Development of a fluorescent nanoprobe based on an amphiphilic single-benzene-based fluorophore for lipid droplet detection and its practical applications.

Lipid droplets (LDs) are crucial biological organelles connected with metabolic pathways in biological systems and diseases. To monitor the locations and accumulation of LDs in lipid-related diseases, the development of a visualization tool for LDs has gained importance. In particular, LD visualization using fluorescent probes has gained attention. Herein, a new fluorescent nanoprobe, BMeS-Ali, is developed that can sense LDs based on an amphiphilic single benzene-based fluorophore (SBBF). BMeS-Ali consists of hydrophilic (-NH2) and hydrophobic (-C12H25) moieties and exists as a micelle nanostructure in aqueous media. BMeS-Ali has a weak fluorescence, but its emission was dramatically enhanced upon exposure to the LD components such as oleic acids (OA) by reassembling its nano-formulation. BMeS-Ali showed a selective LD staining ability and great biocompatibility in cells (cancer cells and stem cells). It also showed a practical sensing ability towards biologically derived lipids and can be applied to the visualization of human fingerprints. We found that the nanoprobe BMeS-Ali has significant potential to serve as a practical dye and sensor for lipids, especially for LD imaging in the biomedical research area and broader industrial applications.

Synthetic Peucedanocoumarin IV Prevents α-Synuclein Neurotoxicity in an Animal Model of Parkinson's Disease.

Pathological protein inclusion formation and propagation are the main causes of neuronal dysfunction in diverse neurodegenerative diseases; therefore, current disease-modifying therapeutic strategies have targeted this disease protein aggregation process. Recently, we reported that peucedanocoumarin III (PCiii) is a promising therapeutic compound with the ability to disaggregate α-synuclein inclusion and protect dopaminergic neurons in Parkinson's disease (PD). Here, we found that trans-4'-acetyl-3'-tigloylkhellactone (racemic peucedanocoumarin IV [PCiv]), a structural isomer of PCiii with a higher synthetic yield presented a strong anti-aggregate activity to a degree comparable to that of PCiii. PCiv retained effective inhibitory function against ß-sheet aggregate-mimic ß23 cytotoxicities and potently prevented α-synucleinopathy in α-synuclein preformed fibril (PFF)-treated mice cortical neurons. In detailed pharmacokinetic profiling of PCiv, oral administration of PCiv in rats exhibited an approximately 97-min half-life and 10% bioavailability. Moreover, tissue distribution analysis revealed favorable profiles of brain penetration with a 6.4 brain-to-plasma concentration ratio. The therapeutic efficacy of PCiv was further evaluated in a sporadic PD mouse model with a combinatorial co-injection of α-synuclein preformed fibril and recombinant adeno-associated virus expressing α-synuclein. Motor dysfunctions induced in this combinatorial α-synucleinopathy PD mouse model was almost completely rescued by PCiv diet administration, and this therapeutic effect is consistent with the marked prevention of dopaminergic neuron loss and suppression of α-synuclein aggregation. Taken together, our translational study suggests that PCiv is advantageous as a therapeutic agent for neurodegenerative diseases, especially with its good synthetic yield, high brain distribution, and anti-aggregate activity. PCiv may be useful in the management of α-synuclein inclusion formation and propagation at different stages of PD.

PLEKHG3 enhances polarized cell migration by activating actin filaments at the cell front.

Cells migrate by directing Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) activities and by polymerizing actin toward the leading edge of the cell. Previous studies have proposed that this polarization process requires a local positive feedback in the leading edge involving Rac small GTPase and actin polymerization with PI3K likely playing a coordinating role. Here, we show that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) is a PI3K-regulated Rho guanine nucleotide exchange factor (RhoGEF) for Rac1 and Cdc42 that selectively binds to newly polymerized actin at the leading edge of migrating fibroblasts. Optogenetic inactivation of PLEKHG3 showed that PLEKHG3 is indispensable both for inducing and for maintaining cell polarity. By selectively binding to newly polymerized actin, PLEKHG3 promotes local Rac1/Cdc42 activation to induce more local actin polymerization, which in turn promotes the recruitment of more PLEKHG3 to induce and maintain cell front. Thus, autocatalytic reinforcement of PLEKHG3 localization to the leading edge of the cell provides a molecular basis for the proposed positive feedback loop that is required for cell polarization and directed migration.

UHRF2, a ubiquitin E3 ligase, acts as a small ubiquitin-like modifier E3 ligase for zinc finger protein 131.

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.

ASCL1-mediated direct reprogramming: converting ventral midbrain astrocytes into dopaminergic neurons for Parkinson's disease therapy.

Parkinson's disease (PD), characterized by dopaminergic neuron degeneration in the substantia nigra, is caused by various genetic and environmental factors. Current treatment methods are medication and surgery; however, a primary therapy has not yet been proposed. In this study, we aimed to develop a new treatment for PD that induces direct reprogramming of dopaminergic neurons (iDAN). Achaete-scute family bHLH transcription factor 1 (ASCL1) is a primary factor that initiates and regulates central nervous system development and induces neurogenesis. In addition, it interacts with BRN2 and MYT1L, which are crucial transcription factors for the direct conversion of fibroblasts into neurons. Overexpression of ASCL1 along with the transcription factors NURR1 and LMX1A can directly reprogram iDANs. Using a retrovirus, GFP-tagged ASCL1 was overexpressed in astrocytes. One week of culture in iDAN convertsion medium reprogrammed the astrocytes into iDANs. After 7 days of differentiation, TH+/TUJ1+ cells emerged. After 2 weeks, the number of mature TH+/TUJ1+ dopaminergic neurons increased. Only ventral midbrain (VM) astrocytes exhibited these results, not cortical astrocytes. Thus, VM astrocytes can undergo direct iDAN reprogramming with ASCL1 alone, in the absence of transcription factors that stimulate dopaminergic neuron development.

Small ubiquitin-like modifier (SUMO) modification of zinc finger protein 131 potentiates its negative effect on estrogen signaling.

Like ubiquitin, small ubiquitin-like modifier (SUMO) covalently attaches to specific target proteins and modulates their functional properties, including subcellular localization, protein dimerization, DNA binding, and transactivation of transcription factors. Diverse transcriptional co-regulator complexes regulate the ability of estrogen receptors to respond to positive and negative acting hormones. Zinc finger protein 131 (ZNF131) is poorly characterized but may act as a repressor of estrogen receptor α (ERα)-mediated trans-activation. Here, we identify ZNF131 as a target for SUMO modification and as a substrate for the SUMO E3 ligase human polycomb protein 2 (hPc2). We report that the SUMO-interacting motif 1 (SIM1) and the C-box of hPc2 are critical regions required for ZNF131 SUMOylation and define the ZNF131 SUMOylation site as lysine 567. We further show that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling and consequently attenuates estrogen-induced cell growth in a breast cancer cell line. Our findings suggest that SUMOylation is a novel regulator of ZNF131 action in estrogen signaling and breast cancer cell proliferation.

Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor α homo-dimerization.

Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ERα and ERß. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ERα and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ERα inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ERα, which consequently inhibits ERα-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ERα-co-repressor.

Challenges involved in cell therapy for Parkinson's disease using human pluripotent stem cells.

Neurons derived from human pluripotent stem cells (hPSCs) provide a valuable tool for studying human neural development and neurodegenerative diseases. The investigation of hPSC-based cell therapy, involving the differentiation of hPSCs into target cells and their transplantation into affected regions, is of particular interest. One neurodegenerative disease that is being extensively studied for hPSC-based cell therapy is Parkinson's disease (PD), the second most common among humans. Various research groups are focused on differentiating hPSCs into ventral midbrain dopaminergic (vmDA) progenitors, which have the potential to further differentiate into neurons closely resembling DA neurons found in the substantia nigra pars compacta (SNpc) after transplantation, providing a promising treatment option for PD. In vivo experiments, where hPSC-derived vmDA progenitor cells were transplanted into the striatum or SNpc of animal PD models, the transplanted cells demonstrated stable engraftment and resulted in behavioral recovery in the transplanted animals. Several differentiation protocols have been developed for this specific cell therapy. However, the lack of a reliable live-cell lineage identification method presents a significant obstacle in confirming the precise lineage of the differentiated cells intended for transplantation, as well as identifying potential contamination by non-vmDA progenitors. This deficiency increases the risk of adverse effects such as dyskinesias and tumorigenicity, highlighting the importance of addressing this issue before proceeding with transplantation. Ensuring the differentiation of hPSCs into the target cell lineage is a crucial step to guarantee precise therapeutic effects in cell therapy. To underscore the significance of lineage identification, this review focuses on the differentiation protocols of hPSC-derived vmDA progenitors developed by various research groups for PD treatment. Moreover, in vivo experimental results following transplantation were carefully analyzed. The encouraging outcomes from these experiments demonstrate the potential efficacy and safety of hPSC-derived vmDA progenitors for PD cell therapy. Additionally, the results of clinical trials involving the use of hPSC-derived vmDA progenitors for PD treatment were briefly reviewed, shedding light on the progress and challenges faced in translating this promising therapy into clinical practice.

Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation.

Human induced pluripotent stem cells (hiPSCs) offer advantages for disease modeling and drug discovery. However, recreating innate cellular pathologies, particularly in late-onset neurodegenerative diseases with accumulated protein aggregates including Parkinson's disease (PD), has been challenging. To overcome this barrier, we developed an optogenetics-assisted α-synuclein (α-syn) aggregation induction system (OASIS) that rapidly induces α-syn aggregates and toxicity in PD hiPSC-midbrain dopaminergic neurons and midbrain organoids. Our OASIS-based primary compound screening with SH-SY5Y cells identified 5 candidates that were secondarily validated with OASIS PD hiPSC-midbrain dopaminergic neurons and midbrain organoids, leading us to finally select BAG956. Furthermore, BAG956 significantly reverses characteristic PD phenotypes in α-syn preformed fibril models in vitro and in vivo by promoting autophagic clearance of pathological α-syn aggregates. Following the FDA Modernization Act 2.0's emphasis on alternative non-animal testing methods, our OASIS can serve as an animal-free preclinical test model (newly termed "nonclinical test") for the synucleinopathy drug development.

Protein kinase A phosphorylates Down syndrome critical region 1 (RCAN1).

The Down syndrome critical region 1 (DSCR1) gene encodes a regulator of the calcineurin 1 (RCAN1) protein, and the elevated levels of RCAN1 are associated with Alzheimer's disease (AD) and Down syndrome (DS). In this report, we found that protein kinase A (PKA) was able to phosphorylate RCAN1 in vitro and in vivo. In addition, we found that the phosphorylation of RCAN1 by PKA caused an increase of RCAN1 expression by increasing of the half-life of the protein. Consistently, the pharmacological inhibition of intracellular PKA using H-89 and the knockdown of the endogenous PKA catalytic subunit with siRNA decreased the expression of RCAN1. Furthermore, the phosphorylation of RCAN1 by PKA enhanced the inhibitory function of RCAN1 on calcineurin-mediated gene transcription. Our data provide the first evidence that PKA acts as an important regulatory component in the control of RCAN1 function through phosphorylation.

Neddylation positively regulates the ubiquitin E3 ligase activity of parkin.

Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP.

Comparison between Wound Closure Methods in the Reversal of Diverting Ileostomy.

Background/Aims: The objective of this study was to determine the more appropriate wound-closure method by comparing the effectiveness of two methods in a group of patients who underwent ileostomy repair. Methods: The study conducted after obtaining the approval of the Institutional Review Board (IRB) included 58 patients ≥19 years of age who underwent ileostomy at the Department of Surgery at the Presbyterian Medical Center. This was a retrospective, single-center trial. Patients who underwent ileostomy closure between January 2011 and September 2017 were assigned to the primary wound-closure (PC, n=25) group and the purse-string wound-closure (PSC, n=33) group. Post-repair complications, such as wound infection, delayed healing, and patient satisfaction related to wound management, were investigated and compared according to the wound-closure method. Results: The PSC group had a significantly lower surgical site infection rate than the PC group (0% vs. 44%, p<0.001). The wound-healing period was also significantly different between the PC and PSC groups (mean 27.18 days vs. 20.96 days, p=0.023). However, the postoperative wound-healing delay of >30 days was not significantly different (39% vs. 20%, p=0.114). In addition, there were no significant differences in the response to questionnaires on patient satisfaction between the two groups. Conclusions: PSC has a lower surgical site infection rate and the wound-healing delay was not very different from that of PC. Therefore, if patients are at risk of wound infection, such as in severe wound contamination, long operating time, and immunocompromised conditions, we should consider PSC as a wound closure method of choice.

Role of post-translational modifications on the alpha-synuclein aggregation-related pathogenesis of Parkinson's disease.

Together with neuronal loss, the existence of insoluble inclusions of alpha-synuclein (α-syn) in the brain is widely accepted as a hallmark of synucleinopathies including Parkinson's disease (PD), multiple system atrophy, and dementia with Lewy body. Because the α-syn aggregates are deeply involved in the pathogenesis, there have been many attempts to demonstrate the mechanism of the aggregation and its potential causative factors including post-translational modifications (PTMs). Although no concrete conclusions have been made based on the previous study results, growing evidence suggests that modifications such as phosphorylation and ubiquitination can alter α-syn characteristics to have certain effects on the aggregation process in PD; either facilitating or inhibiting fibrillization. In the present work, we reviewed studies showing the significant impacts of PTMs on α-syn aggregation. Furthermore, the PTMs modulating α-syn aggregation-induced cell death have been discussed. [BMB Reports 2022; 55(7): 323-335].

ASK1 negatively regulates the 26 S proteasome.

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.

Dyrk1A phosphorylates p53 and inhibits proliferation of embryonic neuronal cells.

Down syndrome (DS) is associated with many neural defects, including reduced brain size and impaired neuronal proliferation, highly contributing to the mental retardation. Those typical characteristics of DS are closely associated with a specific gene group "Down syndrome critical region" (DSCR) on human chromosome 21. Here we investigated the molecular mechanisms underlying impaired neuronal proliferation in DS and, more specifically, a regulatory role for dual-specificity tyrosine-(Y) phosphorylation-regulated kinase 1A (Dyrk1A), a DSCR gene product, in embryonic neuronal cell proliferation. We found that Dyrk1A phosphorylates p53 at Ser-15 in vitro and in immortalized rat embryonic hippocampal progenitor H19-7 cells. In addition, Dyrk1A-induced p53 phosphorylation at Ser-15 led to a robust induction of p53 target genes (e.g. p21(CIP1)) and impaired G(1)/G(0)-S phase transition, resulting in attenuated proliferation of H19-7 cells and human embryonic stem cell-derived neural precursor cells. Moreover, the point mutation of p53-Ser-15 to alanine rescued the inhibitory effect of Dyrk1A on neuronal proliferation. Accordingly, brains from embryonic DYRK1A transgenic mice exhibited elevated levels of Dyrk1A, Ser-15 (mouse Ser-18)-phosphorylated p53, and p21(CIP1) as well as impaired neuronal proliferation. These findings suggest that up-regulation of Dyrk1A contributes to altered neuronal proliferation in DS through specific phosphorylation of p53 at Ser-15 and subsequent p21(CIP1) induction.

AIEgen-based nanoprobe for the ATP sensing and imaging in cancer cells and embryonic stem cells.

A turn-on fluorescent nanoprobe (named AAP-1), based on an aggregation-induced emission luminogen (AIEgen), is disclosed for the detection of adenosine triphosphate (ATP), which is an essential element in the biological system. Organic fluorophore (named TPE-TA) consists of tetraphenylethylene (TPE, sensing and signaling moiety) and mono-triamine (TA, sensing moiety), and it forms an aggregated form in aqueous media as a nanoprobe AAP-1. The nanoprobe AAP-1 has multiple electrostatic interactions as well as hydrophobic interactions with ATP, and it displays superior selectivity toward ATP, reliable sensitivity, with a detection limit around 0.275 ppb, and fast responsive (signal within 10 s). Such a fluorescent probe to monitor ATP has been actively pursued throughout fundamental and translational research areas. In vitro assay and a successful cellular ATP imaging application was demonstrated in cancer cells and embryonic stem cells. We expect that our work warrants further ATP-related studies throughout a variety of fields.

Novel culture system via wirelessly controllable optical stimulation of the FGF signaling pathway for human and pig pluripotency.

Stem cell fate is largely determined by cellular signaling networks and is heavily dependent on the supplementation of exogenous recombinant proteins into culture media; however, uneven distribution and inconsistent stability of recombinant proteins are closely associated with the spontaneous differentiation of pluripotent stem cells (PSCs) and result in significant costs in large-scale manufacturing. Here, we report a novel PSC culture system via wirelessly controllable optical activation of the fibroblast growth factor (FGF) signaling pathway without the need for supplementation of recombinant FGF2 protein, a key molecule for maintaining pluripotency of PSCs. Using a fusion protein between the cytoplasmic region of the FGF receptor-1 and a light-oxygen-voltage domain, we achieved tunable, blue light-dependent activation of FGF signaling in human and porcine PSCs. Our data demonstrate that a highly controllable optical stimulation of the FGF signaling pathway is sufficient for long-term maintenance of PSCs, without the loss of differentiation potential into three germ layers. This culture system will be a cost-effective platform for a large-scale stem cell culture.

Multi-omic analysis of selectively vulnerable motor neuron subtypes implicates altered lipid metabolism in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating disorder in which motor neurons degenerate, the causes of which remain unclear. In particular, the basis for selective vulnerability of spinal motor neurons (sMNs) and resistance of ocular motor neurons to degeneration in ALS has yet to be elucidated. Here, we applied comparative multi-omics analysis of human induced pluripotent stem cell-derived sMNs and ocular motor neurons to identify shared metabolic perturbations in inherited and sporadic ALS sMNs, revealing dysregulation in lipid metabolism and its related genes. Targeted metabolomics studies confirmed such findings in sMNs of 17 ALS (SOD1, C9ORF72, TDP43 (TARDBP) and sporadic) human induced pluripotent stem cell lines, identifying elevated levels of arachidonic acid. Pharmacological reduction of arachidonic acid levels was sufficient to reverse ALS-related phenotypes in both human sMNs and in vivo in Drosophila and SOD1G93A mouse models. Collectively, these findings pinpoint a catalytic step of lipid metabolism as a potential therapeutic target for ALS.

Articulated Structures of D-A Type Dipolar Dye with AIEgen: Synthesis, Photophysical Properties, and Applications.

Articulated structures of naphthalene-based donor (D)-acceptor (A) type dipolar dye and aggregation-induced emission luminogen (AIEgen) based on tetraphenylethylene (TPE) were synthesized, and their photophysical properties were analyzed for the first time. There are many fluorophore backbones, which have dipolar structure and AIEgen. However, there has been neither property analysis nor research that closely articulates DA and AIE through non-conjugation linker. We have therefore prepared two representative fluorophores; DA-AIE series (DA-AIE-M and DA-AIE-D), and characterized their UV/vis absorption and emission properties with quantum chemical calculations. In addition, we utilized the unique photophysical properties of DA-AIE-D for monitoring a trace of dimethyl sulfoxide (DMSO) in aqueous media, including real water samples.

Transcriptional landscape of myogenesis from human pluripotent stem cells reveals a key role of TWIST1 in maintenance of skeletal muscle progenitors.

Generation of skeletal muscle cells with human pluripotent stem cells (hPSCs) opens new avenues for deciphering essential, but poorly understood aspects of transcriptional regulation in human myogenic specification. In this study, we characterized the transcriptional landscape of distinct human myogenic stages, including OCT4::EGFP+ pluripotent stem cells, MSGN1::EGFP+ presomite cells, PAX7::EGFP+ skeletal muscle progenitor cells, MYOG::EGFP+ myoblasts, and multinucleated myotubes. We defined signature gene expression profiles from each isolated cell population with unbiased clustering analysis, which provided unique insights into the transcriptional dynamics of human myogenesis from undifferentiated hPSCs to fully differentiated myotubes. Using a knock-out strategy, we identified TWIST1 as a critical factor in maintenance of human PAX7::EGFP+ putative skeletal muscle progenitor cells. Our data revealed a new role of TWIST1 in human skeletal muscle progenitors, and we have established a foundation to identify transcriptional regulations of human myogenic ontogeny (online database can be accessed in http://www.myogenesis.net/).