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.

PARIS farnesylation prevents neurodegeneration in models of Parkinson's disease.

Accumulation of the parkin-interacting substrate (PARIS; ZNF746), due to inactivation of parkin, contributes to Parkinson's disease (PD) through repression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; PPARGC1A) activity. Here, we identify farnesol as an inhibitor of PARIS. Farnesol promoted the farnesylation of PARIS, preventing its repression of PGC-1α via decreasing PARIS occupancy on the PPARGC1A promoter. Farnesol prevented dopaminergic neuronal loss and behavioral deficits via farnesylation of PARIS in PARIS transgenic mice, ventral midbrain transduction of AAV-PARIS, adult conditional parkin KO mice, and the α-synuclein preformed fibril model of sporadic PD. PARIS farnesylation is decreased in the substantia nigra of patients with PD, suggesting that reduced farnesylation of PARIS may play a role in PD. Thus, farnesol may be beneficial in the treatment of PD by enhancing the farnesylation of PARIS and restoring PGC-1α activity.

α-Synuclein A53T Binds to Transcriptional Adapter 2-Alpha and Blocks Histone H3 Acetylation.

α-Synuclein (α-syn) is a hallmark amyloidogenic protein component of Lewy bodies in dopaminergic neurons affected by Parkinson's disease (PD). Despite the multi-faceted gene regulation of α-syn in the nucleus, the mechanism underlying α-syn crosstalk in chromatin remodeling in PD pathogenesis remains elusive. Here, we identified transcriptional adapter 2-alpha (TADA2a) as a novel binding partner of α-syn using the BioID system. TADA2a is a component of the p300/CBP-associated factor and is related to histone H3/H4 acetylation. We found that α-syn A53T was more preferentially localized in the nucleus than the α-syn wild-type (WT), leading to a stronger disturbance of TADA2a. Indeed, α-syn A53T significantly reduced the level of histone H3 acetylation in SH-SY5Y cells; its reduction was also evident in the striatum (STR) and substantia nigra (SN) of mice that were stereotaxically injected with α-syn preformed fibrils (PFFs). Interestingly, α-syn PFF injection resulted in a decrease in TADA2a in the STR and SN of α-syn PFF-injected mice. Furthermore, the levels of TADA2a and acetylated histone H3 were significantly decreased in the SN of patients with PD. Therefore, histone modification through α-syn A53T-TADA2a interaction may be associated with α-syn-mediated neurotoxicity in PD pathology.

Antioxidant and anti-aging potential of a peptide formulation (Gal2-Pep) conjugated with gallic acid.

Skin is highly vulnerable to premature aging due to external stress, therefore, in this study, a peptide formulation, (galloyl)2-KTPPTTP (Gal2-Pep) was synthesized by combining TPPTTP peptide, and gallic acid (GA). All peptides were synthesized on 2-chlorotrityl chloride resin using solid-phase peptide synthesis (SPPS), and analyzed on an electrospray ionization (ESI)/quadrupole-time-of-flight (Q-TOF) tandem mass spectroscopy (MS) system. Initially, Gal2-Pep showed no toxicity below the concentration 100 µM with cell survival rate of 88% for keratinocytes and fibroblasts. The reactive oxygen species (ROS) scavenging activity of Gal2-Pep was more stable compared to GA alone; and after four weeks at room temperature, its ROS scavenging activity remained higher than 50%. Moreover, the peptide formulation, Gal2-Pep also exhibited elastase inhibitory effect in CCD-1064Sk fibroblast cells. Based on the results of RT-qPCR, it was proved in this study that Gal2-Pep increased the expression of PGC-1α to prevent oxidative stress, and validated its potential as an anti-aging agent through increasing the expression of type I collagen and by decreasing the expression of matrix metalloproteinase-1 (MMP1). The findings obtained reinforce the suggestion that the peptide formulation synthesized in this study could be used as a natural antioxidant and anti-aging agent for its cosmetic applications.

Deubiquitinase USP29 Governs MYBBP1A in the Brains of Parkinson's Disease Patients.

The inactivation of parkin by mutation or post-translational modification contributes to dopaminergic neuronal death in Parkinson's disease (PD). The substrates of parkin, FBP1 and AIMP2, are accumulated in the postmortem brains of PD patients, and it was recently suggested that these parkin substrates transcriptionally activate deubiquitinase USP29. Herein, we newly identified 160 kDa myb-binding protein (MYBBP1A) as a novel substrate of USP29. Knockdown of parkin increased the level of AIMP2, leading to ultimately USP29 and MYBBP1A accumulation in SH-SY5Y cells. Notably, MYBBP1A was downregulated in the ventral midbrain (VM) of Aimp2 knockdown mice, whereas the upregulation of MYBBP1A was observed in the VM of inducible AIMP2 transgenic mice, as well as in the substantia nigra of sporadic PD patients. These results suggest that AIMP2 upregulates USP29 and MYBBP1A in the absence of parkin activity, contributing to PD pathogenesis.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. METHODS: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. RESULTS: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. CONCLUSIONS: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

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.

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.

CKAP2 phosphorylation by CDK1/cyclinB1 is crucial for maintaining centrosome integrity.

Previously, we have reported that CKAP2 is involved in the maintenance of centrosome integrity, thus allowing for proper mitosis in primary hepatocytes. To understand this biological process, we identified the mitosis-specific phosphorylation sites in mouse CKAP2 and investigated CKAP's possible role in cell cycle progression. Because we observed mouse CKAP2 depletion in amplified centrosomes and aberrant chromosomal segregation, which was rescued by ectopic expression of wild-type CKAP2, we focused on the centrosome duplication process among the various aspects of the cell cycle. Among the identified phosphorylation sites, T603 and possibly S608 were phosphorylated by CDK1-cyclin B1 during mitosis, and the ectopic expression of both T603A and S608A mutants was unable to restore the centrosomal abnormalities in CKAP2-depleted cells. These results indicated that the phosphorylation status of CKAP2 during mitosis is critical for controlling both centrosome biogenesis and bipolar spindle formation.

CKAP2 is necessary to ensure the faithful spindle bipolarity in a dividing diploid hepatocyte.

Spindle bipolarity is crucial for segregating chromosome during somatic cell division. Previous studies have suggested that cytoskeleton associated protein 2 (CKAP2) is involved in spindle assembly and chromosome segregation. In this study, we show that CKAP2-depleted primary hepatocytes exhibit over-duplicated centrosomes with disjoined chromosomes from metaphase plate. These cells proceed to apoptosis or multipolar cell division and subsequent apoptotic cell death. In addition, a mouse liver regeneration experiment showed a marked decrease in efficiency of hepatic regeneration in CKAP2-depleted liver. These data suggest a physiological role of CKAP2 in the formation of spindle bipolarity, which is necessary for maintaining chromosomal stability.