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Neuronal regeneration in the central nervous system (CNS) is an important field of research with relevance to all types of neuronal injuries, including neurodegenerative diseases. The glial scar is a result of the astrocyte response to CNS injury. It is made up of many components creating a complex environment in which astrocytes play various key roles. The glial scar is heterogeneous, diverse and its composition depends upon the injury type and location. The heterogeneity of the glial scar observed in different situations of CNS damage and the consequent implications for axon regeneration have not been reviewed in depth. The gap in this knowledge will be addressed in this review which will also focus on our current understanding of central axonal regeneration and the molecular mechanisms involved. The multifactorial context of CNS regeneration is discussed, and we review newly identified roles for components previously thought to solely play an inhibitory role in central regeneration: astrocytes and p75NTR and discuss their potential and relevance for deciding therapeutic interventions. The article ends with a comprehensive review of promising new therapeutic targets identified for axonal regeneration in CNS and a discussion of novel ways of looking at therapeutic interventions for several brain diseases and injuries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Costa, Ribeiro, Sebastião, Muir and Vaz.)

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over "classical" rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Activation of microglia in the spinal cord dorsal horn after peripheral nerve injury contributes to the development of pain hypersensitivity. How activated microglia selectively enhance the activity of spinal nociceptive circuits is not well understood. We discovered that after peripheral nerve injury, microglia degrade extracellular matrix structures, perineuronal nets (PNNs), in lamina I of the spinal cord dorsal horn. Lamina I PNNs selectively enwrap spinoparabrachial projection neurons, which integrate nociceptive information in the spinal cord and convey it to supraspinal brain regions to induce pain sensation. Degradation of PNNs by microglia enhances the activity of projection neurons and induces pain-related behaviors. Thus, nerve injury-induced degradation of PNNs is a mechanism by which microglia selectively augment the output of spinal nociceptive circuits and cause pain hypersensitivity.

Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45 weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45 weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Perineuronal nets (PNNs) are assemblies of extracellular matrix molecules, which surround the cell body and dendrites of many types of neuron and regulate neural plasticity. PNNs are prominently expressed around neurons of the deep cerebellar nuclei (DCN), but their role in adult cerebellar plasticity and behavior is far from clear. Here we show that PNNs in the mouse DCN are diminished during eyeblink conditioning (EBC), a form of associative motor learning that depends on DCN plasticity. When memories are fully acquired, PNNs are restored. Enzymatic digestion of PNNs in the DCN improves EBC learning, but intact PNNs are necessary for memory retention. At the structural level, PNN removal induces significant synaptic rearrangements in vivo, resulting in increased inhibition of DCN baseline activity in awake behaving mice. Together, these results demonstrate that PNNs are critical players in the regulation of cerebellar circuitry and function., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: High levels of positive mental health protect individuals from mental illness. This study investigates longitudinal change in positive mental health as a predictor of mental illness recovery in a cohort group., Methods: Using data from the 1995 and 2005 Midlife in the United States cross-sectional surveys (n = 1,723), logistic regression was used to estimate the odds ratio that individuals diagnosed with a mental illness in 1995 would have recovered in 2005 based on whether their level of positive mental health changed over the 10-year period., Results: Individuals who maintained or gained the highest levels of positive mental health were more than 27.6 and 7.4 times, respectively, more likely to recover when compared to those who maintained the lowest level of positive mental health. Those who maintained or gained moderate levels of positive mental health had more moderate likelihood of recovery, and those whose positive mental health declined to the lowest levels had no significantly different likelihood of recovery compared to participants whose positive mental health remained low., Limitations: This study was limited by the age of the data, and the inability to control for some predictors of recovery., Conclusions: This study suggests that positive mental health may be an important resource for individuals to recover from mental illness and stay mentally healthy. Results point to the need to include positive mental health assessment and interventions into mental health care systems., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Integrated care has been identified as means of managing the demands on the healthcare budget while improving access to and quality of services. It is particularly pertinent to rural health services, which face limited access to specialist and support services. This paper explores the capacity of three rural communities in South Australia to deliver integrated mental health support for older people., Methods: Thirty-one interviews were conducted with local health and social service providers from mental health, community health, general practice, residential aged care, private practice, NGOs and local government as part of a larger action research project on service integration., Results: Participants highlighted differences in service delivery between the communities related to size of the community and access to services. Three structural barriers to delivery of integrated care were identified. These are as follows: fragmentation of governmental responsibility, the current funding climate, and centralisation and standardisation of service delivery., Conclusion: We conclude that despite a focus upon integrated care in mental health policy, many features of current service delivery undermine the flexibility and informal relationships that typically underpin integration in rural communities.

Chondroitinase ABC is a promising preclinical therapy that promotes functional neuroplasticity after CNS injury by degrading extracellular matrix inhibitors. Efficient delivery of chondroitinase ABC to the injured mammalian spinal cord can be achieved by viral vector transgene delivery. This approach dramatically modulates injury pathology and restores sensorimotor functions. However, clinical development of this therapy is limited by a lack of ability to exert control over chondroitinase gene expression. Prior experimental gene regulation platforms are likely to be incompatible with the non-resolving adaptive immune response known to occur following spinal cord injury. Therefore, here we apply a novel immune-evasive dual vector system, in which the chondroitinase gene is under a doxycycline inducible regulatory switch, utilizing a chimeric transactivator designed to evade T cell recognition. Using this novel vector system, we demonstrate tight temporal control of chondroitinase ABC gene expression, effectively removing treatment upon removal of doxycycline. This enables a comparison of short and long-term gene therapy paradigms in the treatment of clinically-relevant cervical level contusion injuries in adult rats. We reveal that transient treatment (2.5 weeks) is sufficient to promote improvement in sensory axon conduction and ladder walking performance. However, in tasks requiring skilled reaching and grasping, only long term treatment (8 weeks) leads to significantly improved function, with rats able to accurately grasp and retrieve sugar pellets. The late emergence of skilled hand function indicates enhanced neuroplasticity and connectivity and correlates with increased density of vGlut1+ innervation in spinal cord grey matter, particularly in lamina III-IV above and below the injury. Thus, our novel gene therapy system provides an experimental tool to study temporal effects of extracellular matrix digestion as well as an encouraging step towards generating a safer chondroitinase gene therapy strategy, longer term administration of which increases neuroplasticity and recovery of descending motor control. This preclinical study could have a significant impact for tetraplegic individuals, for whom recovery of hand function is an important determinant of independence, and supports the ongoing development of chondroitinase gene therapy towards clinical application for the treatment of spinal cord injury.

Olfactory ensheathing cell (OEC) transplantation is a promising strategy for treating spinal cord injury (SCI), as has been demonstrated in experimental SCI models and naturally occurring SCI in dogs. However, the presence of chondroitin sulphate proteoglycans within the extracellular matrix of the glial scar can inhibit efficient axonal repair and limit the therapeutic potential of OECs. Here we have used lentiviral vectors to genetically modify canine OECs to continuously deliver mammalian chondroitinase ABC at the lesion site in order to degrade the inhibitory chondroitin sulphate proteoglycans in a rodent model of spinal cord injury. We demonstrate that these chondroitinase producing canine OECs survived at 4 weeks following transplantation into the spinal cord lesion and effectively digested chondroitin sulphate proteoglycans at the site of injury. There was evidence of sprouting within the corticospinal tract rostral to the lesion and an increase in the number of corticospinal axons caudal to the lesion, suggestive of axonal regeneration. Our results indicate that delivery of the chondroitinase enzyme can be achieved with the genetically modified OECs to increase axon growth following SCI. The combination of these two promising approaches is a potential strategy for promoting neural regeneration following SCI in veterinary practice and human patients.

The present study describes the fabrication of polyaniline-silk fibroin (PASF) nanocomposite-based nerve conduits and their subsequent implantation in a rat sciatic nerve injury model for peripheral nerve regeneration. This is the first in vivo study of polyaniline-based nerve conduits describing the safety and efficacy of the conduits in treating peripheral nerve injuries. The nanocomposite was synthesized by electrospinning a mixture of silk fibroin protein and polyaniline wherein the silk nanofibers were observed to be uniformly coated with polyaniline nanoparticles. Tubular shaped nerve conduits were subsequently formed by multiple rolling of the electrospun sheet over a stainless steel mandrel. The conduits were characterized in vitro for their physico-chemical properties as well as their compatibility with rat Schwann cells. Upon implantation in a 10 mm sciatic nerve injury model, the conduits were evaluated for their neuro-regenerative potential through extensive electrophysiological studies and monitoring of gait pattern over a course of 12 months. Gross examination, histological and ultra-structure analyses of the conduits and the regenerated nerve were also performed to evaluate morphological regeneration of transected nerve. PASF nanocomposite conduits seeded with Schwann cell (cell seeded PASF) exhibited excellent nerve conduction velocity (NCV) (50 m s -1 ), compound muscle action potential (CMAP) (12.8 mV), motor unit potential (MUP) (124 μV), growth of healthy tissue along the nerve gap and thick myelination of axons 12 months after implantation indicating enhanced neuro-regeneration. The excellent functional recovery achieved by animals implanted with cell seeded PASF conduits (86.2% NCV; 80.00% CMAP; 76.07% MUP) are superior to outcomes achieved previously with similar electrically conductive conduits. We believe that the present study would encourage further research in developing electrically active neural implants using synthetic conducting polymers and the in vivo applications of the same.

A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24-72h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75(NGF)) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair., (Copyright © 2016. Published by Elsevier B.V.)

Chondroitin sulphate proteoglycans (CSPGs) are known to be important contributors to the intensely inhibitory environment that prevents tissue repair and regeneration following spinal cord injury. The bacterial enzyme chondroitinase ABC (ChABC) degrades these inhibitory molecules and has repeatedly been shown to promote functional recovery in a number of spinal cord injury models. However, when used to treat more traumatic and clinically relevant spinal contusion injuries, findings with the ChABC enzyme have been inconsistent. We recently demonstrated that delivery of mammalian-compatible ChABC via gene therapy led to sustained and widespread digestion of CSPGs, resulting in significant functional repair of a moderate thoracic contusion injury in adult rats. Here we demonstrate that chondroitinase gene therapy significantly enhances upper limb function following cervical contusion injury, with improved forelimb ladder performance and grip strength as well as increased spinal conduction through the injury site and reduced lesion pathology. This is an important addition to our previous findings as improving upper limb function is a top priority for spinal injured patients. Additionally great importance is placed on replication in the spinal cord injury field. That chondroitinase gene therapy has now been shown to be efficacious in contusion models at either thoracic or cervical level is an important step in the further development of this promising therapeutic strategy towards the clinic., (Copyright © 2015. Published by Elsevier Inc.)

Introduction: An integrated approach to the mental healthcare of older people is advocated across health, aged care and social care sectors. It is not clear, however, how the management of integrated servicing should occur, although interorganisational relations theory suggests a reflective network approach using evaluation feedback. This research will test a network management approach to help regional primary healthcare organisations improve mental health service integration., Methods and Analysis: This mixed methods case study in rural South Australia will test facilitated reflection within a network of health and social care services to determine if this leads to improved integration. Engagement of services will occur through a governance group and a series of three 1-day service stakeholder workshops. Facilitated reflection and evaluation feedback will use information from a review of health sector and local operational policies, a network survey about current service links, gaps and enablers and interviews with older people and their carers about their help seeking journeys. Quantitative and qualitative analysis will describe the policy enablers and explore the current and ideal links between services. The facilitated reflection will be developed to maximise engagement of senior management in the governance group and the service staff at the operational level in the workshops. Benefit will be assessed through indicators of improved service coordination, collective ownership of service problems, strengthened partnerships, agreed local protocols and the use of feedback for accountability., Ethics, Benefits and Dissemination: Ethics approval will deal with the sensitivities of organisational network research where data anonymity is not preserved. The benefit will be the tested utility of a facilitated reflective process for a network of health and social care services to manage linked primary mental healthcare for older people in a rural region. Dissemination will make use of the sectoral networks of the governance group., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatment in vitro induced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.

Transplantation of Schwann cells (SCs) is a promising therapeutic strategy for spinal cord repair. SCs introduced into lesions support axon regeneration, but because these axons do not exit the transplant, additional approaches with SCs are needed. Here, we transplanted SCs genetically modified to secrete a bifunctional neurotrophin (D15A) and chondroitinase ABC (ChABC) into a subacute contusion injury in rats. We examined the effects of these modifications on graft volume, SC number, degradation of chondroitin sulfate proteoglycans (CSPGs), astrogliosis, SC myelination of axons, propriospinal and supraspinal axon numbers, locomotor outcome (BBB scoring, CatWalk gait analysis), and mechanical and thermal sensitivity on the hind paws. D15A secreted from transplanted SCs increased graft volume and SC number and myelinated axon number. SCs secreting ChABC significantly decreased CSPGs, led to some egress of SCs from the graft, and increased propriospinal and 5-HT-positive axons in the graft. SCs secreting both D15A and ChABC yielded the best responses: (1) the largest number of SC myelinated axons, (2) more propriospinal axons in the graft and host tissue around and caudal to it, (3) more corticospinal axons closer to the graft and around and caudal to it, (4) more brainstem neurons projecting caudal to the transplant, (5) increased 5-HT-positive axons in the graft and caudal to it, (6) significant improvement in aspects of locomotion, and (7) improvement in mechanical and thermal allodynia. This is the first evidence that the combination of SC transplants engineered to secrete neurotrophin and chondroitinase further improves axonal regeneration and locomotor and sensory function.

Several diseases and injuries of the central nervous system could potentially be treated by delivery of an enzyme, which might most effectively be achieved by gene therapy. In particular, the bacterial enzyme chondroitinase ABC is beneficial in animal models of spinal cord injury. We have adapted the chondroitinase gene so that it can direct secretion of active chondroitinase from mammalian cells, and inserted it into lentiviral vectors. When injected into adult rat brain, these vectors lead to extensive secretion of chondroitinase, both locally and from long-distance axon projections, with activity persisting for more than 4 weeks. In animals which received a simultaneous lesion of the corticospinal tract, the vector reduced axonal die-back and promoted sprouting and short-range regeneration of corticospinal axons. The same beneficial effects on damaged corticospinal axons were observed in animals which received the chondroitinase lentiviral vector directly into the vicinity of a spinal cord lesion., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury.

Purpose: By using a nerve amputee model of the rat sciatic nerve (Lago and Navarro, 2007), we have tested a strategy for the long-term maintenance of regenerated axons without distal target reinnervation, by grafting Schwann cells (SCs) into a capped silicone chamber containing the ending nerve stump., Methods: The sciatic nerve of rats was transected and repaired with a silicone tube, the distal nerve was again cut at 10 mm and inserted in a capped tube that was filled with saline or with a suspension of cultured SCs. Transplants of SCs obtained from primary cultures have been compared with those of an immortalized SC line (SCTM41) or the same line overexpressing GDNF., Results: The histological results show that nerve fibers were able to regenerate through a short distal nerve segment ending into the capped chamber, and sustain distal branches without degenerating for several months. There was abundant axonal sprouting forming an ending neuroma, and the caliber of myelinated fibers remained far thinner than normal during the 9 months investigated. With a distal transplant of primary SCs there were significantly more regenerated myelinated fibers than in the control group at 9 months, indicating that the grafted cells stimulated the axonal growth response and helped to maintain survival of axon branches. In contrast, axonal regeneration was significantly reduced with grafts of SCTM41 cells, probably due to physical competition between cell proliferation and axonal growth. SCTM41 cells overexpressing GDNF improved the regenerative response with respect to the parent SCTM41 cells, although not to the same extent as the primary SCs., Conclusion: A graft of primary SCs in the capped chamber stimulated axonal growth response and/or maintained survival of axonal branches on the long term in the nerve amputee model.

Purpose: To assess the capacity of a retrovirus-engineered Schwann cell line (SCTM41), transfected with either a glial cell line-derived neurotrophic factor (GDNF) construct or a brain-derived neurotrophic factor (BDNF) construct, to sustain visual function in the dystrophic Royal College of Surgeons (RCS) rat., Methods: Cell suspensions were injected into the subretinal space of the right eye of 3-week-old dystrophic RCS rats through a transscleral approach. The left eye remained as an unoperated control. Sham-surgery animals received injections of carrier medium plus DNase to the right eye. All animals were placed on oral cyclosporine. At 8, 12, 16, and 20 weeks of age, animals were placed in a head-tracking apparatus and screened for their ability to track square-wave gratings at various spatial frequencies (0.125, 0.25, and 0.5 cyc/deg). At the end of the experiment, the animals were perfused and processed for histologic assessment of photoreceptor survival., Results: Animals with SCTM41-GDNF-secreting cells, on average, head tracked longer than animals with SCTM41-BDNF-secreting cells, and both performed better than those injected with the parent SCTM41 line. All tracked longer than sham-surgery or nonsurgical dystrophic eyes. Each cell type demonstrated preservation of photoreceptors up to at least 4 months of age, over and above the sham-surgery control., Conclusions: Engineered Schwann cells sustain retinal structure and function in the dystrophic RCS rat. Cells overexpressing GDNF or BDNF had a greater effect on photoreceptor survival than the parent line or sham surgery. This study demonstrates that ex vivo gene therapy and subsequent cell transplantation can be effective in preserving photoreceptors from the cell death that normally accompanies retinal degeneration.

If gene therapy is to be used to promote axon regeneration after spinal cord injury, a suitable vector for transgene delivery must be obtained. Replication-defective herpes simplex virus (HSV) vectors are promising candidates. We have examined whether they can express a LacZ transgene in injured neurons of adult rat brain. We transected the medial forebrain bundle, injected replication-defective HSV/LacZ vectors close to the lesion site, and looked for transgene expression at 2-14 days after the lesion. The vectors carried the LacZ transgene controlled either by the cytomegalovirus immediate-early promoter (vector CS5) or the HSV latency-associated promoter (vector CS1). CS5 transfected many cells near the lesion at 2 days, but did not give persistent expression at 5 days. CS1, in contrast, labeled many neurons in midbrain regions remote from the injection site at 5 days, and much of this expression remained at 12-14 days. The neurons of most interest were in the substantia nigra pars compacta and parabrachial nuclei, which were axotomized by the lesion. Vector-driven beta-galactosidase expression was detected in neurons in both regions. These were confirmed as axotomized by double immunofluorescence for c-Jun. By 12-14 days, many substantia nigra neurons had disappeared but some transduced neurons remained; there was no net loss of transduced neurons from the parabrachial nuclei. These results show that an HSV vector is capable of transducing axotomized cells in the central nervous system and producing transgene expression in them for at least 2 weeks after injection.

Matrix metalloproteases (MMPs) and tissue inhibitors of metalloproteases (TIMPs) are involved in many cell migration phenomena and produced by many cell types, including neurons and glia. To assess their possible roles in brain injury and regeneration, we investigate their production by glial cells, after brain injury and in tissue culture, and we investigate whether they are capable of digesting known axon-inhibitory proteoglycans. To determine the action of MMPs, we incubated astrocyte conditioned medium with activated MMPs, then did western blots for several chondroitin sulphate proteoglycans. MMP-3 digested all five proteoglycans tested, whereas MMP-2 digested only two and MMP-9 none. To determine whether MMPs or TIMPs are produced by astrocytes in vitro, we tested both primary cultures and astrocyte cell lines by western blotting, and compared them with Schwann cells. All cultures produced at least some MMPs and TIMPs, with no obvious correlation with the ability of axons to grow on those cells. Both MMP-9 and TIMP-3 were regulated by various cytokines. To determine which cells produce MMPs and TIMPs after brain injury, we made lesions of adult rat cortex, and did immunohistochemistry. MMP-2 was seen to be induced in activated astrocytes through the whole thickness of the cortex but not deeper, but MMP-3 was not seen in the injured brain. TIMP-2 and TIMP-3 immunoreactivities were induced in activated astrocytes in deep cortex and the underlying white matter. In situ hybridisation confirmed induction of TIMP-2 in glia as well as neurons, but showed no expression of TIMP-4. These results show that both MMPs and TIMPs are produced by some astrocytes, but TIMP production is particularly strong, especially in deep cortex and white matter which is more inhibitory for axon regeneration. Conversely the MMPs produced may not be adequate to promote migration of cells and axons within the glial scar.

Manipulating the expression of a protein can provide a powerful tool for understanding its function, provided that the protein is expressed at physiologically-significant concentrations. We have developed a simple method to measure (1) the concentration of an overexpressed protein in single cells and (2) the covariation of particular physiological properties with a protein's expression. As an example of how this method can be used, teratocarcinoma cells were transfected with the neuron-specific calcium binding protein calretinin (CR) tagged with green fluorescent protein (GFP). By measuring GFP fluorescence in microcapillaries, we created a standard curve for GFP fluorescence that permitted quantification of CR concentrations in individual cells. Fura-2 measurements in the same cells showed a strong positive correlation between CR-GFP fusion protein expression levels and calcium clearance capacity. This method should allow reliable quantitative analysis of GFP fusion protein expression.

EphA7 is a receptor tyrosine kinase of the Eph family. We have mapped EphA7 immunoreactivity and ligand binding in mouse embryo heads and developing brain. Immunoreactivity for the full-length receptor is found in all the cell populations that express EphA7 mRNA. In particular, it is located on growing axons from EphA7-expressing neurons, both in the trigeminal nerve and in developing brain. In many cases it persists in terminal fields in adult brain. Ligand is detected in a largely complementary distribution in embryos, but is surprisingly weak or undetectable in the target regions of many EphA7-positive axons postnatally.

Astrocytes exclude Schwann cells (SCs) from the central nervous system (CNS) at peripheral nerve entry zones and restrict their migration after transplantation into the CNS. We have modeled the interactions between SCs, astrocytes, and fibroblasts in vitro. Astrocytes and SCs in vitro form separate territories, with sharp boundaries between them. SCs migrate poorly when placed on astrocyte monolayers, but migrate well on various other surfaces such as laminin (LN) and skin fibroblasts. Interactions between individual SCs and astrocytes result in long-lasting adhesive contacts during which the SC is unable to migrate away from the astrocyte. In contrast, SC interactions with fibroblasts are much shorter with less arrest of migration. SCs adhere strongly to astrocytes and other SCs, but less well to substrates that promote migration, such as LN and fibroblasts. SC-astrocyte and SC-SC adhesion is mediated by the calcium-dependent cell adhesion molecule N-cadherin. Inhibition of N-cadherin function by calcium withdrawal, peptides containing the classical cadherin cell adhesion recognition sequence His-Ala-Val, or antibodies directed against this sequence inhibit SC adhesion and increase SC migration on astrocytes. We suggest that N-cadherin-mediated adhesion to astrocytes inhibits the widespread migration of SCs in CNS tissue., (Copyright 1999 Academic Press.)

We have developed a novel Schwann cell line, SCTM41, derived from postnatal sciatic nerve cultures and have stably transfected a clone with a rat glial cell line-derived neurotrophic factor (GDNF) construct. Coculture with this GDNF-secreting clone enhances in vitro survival and fiber growth of embryonic dopaminergic neurons. In the rat unilateral 6-OHDA lesion model of Parkinson's disease, we have therefore made cografts of these cells with embryonic day 14 ventral mesencephalic grafts and assayed for effects on dopaminergic cell survival and process outgrowth. We show that cografts of GDNF-secreting Schwann cell lines improve the survival of intrastriatal embryonic dopaminergic neuronal grafts and improve neurite outgrowth into the host neuropil but have no additional effect on amphetamine-induced rotation. We next looked to see whether bridge grafts of GDNF-secreting SCTM41 cells would promote the growth of axons to their striatal targets from dopaminergic neurons implanted orthotopically into the 6-OHDA-lesioned substantia nigra. We show that such bridge grafts increase the survival of implanted embryonic dopaminergic neurons and promote the growth of axons through the grafts to the striatum.

In an attempt to reconstruct the 6-hydroxydopamine lesioned nigrostriatal system of the adult rat we have combined homotopic grafting of embryonic ventral mesencephalon suspensions with the implantation of long oblique "bridge" grafts of fibroblast growth factor-4-transfected RN-22 schwannoma cells stretching from the site of the neuronal grafts to the striatum. At seven weeks after receiving both grafts, animals were killed and processed for immunohistochemistry against tyrosine hydroxylase. Tyrosine hydroxylase-immunoreactive axons were seen to extend from the nigral grafts, along the bridge graft to the striatum where terminal arborizations could be seen. The retrograde tracer Fluoro-gold was injected intrastriatally in some of the experimental animals and was taken up by grafted neurons confirming their projection to the striatum. In parallel to the anatomical reconstruction of the system, a decrease in amphetamine-induced rotation was demonstrated in those animals receiving both grafts which had received > 98% complete lesions. This decrease was greatest in those animals with the most tyrosine hydroxylase-immunoreactive axons in their bridge grafts. The presence of the bridge graft also led to an increase in neuronal graft survival with twice as many tyrosine hydroxylase-immunoreactive neurons being found in the grafts of those animals that had received both grafts compared to those that had received a neuronal graft but no bridge graft.

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.

A polymerase chain reaction-based subtractive hybridization procedure was applied to cDNAs prepared from mouse insulinoma (beta TC3) and glucagonoma (alpha TC2) cell lines to construct a library of cDNAs that are highly expressed in pancreatic beta-cells. An analysis of 555 randomly chosen clones in the library showed that 80 were derived from abundant mRNAs and were accounted for by 29 distinct sequences. Of these, 17 were identical or homologous to known mammalian cDNAs or expressed sequence tags. Genes known to be highly expressed in beta-cells were represented at a high frequency, namely insulin (15 of 80 clones), islet amyloid polypeptide (8 of 80 clones), proinsulin convertase 1 (6 of 80 clones), and neuropeptide Y (2 of 80 clones). Many of the novel cDNA sequences that were highly represented in the library showed a relative specificity to beta-cells compared with other tissues, including glucagonoma, liver, kidney, brain, 3T3 fibroblasts, and AtT20 corticotrophs, and warrant further investigation. When combined with functional or immunological screening procedures, the approach will be useful for the isolation of beta-cell-specific molecules for immunological and genetic investigations of beta-cell function and pathology.

Calbindin D28K (formerly known as vitamin D-dependent calcium binding protein and referred to here as calbindin) is found in a wide variety of tissues, but only in certain cells within those tissues. Apart from its ability to bind calcium, nothing is known about its function in these cells. To investigate its role we have transfected the chick calbindin cDNA into mouse NIH3T3 fibroblasts and established a new cell line where calbindin is permanently expressed. Immunofluorescence studies show that calbindin is distributed throughout the cytoplasm, and treatment of the cells with cycloheximide shows that it has a relatively long half-life within the cell. Measurements of intracellular calcium concentration using Fura-2 suggest that the presence of calbindin within the cells does not affect the increase in intracellular calcium levels which occurs in response to serum stimulation or the rate at which these return to the basal level, but that it may act as a buffer for the entry of extracellular calcium.

125I-labelled poly(vinylpyrrolidone) was used as a marker of fluid-phase pinocytosis in cultured pig arterial smooth-muscle cells. The rate of pinocytosis was temperature-dependent. A decrease in cellular ATP concentrations as a result of inhibition of either glycolysis or oxidative phosphorylation was associated with a similar decrease in pinocytosis. A microfibrillar-disruptive agent, cytochalasin B, caused a concentration-dependent stimulation of pinocytosis, whereas the microtubular-disruptive agents colchicine and vinblastine decreased pinocytosis to approximately half of control values at all concentrations used. These results indicate that fluid-phase pinocytosis in smooth-muscle cells is dependent on a continuing supply of energy and the integrity of the microtubules. Furthermore, microfilaments appear to exert a certain degree of constraint on pinocytosis, possibly by restricting invagination of the plasma membrane.

Lysosomal overload was induced experimentally in cultured aortic smooth muscle cells by incubation with chloroquine or sucrose. Lysosomal overload was accompanied by a marked reduction in pinocytosis and induced the release of lysosomal contents into the medium. Thus, previously accumulated pinocytic tracer and beta-glucuronidase, a lysosomal enzyme, were released into the medium whereas lactate dehydrogenase, a cytosolic enzyme, was not.