Title

 

Authors

C. Scali,¹I. Ceccarelli¹, R. Remelli¹, D. Franceschini¹, L. Rossini¹, G. Pollio¹, M. Camarri¹, I. Biotti¹, A. Cappelli¹, V. Miragliotta¹, P. Fiengo¹, A. Caricasole¹and G. Gaviraghi¹.
 
¹Siena Biotech S.p.A., Strada del Petriccio e Belriguardo, 35 - 53100 Siena (Italy)

 

Abstract

Huntington’s Disease (HD) is an inherited neurodegenerative disorder characterized by dyskinesia, cognitive impairment and emotional disturbances and associated with a progressive neurodegeneration of medium-spiny neurons in the striatum and with the presence of intracellular Huntingtin (Htt) aggregates in various area of the brain. The mutation responsible for HD has been identified as a CAG expansion in the Htt gene, which is translated into a polyQ stretch at the protein level. Recombinant Adeno Associated Viral (AAV) vectors have been used successfully to transfer genes in a variety of tissues, including the brain, in adult animals. In the present study we have used rAAV9, charged with Exon 1 Htt carrying 17 and 138 CAG repeats (wild type- or m-Htt, respectively) to set up and optimize both in vitro and in vivo model of Huntington’s Disease.

For in vitro model, AAV9-Ex1-GFP-Q138 or, as control, AAV9-Ex1-GFP-Q17 viral preparations have been added to pure cortical neurons prepared from E18 rat embryos and plated in 96-well plates in conditions optimized for imaging analysis with BD Pathway 435. The infection, whose efficiency was about 95%, led to a progressive formation of EM-48 positive aggregates, and subsequent neuronal death, measured as a decrease of NeuN- and phosphoCREB-positive cells (about 50% death at 4 weeks post-infection). Selected reference compounds are being tested for their neuroprotective activity.

For in vivo model, AAV9-Ex1-GFP-Q138 viral preparations have been injected in the right striatum of female rats. The controlateral striatum was injected with AAV9-Ex1-GFP-Q17 and used as control. Twenty-one days after surgery animals were processed for immunohistochemical analysis on brain slices. AAV9-Ex1-GFP-Q138 injection induced the formation of GFP (and EM48) positive Htt aggregates in the entire striatal area, increased GFAP and microglial activation with respect to Q17 injected striatum, and neurodegeneration, as evidenced by a decrease in ChAT- and NeuN-positive neurons in the Q138-injected striatum with respect to Q17. The efficacy of sodium butyrate was tested in this model. MRI and H1 NMR spectroscopy were carried out in a small group of animals, to evaluate the morphologic and metabolic changes induced by mHtt injection.

The AAV9-Ex1-GFP-Q138 HD models may represent a useful preclinical model for the evaluation of neuroprotective approaches in HD.