cyclin-d1 and Spinal-Cord-Injuries

This study focuses on the protective effect of epigenetic silencing of CyclinD1 against spinal cord injury (SCI) using bone marrow-derived mesenchymal stem cells (BMSCs) in rats. Eighty-eight adult female Wistar rats were randomly assigned into the sham group, the control group, the si-CyclinD1 + BMSCs group and the BMSCs group. CyclinD1 protein and mRNA expressions after siRNA transfection were detected by Western blotting and qRT-PCR. The siRNA-CyclinD1 BMSCs were transplanted into rats in the si-CyclinD1 + BMSCs group using stereotaxic method 6 hr after SCI. Hindlimb locomotor performance was determined using inclined plane test and Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Expressions of glial fibrillary acidic protein (GFAP) and nerve growth factor (NGF) were detected by immunohistochemistry. Inclined plane and BBB scores in the control, si-CyclinD1 + BMSCs, and BMSCs groups were significantly lower than the sham group, but these scores were evidently decreased in the control group and increased in the si-CyclinD1 + BMSCs group compared with the BMSCs group. The repair degree of spinal cord tissues of rats in the si-CyclinD1 + BMSCs group was obvious than the BMSCs group. GFAP and NGF protein expressions were markedly decreased in the control, si-CyclinD1 + BMSCs and BMSCs groups when compared with the sham group. GFAP- and NGF-positive cells were significantly increased in the si-CyclinD1 + BMSCs group while decreased in the control group. Our study provides evidence that epigenetic silencing of CyclinD1 using BMSCs might accelerate the repair of SCI in rats.

Topics: Animals; Bone Marrow Cells; Cyclin D1; Disease Models, Animal; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Gene Silencing; Glial Fibrillary Acidic Protein; Humans; Locomotion; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rats; Receptor, Nerve Growth Factor; Recovery of Function; RNA, Small Interfering; Spinal Cord; Spinal Cord Injuries

Large tumor suppressor kinase 1 (LATS1) is a serine/threonine kinase of the AGC kinase family in mammals and involved in various biological processes, it is a key regulator of cell cycle progression. However, the role of LATS1 in central nervous system trauma is still unknown. In present study, we performed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of LATS1 expression in the spinal cord. We found that LATS1 protein levels were significantly decreased at day 1 after injury. Meanwhile, double immunofluorescence staining showed these changes were striking in astrocytes, which were largely proliferated after SCI. In vitro, LATS1 overexpression inhibited astrocyte proliferation. Conversely, LATS1 depletion by siRNA promoted cell proliferation in primary astrocyte. Moreover, LATS1 overexpression reduced cyclin D1 expression and increased the expression of p27

Topics: Animals; Apoptosis Regulatory Proteins; Astrocytes; Cell Proliferation; Cells, Cultured; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Gliosis; Male; Phosphorylation; Protein Serine-Threonine Kinases; Rats, Sprague-Dawley; RNA Interference; Spinal Cord Injuries; YAP-Signaling Proteins

The cell division cycle 6 (CDc6) protein has been primarily investigated as a component of the pre-replicative complex for the initiation of DNA replication. Some studies have shown that CDc6 played a critical role in the development of human carcinoma. However, the expression and roles of CDc6 in the central nervous system remain unknown. We have performed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of CDc6 expression in spinal cord. Western blot have found that CDc6 protein levels first significantly increase, reach a peak at day 3, and then gradually return to normal level at day 14 after SCI. Double immunofluorescence staining showed that CDc6 immunoreactivity was found in neurons, astrocytes, and microglia. Additionally, colocalization of CDc6/active caspase-3 has been detected in neurons and colocalization of CDc6/proliferating cell nuclear antigen has been detected in astrocytes and microglial. In vitro, CDc6 depletion by short interfering RNA inhibits astrocyte proliferation and reduces cyclin A and cyclin D1 protein levels. CDc6 knockdown also decreases neuronal apoptosis. We speculate that CDc6 might play crucial roles in CNS pathophysiology after SCI.

Topics: Animals; Apoptosis; Astrocytes; Caspase 3; Cell Cycle Proteins; Chromosomal Proteins, Non-Histone; Cyclin A; Cyclin D1; Male; Microglia; Neurons; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries

Spinal cord injury (SCI) is a traumatic event that causes a secondary and extended inflammation characterized by infiltration of immune cells, including T lymphocytes, release of pro-inflammatory mediators in the lesion site, and tissue degeneration. Current therapeutic approaches for SCI are limited to glucocorticoids (GC) due to their potent anti-inflammatory activity. GC efficacy resides, in part, in the capability to inhibit NF-κB, T lymphocyte activation, and the consequent cytokine production. In this study, we performed experiments aimed to test the susceptibility of glucocorticoid-induced leucine zipper (GILZ) transgenic (GILZ(TG)) mice, in which GILZ is selectively over-expressed in T lymphocytes, to SCI induction. Consistent with a decreased inflammatory response, GILZ(TG) were less susceptible to SCI as compared to wild-type littermates. Notably, inhibition of NF-κB activation and nuclear translocation, diminished T lymphocytes activation and tissue infiltration, as well as decreased release of cytokines were evident in GILZ(TG) as compared to wild-type mice. Moreover, GILZ(TG) showed a reduced tumor necrosis factor-α, IL-1β, Inductible nitric oxide synthase (iNOS) and nytrotyrosine production, apoptosis, and neuronal tissue damage. Together these results indicate that GILZ mimics the anti-inflammatory effect of GC and represents a potential pharmacological target for modulation of T lymphocyte-mediated immune response in inflammatory disorders, such as SCI.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Cyclin D1; Cytokines; Dinoprostone; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Inflammation; Mice; Mice, Transgenic; Nitric Oxide Synthase Type II; Peroxidase; Signal Transduction; Spinal Cord Injuries; T-Lymphocytes; Transcription Factors

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, E2F1 and PCNA, at various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. Treatment with flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional recovery was significantly improved after SCI from day 7 through day 28. Treatment significantly reduced lesion volume and the number of Iba-1(+) microglia in the preserved tissue and increased the myelinated area of spared white matter as well as the number of CC1(+) oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3, associated with microglial activation and astrocytic reactivity by reduction of GFAP, NG2, and CHL1 expression. Our current study supports the role of cell cycle activation in the pathophysiology of SCI and by using a clinically relevant treatment model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cell Cycle; Cell Cycle Proteins; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; E2F1 Transcription Factor; Flavonoids; Immunohistochemistry; Locomotion; Male; Microfilament Proteins; Microglia; Neurons; Oligodendroglia; Piperidines; Proliferating Cell Nuclear Antigen; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Time Factors