Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Lactate transporter MCT1 in hepatic stellate cells promotes fibrotic collagen expression in nonalcoholic steatohepatitis.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline deficient, high fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Nucleocytoplasmic transport in C9orf72-mediated ALS/FTD.

A GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies indicate that disruption of nucleocytoplasmic transport pathways play a critical role in the pathogenesis of C9orf72-mediated ALS/FTD (C9-ALS). Here, we discuss mechanisms by which C9orf72 mutations cause nucleocytoplasmic transport deficits and contribute to disease pathogenesis. We review the current literature regarding nucleocytoplasmic transport disruption in C9-ALS, and discuss implications and directions for future research.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

OBJECTIVE: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. METHODS: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation. RESULTS: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals. INTERPRETATION: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.

Genetic deletion of the neuronal glutamate transporter, EAAC1, results in decreased neuronal death after pilocarpine-induced status epilepticus.

Excitatory amino acid carrier 1 (EAAC1 also called EAAT3) is a Na(+)-dependent glutamate transporter expressed by both glutamatergic and GABAergic neurons. It provides precursors for the syntheses of glutathione and GABA and contributes to the clearance of synaptically released glutamate. Mice deleted of EAAC1 are more susceptible to neurodegeneration in models of ischemia, Parkinson's disease, and aging. Antisense knock-down of EAAC1 causes an absence seizure-like phenotype. Additionally, EAAC1 expression increases after chemonvulsant-induced seizures in rodent models and in tissue specimens from patients with refractory epilepsy. The goal of the present study was to determine if the absence of EAAC1 affects the sensitivity of mice to seizure-induced cell death. A chemoconvulsant dose of pilocarpine was administered to EAAC1(-/-) mice and to wild-type controls. Although EAAC1(-/-) mice experienced increased latency to seizure onset, no significant differences in behavioral seizure severity or mortality were observed. We examined EAAC1 immunofluorescence 24h after pilocarpine administration and confirmed that pilocarpine causes an increase in EAAC1 protein. Forty-eight hours after induction of seizures, cell death was measured in hippocampus and in cortex using Fluoro-Jade C. Surprisingly, there was ∼2-fold more cell death in area CA1 of wild-type mice than in the corresponding regions of the EAAC1(-/-) mice. Together, these studies indicate that absence of EAAC1 results in either a decrease in pilocarpine-induced seizures that is not detectable by behavioral criteria (surprising, since EAAC1 provides glutamate for GABA synthesis), or that the absence of EAAC1 results in less pilocarpine/seizure-induced cell death, possible explanations as discussed.

Effect of inhibition of spinal cord glutamate transporters on inflammatory pain induced by formalin and complete Freund's adjuvant.

BACKGROUND: Spinal cord glutamate transporters clear synaptically released glutamate and maintain normal sensory transmission. However, their ultrastructural localization is unknown. Moreover, whether and how they participate in inflammatory pain has not been carefully studied. METHODS: Immunogold labeling with electron microscopy was carried out to characterize synaptic and nonsynaptic localization of glutamate transporters in the superficial dorsal horn. Their expression and uptake activity after formalin- and complete Freund's adjuvant (CFA)-induced inflammation were evaluated by Western blot analysis and glutamate uptake assay. Effects of intrathecal glutamate transporter activator (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline and inhibitors (DL-threo-ß-benzyloxyaspartate [TBOA], dihydrokainate, and DL-threo-ß-hydroxyaspartate), or TBOA plus group III metabotropic glutamate receptor antagonist (RS)-α-methylserine-O-phosphate, on formalin- and CFA-induced inflammatory pain were examined. RESULTS: In the superficial dorsal horn, excitatory amino acid carrier 1 is localized in presynaptic membrane, postsynaptic membrane, and axonal and dendritic membranes at nonsynaptic sites, whereas glutamate transporter-1 and glutamate/aspartate transporter are prominent in glial membranes. Although expression of these three spinal glutamate transporters was not altered 1 h after formalin injection or 6 h after CFA injection, glutamate uptake activity was decreased at these time points. Intrathecal (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline had no effect on formalin-induced pain behaviors. In contrast, intrathecal TBOA, dihydrokainate, and DL-threo-ß-hydroxyaspartate reduced formalin-evoked pain behaviors in the second phase. Intrathecal TBOA also attenuated CFA-induced thermal hyperalgesia at 6 h after CFA injection. The antinociceptive effects of TBOA were blocked by coadministration of (RS)-α-methylserine-O-phosphate. CONCLUSION: Our findings suggest that spinal glutamate transporter inhibition relieves inflammatory pain through activation of inhibitory presynaptic group III metabotropic glutamate receptors.

Genetic and functional studies of a missense variant in a glutamate transporter, SLC1A3, in Tourette syndrome.

OBJECTIVE: Abnormalities in neurotransmission within the cortico-striatal-thalamo-cortical circuitry are implicated in the pathogenesis of Tourette syndrome. Glutamate is a major excitatory neurotransmitter and an important member in the cortico-striatal-thalamo-cortical circuitry. To explore the role of glutamatergic neurotransmission in genetic susceptibility of Tourette syndrome, we carried out the genetic and functional characterization of sequence variants in SLC1A3 gene, which encodes the main glutamate transporter in astrocytes in individuals with well-characterized Tourette syndrome (n=256) and normal controls (n=224). METHODS: Exon-containing regions of SLC1A3 gene were screened using capillary electrophoresis-single strand conformation polymorphism followed by direct sequencing. Sequence variants were genotyped by restriction enzyme digestion and studied using glutamate uptake assay and membrane protein pull-down for transporter function. RESULTS: A missense variant involving a highly conserved residue, E219D, was identified in 11 heterozygous individuals with Tourette syndrome and four in the controls. The allele frequency for E219D was 2.4 folds higher in the Tourette syndrome (0.022) compared with the control cohort (0.009) although the difference did not reach statistical significance in the current cohorts (P=0.09). A H-glutamate-uptake assay showed that E219D conveys a significant increase (1.66 fold) in the SLC1A3-mediated glutamate uptake in HEK293 cells. A biotin-mediated membrane pull-down analysis showed a similar increase (1.5 fold) of mutant SLC1A3 protein in the membrane fraction of transfected HEK293 cells compared with that in the wild type controls. CONCLUSION: These results indicate that E219D is a functional SLC1A3 variant that is presented in a small number of individuals with Tourette syndrome. Further studies on possible changes in glutamate transport in the pathogenesis of Tourette syndrome are warranted.

Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS ( approximately 90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.

Respiratory impairment in a mouse model of amyotrophic lateral sclerosis.

Amyothrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of cortical and spinal motoneurons, leading to atrophy of limb, axial, and respiratory muscles. Patients with ALS invariably develop respiratory muscle weakness and most die from pulmonary complications. Overexpression of superoxide dismutase 1 (SOD1) gene mutations in mice recapitulates several of the clinical and pathological characteristics of ALS and is therefore a valuable tool to study this disease. The present study is intended to evaluate an age-dependent progression of respiratory complications in SOD1(G93A) mutant mice. In each animal, baseline measurements of breathing pattern [i.e., breathing frequency and tidal volume (VT)], minute ventilation (VE), and metabolism (i.e., oxygen consumption and carbon dioxide production) were repeatedly sampled at variable time points between 10 and 20 wk of age with the use of whole-body plethysmographic chambers. To further characterize the neurodegeneration of breathing, VE was also measured during 5-min challenges of hypercapnia (5% CO(2)) and hypoxia (10% O(2)). At baseline, breathing characteristics and metabolism remained relatively unchanged from 10 to 14 wk of age. From 14 to 18 wk of age, there were significant (P < 0.05) increases in baseline VT, VE, and the ventilatory equivalent (VE/oxygen consumption). After 18 wk of age, there was a rapid decline in VE due to significant (P < 0.05) reductions in both breathing frequency and VT. Whereas little change in hypoxic VE responses occurred between 10 and 18 wk, hypercapnic VE responses were significantly (P < 0.05) elevated at 18 wk due to an augmented VT response. Like baseline breathing characteristics, hypercapnic VE responses also declined rapidly after 18 wk of age. The phenotypic profile of SOD1(G93A) mutant mice was apparently unique because similar changes in respiration and metabolism were not observed in SOD1 controls. The present results outline the magnitude and time course of respiratory complications in SOD1(G93A) mutant mice as the progression of disease occurs in this mouse model of ALS.

Spinal glutamate uptake is critical for maintaining normal sensory transmission in rat spinal cord.

Glutamate is a major excitatory neurotransmitter in primary afferent terminals and is critical for normal spinal excitatory synaptic transmission. However, little is known about the regulation of synaptically released glutamate in the spinal cord under physiologic conditions. The sodium-dependent, high-affinity glutamate transporters are the primary mechanism for the clearance of synaptically released glutamate. In the present study, we found that intrathecal injection of glutamate transporter blockers DL-threo-beta-benzyloxyaspartate (TBOA) and dihydrokainate produced significant and dose-dependent spontaneous nociceptive behaviors, such as licking, shaking, and caudally directed biting, phenomena similar to the behaviors caused by intrathecal glutamate receptor agonists. Intrathecal TBOA also led to remarkable hypersensitivity in response to thermal and mechanical stimuli. These behavioral responses could be significantly blocked by intrathecal injection of the NMDA receptor antagonists MK-801 and AP-5, the non-NMDA receptor antagonist CNQX or the nitric oxide synthase inhibitor L-NAME. In vivo microdialysis analysis showed short-term elevation of extracellular glutamate concentration in the spinal cord after intrathecal injection of TBOA. Furthermore, topical application of TBOA on the dorsal surface of the spinal cord resulted in a significant elevation of extracellular glutamate concentration demonstrated by in vivo glutamate voltametry. The present study indicates that defective spinal glutamate uptake caused by inhibition of glutamate transporters leads to excessive glutamate accumulation in the spinal cord. The latter results in persistent over-activation of synaptic glutamate receptors, producing spontaneous nociceptive behaviors and sensory hypersensitivity. Our results suggest that glutamate uptake through spinal glutamate transporters is critical for maintaining normal sensory transmission under physiologic conditions.

Toxicity of familial ALS-linked SOD1 mutants from selective recruitment to spinal mitochondria.

One cause of amyotrophic lateral sclerosis (ALS) is mutation in ubiquitously expressed copper/zinc superoxide dismutase (SOD1), but the mechanism of toxicity to motor neurons is unknown. Multiple disease-causing mutants, but not wild-type SOD1, are now demonstrated to be recruited to mitochondria, but only in affected tissues. This is independent of the copper chaperone for SOD1 and dismutase activity. Highly preferential association with spinal cord mitochondria is seen in human ALS for a mutant SOD1 that accumulates only to trace cytoplasmic levels. Despite variable proportions that are successfully imported, nearly constant amounts of SOD1 mutants and covalently damaged adducts of them accumulate as apparent import intermediates and/or are tightly aggregated or crosslinked onto integral membrane components on the cytoplasmic face of those mitochondria. These findings implicate damage from action of spinal cord-specific factors that recruit mutant SOD1 to spinal mitochondria as the basis for their selective toxicity in ALS.