myelin-basic-protein and thymosin-beta(4)

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). No effective remyelination therapies are in use. We hypothesized that thymosin beta4 (Tβ4) is an effective remyelination treatment by promoting differentiation of oligodendrocyte progenitor cells (OPCs), and that the epidermal growth factor receptor (EGFR) signaling pathway contributes to this process. Two demyelination animal models were employed in this study: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE mice were treated daily for 30days, with Tβ4 or saline treatment initiated on the day of EAE onset; and 2) cuprizone diet model, a non-inflammatory demyelination model. The mice were treated daily for 4weeks with Tβ4 or saline after fed a cuprizone diet for 5weeks. Immunofluorescent staining and Western blot were performed to measure the differentiation of OPCs, myelin and axons, respectively. To obtain insight into mechanisms of action, the expression and activation of the EGFR pathway was measured. AG1478, an EGFR inhibitor, was employed in a loss-of-function study. Data revealed that animals in both demyelination models exhibited significant reduction of myelin basic protein (MBP(+)) levels and CNPase(+) oligodendrocytes. Treatment of EAE mice with Tβ4 significantly improved neurological outcome. Double immunofluorescent staining showed that Tβ4 significantly increased the number of newly generated oligodendrocytes identified by BrdU(+)/CNPase(+) cells and MBP(+) mature oligodendrocytes, and reduced axonal damage in the EAE mice compared with the saline treatment. The newly generated mature oligodendrocytes remyelinated axons, and the increased mature oligodendrocytes significantly correlated with functional improvement (r=0.73, p<0.05). Western blot analysis revealed that Tβ4 treatment increased expression and activation of the EGFR pathway. In the cuprizone demyelination model, Tβ4 treatment was confirmed that significantly increased OPC differentiation and remyelination, and increased the expression of EGFR and activated the EGFR pathway in the demyelinating corpus callosum. In cultured OPCs, blockage of the activation of the EGFR pathway with AG1478 abolished the Tβ4-increased OPC differentiation. Collectively, these findings indicate that: 1) Tβ4 increases proliferation of OPCs and the maturation of OPCs to myelinating oligodendrocytes which in concert, likely contribute to the beneficial effect of Tβ4 on EAE, 2) EGFR upregul

Topics: Animals; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chelating Agents; Cuprizone; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Enzyme Inhibitors; ErbB Receptors; Female; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Myelin Proteolipid Protein; Oligodendroglia; Peptide Fragments; Proteoglycans; Quinazolines; Stem Cells; Thymosin; Time Factors; Tyrphostins

Thymosin β4 (Tβ4), a G-actin-sequestering peptide, improves neurological outcome in rat models of neurological injury. Tissue inflammation results from neurological injury, and regulation of the inflammatory response is vital for neurological recovery. The innate immune response system, which includes the Toll-like receptor (TLR) proinflammatory signaling pathway, regulates tissue injury. We hypothesized that Tβ4 regulates the TLR proinflammatory signaling pathway. Because oligodendrogenesis plays an important role in neurological recovery, we employed an in vitro primary rat embryonic cell model of oligodendrocyte progenitor cells (OPCs) and a mouse N20.1 OPC cell line to measure the effects of Tβ4 on the TLR pathway. Cells were grown in the presence of Tβ4, ranging from 25 to 100 ng/ml (RegeneRx Biopharmaceuticals Inc., Rockville, MD), for 4 days. Quantitative real-time PCR data demonstrated that Tβ4 treatment increased expression of microRNA-146a (miR-146a), a negative regulator the TLR signaling pathway, in these two cell models. Western blot analysis showed that Tβ4 treatment suppressed expression of IL-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two proinflammatory cytokines of the TLR signaling pathway. Transfection of miR-146a into both primary rat embryonic OPCs and mouse N20.1 OPCs treated with Tβ4 demonstrated an amplification of myelin basic protein (MBP) expression and differentiation of OPC into mature MBP-expressing oligodendrocytes. Transfection of anti-miR-146a nucleotides reversed the inhibitory effect of Tβ4 on IRAK1 and TRAF6 and decreased expression of MBP. These data suggest that Tβ4 suppresses the TLR proinflammatory pathway by up-regulating miR-146a.

Topics: Animals; Cell Differentiation; Cytokines; Hep G2 Cells; Humans; Inflammation; Interleukin-1 Receptor-Associated Kinases; Mice; MicroRNAs; Myelin Basic Protein; Oligodendroglia; Rats; Rats, Wistar; Signal Transduction; Thymosin; TNF Receptor-Associated Factor 6; Toll-Like Receptors; Up-Regulation

Thymosin β4 (Tβ4) has many physiological functions that are highly relevant to spinal cord injury (SCI), including neuronal survival, anti-inflammation, wound repair promotion, and angiogenesis. The present study investigated the therapeutic value of Tβ4 in SCI, with a focus on its neuroprotective, anti-inflammatory, and vasculoprotective properties. Tβ4 or a saline control was administered by intraperitoneal injection 30 min, 3 days, or 5 days after SCI with mild compression in rat. Locomotor recovery was tested with the Basso-Beattie-Bresnahan scale and a footprint analysis. All behavioral assessments were markedly improved with Tβ4 treatment. Histological examination at 7 days post injury showed that the numbers of surviving neurons and oligodendrocytes were significantly increased in Tβ4-treated animals compared to saline-treated controls. Levels of myelin basic protein, a marker of mature oligodendrocytes, in Tβ4-treated rats were 57.8% greater than those in saline-treated controls. The expression of ED1, a marker of activated microglia/macrophages, was reduced by 36.9% in the Tβ4-treated group compared to that of the saline-treated group. Tβ4 treatment after SCI was also associated with a significant decrease in pro-inflammatory cytokine gene expression and a significant increase in the mRNA levels of IL-10 compared to the control. Moreover, the size of lesion cavity delineated by astrocyte scar in the injured spinal cord was markedly reduced in Tβ4-treated animals compared to saline-treated controls. Given the known safety of Tβ4 in clinical trials and its beneficial effects on SCI recovery, the results of this study suggested that Tβ4 is a good candidate for SCI treatment in humans.

Topics: Animals; Cell Survival; Cytokines; Disease Models, Animal; Interleukin-10; Macrophages; Male; Microglia; Myelin Basic Protein; Neuroimmunomodulation; Neurons; Neuroprotective Agents; Oligodendroglia; Random Allocation; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Thymosin; Time Factors

Thymosin β4 (Tβ4) is a 5K actin binding peptide. Tβ4 improves neurological outcome in a rat model of embolic stroke and research is now focused on optimizing its dose for clinical trials. The purpose of this study was to perform a dose-response study of Tβ4 to determine the optimal dose of neurological improvement in a rat model of embolic stroke.. Male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAo). Rats were divided into 4 groups of 10 animals/group: control, 2, 12 and 18 mg/kg. Tβ4 was administered intraperitoneally 24h after MCAo and then every 3 days for 4 additional doses in a randomized controlled fashion. Neurological tests were performed after MCAo and before treatment and up to 8 weeks after treatment. The rats were sacrificed 56 days after MCAo and lesion volumes measured. Generalized estimating equation was used to compare the treatment effect on long term functional recovery at day 56. A quartic regression model was used for an optimal dose determination.. Tβ4 significantly improved neurological outcome at dose of 2 and 12 mg/kg at day 14 and extended to day 56 (p-values <0.05). The higher dose of 18 mg/kg did not show significant improvement. The estimated optimal dose of 3.75 mg/kg would provide optimal neurological improvement.. This study shown that Tβ4 significantly improved the long term neurological functional recovery at day 56 after MCAo with an optimal dose of 3.75 mg/kg. These results provide preclinical data for human clinical trials.

Topics: Acute Disease; Adenomatous Polyposis Coli; Animals; Brain; Bromodeoxyuridine; Disease Models, Animal; Dose-Response Relationship, Drug; Infarction, Middle Cerebral Artery; Male; Myelin Basic Protein; Neuroimaging; Neurologic Examination; Rats; Stroke; Thymosin; Time Factors; Treatment Outcome; Versicans