Lentiviral delivery of meteorin protects striatal neurons against excitotoxicity and reverses motor deficits in the quinolinic acid rat model.

Meteorin is a newly discovered secreted protein involved in both glial and neuronal cell differentiation, as well as in cerebral angiogenesis during development; but effects in the adult nervous system are unknown. The growth factor-like properties and expression of Meteorin during the development of the nervous system raises the possibility that it might possess important neuroprotective or regenerative capabilities. This report is the first demonstration that Meteorin has potent neuroprotective effects in vivo. Lentiviral-mediated striatal delivery of Meteorin to rats two weeks prior to injections of quinolinic acid (QA) dramatically reduced the loss of striatal neurons. The cellular protection afforded by Meteorin was associated with normalization of neurological performance on spontaneous forelimb placing and cylinder behavioral tests and a complete protection against QA-induced weight loss. These benefits were comparable in magnitude to those obtained with lentiviral-mediated delivery of ciliary neurotrophic factor (CNTF), a protein with known neuroprotective properties in the same model system. In naive animals, endogenous levels of both Meteorin and CNTF were increased in glial cells in response to QA lesion indicating that Meteorin may exert its protective effects as part of the reactive gliosis cascade in the injured brain. In summary, these data demonstrate that Meteorin strongly protects striatal neurons and deserves additional evaluation as a novel therapeutic for the treatment of neurological disorders with an excitotoxic component such as Huntington's Disease.

Parkinson-like neurodegeneration induced by targeted overexpression of alpha-synuclein in the nigrostriatal system.

Recombinant adeno-associated viral vectors display efficient tropism for transduction of the dopamine neurons of the substantia nigra. Taking advantage of this unique property of recombinant adeno-associated viral vectors, we expressed wild-type and A53T mutated human alpha-synuclein in the nigrostriatal dopamine neurons of adult rats for up to 6 months. Cellular and axonal pathology, including alpha-synuclein-positive cytoplasmic inclusions and swollen, dystrophic neurites similar to those seen in brains from patients with Parkinson's disease, developed progressively over time. These pathological alterations occurred preferentially in the nigral dopamine neurons and were not observed in other nondopaminergic neurons transduced by the same vectors. The degenerative changes were accompanied by a loss of 30-80% of the nigral dopamine neurons, a 40-50% reduction of striatal dopamine, and tyrosine hydroxylase levels that was fully developed by 8 weeks. Significant motor impairment developed in those animals in which dopamine neuron cell loss exceeded a critical threshold of 50-60%. At 6 months, signs of cell body and axonal pathology had subsided, suggesting that the surviving neurons had recovered from the initial insult, despite the fact that alpha-synuclein expression was maintained at a high level. These results show that nigral dopamine neurons are selectively vulnerable to high levels of either wild-type or mutant alpha-synuclein, pointing to a key role for alpha-synuclein in the pathogenesis of Parkinson's disease. Targeted overexpression of alpha-synuclein in the nigrostriatal system may provide a new animal model of Parkinson's disease that reproduces some of the cardinal pathological, neurochemical, and behavioral features of the human disease.

Low-level tyrosine hydroxylase (TH) expression allows for the generation of stable TH+ cell lines of human neural stem cells.

Genetic engineering of neurotransmitter metabolic routes is important for the development of neurotransmitter-producing cells for the ex vivo gene therapy of many CNS diseases. Human neural stem cells (hNSCs) are excellent candidates to serve this role, but, for the case of Parkinson's disease, the cells do not normally express the rate-limiting dopamine (DA) synthesis enzyme tyrosine hydroxylase (TH), and are not equipped with the detoxifying mechanisms needed to prevent the neurotoxicity associated with the DA phenotype. In this study we have examined the capacity of hNSCs for ectopic expression of human TH. High-level TH expression (from viral promoters) leads to growth arrest and hNSC death (associated with an increase in p53 expression and nuclear fragmentation), which can be counteracted by treatment with a pan-caspase inhibitor. As a consequence, stable TH-expressing hNSC sublines could not be derived using viral promoters. In contrast, moderate TH expression (from a human housekeeping promoter, polyubiquitin gene), allows for stable TH+ subclone derivation, seemingly originating from low-expressing cells. Our results are thus compatible with the view that stable TH-expressing hNSC lines can be generated if TH expression levels are kept at a moderate level, and that the goal normally set of aiming at high-level TH expression may need to be reconsidered. These results may be relevant for the generation of TH/DA-producing human neural cells for in vitro and neurotransplantation research in Parkinson's disease.

Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites.

The development of a set of synthetic mammalian promoters with different specific activities is described. The library is based on a synthetic promoter, JeT, constructed as a 200 bp chimeric promoter built from fragments of the viral SV40 early promoter and the human beta-actin and ubiquitin C promoters. The JeT promoter was made by separating the included consensus boxes by the same distances in base pairs as found in the wild-type promoters, thus preserving transcription factor interaction. The resulting promoter was shown to drive reporter expression to high levels in enhanced green fluorescent protein and secreted alkaline phosphatase reporter assays. By replacing sequences separating the transcription factor binding sites with randomized sequences of the same length, sets of new promoters with different strengths, spanning a 10-fold range of transcriptional activity in cell culture, was obtained. The measured activity of each promoter in the library was highly specific and reproducible when tested in HiB5 and ARPE-19 cell culture.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.

Meteorin reverses hypersensitivity in rat models of neuropathic pain.

Neuropathic pain is caused by a lesion or disease to the somatosensory nervous system and current treatment merely reduces symptoms. Here, we investigate the potential therapeutic effect of the neurotrophic factor Meteorin on multiple signs of neuropathic pain in two distinct rat models. In a first study, two weeks of intermittent systemic administration of recombinant Meteorin led to a dose-dependent reversal of established mechanical and cold hypersensitivity in rats after photochemically-induced sciatic nerve injury. Moreover, analgesic efficacy lasted for at least one week after treatment cessation. In rats with a chronic constriction injury (CCI) of the sciatic nerve, five systemic injections of Meteorin over 9 days dose-dependently reversed established mechanical and thermal hypersensitivity as well as weight bearing deficits taken as a surrogate marker of spontaneous pain. The beneficial effects of systemic Meteorin were sustained for at least three weeks after treatment ended and no adverse side effects were observed. Pharmacokinetic analysis indicated that plasma Meteorin exposure correlated well with dosing and was no longer detectable after 24 hours. This pharmacokinetic profile combined with a delayed time of onset and prolonged duration of analgesic efficacy on multiple parameters suggests a disease-modifying mechanism rather than symptomatic pain relief. In sciatic nerve lesioned rats, delivery of recombinant Meteorin by intrathecal injection was also efficacious in reversing mechanical and cold hypersensitivity. Together, these data demonstrate that Meteorin represents a novel treatment strategy for the effective and long lasting relief from the debilitating consequences of neuropathic pain.

Increased in vitro and in vivo transgene expression levels mediated through cis-acting elements.

BACKGROUND: Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. METHODS: Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. RESULTS: Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. CONCLUSION: The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.