Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

Abstract

Clostridial neurotoxins have assumed increasing importance in clinical application. The toxin's light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health. To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed. LC expressed in differentiated neuronal PC12 cells was shown to induce time-and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products. LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso-Beattie-Bresnahan (BBB) scale (Po0.001) and rotarod assay (Po0.003).Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats. At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected. Spontaneous functional recovery was observed to parallel the cessation of LC gene expression. These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.

Anatomically discrete functional effects of adenoviral clostridial light chain gene-based synaptic inhibition in the midbrain

“…6 However, in situ reduction of VAMP-1 has not previously been demonstrated in brain parenchyma. Immunohistochemistry for VAMP-1 was performed in sections identified to contain AdLC-or AdGFP-mediated gene expression.…”

“…Immunohistochemistry and Western blots revealed a focal reduction in VAMP-1 levels in regions of LC expression within the dSC/DpMe, consistent with the previous demonstration of in vitro time and concentration-dependent rat brain VAMP-1 digestion. 6 These findings suggest that LC mediated VAMP-1 digestion can achieve targeted neural inhibition. LC is the enzymatically active fragment of Tetanus Toxin.…”

“…6 Spinal cord LC expression resulted in hindlimb sensorimotor dysfunction localized by electrophysiology to the neuromuscular junction. Because of the binding and transport properties of Hc, the spinal motor system is the endogenous target of clostridial neurotoxins.…”

“…6 In short, the 1496 bp synthetic gene encoding the light chain of tetanus toxin protein (LC) was cloned into a shuttle vector between the CMV promoter, and an IRES-GFP sequence. AdLC was produced through Cremediated in vitro recombination.…”

“…Our laboratory has recently demonstrated that direct injection of a first-generation adenoviral vector expressing LC (AdLC) induces synaptic inhibition in spinal cord motor neurons. 6 LC expression resulted in the digestion of synaptobrevin (VAMP-1), a protein critical for synaptic vesicle docking. The time course of synaptic inhibition paralleled that of gene expression, which diminished over the second and third weeks after injection.…”