Motor rehabilitation as a therapeutic tool for spinal cord injury: New perspectives in immunomodulation.

Traumatic spinal cord injury (SCI) is a devastating condition that significantly impacts motor, sensory and autonomic function in patients. Despite advances in therapeutic approaches, there is still no curative therapy currently available. Neuroinflammation is a persisting event of the secondary injury phase of SCI that affects functional recovery, and modulation of the inflammatory response towards a beneficial anti-inflammatory state can improve recovery in preclinical SCI models. In human SCI patients, rehabilitative exercise, or motor rehabilitation as we will refer to it from here on out, remains the cornerstone of treatment to increase functional capacity and prevent secondary health implications. Motor rehabilitation is known to have anti-inflammatory effects; however, current literature is lacking in the description of the effect of motor rehabilitation on inflammation in the context of SCI. Understanding the effect on different inflammatory markers after SCI should enable the optimization of motor rehabilitation as a therapeutic regime. This review extensively describes the effect of motor rehabilitation on selected inflammatory mediators in both preclinical and human SCI studies. Additionally, we summarize how the type, duration, and intensity of motor rehabilitation can affect the inflammatory response after SCI. In doing so, we introduce a new perspective on how motor rehabilitation can be optimized as an immunomodulatory therapy to improve patient outcome after SCI.

An In Vitro and Ex Vivo Analysis of the Potential of GelMA Hydrogels as a Therapeutic Platform for Preclinical Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating condition with no curative therapy currently available. Immunomodulation can be applied as a therapeutic strategy to drive alternative immune cell activation and promote a proregenerative injury microenvironment. Locally injected hydrogels carrying immunotherapeutic cargo directly to injured tissue offer an encouraging treatment approach from an immunopharmacological perspective. Gelatin methacrylate (GelMA) hydrogels are promising in this regard, however, detailed analysis on the immunogenicity of GelMA in the specific context of the SCI microenvironment is lacking. Here, the immunogenicity of GelMA hydrogels formulated with a translationally relevant photoinitiator is analyzed in vitro and ex vivo. 3% (w/v) GelMA, synthesized from gelatin type-A, is first identified as the optimal hydrogel formulation based on mechanical properties and cytocompatibility. Additionally, 3% GelMA-A does not alter the expression profile of key polarization markers in BV2 microglia or RAW264.7 macrophages after 48 h. Finally, it is shown for the first time that 3% GelMA-A can support the ex vivo culture of primary murine organotypic spinal cord slices for 14 days with no direct effect on glial fibrillary acidic protein (GFAP+ ) astrocyte or ionized calcium-binding adaptor molecule 1 (Iba-1+ ) microglia reactivity. This provides evidence that GelMA hydrogels can act as an immunotherapeutic hydrogel-based platform for preclinical SCI.

A finite element model of contusion spinal cord injury in rodents.

Traumatic spinal cord injuries result from high impact forces acting on the spine and are proceeded by an extensive secondary inflammatory response resulting in motor, sensory, and autonomic dysfunction. Experimental in vivo traumatic spinal cord injuries in rodents using a contusion model have been extremely useful in elucidating the underlying pathophysiology of these injuries. However, the relationship between the pathophysiology and the biomechanical factors is still not well understood. Therefore, the aim of this research is to provide a comprehensive analysis of the biomechanics of traumatic spinal cord injury in a rat contusion model. This is achieved through the development and validation of a finite element model of the thoracic rat spinal cord and subsequently simulating controlled cortical impact-induced traumatic spinal cord injury. The effects of impactor velocity, depth, and geometry on the resulting stresses and strains within the spinal cord are investigated. Our results show that increasing impactor depth results in larger stresses and strains within the spinal cord tissue as expected. Further, for the first time ever our results show that impactor geometry (spherical versus cylindrical) plays an important role in the distribution and magnitude of stresses and strains within the cord. Therefore, finite element modelling can be a powerful tool used to predict stresses and strains that occur in spinal cord tissue during trauma.

Functional hydrogels as therapeutic tools for spinal cord injury: New perspectives on immunopharmacological interventions.

Spinal cord injury (SCI) is a complex medical and psychological challenge for which there is no curative therapy currently available. Despite major progress in pharmacological and surgical approaches, clinical trials for SCI patients have been uniformly disappointing thus far as there are many practical and biological issues yet to be resolved. Neuroinflammation is a critical event of the secondary injury phase after SCI, and recent research strategies have focused on modulating the immune response after injury to provide a more favorable recovery environment. Biomaterials can serve this purpose by providing physical and trophic support to the injured spinal cord after SCI. Of all potential biomaterials, functional hydrogels are emerging as a key component in novel treatment strategies for SCI, including controlled and localized delivery of immunomodulatory therapies to drive polarization of immune cells towards a pro-regenerative phenotype. Here, we extensively review recent developments in the use of functional hydrogels as immunomodulatory therapies for SCI. We briefly describe physicochemical properties of hydrogels and demonstrate how advanced fabrication methods lead to the required heterogeneity and hierarchical arrangements that increasingly mimic complex spinal cord tissue. We then summarize potential SCI therapeutic modalities including: (i) hydrogels alone; (ii) hydrogels as cellular or (iii) bioactive molecule delivery vehicles, and; (iv) combinatorial approaches. By linking the structural properties of hydrogels to their functions in treatment with particular focus on immunopharmacological stimuli, this may accelerate further development of functional hydrogels for SCI, and indeed next-generation central nervous system regenerative therapies.

Evidence for an interaction between Golli and STIM1 in store-operated calcium entry.

SOCCs (store-operated Ca(2+) channels) are highly selective ion channels that are activated upon release of Ca(2+) from intracellular stores to regulate a multitude of diverse cellular functions. It was reported previously that Golli-BG21, a member of the MBP (myelin basic protein) family of proteins, regulates SOCE (store-operated Ca(2+) entry) in T-cells and oligodendrocyte precursor cells, but the underlying mechanism for this regulation is unknown. In the present study we have discovered that Golli can directly interact with the ER (endoplasmic reticulum) Ca(2+)-sensing protein STIM1 (stromal interaction molecule 1). Golli interacts with the C-terminal domain of STIM1 in both in vitro and in vivo binding assays and this interaction may be modulated by the intracellular Ca(2+) concentration. Golli also co-localizes with full-length STIM1 and Orai1 complexes in HeLa cells following Ca(2+) store depletion. Overexpression of Golli reduces SOCE in HeLa cells, but this inhibition is overcome by overexpressing STIM1. We therefore suggest that Golli binds to STIM1-Orai1 complexes to negatively regulate the activity of SOCCs.

The amyloid precursor protein intracellular domain(AICD) disrupts actin dynamics and mitochondrial bioenergetics.

The amyloid precursor protein (APP) is critically involved in the pathogenesis of Alzheimer's disease, and is strongly up-regulated in response to traumatic, metabolic, or toxic insults to the nervous system. The processing of APP by gamma/epsilon-secretase activity results in the generation of the APP intracellular domain (AICD). Previously, we have shown that AICD induces the expression of genes (transgelin, alpha2-actin) with functional roles in actin organization and dynamics and demonstrated that the induction of AICD and its co-activator Fe65 (AICD/Fe65) resulted in a loss of organized filamentous actin structures within the cell. As mitochondrial function is thought to be reliant on ordered actin dynamics, we examined mitochondrial function in human SHEP neuroblastoma cells inducibly expressing AICD/Fe65. Confocal analysis of the mitochondrial membrane potential (DeltaPsim) identified a significant decrease in the DeltaPsim in the AICD50/Fe65 over-expressing cells. This was paralleled by significantly reduced ATP levels and decreased basal superoxide production. Overexpression of the proposed AICD target gene transgelin in SHEP-SF parental cells and primary neurons was sufficient to destabilize actin filaments, depolarize DeltaPsim, and significantly alter mitochondrial distribution and morphology. Our data demonstrate that the induction of AICD/Fe65 or transgelin significantly alters actin dynamics and mitochondrial function in neuronal cells.

Role of phosphoinositides in STIM1 dynamics and store-operated calcium entry.

Ca2+ entry through store-operated Ca2+ channels involves the interaction at ER-PM (endoplasmic reticulum-plasma membrane) junctions of STIM (stromal interaction molecule) and Orai. STIM proteins are sensors of the luminal ER Ca2+ concentration and, following depletion of ER Ca2+, they oligomerize and translocate to ER-PM junctions where they form STIM puncta. Direct binding to Orai proteins activates their Ca2+ channel function. It has been suggested that an additional interaction of the C-terminal polybasic domain of STIM1 with PM phosphoinositides could contribute to STIM1 puncta formation prior to binding to Orai. In the present study, we investigated the role of phosphoinositides in the formation of STIM1 puncta and SOCE (store-operated Ca2+ entry) in response to store depletion. Treatment of HeLa cells with inhibitors of PI3K (phosphatidylinositol 3-kinase) and PI4K (phosphatidylinositol 4-kinase) (wortmannin and LY294002) partially inhibited formation of STIM1 puncta. Additional rapid depletion of PtdIns(4,5)P2 resulted in more substantial inhibition of the translocation of STIM1-EYFP (enhanced yellow fluorescent protein) into puncta. The inhibition was extensive at a concentration of LY294002 (50 microM) that should primarily inhibit PI3K, consistent with a major role for PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in puncta formation. Depletion of phosphoinositides also inhibited SOCE based on measurement of the rise in intracellular Ca2+ concentration after store depletion. Overexpression of Orai1 resulted in a recovery of translocation of STMI1 into puncta following phosphoinositide depletion and, under these conditions, SOCE was increased to above control levels. These observations support the idea that phosphoinositides are not essential but contribute to STIM1 accumulation at ER-PM junctions with a second translocation mechanism involving direct STIM1-Orai interactions.

Modulation of gene expression and cytoskeletal dynamics by the amyloid precursor protein intracellular domain (AICD).

Amyloidogenic processing of the amyloid precursor protein (APP) results in the generation of beta-amyloid, the main constituent of Alzheimer plaques, and the APP intracellular domain (AICD). Recently, it has been demonstrated that AICD has transactivation potential; however, the targets of AICD-dependent gene regulation and hence the physiological role of AICD remain largely unknown. We analyzed transcriptome changes during AICD-dependent gene regulation by using a human neural cell culture system inducible for expression of AICD, its coactivator FE65, or the combination of both. Induction of AICD was associated with increased expression of genes with known function in the organization and dynamics of the actin cytoskeleton, including alpha2-Actin and Transgelin (SM22). AICD target genes were also found to be differentially regulated in the frontal cortex of Alzheimer's disease patients compared with controls as well as in AICD/FE65 transiently transfected murine cortical neurons. Confocal image analysis of neural cells and cortical neurons expressing both AICD and FE65 confirmed pronounced changes in the organization of the actin cytoskeleton, including the destabilization of actin fibers and clumping of actin at the sites of cellular outgrowth. Our data point to a role of AICD in developmental and injury-related cytoskeletal dynamics in the nervous system.

Phosphorylation of cysteine string protein on Serine 10 triggers 14-3-3 protein binding.

Cysteine string protein (CSP) is a neuronal chaperone that maintains normal neurotransmitter exocytosis and is essential for preventing presynaptic neurodegeneration. CSP is phosphorylated in vivo on a single residue, Ser10, and this phosphorylation regulates its cellular functions, although the molecular mechanisms involved are unclear. To identify novel phosphorylation-specific binding partners for CSP, we used a pull-down approach using synthetic peptides and recombinant proteins. A single protein band was observed to bind specifically to a Ser10-phosphorylated CSP peptide (residues 4-14) compared to a non-phosphorylated peptide. This band was identified as 14-3-3 protein of various isoforms using mass spectrometry and Western blotting. PKA phosphorylation of full-length CSP protein stimulated 14-3-3 binding, and this was abolished in a Ser10-Ala mutant CSP, confirming the binding site as phospho-Ser10. As both CSP and 14-3-3 proteins are implicated in neurotransmitter exocytosis and neurodegeneration, this novel phosphorylation-dependent interaction may help maintain the functional integrity of the synapse.

ATP depletion induces translocation of STIM1 to puncta and formation of STIM1-ORAI1 clusters: translocation and re-translocation of STIM1 does not require ATP.

Depletion of the endoplasmic reticulum (ER) calcium store triggers translocation of stromal interacting molecule one (STIM1) to the sub-plasmalemmal region and formation of puncta-structures in which STIM1 interacts and activates calcium channels. ATP depletion induced the formation of STIM1 puncta in PANC1, RAMA37, and HeLa cells. The sequence of events triggered by inhibition of ATP production included a rapid decline of ATP, depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and a slow calcium leak from the ER followed by formation of STIM1 puncta. STIM1 puncta induced by ATP depletion were co-localized with clusters of ORAI1 channels. STIM1-ORAI1 clusters that developed as a result of ATP depletion were very poor mediators of Ca(2+) influx. Re-translocation of STIM1 from puncta back to the ER was observed during total ATP depletion. We can therefore conclude that STIM1 translocation and re-translocation as well as formation of STIM1-ORAI1 clusters occur in an ATP-independent fashion and under conditions of PI(4,5)P(2) depletion.