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.

Cerebellar dysfunction and schizophrenia-like behavior in Ebp1-deficient mice.

Cerebellar deficits with Purkinje cell (PCs) loss are observed in several neurologic disorders. However, the underlying mechanisms as to how the cerebellum is affected during development remain unclear. Here we demonstrated that specific inactivation of murine Ebp1 in the central nervous system causes a profound neuropathology characterized by reduced cerebellar volume and PCs loss with abnormal dendritic development, leading to phenotypes including motor defects and schizophrenia (SZ)-like behaviors. Loss of Ebp1 leads to untimely gene expression of Fbxw7, an E3 ubiquitin ligase, resulting in aberrant protein degradation of PTF1A, thereby eliciting cerebellar defects. Reinstatement of Ebp1, but not the Ebp1-E183Ter mutant found in SZ patients, reconstituted cerebellar architecture with increased PCs numbers and improved behavioral phenotypes. Thus, our findings indicate a crucial role for EBP1 in cerebellar development, and define a molecular basis for the cerebellar contribution to neurologic disorders such as SZ.

Roles of ErbB3-binding protein 1 (EBP1) in embryonic development and gene-silencing control.

ErbB3-binding protein 1 (EBP1) is implicated in diverse cellular functions, including apoptosis, cell proliferation, and differentiation. Here, by generating genetic inactivation of Ebp1 mice, we identified the physiological roles of EBP1 in vivo. Loss of Ebp1 in mice caused aberrant organogenesis, including brain malformation, and death between E13.5 and 15.5 owing to severe hemorrhages, with massive apoptosis and cessation of cell proliferation. Specific ablation of Ebp1 in neurons caused structural abnormalities of brain with neuron loss in [Nestin-Cre; Ebp1flox/flox ] mice. Notably, global methylation increased with high levels of the gene-silencing unit Suv39H1/DNMT1 in Ebp1-deficient mice. EBP1 repressed the transcription of Dnmt1 by binding to its promoter region and interrupted DNMT1-mediated methylation at its target gene, Survivin promoter region. Reinstatement of EBP1 into embryo brain relived gene repression and rescued neuron death. Our findings uncover an essential role for EBP1 in embryonic development and implicate its function in transcriptional regulation.

SIAH1 ubiquitin ligase mediates ubiquitination and degradation of Akt3 in neural development.

Akt signaling is an important regulator of neural development, but the distinctive function of Akt isoforms in brain development presents a challenge. Here we show Siah1 as an ubiquitin ligase that preferentially interacts with Akt3 and facilitates ubiquitination and degradation of Akt3. Akt3 is enriched in the axonal shaft and branches but not growth cone tips, where Siah1 is prominently present. Depletion of Siah1 enhanced Akt3 levels in the soma and axonal tips, eliciting multiple branching. Brain-specific somatic mutation in Akt3-E17K escapes from Siah1-mediated degradation and causes improper neural development with dysmorphic neurons. Remarkably, coexpression of Siah1 with Akt3-WT restricted disorganization of neural development is caused by Akt3 overexpression, whereas forced expression of Siah1 with the Akt3-E17K mutant fails to cope with malformation of neural development. These findings demonstrate that Siah1 limits Akt3 turnover during brain development and that this event is essential for normal organization of the neural network.

Dysregulation of Epigenetic Control Contributes to Schizophrenia-Like Behavior in Ebp1+/- Mice.

Dysregulation of epigenetic machinery can cause a variety of neurological disorders associated with cognitive abnormalities. In the hippocampus of postmortem Schizophrenia (SZ) patients, the most notable finding is the deregulation of GAD67 along with differential regulation of epigenetic factors associated with glutamate decarboxylase 67 (GAD67) expression. As we previously reported, ErbB3-binding protein 1 (EBP1) is a potent epigenetic regulator. EBP1 can induce repression of Dnmt1, a well-studied transcriptional repressor of GAD67. In this study, we investigated whether EBP1 contributes to the regulation of GAD67 expression in the hippocampus, controlling epigenetic machinery. In accordance with SZ-like behaviors in Ebp1(+/-) mice, heterozygous deletion of EBP1 led to a dramatic reduction of GAD67 expression, reflecting an abnormally high level of Dnmt1. Moreover, we found that EBP1 binds to the promoter region of HDAC1, which leads to histone deacetylation of GAD67, and suppresses histone deacetylase 1 (HDAC1) expression, inversely mirroring an unusually high level of HDAC1 in Ebp1(+/-) mice. However, EBP1 mutant (p.Glu 183 Ter) found in SZ patients did not elevate the expression of GAD67, failing to suppress Dnmt1 and/or HDAC1 expression. Therefore, this data supports the hypothesis that a reduced amount of EBP1 may contribute to an etiology of SZ due to a loss of transcriptional inhibition of epigenetic repressors, leading to a decreased expression of GAD67.

B23/Nucleophosmin promotes reconstitution of synaptic path in hippocampus after injury.

B23, also known as nucleophosmin (NPM), is multifunctional protein directly implicated in cell proliferation, cell cycle progression, and cell survival. In the current study, in addition to confirming its anti-apoptotic function in neuronal survival, we demonstrated that the spatial-temporal expression profile of B23 during development of hippocampal neurons is high in the embryonic stage, down-regulated after birth, and preferentially localized at the tips of growing neuritis and branching points. Overexpression of B23 promotes axon growth with abundant branching points in growing hippocampal neurons, but depletion of B23 impairs axon growth, leading to neuronal death. Following injury to the trisynaptic path in hippocampal slice, overexpression of B23 remarkably increased the number and length of regenerative fibers in the mossy fiber path. Our study suggests that B23 expression in developing neurons is essential for neuritogenesis and axon growth and that up-regulation of B23 may be a strategy for enhancing the reconstitution of synaptic paths after injury to hippocampal synapses.

Strategies to enhance paracrine potency of transplanted mesenchymal stem cells in intractable neonatal disorders.

Mesenchymal stem cell (MSC) transplantation represents the next breakthrough in the treatment of currently intractable and devastating neonatal disorders with complex multifactorial etiologies, including bronchopulmonary dysplasia, hypoxic ischemic encephalopathy, and intraventricular hemorrhage. Absent engraftment and direct differentiation of transplanted MSCs, and the "hit-and-run" therapeutic effects of these MSCs suggest that their pleiotropic protection might be attributable to paracrine activity via the secretion of various biologic factors rather than to regenerative activity. The transplanted MSCs, therefore, exert their therapeutic effects not by acting as "stem cells," but rather by acting as "paracrine factors factory." The MSCs sense the microenvironment of the injury site and secrete various paracrine factors that serve several reparative functions, including antiapoptotic, anti-inflammatory, antioxidative, antifibrotic, and/or antibacterial effects in response to environmental cues to enhance regeneration of the damaged tissue. Therefore, the therapeutic efficacy of MSCs might be dependent on their paracrine potency. In this review, we focus on recent investigations that elucidate the specifically regulated paracrine mechanisms of MSCs by injury type and discuss potential strategies to enhance paracrine potency, and thus therapeutic efficacy, of transplanted MSCs, including determining the appropriate source and preconditioning strategy for MSCs and the route and timing of their administration.

Chaperone-E3 Ligase Complex HSP70-CHIP Mediates Ubiquitination of Ribosomal Protein S3.

In addition to its role in ribosome biogenesis, ribosomal protein S3 (RPS3), a component of the 40S ribosomal subunit, has been suggested to possess several extraribosomal functions, including an apoptotic function. In this study, we demonstrated that in the mouse brain, the protein levels of RPS3 were altered by the degree of nutritional starvation and correlated with neuronal apoptosis. After endurable short-term starvation, the apoptotic function of RPS3 was suppressed by Akt activation and Akt-mediated T70 phosphorylation, whereas after prolonged starvation, the protein levels of RPS3 notably increased, and abundant neuronal death occurred. These events coincided with ubiquitination and subsequent degradation of RPS3, controlled by HSP70 and the cochaperone E3 ligase: carboxy terminus of heat shock protein 70-interacting protein (CHIP). Thus, our study points to an extraribosomal role of RPS3 in balancing neuronal survival or death depending on the degree of starvation through CHIP-mediated polyubiquitination and degradation.

Opposing roles of the two isoforms of ErbB3 binding protein 1 in human cancer cells.

The different functions of the two isoforms of ErbB3 binding protein 1 (Ebp1), p48 and p42, have recently become the focus of interest as they reveal contradictory roles in cell growth promoting ability. The conformational change that crystal structure of p42 was shown to lack α helices at the amino-terminus present in p48 represents the differential binding partners and protein modifications of two Ebp1 isoforms. N-terminal specific phosphorylation by CDK2 and deregulation of the p53 tumor suppressor through specific interaction with HDM2 and Akt activation is postulated to contribute to p48-mediated tumorigenesis. The short isoform p42 Ebp1, which is actual binding partner of ErbB3 has been implicated as a tumor suppressor with many binding partners such as Rb, HDAC2, Sin3A and the p85 subunit of PI3K with HSP70/CHIP, inhibiting its own antiproliferative activity or inhibiting PI3K activity. The aim of the current review is to provide a summary on distinctive cellular functions of two Ebp1 proteins and their molecular partners that might be responsible for the unique functions of each isoform of Ebp1.

Phosphorylation of the N-terminal domain of p48 Ebp1 by CDK2 is required for tumorigenic function of p48.

The long isoform of ErbB3 binding protein 1 (Ebp1), p48, strongly promotes tumorigenesis of glioblastoma, accelerating cell proliferation and transformation, while the short isoform, p42, which lacks the N-terminal 54 amino acids, inhibits tumor growth. However, it is unclear if the N-terminal domain of p48 regulates the oncogenic function of p48. Here, we show that p48, but not p42, interacts with cyclin-dependent kinase 2 (CDK2) through its N-terminal domain, resulting in the specific phosphorylation of serine 34 of p48. Overexpression of wild-type p48 greatly enhanced tumor cell growth, whereas phospho-ablated mutant S34A of p48, which is mutated at the CDK2 phosphorylation site, antagonizes cell proliferation and transformation. Moreover, phospho-ablated mutant S34A abrogated the ability of p48 to accelerate tumor cell growth in a mouse engraft model. Thus, our findings indicate that p48Ebp1 acts as an oncoprotein through selective interaction and/or modification of the N-terminal domain that does not exist in its short isoform p42.

PA2G4/EBP1 ubiquitination by PRKN/PARKIN promotes mitophagy protecting neuron death in cerebral ischemia.

Cerebral ischemia induces massive mitochondrial damage, leading to neuronal death. The elimination of damaged mitochondria via mitophagy is critical for neuroprotection. Here we show that the level of PA2G4/EBP1 (proliferation-associated 2G4) was notably increased early during transient middle cerebral artery occlusion and prevented neuronal death by eliciting cerebral ischemia-reperfusion (IR)-induced mitophagy. Neuron-specific knockout of Pa2g4 increased infarct volume and aggravated neuron loss with impaired mitophagy and was rescued by introduction of adeno-associated virus serotype 2 expressing PA2G4/EBP1. We determined that PA2G4/EBP1 is ubiquitinated on lysine 376 by PRKN/PARKIN on the damaged mitochondria and interacts with receptor protein SQSTM1/p62 for mitophagy induction. Thus, our study suggests that PA2G4/EBP1 ubiquitination following cerebral IR-injury promotes mitophagy induction, which may be implicated in neuroprotection.Abbreviations: AAV: adeno-associated virus; ACTB: actin beta; BNIP3L/NIX: BCL2 interacting protein 3 like; CA1: Cornu Ammonis 1; CASP3: caspase 3; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; PA2G4/EBP1: proliferation-associated 2G4; FUNDC1: FUN14 domain containing 1; IB: immunoblotting; ICC: immunocytochemistry; IHC: immunohistochemistry; IP: immunoprecipitation; MCAO: middle cerebral artery occlusion; MEF: mouse embryonic fibroblast; OGD: oxygen-glucose deprivation; PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced kinase 1; RBFOX3/NeuN: RNA binding fox-1 homolog 3; SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; TUBB: tubulin beta class I; WT: wild-type.

Exploration of drug resistance mechanisms in triple negative breast cancer cells using a microfluidic device and patient tissues.

Chemoresistance is a major cause of treatment failure in many cancers. However, the life cycle of cancer cells as they respond to and survive environmental and therapeutic stress is understudied. In this study, we utilized a microfluidic device to induce the development of doxorubicin-resistant (DOXR) cells from triple negative breast cancer (TNBC) cells within 11 days by generating gradients of DOX and medium. In vivo chemoresistant xenograft models, an unbiased genome-wide transcriptome analysis, and a patient data/tissue analysis all showed that chemoresistance arose from failed epigenetic control of the nuclear protein-1 (NUPR1)/histone deacetylase 11 (HDAC11) axis, and high NUPR1 expression correlated with poor clinical outcomes. These results suggest that the chip can rapidly induce resistant cells that increase tumor heterogeneity and chemoresistance, highlighting the need for further studies on the epigenetic control of the NUPR1/HDAC11 axis in TNBC.

Butein derivatives prevent obesity and improve insulin resistance through the induction of energy expenditure in high-fat diet-fed obese mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization.

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

ErbB3 binding protein 1 contributes to adult hippocampal neurogenesis by modulating Bmp4 and Ascl1 signaling.

Neural stem cells (NSCs) in the adult hippocampus divide infrequently; the endogenous molecules modulating adult hippocampal neurogenesis (AHN) remain largely unknown. Here, we show that ErbB3 binding protein 1 (Ebp1), which plays important roles in embryonic neurodevelopment, acts as an essential modulator of adult neurogenic factors. In vivo analysis of Ebp1 neuron depletion mice showed impaired AHN with a low number of hippocampal NSCs and neuroblasts. Ebp1 leads to transcriptional repression of Bmp4 and suppression of Ascl1 promoter methylation in the dentate gyrus of the adult hippocampus reflecting an unusually high level of Bmp4 and low Ascl1 level in neurons of Ebp1-deficient mice. Therefore, our findings suggests that Ebp1 could act as an endogenous modulator of the interplay between Bmp4 and Ascl1/Notch signaling, contributing to AHN.

Akt2 and nucleophosmin/B23 function as an oncogenic unit in human lung cancer cells.

The signaling network of protein kinase B(PKB)/Akt has been implicated in survival of lung cancer cells. However, understanding the relative contribution of the different isoform of Akt network is nontrival. Here, we report that Akt2 is highly expressed in human lung adenocarcinoma cell line A549 cells. Suppression of Akt2 expression in A549 cells results in notable inhibition of cell poliferation, soft agar growth, and invasion, accompanying by a decrease of nucleophosmin/B23 protein. Overexpression of Akt1 restores cancerous growth of A549 cells in B23-knockdown (KD) cells while Akt2 overexpression did not restore proliferating potential in cells with downregulated B23, thus suggesting Akt2 requires B23 to drive proliferation of lung cancer cell. Loss of functional Akt2 and B23 has similar defects on cell proliferation, apoptotic resistance and cell cycle regulation, while loss of Akt1 has less defects on cell proliferation, survival and cell cycle progression in A549 cells. Moreover, overexpression of B23 rescues the proliferative block induced as a consequence of loss of Akt2. Thus our data suggest that Akt2/B23 functions as an oncogenic unit to drive tumorigenesis of A549 lung cancer cells.

Identification and evaluation of midbrain specific longevity-related genes in exceptionally long-lived but healthy mice.

Brain aging is a complex biological process that is affected by both genetic background and environment. The transcriptomic analysis of aged human and rodent brains has been applied to identify age-associated molecular and cellular processes for which intervention could possibly restore declining brain functions induced by aging. However, whether these age-associated genetic alterations are indeed involved in the healthy aging of the brain remains unclear. We herein characterized a naturally occurring, extremely long-lived (34 months of age) but healthy mouse group retaining well-preserved motor functions. Strikingly, these long-lived mice maintained tyrosine hydroxylase expression and dopaminergic fiber densities, even in the presence of persistent neuroinflammation and expression of aging markers. Combined with Endeavor gene prioritization, we identified the following midbrain-specific longevity-associated genes in the midbrain of these mice: aimp2, hexb, cacybp, akt2, nrf1, axin1, wwp2, sp2, dnajb9, notch, traf7, and lrp1. A detailed biochemical analysis of the midbrain of these long-lived mice confirmed the increased expression of Nrf1 and the activation of Akt1 and 2. Interestingly, dopaminergic neuroprotective and age-associated E3 ubiquitin ligase parkin expression was retained at high levels in the aforementioned midbrains, possibly supporting the suppression of its toxic substrates AIMP2 and PARIS. In contrast, the 24-month-old mice with dopaminergic neurite deficits failed to maintain parkin expression in the midbrain. AIMP2-induced cytotoxicity, mitochondrial stress, and neurite toxicity can be prevented by overexpression of parkin, Akt1, and Nrf1 in SH-SY5Y and PC12 cells, and basal expression of parkin, Akt1, and Nrf1 is required for maintenance of mitochondrial function and neurite integrity in PC12 cells. Taken together, this longevity-associated pathway could be a potential target of intervention to maintain nigrostriatal dopaminergic fibers and motor ability to ensure healthy longevity.

Ribosomal protein S3, a new substrate of Akt, serves as a signal mediator between neuronal apoptosis and DNA repair.

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.

Neuroprotective effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against oxidative stress.

Oxidative stress, glutamate excitotoxicity, and inflammation are the important pathological mechanisms in neurodegenerative diseases. Recently, we reported that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects rat glial cells against glutamate-induced excitotoxicity. In this study, we report the effects of 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride on primary cultured cortical astrocytes after exposure to hydrogen peroxide (H2O2). Pretreatment of cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride prior to H2O2 exposure attenuated the H2O2-induced reductions in cell survival and superoxide dismutase, catalase, glutathione, and glutathione peroxidase activities. It also reduced H2O2-induced increases in reactive oxygen species levels, malondialdehyde content, and production of nitric oxide. These effects were all concentration-dependent. Our results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride protects against oxidative stress.

Nuclear Akt interacts with B23/NPM and protects it from proteolytic cleavage, enhancing cell survival.

B23/NPM is a major nucleolar phosphoprotein that has a critical role in cell proliferation and cell death. Here, we show that it forms a complex with Akt on growth factor (GF) stimulation in both the cytoplasm and the nucleus, for which Akt activation is indispensable. The C terminus of B23 (239-294 residues) potently binds pleckstrin homology (PH) domain of Akt. Akt binding to B23 protects it from proteolytic degradation by caspase-3, leading to the up-regulation of cell survival. Interestingly, unsumoylated B23 K263R, but not wild-type B23, strongly interacts with Akt in the nucleoplasm in the absence of GFs. Furthermore, we show that Akt2, but not other isoforms, specifically regulates B23 sumoylation and protein stability. Also, nuclear Akt regulates the cell cycle progression activity of B23. Therefore, our findings support that nuclear Akt binds and stabilizes B23 in the nucleoplasm, and regulates its activities in cell survival and cell cycle.