Histological examinations, gene expression analyses, and genetic animal models were utilized to determine the effect and mechanism by which PM2.5 exposure promotes hepatic fibrosis. Results Inhalation exposure to concentrated ambient PM2.5 induces hepatic fibrosis in mice under the normal chow or high-fat diet. Mice after PM2.5 exposure displayed increased expression of collagens in liver tissues. Exposure to PM2.5 led to activation of the transforming growth factor β (TGFβ)-SMAD3 signaling, suppression of peroxisome proliferator-activated receptor γ (PPARγ), and expression of collagens in hepatic stellate cells. NADPH oxidase plays a critical role in PM2.5-induced liver fibrogenesis. Conclusions Exposure to PM2.5 exerts discernible effects on promoting hepatic fibrogenesis. NADPH oxidase mediates the effects of PM2.5 exposure on promoting hepatic fibrosis.

Spinal cord transection results in severe neurological sequelae, and to date, there is no effective treatment. Because of the limited capacity for axonal regeneration in the spinal cord, recovery is minimal. Recently, efforts have been made to establish, by grafting neural tissue, a functional relay-station between the severed stumps of the injured cord. Previously, we used co-transplantation of neural stem cells (NSCs) and Schwann cells (SCs) to improve functional recovery of transected spinal cord. However, this effort has been partially impeded by limited neuronal differentiation of transplanted NSCs. To circumvent this problem, we have pre-differentiated NSCs toward neurons in vitro with the application of retinoic acid (RA) prior to cell grafting. Further, we genetically modified SCs to overexpress human neurotrophin-3 (hNT-3). When these cells were co-transplanted into the transected spinal cord of rats, injured animals had partial improvement (both functionally and structurally), including improved Basso, Beattie, and Bresnahan (BBB) scores, increased axonal regeneration/remyelination, and reduced neuronal loss. However, this pre-differentiation of NSCs in vitro only mildly improved neuronal differentiation of NSCs in vivo.