In Vivo Assessment of Cell Death and Nigrostriatal Pathway Integrity Following Continuous Expression of C3 Transferase

Abstract

The bacterial exoenzyme C3 transferase (C3) irreversibly inhibits RhoA GTPase leading to stimulation of axonal outgrowth in injured neurons. C3 has been used successfully in models of neurotrauma and shows promise as an option to support cell survival and axonal growth of dopaminergic (DA) neurons in Parkinson's disease (PD) cell therapy. Whether the continuous expression of C3 in DA neurons is well-tolerated is unknown. To assess the potential neurotoxicity of sustained expression of C3 in DA neurons, we generated Cre recombinase-dependent adeno-associated viral vectors (AAV) for targeted C3 delivery to DA neurons of the mouse substantia nigra pars compacta (SNc). The effect of continuous expression of C3 on DA neurons was assessed by immunohistochemistry and compared to that of Enhanced Yellow Fluorescent Protein (EYFP) as negative controls. We did not find significant reduction of tyrosine hydroxylase (TH) expression levels nor the presence of cleaved activated caspase 3. Astrocytic activation as determined by GFAP expression was comparable to EYFP controls. To evaluate the impact of C3 expression on striatal terminals of the nigrostriatal pathway, we compared the rotational behavior of wildtype mice injected unilaterally with either C3 or 6-hydroxydopamine (6-OHDA). Mice injected with C3 exhibited similar ipsiversive rotations to the site of injection in comparison to control mice injected with EYFP and significantly fewer ipsiversive rotations compared to 6-OHDA lesioned mice. Non-significant difference between C3 and EYFP controls in behavioral and histological analyses demonstrate that transduced DA neurons express C3 continuously without apparent adverse effects, supporting the use of C3 in efficacy studies targeting DA neurons.

Keywords: C3 transferase; Cre recombinase; RhoA; dopaminergic neurons; rodent behavior; tyrosine hydroxylase.