Combinational Spinal GAD65 Gene Delivery and Systemic GABA-Mimetic Treatment for Modulation of Spasticity

receptor agonist), while effective in modulating spasticity is associated with major side effects such as general sedation and progressive tolerance development. The goal of the present study was to assess if a combined therapy composed of spinal segment-specific upregulation of GAD65 (glutamate decarboxylase) gene once combined with systemic treatment with tiagabine (GABA uptake inhibitor) will lead to an antispasticity effect and whether such an effect will only be present in GAD65 gene over-expressing spinal segments.Adult Sprague-Dawley (SD) rats were exposed to transient spinal ischemia (10 min) to induce muscle spasticity. Animals then received lumbar injection of HIV1-CMV-GAD65 lentivirus (LVs) targeting ventral α-motoneuronal pools. At 2–3 weeks after lentivirus delivery animals were treated systemically with tiagabine (4, 10, 20 or 40 mg/kg or vehicle) and the degree of spasticity response measured. In a separate experiment the expression of GAD65 gene after spinal parenchymal delivery of GAD65-lentivirus in naive minipigs was studied. Spastic SD rats receiving spinal injections of the GAD65 gene and treated with systemic tiagabine showed potent and tiagabine-dose-dependent alleviation of spasticity. Neither treatment alone (i.e., GAD65-LVs injection only or tiagabine treatment only) had any significant antispasticity effect nor had any detectable side effect. Measured antispasticity effect correlated with increase in spinal parenchymal GABA synthesis and was restricted to spinal segments overexpressing GAD65 gene.These data show that treatment with orally bioavailable GABA-mimetic drugs if combined with spinal-segment-specific GAD65 gene overexpression can represent a novel and highly effective anti-spasticity treatment which is associated with minimal side effects and is restricted to GAD65-gene over-expressing spinal segments

A Transgenic Minipig Model of Huntington\u27s Disease

Background: Some promising treatments for Huntington\u27s disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. Objective: To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1–548 under the control of human HTT promoter. Methods: Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. Results: One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. Conclusions: The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study

Spinal parenchymal injections of HIV1-CMV-GAD65-GFP lentivirus leads to increased GAD65 expression in infected astrocytes in rat and minipig and is associated with increased extracellular GABA release after tiagabine treatment in rats with ischemic spasticity.

<p>(<b>A–C</b>) Immunofluorescence images taken from a transverse lumbar spinal cord section of a spastic rat at 3 weeks after spinal injection of HIV1-CMV-GAD65-GFP lentivirus. Sections were stained with GFP, GAD65 and GFAP antibody. (<b>D, E</b>) Confocal images demonstrating the localization of GAD65-GFP (green) expressing processes in HIV1-CMV-GAD65-GFP-infected cells surrounding VGLUT1 (red)-IR primary afferent terminals in the vicinity of persisting CHAT (blue)-IR α-motoneurons. (<b>F</b>) Western blot analysis for GAD65 in spinal cord homogenate taken from lumbar spinal parenchyma of naive-control (column 1) spastic non-treated (columns 2 and 3) and spastic HIV1-CMV-GAD65-GFP-injected animal (column 4). (<b>G</b>) Extracellular GABA concentration measured by intraparenchymal microdialysis in lumbar gray matter in naive (n = 6), ischemic-spastic (n = 6), ischemic-spastic-HIV1-CMV-GFP (n = 6) and ischemic-spastic-HIV1-CMV-GAD65-GFP (n = 6) lentivirus-injected animals before and after systemic tiagabine (40 mg/kg) injection. A significant increase in extracellular GABA concentration was measured at 20–40 min after tiagabine administration in naive animals and ischemic-spastic animals previously injected spinally with HIV1-CMV-GAD65-GFP lentivirus (P<0.05; paired <i>t</i> test). (<b>H</b>) Confocal images of transverse spinal cord section taken from a minipig lumbar spinal cord at 2 months after spinal HIV1-CMV-GAD65-GFP injections and stained with GFP, GAD65 and CHAT antibody.</p

Loss of segmental inhibitory GABA-ergic interneurons and increased expression of GABA B R1+R2 receptor in α-motoneurons after transient spinal cord ischemia is associated with the development of chronic muscle spasticity.

<p>(<b>A, B</b>) Transverse spinal cord sections taken from L2–L5 segments in control (<b>A</b>) or spinal ischemia-induced-spastic rat (<b>B</b>) at 24 h after intrathecal colchicine injection and stained for GABA. Note an apparent loss of GABA-ergic interneurons in the intermediate zone in spastic rat (<b>B</b>; red circle). (<b>C–F</b>) Loss of GABA-ergic interneurons corresponds with loss of GABA-IR and GAD65-IR boutons on membranes of persisting CHAT-IR α-motoneurons in animals with ischemic spasticity (white arrows). (<b>G, H</b>) Western blotting for GAD65 and GAD67 in lumbar spinal cord samples taken from control animals (n = 5–6) or animals with developed ischemic spasticity (n = 5–6), (*P = 0.017; **P = 0.045, unpaired <i>t</i>-test). (<b>I–N</b>) In comparison to control animals, an upregulation in GABA B R1+R2 receptors in lumbar α-motoneurons was identified in animals with spasticity (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1I</b></a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1J</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1L</b></a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1M</b></a>). Quantitative densitometric analysis showed significantly increased densities for both receptor subunits in spastic animals (*-P<0.05; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1K</b></a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030561#pone-0030561-g001" target="_blank"><b>Fig. 1N</b></a>). (<b>O, P</b>) Measurement of EMG activity in gastrocnemius muscle and corresponding ankle resistance during computer-controlled ankle rotation (45°/3 sec) in awake control sham-operated animals (<b>O</b>) and in animals with ischemic spasticity (<b>P</b>).</p

Effective suppression of spasticity after combined therapy with systemic tiagabine and intrathecal injection of GABA or spinal parenchymal GAD65 gene delivery.

<p>(<b>A</b>) EMG responses recorded from gastrocnemius muscle in spastic animals during computer-controlled ankle dorsiflexion before and after systemic treatment with tiagabine (40 mg/kg; i.p.; n = 6), intrathecal GABA (1 mg; IT; n = 6) or combined treatment with tiagabine+IT GABA (n = 6). (<b>B</b>) Time-course of ankle resistance measured during ankle dorsiflexion at baseline and then in 5-min intervals up to 80 min after treatments (* P<0.01; one-way analysis of variance-ANOVA, Bonferroni's posthoc test; MPE-maximum positive effect). (<b>C</b>) EMG responses recorded from the gastrocnemius muscle in spastic animals previously injected spinally with HIV1-CMV-GFP (control; n = 6) or HIV1-CMV-GAD65 (n = 6) lentivirus and then treated with systemic 10 mg/kg or 40 mg/kg tiagabine. (<b>D</b>) Time-course of anti-spastic effect after tiagabine treatment expressed as % of maximum possible effect in measured ankle resistance in HIV1-CMV-GFP or HIV1-CMV-GAD65-GFP lentivirus-injected animals (* P<0.01; one-way analysis of variance-ANOVA, Bonferroni's posthoc test; MPE-maximum positive effect). (<b>E</b>) Changes in H-wave amplitudes recorded from interdigital muscles of the lower extremity during high frequency (20 Hz) sciatic nerve stimulation in animals previously injected spinally with HIV1-CMV-GFP or HIV1-CMV-GAD65 lentivirus and then treated with 40 mg/kg tiagabine. (<b>F</b>) Time-course of changes in H-wave amplitudes before and up to 90 min after tiagabine administration (red line-P<0.05; unpaired <i>t</i> test).</p

Infection of rat primary spinal cord culture with HIV1-CMV-GAD65 or HIV1-CMV-GAD65-GFP lentivirus leads to a preferential astrocyte GAD65 expression and release of biologically active GABA.

<p>(<b>A</b>) Rat spinal cord primary culture infected with HIV1-CMV-GAD65-GFP lentivirus and stained with anti-GFP antibody at 4 days after lentivirus infection. (<b>B–D</b>) Co-staining of HIV1-CMV-GAD65-GFP-infected cells with GAD65 antibody showed preferential GAD65 expression in GFP-IR cells. (<b>E–G</b>) Colocalization of GFP-IR with GFAP-IR in HIV1-CMV-GAD65-GFP-infected astrocytes at 14 days after infection. (<b>H</b>) Western blotting for GFP or GAD65 in cell lysates taken from rat primary spinal cord culture infected with HIV1-CMV-GFP (control), HIV1-CMV-GAD65-GFP, and HIV1-CMV-GAD65 lentivirus. (<b>I</b>) Extracellular GABA release measured in cell culture media taken from rat primary spinal cord culture 3–14 days after HIV1-CMV-GFP (control) or HIV1-CMV-GAD65-GFP lentivirus injection. (<b>J</b>) Progressive increase in extracellular GABA release measured in Ca²⁺-free media 1–3 hrs after cell culture wash in HIV1-CMV-GAD65-GFP but not in HIV1-CMV-GFP (control) lentivirus-infected cells (* P<0.01; paired <i>t</i> test). (<b>K</b>) Human fetal spinal cord astrocytes infected with HIV1-CMV-GAD65-GFP lentivirus and stained with anti-GFP antibody at 7 days after lentivirus infection. (<b>L</b>) Changes in whole-cell inward current in cultured human NT neurons after bath application of human astrocyte-HIV1-CMV-GAD65-GFP-conditioned media, (<b>M</b>) 50 µM GABA or (<b>N</b>) human astrocyte-HIV1-CMV-GFP-conditioned media (control); (neurons clamped at holding potential (-) 60 mV).</p