Age-Dependent Changes in the Proteome Following Complete Spinal Cord Transection in a Postnatal South American Opossum (Monodelphis domestica)

Recovery from severe spinal injury in adults is limited, compared to immature animals who demonstrate some capacity for repair. Using laboratory opossums (Monodelphis domestica), the aim was to compare proteomic responses to injury at two ages: one when there is axonal growth across the lesion and substantial behavioural recovery and one when no axonal growth occurs. Anaesthetized pups at postnatal day (P) 7 or P28 were subjected to complete transection of the spinal cord at thoracic level T10. Cords were collected 1 or 7 days after injury and from age-matched controls. Proteins were separated based on isoelectric point and subunit molecular weight; those whose expression levels changed following injury were identified by densitometry and analysed by mass spectrometry. Fifty-six unique proteins were identified as differentially regulated in response to spinal transection at both ages combined. More than 50% were cytoplasmic and 70% belonged to families of proteins with characteristic binding properties. Proteins were assigned to groups by biological function including regulation (40%), metabolism (26%), inflammation (19%) and structure (15%). More changes were detected at one than seven days after injury at both ages. Seven identified proteins: 14-3-3 epsilon, 14-3-3 gamma, cofilin, alpha enolase, heart fatty acid binding protein (FABP3), brain fatty acid binding protein (FABP7) and ubiquitin demonstrated age-related differential expression and were analysed by qRT-PCR. Changes in mRNA levels for FABP3 at P7+1day and ubiquitin at P28+1day were statistically significant. Immunocytochemical staining showed differences in ubiquitin localization in younger compared to older cords and an increase in oligodendrocyte and neuroglia immunostaining following injury at P28. Western blot analysis supported proteomic results for ubiquitin and 14-3-3 proteins. Data obtained at the two ages demonstrated changes in response to injury, compared to controls, that were different for different functional protein classes. Some may provide targets for novel drug or gene therapies

Selective inhibition of ASIC1a confers functional and morphological neuroprotection following traumatic spinal cord injury

Tissue loss after spinal trauma is biphasic, with initial mechanical/haemorrhagic damage at the time of impact being followed by gradual secondary expansion into adjacent, previously unaffected tissue. Limiting the extent of this secondary expansion of tissue damage has the potential to preserve greater residual spinal cord function in patients. The acute tissue hypoxia resulting from spinal cord injury (SCI) activates acid-sensing ion channel 1a (ASIC1a). We surmised that antagonism of this channel should provide neuroprotection and functional preservation after SCI. We show that systemic administration of the spider-venom peptide PcTx1, a selective inhibitor of ASIC1a, improves locomotor function in adult Sprague Dawley rats after thoracic SCI. The degree of functional improvement correlated with the degree of tissue preservation in descending white matter tracts involved in hind limb locomotor function. Transcriptomic analysis suggests that PcTx1-induced preservation of spinal cord tissue does not result from a reduction in apoptosis, with no evidence of down-regulation of key genes involved in either the intrinsic or extrinsic apoptotic pathways. We also demonstrate that trauma-induced disruption of blood-spinal cord barrier function persists for at least 4 days post-injury for compounds up to 10 kDa in size, whereas barrier function is restored for larger molecules within a few hours. This temporary loss of barrier function provides a " " through which systemically administered drugs have unrestricted access to spinal tissue in and around the sites of trauma. Taken together, our data provide evidence to support the use of ASIC1a inhibitors as a therapeutic treatment for SCI. This study also emphasizes the importance of objectively grading the functional severity of initial injuries (even when using standardized impacts) and we describe a simple scoring system based on hind limb function that could be adopted in future studies

Expression and cellular distribution of ubiquitin in response to injury in the developing spinal cord of Monodelphis domestica

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

Cellular specificity of the bloodCSF barrier for albumin transfer across the choroid plexus epithelium

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the bloodCSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse bloodCSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells.HEALTH-F2-2009-24177

Weight-Bearing Locomotion in the Developing Opossum, <i>Monodelphis domestica</i> following Spinal Transection: Remodeling of Neuronal Circuits Caudal to Lesion

<div><p>Complete spinal transection in the mature nervous system is typically followed by minimal axonal repair, extensive motor paralysis and loss of sensory functions caudal to the injury. In contrast, the immature nervous system has greater capacity for repair, a phenomenon sometimes called the infant lesion effect. This study investigates spinal injuries early in development using the marsupial opossum <i>Monodelphis domestica</i> whose young are born very immature, allowing access to developmental stages only accessible <i>in utero</i> in eutherian mammals. Spinal cords of <i>Monodelphis</i> pups were completely transected in the lower thoracic region, T10, on postnatal-day (P)7 or P28 and the animals grew to adulthood. In P7-injured animals regrown supraspinal and propriospinal axons through the injury site were demonstrated using retrograde axonal labelling. These animals recovered near-normal coordinated overground locomotion, but with altered gait characteristics including foot placement phase lags. In P28-injured animals no axonal regrowth through the injury site could be demonstrated yet they were able to perform weight-supporting hindlimb stepping overground and on the treadmill. When placed in an environment of reduced sensory feedback (swimming) P7-injured animals swam using their hindlimbs, suggesting that the axons that grew across the lesion made functional connections; P28-injured animals swam using their forelimbs only, suggesting that their overground hindlimb movements were reflex-dependent and thus likely to be generated locally in the lumbar spinal cord. Modifications to propriospinal circuitry in P7- and P28-injured opossums were demonstrated by changes in the number of fluorescently labelled neurons detected in the lumbar cord following tracer studies and changes in the balance of excitatory, inhibitory and neuromodulatory neurotransmitter receptors’ gene expression shown by qRT-PCR. These results are discussed in the context of studies indicating that although following injury the isolated segment of the spinal cord retains some capability of rhythmic movement the mechanisms involved in weight-bearing locomotion are distinct.</p></div

Limb placement phase lags.

<p>Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9,grey bars) opossums. <b><i>A</i></b>: Schematic examples showing calculation method for girdle, diagonal and ipsilateral phase lags. These examples are taken from the control animal in Fig. 1B. <b><i>B</i></b>: Forelimb and hindlimb girdle phase lags <b><i>C</i></b>: Diagonal phase lags. <b><i>D</i></b>: Ipsilateral phase lags. Mean ± sem; *<i>P</i>≤0.05 vs control; ^#<i>P</i>≤0.05 vs P7-inj, by One-way ANOVA.</p

Morphometric measurements of <i>Monodelphis</i> spinal cords.

<p>Control (n = 5, white circles), P7-injured (n = 3, black circles) and P28-injured (n = 4, grey circles) opossum spinal cords. <b><i>A</i></b>: Representative whole mounts of lower cervical and thoracic spinal cords from control (top), P7-injured (middle) and P28-injured (bottom) <i>Monodelphis</i>. <b><i>B</i></b>: Whole spinal cord cross-sectional area along the length of the spinal cord. <b><i>C</i></b>: Grey matter area. <b><i>D</i></b>: White matter area. Mean ± sem. *<i>P</i>≤0.05 vs control. Stars denoting significance appear immediately <i>above</i> data for P7-injured animals or immediately <i>below</i> data for P28-injured animals.</p

Silver stained gels and western blots of ubiquitin showing cross-reactivity of anti-ubiquitin antibody.

<p>Silver stained (SS) bands identified by mass spectrometry showing two protein bands. Higher band was identified as a fusion protein containing ubiquitin at the N-terminus and a ribosomal protein L40 at the C-terminus and the lower one was identified as mono-ubiquitin. Western blots (WB) of ubiquitin showing that the anti-ubiquitin antibody used only cross-reacts with a single band, as demonstrated in both Fractions 7 and 8 (black arrows).</p

Primers used in this study.

<p>Designed with reference to <i>Monodelphis domestica</i> database <b>(Ensembl database MonDom5</b><a href="http://www.ensembl.org/Monodelphis_domestica" target="_blank">http://www.ensembl.org/Monodelphis_domestica</a><b>)</b>.</p

Behavioural assessments.

<p>Control (n = 11, white bars), P7-injured (n = 7, black bars) and P28-injured (n = 9, grey bars) opossums. <b><i>A</i></b>: BBB Locomotor rating scores. <b><i>B</i></b>: Representative gait traces from treadmill locomotion. <b><i>C</i></b>: Regularity index. <i>D</i>: Step duration. <i>E</i>: Stance duration. <i>F</i>: Swing duration. Mean ± sem; *<i>P</i>≤0.05 vs control; ^#<i>P</i>≤0.05 vs P7-inj, by One-way ANOVA.</p