Transforming growth factor-β1 impairs neuropathic pain through pleiotropic effects

<p>Abstract</p> <p>Background</p> <p>Understanding the underlying mechanisms of neuropathic pain caused by damage to the peripheral nervous system remains challenging and could lead to significantly improved therapies. Disturbance of homeostasis not only occurs at the site of injury but also extends to the spinal cord and brain involving various types of cells. Emerging data implicate neuroimmune interaction in the initiation and maintenance of chronic pain hypersensitivity.</p> <p>Results</p> <p>In this study, we sought to investigate the effects of TGF-β1, a potent anti-inflammatory cytokine, in alleviating nerve injury-induced neuropathic pain in rats. By using a well established neuropathic pain animal model (partial ligation of the sciatic nerve), we demonstrated that intrathecal infusion of recombinant TGF-β1 significantly attenuated nerve injury-induced neuropathic pain. TGF-β1 treatment not only prevents development of neuropathic pain following nerve injury, but also reverses previously established neuropathic pain conditions. The biological outcomes of TGF-β1 in this context are attributed to its pleiotropic effects. It inhibits peripheral nerve injury-induced spinal microgliosis, spinal microglial and astrocytic activation, and exhibits a powerful neuroprotective effect by preventing the induction of ATF3⁺neurons following nerve ligation, consequently reducing the expression of chemokine MCP-1 in damaged neurons. TGF-β1 treatment also suppresses nerve injury-induced inflammatory response in the spinal cord, as revealed by a reduction in cytokine expression.</p> <p>Conclusion</p> <p>Our findings revealed that TGF-β1 is effective in the treatment of neuropathic by targeting both neurons and glial cells. We suggest that therapeutic agents such as TGF-β1 having multipotent effects on different types of cells could work in synergy to regain homeostasis in local spinal cord microenvironments, therefore contributing to attenuate neuropathic pain.</p

MALDI-TOF High Mass Calibration up to 200 kDa Using Human Recombinant 16 kDa Protein Histidine Phosphatase Aggregates

Background: Protein histidine phosphatase (PHP) is an enzyme which removes phosphate groups from histidine residues. It was described for vertebrates in the year 2002. The recombinant human 16 kDa protein forms multimeric complexes in physiological buffer and in the gas phase. High-mass calibration in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has remained a problem due to the lack of suitable standards. Large proteins can hardly be freed of their substructural microheterogeneity by classical purification procedures so that their use as calibrants is limited. A small adduct-forming protein of validated quality is a valuable alternative for that purpose. Methodology/Principal Findings: Three major PHP clusters of,113, 209 and.600 kDa were observed in gel filtration analysis. Re-chromatography of the monomer peak showed the same cluster distribution. The tendency to associate was detected also in MALDI-TOF MS measuring regular adducts up to 200 kDa. Conclusions/Significance: PHP forms multimers consisting of up to more than 35 protein molecules. In MALDI-TOF MS it generates adduct ions every 16 kDa. The protein can be produced with high quality so that its use as calibration compoun

L-arginine Supplementation Improves Responses to Injury and Inflammation in Dextran Sulfate Sodium Colitis

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y+ cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS−/− mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity

Equivalence of Conventionally-Derived and Parthenote-Derived Human Embryonic Stem Cells

As human embryonic stem cell (hESC) lines can be derived via multiple means, it is important to determine particular characteristics of individual lines that may dictate the applications to which they are best suited. The objective of this work was to determine points of equivalence and differences between conventionally-derived hESC and parthenote-derived hESC lines (phESC) in the undifferentiated state and during neural differentiation.hESC and phESC were exposed to the same expansion conditions and subsequent neural and retinal pigmented epithelium (RPE) differentiation protocols. Growth rates and gross morphology were recorded during expansion. RTPCR for developmentally relevant genes and global DNA methylation profiling were used to compare gene expression and epigenetic characteristics. Parthenote lines proliferated more slowly than conventional hESC lines and yielded lower quantities of less mature differentiated cells in a neural progenitor cell (NPC) differentiation protocol. However, the cell lines performed similarly in a RPE differentiation protocol. The DNA methylation analysis showed similar general profiles, but the two cell types differed in methylation of imprinted genes. There were no major differences in gene expression between the lines before differentiation, but when differentiated into NPCs, the two cell types differed in expression of extracellular matrix (ECM) genes.These data show that hESC and phESC are similar in the undifferentiated state, and both cell types are capable of differentiation along neural lineages. The differences between the cell types, in proliferation and extent of differentiation, may be linked, in part, to the observed differences in ECM synthesis and methylation of imprinted genes

Differential Spatial Expression and Subcellular Localization of CtBP Family Members in Rodent Brain

C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol

Gene expression profiling of the astrocyte transcriptome in multiple sclerosis normal appearing white matter reveals a neuroprotective role

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression. Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC ≥ 1.5 and p-value ≤ 0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in M

MPP+-induced toxicity in the presence of dopamine is mediated by COX-2 through oxidative stress

Accumulating evidence suggests that endogenous dopamine may act as a neurotoxin and thereby participate in the pathophysiology of Parkinson’s disease (PD). Cyclooxygenase-2 (COX-2) has been implicated in the pathogenesis of PD due to its ability to generate reactive oxygen species (ROS). Inhibition of COX-2 leads to neuroprotection by preventing the formation of dopamine-quinone. In this study, we examined whether dopamine mediates 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity in primary ventral mesencephalic (VM) neurons, an in vitro model of PD, and if so, whether the protective effects of COX-2 inhibitors on dopamine mediated MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis result from the reduction of ROS. Reserpine, a dopamine-depleting agent, significantly reduced VM neurotoxicity induced by MPP+, whereas dopamine had an additive effect on MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis. However, inhibition of COX-2 by a selective COX-2 inhibitor (DFU) or ibuprofen significantly attenuated MPP+-induced VM cell toxicity and VM dopaminergic cell apoptosis, which was accompanied by a decrease in ROS production in VM dopaminergic neurons. These results suggest that dopamine itself mediates MPP+-induced VM neurotoxicity and VM dopaminergic cell apoptosis in the presence of COX-2