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.

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.

Inflammatory bowel disease (IBD) is characterized by abnormal immune responses, including elevated proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) in the gastrointestinal (GI) tract. This study presents the synthesis and anti-inflammatory evaluation of 2,4,5-trimethylpyridin-3-ol analogues, which exhibit dual inhibition of TNFα- and IL-6-induced inflammation. Analysis using <i>in silico</i> methods, including 3D shape-based target identification, modeling, and docking, identified G protein-coupled estrogen receptor 1 (GPER) as the molecular target for the most effective analogue, <b>6</b>-<b>26</b>, which exhibits remarkable efficacy in ameliorating inflammation and restoring colonic mucosal integrity. This was further validated by surface plasmon resonance (SPR) assay results, which showed direct binding to GPER, and by the results showing that GPER knockdown abolished the inhibitory effects of <b>6</b>-<b>26</b> on TNFα and IL-6 actions. Notably, <b>6</b>-<b>26</b> displayed no cytotoxicity, unlike G1 and G15, a well-known GPER agonist and an antagonist, respectively, which induced necroptosis independently of GPER. These findings suggest that the GPER-selective compound <b>6</b>-<b>26</b> holds promise as a therapeutic candidate for IBD.

Cellular prion protein (PrP^C) is a glycoprotein tethered to the plasma membrane via a GPI-anchor, and it plays a crucial role in prion diseases by undergoing conformational change to PrP^Sc. To generate a knock-in (KI) mouse model expressing bank vole PrP^C (BVPrP^C), a KI targeting construct was designed. However, a Prnp gene sequence that encodes PrP^C lacking seven C-terminal amino acid residues of the GPI-anchoring signal sequence (GPI-SS) was unintentionally introduced into the construct. The resulting KIBVPrP248 mice exhibited very low PrP^C expression and resistance to prion infection. To investigate the underlying mechanism of reduced PrP^C expression, RK13 cells expressing either full-length GPI-SS (BVPrP255) or truncated GPI-SS (BVPrP248) and KIBVPrP248 mice were analyzed. In RK13-BVPrP248 cells, PrP^C protein levels were nearly ten-fold lower than in RK13-BVPrP255 cells, mimicking the extremely low PrP^C expression of the KIBVPrP248 mice. The abundance, stability, and translational efficiency of the Prnp mRNA were not the primary causes for the low PrP^C expression in RK13-BVPrP248 cells. A pharmacological analysis revealed that BVPrP248 underwent enhanced degradation via the ER-associated degradation pathway, with increased PrP ubiquitination detected in both the cell and animal models. An immunofluorescence analysis showed that BVPrP248 was mislocalized to the ER, co-localizing with Grp78, an ER chaperone. Although mislocalization of BVPrP248 under the transient overexpression condition led to mild activation of the unfolded protein response in RK13-BVPrP248 cells, low-level chronic expression of BVPrP248 in stable transfectants and KIBVPrP248 mice did not facilitate such events. These findings suggested that the C-terminal GPI-SS of PrP^C plays a critical role in PrP^C biogenesis.

Prion diseases, known as a group of fatal neurodegenerative disorders caused by prions, remain incurable despite extensive research efforts. In a recent study, crude extract from <i>Curcuma phaeocaulis</i> Valeton (<i>Cp</i>) showed promising anti-prion efficacy in in vitro and in vivo models, prompting further investigation into their active compounds. We endeavored to identify the chemical constituents of the <i>Cp</i> extract and discover potential anti-prion agents. With the use of centrifugal partition chromatography (CPC), major constituents were isolated from the <i>n</i>-hexane (HX) fraction of the extract in a single step. Spectroscopic analysis confirmed the presence of curcumenone, curcumenol, and furanodienone. Subsequent efficacy testing in a cell culture model of prion disease identified curcumenol and furanodienone as active compounds. This study underscores the potential of natural products in the search for effective treatments against prion diseases.

Cellular prion protein (PrPC) encoded at Prnp gene is well-known to form a misfolded isoform, termed scrapie PrP (PrPSC) that cause transmissible degenerative diseases in central nervous system. The physiological role of PrPC has been proposed by many studies, showing that PrPC interacts with various intracellular, membrane, and extracellular molecules including mitochondrial inner membrane as a scaffold. PrPC is expressed in most cell types including reproductive organs. Numerous studies using PrPC knockout rodent models found no obvious phenotypic changes, in particular the clear phenotypes in development and reproduction have not demonstrated in these knockout models. However, various roles of PrPC have been evaluated at the cellular levels. In this review, we summarized the known roles of PrPC in various cell types and tissues with a special emphasis on those involved in reproduction.

Prion protein (PrP) is highly conserved and is expressed in most tissues in a developmental stage-specific manner. Glycosylated cellular prion protein (PrP^C) is found in most cells and subcellular areas as a physiological regulating molecule. On the other hand, the amyloid form of PrP^C, scrapie PrP (PrP^SC), causes transmissible pathogenesis in the central nervous system and induces degeneration of the nervous system. Although many amyloids are reversible and critical in determining the fate, differentiation, and physiological functions of cells, thus far, PrP^SC originating from PrP^C is not. Although many studies have focused on disorders involving PrP^C and the deletion mammalian models for PrP^C have no severe phenotype, it has been suggested that PrP^C has a role in normal development. It is conserved and expressed from gametes to adult somatic cells. In addition, severe developmental phenotypes appear in PrP null zebrafish embryos and in various mammalian cell model systems. In addition, it has been well established that PrP^C is strongly involved in the stemness and differentiation of embryonic stem cells and progenitors. Thus far, many studies on PrP^C have focused mostly on disease-associated conditions with physiological roles as a complex platform but not on development. The known roles of PrP^C depend on the interacting molecules through its flexible tail and domains. PrP^C interacts with membrane, and various intracellular and extracellular molecules. In addition, PrP^C and amyloid can stimulate signaling pathways differentially. In this review, we summarize the function of prion protein and discuss its role in development.

Prions are infectious protein particles known to cause prion diseases. The biochemical entity of the pathogen is the misfolded prion protein (PrP^Sc) that forms insoluble amyloids to impair brain function. PrP^Sc interacts with the non-pathogenic, cellular prion protein (PrP^C) and facilitates conversion into a nascent misfolded isoform. Several small molecules have been reported to inhibit the aggregation of PrP^Sc but no pharmacological intervention was well established thus far. We, here, report that acylthiosemicarbazides inhibit the prion aggregation. Compounds <b>7x</b> and <b>7y</b> showed almost perfect inhibition (EC₅₀ = 5 µM) in prion aggregation formation assay. The activity was further confirmed by atomic force microscopy, semi-denaturing detergent agarose gel electrophoresis and real-time quaking induced conversion assay (EC₅₀ = 0.9 and 2.8 µM, respectively). These compounds also disaggregated pre-existing aggregates <i>in vitro</i> and one of them decreased the level of PrP^Sc in cultured cells with permanent prion infection, suggesting their potential as a treatment platform. In conclusion, hydroxy-2-naphthoylthiosemicarbazides can be an excellent scaffold for the discovery of anti-prion therapeutics.

Cell culture systems derived from the progenitor cells of human patients have many advantages over animal models for therapeutic drug testing and studies of disease pathogenesis. Here we describe a three-dimensional (3D) spheroid co-culture system of neurons and astrocytes derived from induced pluripotent stem cells-neural precursor cells (iPSCs-NPCs) of Alzheimer's disease (AD) patients or healthy individuals that can provide information on drug efficacy unobtainable by 2D co-culture or monoculture approaches. iPSCs-NPCs of healthy controls or AD patients were seeded onto 96-well U-bottom plates and incubated with neuronal differentiation medium for one week and with astrocytic medium for two weeks to replicate the temporal order of cell maturation during brain development. These 3D spheroid models expressed marker proteins for mature neurons and astrocytes. In particular, patient-derived spheroids showed beta-amyloid (Aβ) accumulation as revealed by thioflavin T (ThT) staining and ELISA. Aggregation of Aβ induced caspase activation and cell death, while the neuroprotectants nordihydroguaiaretic acid (NDGA) and curcumin (CU) reduced the levels of both ThT and caspase staining. Taken together, these results demonstrate the feasibility of our 3D spheroids combined with ThT and caspase staining as a patient-based anti-AD drug screening platform.