Reversible unilateral nigrostriatal pathway inhibition induced through expression of adenovirus-mediated clostridial light chain gene in the substantia nigra.

Clostridial light chain (LC) inhibits synaptic transmission by digesting a vesicle-docking protein, synaptobrevin, without killing neurons. We here report the feasibility of creating a rat hemiparkinsonism model through LC gene expression in the substantia nigra (SN), inhibiting nigrostriatal transmission. 40 adult Sprague Dawley rats were divided into four groups for SN injections of PBS, 6-hydroxydopamine (6-OHDA), or adenoviral vectors for the expression of LC (AdLC), or GFP (AdGFP). Amphetamine and apomorphine induced rotations were assessed before and after SN injection, revealing significant rotational alterations at 8 or 10 days after injection in both AdLC and 6-OHDA but not PBS and AdGFP groups. Induced rotation recovered by one month in AdLC rats but persisted in 6-OHDA rats. Histological analysis of the SN revealed LC and GFP expression with corresponding synaptobrevin depletion in the LC, but not the GFP groups. Tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunohistochemistry (IHC) showed markedly decreased staining in ipsilateral SN and striatum in 6-OHDA but not AdLC or AdGFP rats. Similarly, compared with contralateral, ipsilateral striatal dopamine level only decreased in 6-OHDA but not AdLC, AdGFP, or PBS treated rats. Thus, LC expression induces nigral synaptobrevin depletion with resulting inhibition of nigrostriatal synaptic transmission. Unlike 6-OHDA, LC expression inhibits synaptic activity without killing neurons. This approach, therefore, represents a potentially reversible means of nigrostriatal pathway inhibition as a model for Parkinson's disease. Such a model might facilitate transient and controlled nigral inhibition for studying striatal recovery, dopaminergic re-innervation, and normalization of striatal receptors following the recovery of nigrostriatal transmission.

X-Linked inhibitor of apoptosis protein gene-based neuroprotection for the peripheral nervous system.

OBJECTIVE: The recently discovered X-linked inhibitor of apoptosis protein (XIAP) is among the most potent inhibitors of programmed cell death. In the current experiment, we examine the potential of adenoviral XIAP gene delivery to protect neurons of the peripheral nervous system using in vitro models of amyotrophic lateral sclerosis (ALS) and diabetic neuropathy. METHODS: XIAP complementary deoxyribonucleic acid was fused in frame with the green fluorescent protein sequence and cloned into a first generation adenoviral vector. The impact of XIAP gene expression on glutamate-induced apoptosis was measured in the neuronal SH-SY5Y cell line with immunohistochemistry for active caspase-3 and with cell density assays. Next, the effect of XIAP expressing neurons on the survival of uninfected neighboring neurons was measured. Finally, the impact of XIAP gene expression on glutamate-induced apoptosis was assessed in embryonic motor neuron and dorsal root ganglion cultures. RESULTS: XIAP gene expression reduced the percentage of active caspase-3 positive SH-SY5Y neurons and preserved cell density after glutamate exposure. In heterogeneously infected cultures, cells infected with XIAP were protected, but uninfected neighboring cells were not. In primary E15 models, inhibition of proapoptotic effects was demonstrated after glutamate insult in motor neurons and glucose insult in dorsal root ganglion cells. CONCLUSION: XIAP gene delivery through the neurosurgical delivery of viral vectors may provide a means for neuroprotection in ALS and diabetic neuropathy.

Neuroprotective adeno-associated virus Bcl-xL gene transfer in models of motor neuron disease.

Recent work implicates excitotoxicity-induced apoptosis as the mechanism triggering motor neuron death in amyotrophic lateral sclerosis (ALS). Our laboratory has previously utilized glutamate excitotoxicity in vitro to study this process. The present experiment tests whether overexpression of the gene for Bcl-xL can inhibit excitotoxicity in this model system. To track Bcl-xL expression, the gene for green fluorescent protein (GFP) was inserted in-frame, upstream of the Bcl-xL gene. The GFP-Bcl-xL gene was then cloned into an adeno-associated viral (AAV2) vector. GFP expression in both SH-SY5Y and embryonic day 15 (E15) motor neurons (MNs) peaked 48 hours after infection. Bcl-xL expression in SH-SY5Y cells significantly reduced terminal deoxy-UTP nick-end labeling (TUNEL)-positive cells and maintained cell density after glutamate exposure. Similarly, Bcl-xL expression inhibited the development of TUNEL staining in E15 MNs and supported cell density after glutamate exposure. These findings suggest that AAV-mediated expression of genes for antiapoptotic proteins may provide a means for ALS gene therapy.

Cervical spinal cord delivery of a rabies G protein pseudotyped lentiviral vector in the SOD-1 transgenic mouse. Invited submission from the Joint Section Meeting on Disorders of the Spine and Peripheral Nerves, March 2004.

OBJECT: Lentiviral vectors may constitute a vehicle for long-term therapeutic gene expression in the spinal cord. In amyotrophic lateral sclerosis, spinal cord sclerosis and altered axonal transport pose barriers to therapeutic gene distribution. In the present study the authors characterize gene expression distribution and the behavioral impact of the rabies G (RabG) protein pseudotyped lentiviral vector EIAV.LacZ through cervical spinal cord injection in control and Cu/Zn superoxide dismutase-1 (SOD-1) transgenic mice. METHODS: Seven-week-old SOD-1 transgenic mice and their wild-type littermates underwent exposure of the cervicomedullary junction and microinjection of RabG.EIAV.LacZ or vehicle. The Basso-Beattie-Bresnahan locomotor score, grip strength meter, and Rotarod assays were used to assess the effects of disease progression, spinal cord microinjection, and lentiviral gene expression. Spinal cords were removed when the mice were in the terminal stage of the disease. The distribution of LacZ gene expression was histologically evaluated and quantified. Direct cervical spinal cord microinjection of RabG.EIAV.LacZ results in extensive central nervous system uptake in SOD-1 transgenic mice; these findings were statistically similar to those in wild-type mice (p > 0.05). Gene expression lasts for the duration of the animal's survival (132 days). The SOD-1 mutation does not prevent retrograde axonal transport of the vector. Three behavioral assays were used to demonstrate that long-term gene expression does not alter sensorimotor function. In comparison with normative data, vector injection and transgene expression do not accelerate disease progression. CONCLUSIONS: Direct spinal cord injection of RabG.EIAV vectors represents a feasible method for delivering therapeutic genes to upper cervical spinal cord and brainstem motor neurons. Distribution is not affected by the SOD-1 mutation or disease phenotype.

Molecular biology and gene therapy in the treatment of chronic pain.

Technologic advancements have made cell type-specific targeting, expression control, and safe and stable gene transfer possible. Animal research has provided increasing experience with gene transfer to the nervous system and sensory neurons in particular. Gene-based neuromodultion can be achieved through neuronal delivery of transgenes capable of altering synaptic function. Alternatively, ex vivo gene transfer can be used to create cell lines capable of secreting analgesic neurepeptides. Translatation of these grafts and direct gene-based neuromoduation can be applied to the control of pain and the root causes of pain. These approaches combine anatomic and pharmacologic specificity. As the technology continues to improve, clinical application of cellular and molecular pain control is likely.