A40 Modulation of DARPP32 homeostasis by htt protein in derivatives of disease-specific and control human pluripotent stem cells

Abstract

In Huntington disease (HD), the most affected cells are the GABA-releasing medium spiny neurons (MSN) of the striatum, the subcortical brain structure that controls body movement. DARPP32 (dopamine- and 3’,5’-cyclic adenosine monophosphate-regulated phosphoprotein, 32 kDa) is a class defining protein marker for striatal MSNs and a central mediator of dopamine signalling and other first messengers in these cells. DARPP-32 is expressed in 97% of the MSNs, several cortical layers, and cerebellar Purkinje cells. This protein has the capacity to function as either a kinase or a phosphatase inhibitor, depending on the phosphorylation state of key amino acid residues. DARPP32 is highly down-regulated in the striatum of HD patient and in the majority of mouse models of HD. The rational of our work is to use derivatives of several types of HD and WT-human Pluripotent Stem Cell (hPSC) lines challenged or not with HTT-targeting RNA interference (shRNA lentiviral vectors) or HTT-targeting CRISPR-Cas9 complex to decipher possible wt-HTT dosage, mut-HTT dominant-negative, mut-HTT de novo toxic or CAG repeat length effects on DARPP32 homeostasis. Using lentiviral vectors, we investigated the role of HTT isoforms on DARPP-32 protein levels measured by Western Blot and immunocytochemistry. We observed changes in DARPP32 and phopsho-DARPP32 protein level in hPSC derivatives treated with shHTT lentivirus. Next, we generated a series of CRISPER-CAS9 HTT-gene edited clones of HD-iPSC and WT-iPSC. Either or both allele of HTT gene was inactivated in those cells and the MSN derived from those clones. Exploration of DARPP32 protein levels in undifferentiated cells and MSN derived from these clones is ongoing. This study should help understand the role of HTT isoforms on human MSN homeostasis.