Composite Fibrin/Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs.

Biomaterials may enhance neural repair after spinal cord injury (SCI) and testing their functionality in large animals is essential to achieve successful clinical translation. This work developed a porcine contusion/compression SCI model to investigate the consequences of myelotomy and implantation of fibrin gel containing biofunctionalized carbon microfibers (MFs). Fourteen pigs were distributed in SCI, SCI/myelotomy, and SCI/myelotomy/implant groups. An automated device was used for SCI. A dorsal myelotomy was performed on the lesion site at 1 day post-injury for removing cloths and devitalized tissue. Bundles of MFs coated with a conducting polymer and cell adhesion molecules were embedded in fibrin gel and used to bridge the spinal cord cavity. Reproducible lesions of about 1 cm in length were obtained. Myelotomy and lesion debridement caused no further neural damage compared to SCI alone but had little positive effect on neural regrowth. The MFs/fibrin gel implant facilitated axonal sprouting, elongation, and alignment within the lesion. However, the implant also increased lesion volume and was ineffective in preventing fibrosis, thus precluding functional neural regeneration. Our results indicate that myelotomy and lesion debridement can be advantageously used for implanting MF-based scaffolds. However, the implants need refinement and pharmaceuticals will be necessary to limit scarring.

CPEB1 is overexpressed in neurons derived from Down syndrome IPSCs and in the hippocampus of the mouse model Ts1Cje.

Trisomy 21, also known as Down syndrome (DS), is the most frequent genetic cause of intellectual impairment. In mouse models of DS, deficits in hippocampal synaptic plasticity have been observed, in conjunction with alterations to local dendritic translation that are likely to influence plasticity, learning and memory. Here we show that expression of a local translational regulator, the Cytoplasmic Polyadenylation Element Binding Protein 1 (CPEB1), is enhanced in hippocampal neurons from the Ts1Cje DS mouse model. Interestingly, this protein, which is also involved in dendritic mRNA transport, is overexpressed in dendrites of neurons derived from DS human induced pluripotent stem cells (hIPSCs). Moreover, there is an increase in the mRNA levels of α-Calmodulin Kinase II (α-CaMKII) and Microtubule-associated protein 1B (MAP1B), two dendritic mRNAs, in Ts1Cje synaptoneurosomes. Taking into account the fundamental role of CPEB1 protein and its target mRNAs in synaptic plasticity, these data could be relevant to the intellectual impairment in the context of DS.

Stability of Conducting Polymer-Coated Carbon Microfibers for Long-Term Electrical Stimulation of Injured Neural Tissue.

Electroactive microfiber-based scaffolds aid neural tissue repair. Carbon microfibers (CMFs) coated with the conducting polymer poly(3,4-ethylenedioxythiophene) doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] (PEDOT:PSS-co-MA) provide efficient support and guidance to regrowing axons across spinal cord lesions in rodents and pigs. We investigated the electrical and structural performance of PEDOT:PSS-co-MA-coated carbon MFs (PCMFs) for long-term, biphasic electrical stimulation (ES). Chronopotentiometry and electrochemical impedance spectroscopy (EIS) allowed the characterization of charge transfer in PCMFs during ES in vitro, and morphological changes were assessed by scanning electron microscopy (SEM). PCMFs that were 4 mm long withstood two-million-biphasic pulses without reaching cytotoxic voltages, with a 6 mm length producing optimal results. Although EIS and SEM unveiled some polymer deterioration in the 6 mm PCMFs, no significant changes in voltage excursions appeared. For the preliminary testing of the electrical performance of PCMFs in vivo, we used 12 mm long, 20-microfiber assemblies interconnected by metallic microwires. PCMFs-assemblies were implanted in two spinal cord-injured pigs and submitted to ES for 10 days. A cobalt-alloy interconnected assembly showed safe voltages for about 1.5 million-pulses and was electrically functional at 1-month post-implantation, suggesting its suitability for sub-chronic ES, as likely required for spinal cord repair. However, improving polymer adhesion to the carbon substrate is still needed to use PCMFs for prolonged ES.

An increase in basal BDNF provokes hyperactivation of the Akt-mammalian target of rapamycin pathway and deregulation of local dendritic translation in a mouse model of Down's syndrome.

As in other diseases associated with mental retardation, dendrite morphology and synaptic plasticity are impaired in Down's syndrome (DS). Both these features of neurons are critically influenced by BDNF, which regulates local dendritic translation through phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (mTOR) and Ras-ERK signaling cascades. Here we show that the levels of BDNF and phosphorylated Akt-mTOR (but not Ras-ERK) pathway proteins are augmented in hippocampal dendrites of Ts1Cje mice, a DS model. Consequently, the rate of local dendritic translation is abnormally high and the modulatory effect of exogenous BDNF is lost. Interestingly, rapamycin (a Food and Drug Administration-approved drug) restores normal levels of phosphorylated Akt-mTOR proteins and normal rates of local translation in Ts1Cje neurons, opening new therapeutic perspectives for DS. The NMDAR inhibitors APV, MK-801, and memantine also restore the normal levels of phospho-mTOR in dendrites of Ts1Cje hippocampal neurons. We propose a model to explain how BDNF-mediated regulation of local translation is lost in the Ts1Cje hippocampus through the establishment of a glutamatergic positive-feedback loop. Together, these findings help elucidate the mechanisms underlying altered synaptic plasticity in DS.

NMDA-mediated regulation of DSCAM dendritic local translation is lost in a mouse model of Down's syndrome.

Down's syndrome cell adhesion molecule (DSCAM) belongs to the Down's syndrome critical region of human chromosome 21, and it encodes a cell adhesion molecule involved in dendrite morphology and neuronal wiring. Although the function of DSCAM in the adult brain is unknown, its expression pattern suggests a role in synaptic plasticity. Local mRNA translation is a key process in axonal growth, dendritogenesis, and synaptogenesis during development, and in synaptic plasticity in adulthood. Here, we report the dendritic localization of DSCAM mRNA in the adult mouse hippocampus, where it associates with CPEB1 [cytoplasmic polyadenylation element (CPE) binding protein 1], an important regulator of mRNA transport and local translation. We identified five DSCAM isoforms produced by alternative polyadenylation bearing different combinations of regulatory CPE motifs. Overexpression of DSCAM in hippocampal neurons inhibited dendritic branching. Interestingly, dendritic levels of DSCAM mRNA and protein were increased in hippocampal neurons from Ts1Cje mice, a model of Down's syndrome. Most importantly, DSCAM dendritic translation was rapidly induced by NMDA in wild-type, but not in Ts1Cje neurons. We propose that impairment of the NMDA-mediated regulation of DSCAM translation may contribute to the alterations in dendritic morphology and/or synaptic plasticity in Down's syndrome.

Local translation of dendritic RhoA revealed by an improved synaptoneurosome preparation.

Changes in dendritic spine morphology, a hallmark of synaptic plasticity, involve remodeling of the actin cytoskeleton, a process that is regulated by Rho GTPases. RhoA, a member of this GTPase family, segregates to dendrites in differentiated neurons. Given the emerging role of dendritic mRNA local translation in synaptic plasticity, we have assessed the possible localization and translation of RhoA mRNA at dendrites. At this end, we have developed and describe here in detail an improved method for isolating hippocampal and neocortical mouse synaptoneurosomes. This synaptoneurosomal preparation is much more enriched in synaptic proteins than those obtained in former methods, exhibits bona fide electron microscopy pre- and postsynaptic morphologies, contains abundant dendritic mRNAs, and is competent for activity-regulated protein synthesis. Using this preparation, we have found that RhoA mRNA is dendritically localized and its local translation is enhanced by BDNF stimulation. These findings suggest that some of the known functions of RhoA on spine morphology may be mediated by regulating its local translation.

Glial progenitor cell migration promotes CNS axon growth on functionalized electroconducting microfibers.

Electroactive systems that promote directional axonal growth and migration of glial progenitor cells (GPC) are needed for the treatment of neurological injuries. We report the functionalization of electroconducting microfibers with multiple biomolecules that synergistically stimulate the proliferation and migration of GPC, which in turn induce axonal elongation from embryonic cerebral cortex neurons. PEDOT doped with poly[(4-styrenesulfonic acid)-co-(maleic acid)] was synthesized on carbon microfibers and used for covalent attachment of molecules to the electroactive surface. The molecular complexes that promoted GPC proliferation and migration, followed by axonal extension, were composed of polylysine, heparin, basic fibroblast growth factor (bFGF), and matricellular proteins; the combination of bFGF with vitronectin or fibronectin being indispensable for sustained glial and axonal growth. The rate of glial-induced axonal elongation was about threefold that of axons growing directly on microfibers functionalized with polylysine alone. Electrical stimuli applied through the microfibers released bFGF and fibronectin from the polymer surface, consequently reducing GPC proliferation and promoting their differentiation into astrocytes, without causing cell detachment or toxicity. These results suggest that functionalized electroactive microfibers may provide a multifunctional tool for controlling neuron-glia interactions and enhancing neural repair. STATEMENT OF SIGNIFICANCE: We report a multiple surface functionalization strategy for electroconducting microfibers (MFs), in order to promote proliferation and guided migration of glial precursor cells (GPC) and consequently create a permissive substrate for elongation of central nervous system (CNS) axons. GPC divided and migrated extensively on the functionalized MFs, leading to fast elongation of embryonic cerebral cortex axons. The application of electric pulses thorough the MFs controlled glial cell division and differentiation. The functionalized MFs provide an advanced tool for neural tissue engineering and for controlling neuron-glial interactions. CNS axonal growth associated to migratory glial precursors, together with the possibility of directing glial differentiation by electrical stimuli applied through the MFs, open a new research avenue to explore for CNS repair.

Biofunctionalized PEDOT-coated microfibers for the treatment of spinal cord injury.

Poly(3, 4-ethylenedioxythiophene)-coated carbon microfibers (PEDOT-MFs) hold promise for developing advanced neuroprostheses and neural repair devices. We investigated the chronic cellular responses to PEDOT-MFs implanted into the uninjured and the transected rat spinal cord, and compared the effects of polymer surface biofunctionalization with covalently attached polylysine (PLL) or a multimolecular complex of PLL, heparin, basic fibroblast growth factor (bFGF), and fibronectin. An alginate gel was used to facilitate microfiber implantation and reduce connective tissue scarring after spinal cord injury (SCI). PLL/heparin/bFGF/fibronectin-functionalized PEDOT-MFs showed excellent integration within the uninjured and injured spinal cord, frequently establishing contact with neuronal somas, axons, dendrites and glial cells, accompanied by very little or absent scarring response. On the contrary, non-functionalized and PLL-functionalized microfibers provoked inflammation and fibrosis with loss of neural elements in the surrounding tissue. Within the lesion, the PEDOT-MFs by themselves facilitated longitudinal alignment of migratory cells and growing axons, and their modification with PLL/heparin/bFGF/fibronectin promoted tissue healing, enhancing blood vessel formation and axonal regeneration without increasing inflammation. These results support the incorporation of biofunctionalized electroconducting microfibers in neuro-electronic interfaces and lesion-bridging systems for the treatment of SCI.

Deregulated mTOR-mediated translation in intellectual disability.

Local translation of dendritic mRNAs is a key aspect of dendrite and spine morphogenesis and synaptic plasticity, two phenomena generally compromised in intellectual disability disorders. Mammalian target of rapamycin (mTOR) is a protein kinase involved in a plethora of functions including dendritogenesis, plasticity and the regulation of local translation. Hence, this kinase may well be implicated in intellectual disability. Hyperactivation of mTOR has been recently reported in mouse models of Fragile X and tuberous sclerosis, two important causes of intellectual disability. Moreover, local dendritic translation seems to be increased in Fragile X syndrome. Recent findings show that the mTOR pathway is also deregulated in murine models of Rett's syndrome and Down's syndrome. As in Fragile X, local dendritic translation seems to be abnormally active in Down's syndrome mice, while rapamycin, a Food and Drug Administration-approved mTOR inhibitor, restores normal rates of translation. Rapamycin administration in tuberous sclerosis mice rescues deficits in behavior and synaptic plasticity. Indeed, mTOR-dependent deregulation of local translation may be a common trait in different intellectual deficiencies, suggesting that mTOR inhibitors may have significant therapeutic potential for the treatment of diverse forms of cognitive impairment.