Huntington disease (HD) is a neurodegenerative disease caused by the expression of a mutant form of HTT (huntingtin; mHTT), caused by an abnormal expansion of polyglutamine in HTT. In HD, macroautophagy/autophagy dysfunction can cause mHTT accumulation. Moreover, the promotion of autophagy is considered a therapeutic strategy for the treatment of HD. ZKSCAN3 (zinc finger with KRAB And SCAN domains 3) has been identified as a transcriptional repressor of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal functions. In this study, we conducted CRISPR-Cas9-based gene ablation to disrupt ZKSCAN3 in HD animal models and HD patient-induced pluripotent stem cell (iPSC) -derived three-dimensional (3D) spheroids. In animal models of HD, targeted in vivo <i>zkscan3</i> ablation via a single adeno-associated virus (AAV) mediated CRISPR-Cas9 approach resulted in reduced mHTT levels, leading to improvements in both behavioral symptoms and the brain environment. Furthermore, CRISPR-Cas9 mediated ablation of ZKSCAN3 in 3D spheroids from HD patient-derived iPSC resulted in increased autophagy and lysosomal function, along with reduced mHTT accumulation. Specifically, in iPSC-derived neurons from HD patients, ZKSCAN3-depleted neurons demonstrated increased lysosomal function and reduced oxidative stress compared to controls. Additionally, transcriptional analysis of ZKSCAN3-edited neurons revealed an increased expression of genes involved in synaptic function and transporter activity. Taken together, these results suggest that in HD treatment strategies for improving neuronal function and the brain environment, ZKSCAN3 downregulation in neurons by autophagy activation may improve the brain environment through neuronal self-repair.<b>Abbreviations:</b> 2D: two-dimensional; 3D: three-dimensional; 4-HNE: 4-hydroxynonenal; AAV: adeno-associated virus; AD: Alzheimer disease; Aβ: beta-amyloid; DAPI: 4,6-diamidino-2-phenylindole; GFP: green fluorescent protein; HD: Huntington disease; HTT: huntingtin; IXMC: ImageXpress microconfocal high-content imaging system; Indel: insertion or deletion; iPSC: induced pluripotent stem cell; LAMP1: lysosomal-associated membrane protein 1; mHTT: mutant huntingtin; NPCs: neural precursor cells; RBFOX3/NeuN: RNA binding fox-1 homolog 3; PD: Parkinson disease; RNP: ribonucleoprotein; sgRNAs: single guide RNAs; ST: striatum; TFEB: transcription factor EB; TUBB3/Tuj-1: tubulin beta 3 class III; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Prions are infective agents composed of abnormally folded prion proteins (PrP^Sc), which are pathogenic isoforms of normal cellular prion proteins (PrP^C) that cause incurable, transmissible, neurodegenerative conditions in mammals called prion diseases. The spread of PrP^Sc within a host is facilitated by the lymphoreticular system, which uptakes and propagates PrP^Sc in the periphery and transmits them to the central nervous system. Our previous study showed that poly-l-arginine (PLR), a cationic amino acid polymer, inhibits PrP^Sc accumulation in neuroblastoma cells with persistent prion infection (ScN2a). Here, we report the beneficial effect of PLR against prions. In the in vitro prion infection experiment, PLR efficiently reduced the titer of prions inoculated to infect cultured N2a cells. In animal experiments, PLR inhibited the accumulation of PrP^Sc in the spleens of mice intraperitoneally inoculated with prions during asymptomatic periods. Prophylactic administration of PLR significantly prolonged incubation periods in mice intraperitoneally infected with prions, mitigating vacuolation and astrogliosis, although PrP^Sc level was not dramatically reduced in the brain. However, PrP^Sc level was reduced and the marginal zone distortion associated with prion infection was prevented in spleens of mice that was intraperitoneally infected with prions and received PLR, even at the terminal stage. Expression of follicular dendritic cell (FDC)-M1 antigens, a marker of FDC activation, and the level of PrP^Sc colonized within the white pulp of the spleens, as well as co-localization of FDC-M1 antigens and PrP^Sc, were reduced in these mice during the course of disease, suggesting that PLR counteracts the ability of FDCs that support PrP^Sc propagation in the spleen. Overall, prophylactically administered PLR suppresses prions in vivo, presumably through cellular control of pathological processes that occur in the spleen and eventually delay prion spread to the brain. This study presents implications for modulating the progress of prion diseases acquired peripherally.