Effects of Reducing Suppressors of Cytokine Signaling-3 (SOCS3) Expression on Dendritic Outgrowth and Demyelination after Spinal Cord Injury

Abstract

Suppressors of cytokine signaling-3 (SOCS3) is associated with limitations of nerve growth capacity after injury to the central nervous system. Although genetic manipulations of SOCS3 can enhance axonal regeneration after optic injury, the role of SOCS3 in dendritic outgrowth after spinal cord injury (SCI) is still unclear. The present study investigated the endogenous expression of SOCS3 and its role in regulating neurite outgrowth in vitro. Interleukin-6 (IL-6) induces SOCS3 expression at the mRNA and protein levels in neuroscreen-1 (NS-1) cells. In parallel to SOCS3 expression, IL-6 induced tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) in NS-1 cells. Lentiviral delivery of short hairpin RNA (shSOCS3) (Lenti-shSOCS3) to decrease SOCS3 expression into NS-1 cells enhanced IL-6-induced tyrosine phosphorylation of STAT3 (P-STAT3 Tyr705) and promoted neurite outgrowth. In addition, we determined if reduction of SOCS3 expression by microinjection of Lenti-shSOCS3 into spinal cord enhances dendrite outgrowth in spinal cord neurons after SCI. Knocking down of SOCS3 in spinal cord neurons with Lenti-shSOCS3 increased complete SCI-induced P-STAT3 Tyr705. Immunohistochemical analysis showed that complete SCI induced a significant reduction of microtubule association protein 2-positive (MAP-2+) dendrites in the gray and white matter at 1 and 4 weeks after injury. The SCI-induced reduction of MAP-2+ dendrites was inhibited by infection with Lenti-shSOCS3 in areas both rostral and caudal to the lesion at 1 and 4 weeks after complete SCI. Furthermore, shSOCS3 treatment enhanced up-regulation of growth associated protein-43 (GAP-43) expression, which co-localized with MAP-2+ dendrites in white matter and with MAP-2+ cell bodies in gray matter, indicating Lenti-shSOCS3 may induce dendritic regeneration after SCI. Moreover, we demonstrated that Lenti-shSOCS3 decreased SCI-induced demyelination in white matter of spinal cord both rostral and caudal to the injury site 1 week post-injury, but not rostral to the injury at 4 weeks post-injury. Importantly, similar effects as Lenti-shSOCS3 on increasing MAP-2+ intensity and dendrite length, and preventing demyelination were observed when a second shSOCS3 (Lenti-shSOCS3 #2) was applied to rule out the possibilities of off target effects of shRNA. Collectively, these results suggest that knocking down of SOCS3 enhances dendritic regeneration and prevents demyelination after SCI.

Mitochondrial STAT3 is negatively regulated by SOCS3 and upregulated after spinal cord injury

“…Lentiviral particles were generated by calcium phosphate-mediated co-transfection of HEK-293T cells with pLVX, SOCS3/pLVX, pGipz, or shSOCS3/pGipz, together with psPAX2 (packaging plasmid) and pMD2G (envelope plasmid). Virus was collected after 72 h and titers up to 3–4×10 7 infectious units/ml were obtained (Park et al, 2014; Park et al, 2015). SH-SY5Y cells were infected with Lentivirus delivering pLVX, SOCS3/pLVX, pGipz, or shSOCS3/pGipz, sorted by FACS analysis using green fluorescence protein (GFP) expression, and stored at −80°C (Park et al, 2012).…”

“…Expression of SOCS3 in neurons plays a negative role in regulating cell survival and neurite outgrowth and is upregulated after SCI (Miao et al, 2006; Park et al, 2014; Park et al, 2015). Following axonal injury, SOCS3 is upregulated rapidly in injured neurons (Fischer et al, 2004; Park et al, 2009) by cytokines of the interleukin-6 (IL-6) family via STAT3 activation (Baker et al, 2009).…”

“…These results coincided with our previous findings that T8 spinal cord complete transection induces both SOCS3 and P-STAT3 Tyr705 (P-STAT3 Tyr) in vivo . Knocking down SOCS3 reduced spinal cord injury (SCI)-induced neuronal death and demyelination (Park et al, 2014; Park et al, 2015). Furthermore, knocking down SOCS3 with Lentiviral-delivered short hairpin RNAs (shRNAs) (shSOCS3) protected neuronal morphological features and enhanced dendritic regeneration after SCI (Park et al, 2014; Park et al, 2015), which might be attributable to increased P-STAT3 Tyr.…”

“…Although a number of studies have reported that SOCS3 inhibits P-STAT3 Tyr as a negative regulator to terminate signaling (Miao et al, 2006; Newbern et al, 2009; Park et al, 2014; Park et al, 2015), regulation of P-STAT3 Ser by SOCS3 is poorly understood. The present study addressed these issues, using immunoblot analyses of P-STAT3 Ser levels in SH-SY5Y neuroblastoma cells after stimulation with Oncostatin M (OSM), a member of the IL-6 cytokine family.…”

Advances in peripheral nerve regeneration as it relates to VCA

“…The same team also showed that suppression of the SOCS3 pathway has a neuroprotective effect. 86 Reducing SOCS3 expression after spinal cord injury led to reduced demyelination in areas distal to the injury.…”