Delayed cell cycle pathway modulation facilitates recovery after spinal cord injury

Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 05/2012; 11(9):1782-95. DOI: 10.4161/cc.20153
Source: PubMed


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.

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Available from: Michael Dinizo, Mar 17, 2014
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    • "Cell cycle proteins are also expressed in other cell types of the CNS [53,54], such as oligodendrocytes and infiltrating Schwann cells, which contribute to myelin repair in the injured spinal cord [55]. We recently reported increases in the myelinated white matter area and expression of myelin basic protein in flavopiridol-treated injured rats [24]. However, it remains unclear whether cell cycle inhibition increases remyelinated axons by oligodendrocytes and Schwann cells, or reduces chronic progressive demyelination. "
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    ABSTRACT: Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs) inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins - including cyclin D1 and E, CDK4, E2F5 and PCNA - for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX - a key component of NADPH oxidase - up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar formation, chronic inflammation and further tissue loss.
    Journal of Neuroinflammation 07/2012; 9(1):169. DOI:10.1186/1742-2094-9-169 · 5.41 Impact Factor
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    ABSTRACT: Smad ubiquitination regulatory factor 1 (Smurf1) is one of members of the Hect family of proteins, which also includes the ubiquitin E3-type ligases Nedd3 and E6-AP. As an E3 ligase, Smurf1 selectively interacts with receptor-regulated Smads to trigger their ubiquitination and degradation. Recently, a report indicates that Smurf1 can inhibit apoptosis by regulating p53 negatively, which depends on the effect of Smurf1 stabilizing the MDM2-MDMX complex. However, the roles of Smurf1 in central nervous system injury remain to be unknown. In our study, we finished acute spinal cord injury (SCI) in adult rats to research the protein expression and cellular localization of Smurf1 in spinal cord. Western blot analysis showed that Smurf1 was low expressed in normal spinal cord. It was increased at 6 h after SCI, peaked at 1 day, remained for 3 days, and then declined gradually during the following days. Immunohistochemistry further confirmed that Smurf1 immunoactivity was expressed at low levels in the gray matter and white matter in normal condition and increased after SCI. Double immunofluorescence staining showed that Smurf1 was co-expressed NeuN (neuronal marker), CNPase (oligodendroglial marker), and active caspase-3 at 1 day post-injury. Additionally, p53 and MDM2 levels were up-regulated after SCI consistently. All these findings suggest that Smurf1 might be involved in the pathophysiology of spinal cord after SCI.
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    ABSTRACT: Spinal cord injury (SCI) causes not only sensorimotor and cognitive deficits, but frequently also severe chronic pain that is difficult to treat (SCI pain). We previously showed that hyperesthesia, as well as spontaneous pain induced by electrolytic lesions in the rat spinothalamic tract, is associated with increased spontaneous and sensory-evoked activity in the posterior thalamic nucleus (PO). We have also demonstrated that rodent impact SCI increases cell cycle activation (CCA) in the injury region and that post-traumatic treatment with cyclin dependent kinase inhibitors reduces lesion volume and motor dysfunction. Here we examined whether CCA contributes to neuronal hyperexcitability of PO and hyperpathia after rat contusion SCI, as well as to microglial and astroglial activation (gliopathy) that has been implicated in delayed SCI pain. Trauma caused enhanced pain sensitivity, which developed weeks after injury and was correlated with increased PO neuronal activity. Increased CCA was found at the thoracic spinal lesion site, the lumbar dorsal horn, and the PO. Increased microglial activation and cysteine-cysteine chemokine ligand 21 expression was also observed in the PO after SCI. In vitro, neurons co-cultured with activated microglia showed up-regulation of cyclin D1 and cysteine-cysteine chemokine ligand 21 expression. In vivo, post-injury treatment with a selective cyclin dependent kinase inhibitor (CR8) significantly reduced cell cycle protein induction, microglial activation, and neuronal activity in the PO nucleus, as well as limiting chronic SCI-induced hyperpathia. These results suggest a mechanistic role for CCA in the development of SCI pain, through effects mediated in part by the PO nucleus. Moreover, cell cycle modulation may provide an effective therapeutic strategy to improve reduce both hyperpathia and motor dysfunction after SCI.
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