Expression and Cellular Distribution of Ubiquitin in Response to Injury in the Developing Spinal Cord of Monodelphis domestica

Abstract

Ubiquitin, an 8.5 kDa protein associated with the proteasome degradation pathway has been recently identified as differentially expressed in segment of cord caudal to site of injury in developing spinal cord. Here we describe ubiquitin expression and cellular distribution in spinal cord up to postnatal day P35 in control opossums (Monodelphis domestica) and in response to complete spinal transection (T10) at P7, when axonal growth through site of injury occurs, and P28 when this is no longer possible. Cords were collected 1 or 7 days after injury, with age-matched controls and segments rostral to lesion were studied. Following spinal injury ubiquitin levels (western blotting) appeared reduced compared to controls especially one day after injury at P28. In contrast, after injury mRNA expression (qRT-PCR) was slightly increased at P7 but decreased at P28. Changes in isoelectric point of separated ubiquitin indicated possible post-translational modifications. Cellular distribution demonstrated a developmental shift between earliest (P8) and latest (P35) ages examined, from a predominantly cytoplasmic immunoreactivity to a nuclear expression; staining level and shift to nuclear staining was more pronounced following injury, except 7 days after transection at P28. After injury at P7 immunostaining increased in neurons and additionally in oligodendrocytes at P28. Mass spectrometry showed two ubiquitin bands; the heavier was identified as a fusion product, likely to be an ubiquitin precursor. Apparent changes in ubiquitin expression and cellular distribution in development and response to spinal injury suggest an intricate regulatory system that modulates these responses which, when better understood, may lead to potential therapeutic targets.

NGF-Dependent Changes in Ubiquitin Homeostasis Trigger Early Cholinergic Degeneration in Cellular and Animal AD-Model

“…in 0.2% B27 media in the presence of exogenous NGF (100 ng/ml) from plating ( t 0 ) were deprived of this trophic support for different periods of time (-1.5, -3, -6 h) and Western blotting was carried out on whole-cell lysates by probing with anti-pan ubiquitin antibody (Z0458 Dako) (Emmerich and Cohen, 2015). This polyclonal antibody is reported to detect both conjugated-ubiquitin and free ubiquitin (Myeku and Figueiredo-Pereira, 2011; Noor et al, 2013; Emmerich and Cohen, 2015) with similar affinity for Lys48- and Lys63-ubiquitinated substrates (Myeku and Figueiredo-Pereira, 2011). Following short-term removal of neurotrophin, we detected a progressive decrease in immunoreactivity of polyubiquitin chains-conjugates (Figures 1A,B) whose degradation -especially of those formed with ubiquitin lysine 48 (K48) linkage- generally relies on the 26S proteasome (Thrower et al, 2000; Grice and Nathan, 2016) and is canonically used as an indirect cellular indicator of activation in proteasomal function(s) (Myeku et al, 2011).…”

The Ubiquitin-Proteasome System: Potential Therapeutic Targets for Alzheimer’s Disease and Spinal Cord Injury

“…Several lines of evidence link ubiquitin, Uch-L1 and E3 ubiquitin ligases to survival of such interneurons. Specifically, spinal cord interneurons with increased ubiquitin and Uch-L1 expression have been reported to have greater cell survival after SCI (Noor et al, 2013 ). Similarly, ubiquitin and Uch-L1 up-regulation has also been observed in complete spinal cord transection (Fry et al, 2003 ; Lane et al, 2007 ).…”

“…For example, following spinal cord transection in Monodelphis domestica, axonal regeneration and regrowth of neuronal tissue was found at the injury site in animals injured at 7 days after birth (P7), which was associated with upregulation of ubiquitin mRNA and protein expression. However if injury occurred at postnatal 28 day (p28), little or no upregulation in ubiquitin expression occurred and no neuronal regeneration was observed (Ji et al, 2008 ; Yan et al, 2010 ; Noor et al, 2013 ). The levels of mono-ubiquitin were decreased at sites both caudal and rostral to the SCI injury site in aged animals, which also have reduced neuroregenerative capabilities; the magnitude of the decrease was found to be age-related.…”

“…For example, in motor neurons or interneurons of the ventral horn, ubiquitin is significantly increased in the cytosol after injury of postnatal 7 day (p7) mice which demonstrate significant neuroregenerative capabilities. By contrast, in mature mice, where the capacity of neuron regeneration is lost, there is minimal change in ubiquitin levels in the cytosol of ventral horn neurons, in which ubiquitin was primarily localized to the nuclei (Noor et al, 2011 , 2013 ). These findings suggest that the growth inhibitory or stimulatory factors that explain the difference in neuroregenerative capacity between young and old mice regulate ubiquitin expression and localization and support the view that certain levels of ubiquitin in injured neurons are essential for neuron regeneration.…”

Rapid recovery and altered neurochemical dependence of locomotor central pattern generation following lumbar neonatal spinal cord injury

“…; Noor et al . ). To obtain more comprehensive information about the enhanced plasticity that occurs in the neonatal spinal cord, we developed a neonatal mouse model for spinal cord injury in which standardized injuries can be made and a variety of methodologies ranging from genetic to cellular to network to behavioural can be implemented (Boulland et al .…”