PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson's disease.

A hallmark of Parkinson's disease (PD) is the preferential loss of substantia nigra dopamine neurons. Here, we identify a new parkin interacting substrate, PARIS (ZNF746), whose levels are regulated by the ubiquitin proteasome system via binding to and ubiquitination by the E3 ubiquitin ligase, parkin. PARIS is a KRAB and zinc finger protein that accumulates in models of parkin inactivation and in human PD brain. PARIS represses the expression of the transcriptional coactivator, PGC-1α and the PGC-1α target gene, NRF-1 by binding to insulin response sequences in the PGC-1α promoter. Conditional knockout of parkin in adult animals leads to progressive loss of dopamine (DA) neurons in a PARIS-dependent manner. Moreover, overexpression of PARIS leads to the selective loss of DA neurons in the substantia nigra, and this is reversed by either parkin or PGC-1α coexpression. The identification of PARIS provides a molecular mechanism for neurodegeneration due to parkin inactivation.

PARIS undergoes liquid-liquid phase separation and poly(ADP-ribose)-mediated solidification.

ZNF746 was identified as parkin-interacting substrate (PARIS). Investigating its pathophysiological properties, we find that PARIS undergoes liquid-liquid phase separation (LLPS) and amorphous solid formation. The N-terminal low complexity domain 1 (LCD1) of PARIS is required for LLPS, whereas the C-terminal prion-like domain (PrLD) drives the transition from liquid to solid phase. In addition, we observe that poly(ADP-ribose) (PAR) strongly binds to the C-terminus of PARIS near the PrLD, accelerating its LLPS and solidification. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PAR formation leads to PARIS oligomerization in human iPSC-derived dopaminergic neurons that is prevented by the PARP inhibitor, ABT-888. Furthermore, SDS-resistant PARIS species are observed in the substantia nigra (SN) of aged mice overexpressing wild-type PARIS, but not with a PAR binding-deficient PARIS mutant. PARIS solidification is also found in the SN of mice injected with preformed fibrils of α-synuclein (α-syn PFF) and adult mice with a conditional knockout (KO) of parkin, but not if α-syn PFF is injected into mice deficient for PARP1. Herein, we demonstrate that PARIS undergoes LLPS and PAR-mediated solidification in models of Parkinson's disease.

Exploring the Pathological Effect of Aß42 Oligomers on Neural Networks in Primary Cortical Neuron Culture.

Alzheimer's disease (AD) is a multifactorial disorder that affects cognitive functioning, behavior, and neuronal properties. The neuronal dysfunction is primarily responsible for cognitive decline in AD patients, with many causal factors including plaque accumulation of Aß42. Neural hyperactivity induced by Aß42 deposition causes abnormalities in neural networks, leading to alterations in synaptic activity and interneuron dysfunction. Even though neuroimaging techniques elucidated the underlying mechanism of neural connectivity, precise understanding at the cellular level is still elusive. Previous multielectrode array studies have examined the neuronal network modulation in in vitro cultures revealing the relevance of ion channels and the chemical modulators in the presence of Aß42. In this study, we investigated neuronal connectivity and dynamic changes using a high-density multielectrode array, particularly looking at network-wide parameter changes over time. By comparing the neuronal network between normal and Aß42treated neuronal cultures, it was possible to discover the direct pathological effect of the Aß42 oligomer altering the network characteristics. The detrimental effects of the Aß42 oligomer included not only a decline in spike activation but also a qualitative impairment in neural connectivity as well as a disorientation of dispersibility. As a result, this will improve our understanding of how neural networks are modified during AD progression.

Identification of Three Human POLH Germline Variants Defective in Complementing the UV- and Cisplatin-Sensitivity of POLH-Deficient Cells.

DNA polymerase (pol) η is responsible for error-free translesion DNA synthesis (TLS) opposite ultraviolet light (UV)-induced cis-syn cyclobutane thymine dimers (CTDs) and cisplatin-induced intrastrand guanine crosslinks. POLH deficiency causes one form of the skin cancer-prone disease xeroderma pigmentosum variant (XPV) and cisplatin sensitivity, but the functional impacts of its germline variants remain unclear. We evaluated the functional properties of eight human POLH germline in silico-predicted deleterious missense variants, using biochemical and cell-based assays. In enzymatic assays, utilizing recombinant pol η (residues 1-432) proteins, the C34W, I147N, and R167Q variants showed 4- to 14-fold and 3- to 5-fold decreases in specificity constants (kcat/Km) for dATP insertion opposite the 3'-T and 5'-T of a CTD, respectively, compared to the wild-type, while the other variants displayed 2- to 4-fold increases. A CRISPR/Cas9-mediated POLH knockout increased the sensitivity of human embryonic kidney 293 cells to UV and cisplatin, which was fully reversed by ectopic expression of wild-type pol η, but not by that of an inactive (D115A/E116A) or either of two XPV-pathogenic (R93P and G263V) mutants. Ectopic expression of the C34W, I147N, and R167Q variants, unlike the other variants, did not rescue the UV- and cisplatin-sensitivity in POLH-knockout cells. Our results indicate that the C34W, I147N, and R167Q variants-substantially reduced in TLS activity-failed to rescue the UV- and cisplatin-sensitive phenotype of POLH-deficient cells, which also raises the possibility that such hypoactive germline POLH variants may increase the individual susceptibility to UV irradiation and cisplatin chemotherapy.

Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

Aberrant activation of the non-receptor kinase c-Abl is implicated in the development of pathogenic hallmarks of Parkinson's disease, such as α-synuclein aggregation and progressive neuronal loss. c-Abl-mediated phosphorylation and inhibition of parkin ligase function lead to accumulation of parkin interacting substrate (PARIS) that mediates α-synuclein pathology-initiated dopaminergic neurodegeneration. Here we show that, in addition to PARIS accumulation, c-Abl phosphorylation of PARIS is required for PARIS-induced cytotoxicity. c-Abl-mediated phosphorylation of PARIS at Y137 (within the Krüppel-associated box domain) drives its association with KAP1 and the repression of genes with diverse functions in pathways such as chromatin remodelling and p53-dependent cell death. One phosphorylation-dependent PARIS target, MDM4 (a p53 inhibitor that associates with MDM2; also known as MDMX), is transcriptionally repressed in a histone deacetylase-dependent manner via PARIS binding to insulin response sequence motifs within the MDM4 promoter. Virally induced PARIS transgenic mice develop c-Abl activity-dependent Parkinson's disease features such as motor deficits, dopaminergic neuron loss and neuroinflammation. PARIS expression in the midbrain resulted in c-Abl activation, PARIS phosphorylation, MDM4 repression and p53 activation, all of which are blocked by the c-Abl inhibitor nilotinib. Importantly, we also observed aberrant c-Abl activation and PARIS phosphorylation along with PARIS accumulation in the midbrain of adult parkin knockout mice, implicating c-Abl in recessive Parkinson's disease. Inhibition of c-Abl or PARIS phosphorylation by nilotinib or Y137F-PARIS expression in adult parkin knockout mice blocked MDM4 repression and p53 activation, preventing motor deficits and dopaminergic neurodegeneration. Finally, we found correlative increases in PARIS phosphorylation, MDM4 repression and p53 activation in post-mortem Parkinson's disease brains, pointing to clinical relevance of the c-Abl-PARIS-MDM4-p53 pathway. Taken together, our results describe a novel mechanism of epigenetic regulation of dopaminergic degeneration downstream of pathological c-Abl activation in Parkinson's disease. Since c-Abl activation has been shown in sporadic Parkinson's disease, PARIS phosphorylation might serve as both a useful biomarker and a potential therapeutic target to regulate neuronal loss in Parkinson's disease.

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.

Robust kinase- and age-dependent dopaminergic and norepinephrine neurodegeneration in LRRK2 G2019S transgenic mice.

Mutations in LRRK2 are known to be the most common genetic cause of sporadic and familial Parkinson's disease (PD). Multiple lines of LRRK2 transgenic or knockin mice have been developed, yet none exhibit substantial dopamine (DA)-neuron degeneration. Here we develop human tyrosine hydroxylase (TH) promoter-controlled tetracycline-sensitive LRRK2 G2019S (GS) and LRRK2 G2019S kinase-dead (GS/DA) transgenic mice and show that LRRK2 GS expression leads to an age- and kinase-dependent cell-autonomous neurodegeneration of DA and norepinephrine (NE) neurons. Accompanying the loss of DA neurons are DA-dependent behavioral deficits and α-synuclein pathology that are also LRRK2 GS kinase-dependent. Transmission EM reveals that that there is an LRRK2 GS kinase-dependent significant reduction in synaptic vesicle number and a greater abundance of clathrin-coated vesicles in DA neurons. These transgenic mice indicate that LRRK2-induced DA and NE neurodegeneration is kinase-dependent and can occur in a cell-autonomous manner. Moreover, these mice provide a substantial advance in animal model development for LRRK2-associated PD and an important platform to investigate molecular mechanisms for how DA neurons degenerate as a result of expression of mutant LRRK2.

Parkin interacting substrate zinc finger protein 746 is a pathological mediator in Parkinson's disease.

α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson's disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson's disease, there is evidence that parkin is inactivated in sporadic Parkinson's disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson's disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson's disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson's disease and related α-synucleinopathies.

Brain Endothelial P-Glycoprotein Level Is Reduced in Parkinson's Disease via a Vitamin D Receptor-Dependent Pathway.

The progressive neurodegeneration in Parkinson's disease (PD) is accompanied by neuroinflammation and endothelial vascular impairment. Although the vitamin D receptor (VDR) is expressed in both dopamine neurons and brain endothelial cells, its role in the regulation of endothelial biology has not been explored in the context of PD. In a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, we observed reduced transcription of the VDR and its downstream target genes, CYP24 and MDR1a. The 6-OHDA-induced transcriptional repression of these genes were recovered after the VDR ligand-1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment. Similarly, reduced vascular protein expression of P-glycoprotein (P-gp), encoded by MDR1a, after 6-OHDA administration was reversed by 1,25(OH)2D3. Moreover, marked reduction of endothelial P-gp expression with concomitant α-synuclein aggregation was found in a combinatorial AAV-αSyn/αSyn preformed fibril (PFF) injection mouse model and postmortem PD brains. Supporting the direct effect of α-synuclein aggregation on endothelial biology, PFF treatment of human umbilical vein endothelial cells (HUVECs) was sufficient to induce α-synuclein aggregation and repress transcription of the VDR. PFF-induced P-gp downregulation and impaired functional activity in HUVECs completely recovered after 1,25(OH)2D3 treatment. Taken together, our results suggest that a dysfunctional VDR-P-gp pathway could be a potential target for the maintenance of vascular homeostasis in PD pathological conditions.

Rhododendrin-Induced RNF146 Expression via Estrogen Receptor ß Activation is Cytoprotective Against 6-OHDA-Induced Oxidative Stress.

Ring finger protein 146 (RNF146) is an E3 ubiquitin ligase whose activity prevents poly (ADP-ribose) polymerase 1 (PARP1)-dependent neurodegeneration in Parkinson's disease (PD). Previously, we reported that rhododendrin is a chemical inducer that increases RNF146 expression. However, the molecular mechanism of rhododendrin-induced RNF146 expression is largely unknown and its translational application for the treatment of Parkinson's disease remains unexplored. Here we found that rhododendrin increased RNF146 expression via estrogen receptor ß (ERß) activation. Rhododendrin stimulated ERß nuclear translocation and binding to the RNF146 promoter, thereby enhancing its transcription. Rhododendrin is cytoprotective against 6-hydroxydopamine (6-OHDA)-induced cell death, which is largely dependent on ERß activity and RNF146 expression. Finally, we demonstrated that rhododendrin treatment resulted in RNF146 expression in dopaminergic neurons in mice. Moreover, dopaminergic neuron viability was markedly enhanced by pretreatment with rhododendrin in 6-OHDA-induced mouse models for PD. Our findings indicate that estrogen receptor activation plays a neuroprotective role and that rhododendrin could be a potential therapeutic agent in preventing PARP1-dependent dopaminergic cell loss in PD.

Therapeutic Evaluation of Synthetic Peucedanocoumarin III in an Animal Model of Parkinson's Disease.

The motor and nonmotor symptoms of Parkinson's disease (PD) correlate with the formation and propagation of aberrant α-synuclein aggregation. This protein accumulation is a pathological hallmark of the disease. Our group recently showed that peucedanocoumarin III (PCIII) possesses the ability to disaggregate ß sheet aggregate structures, including α-synuclein fibrils. This finding suggests that PCIII could be a therapeutic lead compound in PD treatment. However, the translational value of PCIII and its safety information have never been explored in relevant animal models of PD. Therefore, we first designed and validated a sequence of chemical reactions for the large scale organic synthesis of pure PCIII in a racemic mixture. The synthetic PCIII racemate facilitated clearance of repeated ß sheet aggregate (ß23), and prevented ß23-induced cell toxicity to a similar extent to that of purified PCIII. Given these properties, the synthetic PCIII's neuroprotective function was assessed in 6-hydroxydopamine (6-OHDA)-induced PD mouse models. The PCIII treatment (1 mg/kg/day) in a 6-OHDA-induced PD mouse model markedly suppressed Lewy-like inclusions and prevented dopaminergic neuron loss. To evaluate the safety profiles of PCIII, high dose PCIII (10 mg/kg/day) was administered intraperitoneally to two-month-old mice. Following 7 days of PCIII treatment, PCIII distributed to various tissues, with substantial penetration into brains. The mice that were treated with high dose PCIII had no structural abnormalities in the major organs or neuroinflammation. In addition, high dose PCIII (10 mg/kg/day) in mice had no adverse impact on motor function. These findings suggest that PCIII has a relatively high therapeutic index. Given the favorable safety features of PCIII and neuroprotective function in the PD mouse model, it may become a promising disease-modifying therapy in PD to regulate pathogenic α-synuclein aggregation.

PARIS reprograms glucose metabolism by HIF-1α induction in dopaminergic neurodegeneration.

Our previous study found that PARIS (ZNF746) transcriptionally suppressed transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) in the substantia nigra (SN) of AAV-PARIS injected mice. In this study, we revealed that PARIS overexpression reprogrammed glucose metabolic pathway, leading to the increment of glycolytic proteins along with TKT reduction in the SN of AAV-PARIS injected mice. Knock-down of TKT in differentiated SH-SY5Y cells led to an increase of glycolytic enzymes and decrease of PPP-related enzymes whereas overexpression of TKT restored PARIS-mediated glucose metabolic shift, suggesting that glucose metabolic alteration by PARIS is TKT-dependent. Inhibition of PPP by either PARIS overexpression or TKT knock-down elevated the level of H2O2, and diminished NADPH and GSH levels, ultimately triggering the induction of HIF-1α, a master activator of glycolysis. In addition, TKT inhibition by stereotaxic injection of oxythiamine demonstrated slight decrement of dopaminergic neurons (DNs) in SN but not cortical neurons in the cortex, suggesting that TKT might be a survival factor of DNs. In differentiated SH-SY5Y, cell toxicity by GFP-PARIS was partially restored by introduction of Flag-TKT and siRNA-HIF-1α. We also observed the increase of HIF-1α and glycolytic hexokinase 2 in the SN of Parkinson's disease patients. Taken together, these results suggest that PARIS accumulation might distort the balance of glucose metabolism, providing clues for understanding mechanism underlying selective DNs death by PARIS.

Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration.

Mutations in parkin lead to early-onset autosomal recessive Parkinson's disease (PD) and inactivation of parkin is thought to contribute to sporadic PD. Adult knockout of parkin in the ventral midbrain of mice leads to an age-dependent loss of dopamine neurons that is dependent on the accumulation of parkin interacting substrate (PARIS), zinc finger protein 746 (ZNF746), and its transcriptional repression of PGC-1α. Here we show that adult knockout of parkin in mouse ventral midbrain leads to decreases in mitochondrial size, number, and protein markers consistent with a defect in mitochondrial biogenesis. This decrease in mitochondrial mass is prevented by short hairpin RNA knockdown of PARIS. PARIS overexpression in mouse ventral midbrain leads to decreases in mitochondrial number and protein markers and PGC-1α-dependent deficits in mitochondrial respiration. Taken together, these results suggest that parkin loss impairs mitochondrial biogenesis, leading to declining function of the mitochondrial pool and cell death.

Identification of transketolase as a target of PARIS in substantia nigra.

Recently, PARIS (ZNF746) is introduced as authentic substrate of parkin and transcriptionally represses PGC-1α by binding to insulin responsive sequences (IRSs) in the promoter of PGC-1α. The overexpression of PARIS selectively leads to the loss of dopaminergic neurons (DN) and mitochondrial abnormalities in the substantia nigra (SN) of Parkinson's disease (PD) models. To identify PARIS target molecules altered in SN region-specific manner, LC-MS/MS-based quantitative proteomic analysis is employed to investigate proteomic alteration in the cortex, striatum, and SN of AAV-PARIS injected mice. Herein, we find that the protein and mRNA of transketolase (TKT), a key enzyme in pentose phosphate pathway (PPP) of glucose metabolism, is exclusively decreased in the SN of AAV-PARIS mice. PARIS overexpression suppresses TKT transcription via IRS-like motif in the TKT promoter. Moreover, the reduction of TKT by PARIS is found in primary DN but not in cortical neurons, suggesting that PARIS-medicated TKT suppression is cell type-dependent. Interestingly, we observe the reduced level of TKT in the SN of PD patients but not in the cortex. These findings indicate that TKT might be a SN-specific target of PARIS, providing new clues to understand the mechanism underlying selective DNs death in PD.

CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation.

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

Protein microarray characterization of the S-nitrosoproteome.

Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO's actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.

Iduna is a poly(ADP-ribose) (PAR)-dependent E3 ubiquitin ligase that regulates DNA damage.

Ubiquitin mediated protein degradation is crucial for regulation of cell signaling and protein quality control. Poly(ADP-ribose) (PAR) is a cell-signaling molecule that mediates changes in protein function through binding at PAR binding sites. Here we characterize the PAR binding protein, Iduna, and show that it is a PAR-dependent ubiquitin E3 ligase. Iduna's E3 ligase activity requires PAR binding because point mutations at Y156A and R157A eliminate Iduna's PAR binding and Iduna's E3 ligase activity. Iduna's E3 ligase activity also requires an intact really interesting new gene (RING) domain because Iduna possessing point mutations at either H54A or C60A is devoid of ubiquitination activity. Tandem affinity purification reveals that Iduna binds to a number of proteins that are either PARsylated or bind PAR including PAR polymerase-1, 2 (PARP1, 2), nucleolin, DNA ligase III, KU70, KU86, XRCC1, and histones. PAR binding to Iduna activates its E3 ligase function, and PAR binding is required for Iduna ubiquitination of PARP1, XRCC1, DNA ligase III, and KU70. Iduna's PAR-dependent ubiquitination of PARP1 targets it for proteasomal degradation. Via PAR binding and ubiquitin E3 ligase activity, Iduna protects against cell death induced by the DNA damaging agent N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and rescues cells from G1 arrest and promotes cell survival after γ-irradiation. Moreover, Iduna facilitates DNA repair by reducing apurinic/apyrimidinic (AP) sites after MNNG exposure and facilitates DNA repair following γ-irradiation as assessed by the comet assay. These results define Iduna as a PAR-dependent E3 ligase that regulates cell survival and DNA repair.

Metal-enhanced fluorescence biosensor integrated in capillary flow-driven microfluidic cartridge for highly sensitive immunoassays.

Point-of-care testing (POCT) for low-concentration protein biomarkers remains challenging due to limitations in biosensor sensitivity and platform integration. This study addresses this gap by presenting a novel approach that integrates a metal-enhanced fluorescence (MEF) biosensor within a capillary flow-driven microfluidic cartridge (CFMC) for the ultrasensitive detection of the Parkinson's disease biomarker, aminoacyl-tRNA synthetase complex interacting multi-functional protein 2 (AIMP-2). Crucial point to this approach is the orientation-controlled immobilization of capture antibody on a nanodimple-structured MEF substrate within the CFMC. This strategy significantly enhances fluorescence signals without quenching, enabling accurate quantification of low-concentration AIMP-2 using a simple digital fluorescence microscope with a light-emitting diode excitation source and a digital camera. The resulting platform exhibits exceptional sensitivity, achieving a limit of detection in the pg/mL range for AIMP-2 in human serum. Additionally, the CFMC design incorporates a capillary-driven passive sample transport mechanism, eliminating the need for external pumps and further simplifying the detection process. Overall, this work demonstrates the successful integration of MEF biosensing with capillary microfluidics for point-of-care applications.

Phosphorylation by the c-Abl protein tyrosine kinase inhibits parkin's ubiquitination and protective function.

Mutations in PARK2/Parkin, which encodes a ubiquitin E3 ligase, cause autosomal recessive Parkinson disease (PD). Here we show that the nonreceptor tyrosine kinase c-Abl phosphorylates tyrosine 143 of parkin, inhibiting parkin's ubiquitin E3 ligase activity and protective function. c-Abl is activated by dopaminergic stress and by dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP(+)) in vitro and in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), leading to parkin inactivation, accumulation of the parkin substrates aminoacyl-tRNA synthetase-interacting multifunctional protein type 2 (AIMP2) (p38/JTV-1) and fuse-binding protein 1 (FBP1), and cell death. STI-571, a c-Abl-family kinase inhibitor, prevents the phosphorylation of parkin, maintaining parkin in a catalytically active and protective state. STI-571's protective effects require parkin, as shRNA knockdown of parkin prevents STI-571 protection. Conditional knockout of c-Abl in the nervous system also prevents the phosphorylation of parkin, the accumulation of its substrates, and subsequent neurotoxicity in response to MPTP intoxication. In human postmortem PD brain, c-Abl is active, parkin is tyrosine-phosphorylated, and AIMP2 and FBP1 accumulate in the substantia nigra and striatum. Thus, tyrosine phosphorylation of parkin by c-Abl is a major posttranslational modification that inhibits parkin function, possibly contributing to pathogenesis of sporadic PD. Moreover, inhibition of c-Abl may be a neuroprotective approach in the treatment of PD.

ATF4-activated parkin induction contributes to deferasirox-mediated cytoprotection in Parkinson's disease.

The E3 ubiquitin ligase parkin plays neuroprotective functions in the brain and the deficits of parkin's ligase function in Parkinson's disease (PD) is associated with reduced survival of dopaminergic neurons. Thus, compounds enhancing parkin expression have been developed as potential neuroprotective agents that prevent ongoing neurodegeneration in PD environments. Besides, iron chelators have been shown to have neuroprotective effects in diverse neurological disorders including PD. Although repression of iron accumulation and oxidative stress in brains has been implicated in their marked neuroprotective potential, molecular mechanisms of iron chelator's neuroprotective function are largely unexplored. Here, we show that the iron chelator deferasirox provides cytoprotection against oxidative stress through enhancing parkin expression under basal conditions. Parkin expression is required for cytoprotection against oxidative stress in SH-SY5Y cells with deferasirox treatment as confirmed by abolished deferasirox's cytoprotective effect after parkin knockdown by shRNA. Similar to the previously reported parkin inducing compound diaminodiphenyl sulfone, deferasirox-mediated parkin expression was induced by activation of the PERK-ATF4 pathway, which is associated with and stimulated by mild endoplasmic reticulum stress. The translational potential of deferasirox for PD treatment was further evaluated in cultured mouse dopaminergic neurons. There was a robust ATF4 activation and parkin expression in response to deferasirox treatment in dopaminergic neurons under basal conditions. Consequently, the enhanced parkin expression by deferasirox provided substantial neuroprotection against 6-hydroxydopamine-induced oxidative stress. Taken together, our study results revealed a novel mechanism through which an iron chelator, deferasirox induces neuroprotection. Since parkin function in the brain is compromised in PD and during aging, maintenance of parkin expression through the iron chelator treatment could be beneficial by increasing dopaminergic neuronal survival.