Zhiqi Song

REST alleviates neurotoxic prion peptide-induced synaptic abnormalities, neurofibrillary degeneration and neuronal death partially via LRP6-mediated Wnt-β-catenin signaling

Prion diseases are a group of infectious neurodegenerative diseases characterized by multiple neuropathological hallmarks including synaptic damage, spongiform degeneration and neuronal death. The factors and mechanisms that maintain cellular morphological integrity and protect against neurodegeneration in prion diseases are still unclear. Here we report that after stimulation with the neurotoxic PrP106-126 fragment in primary cortical neurons, REST translocates from the cytoplasm to the nucleus and protects neurons from harmful effects of PrP106-126. Overexpression of REST reduces pathological damage and abnormal biochemical alterations of neurons induced by PrP106-126 and maintains neuronal viability by stabilizing the level of pro-survival protein FOXO1 and inhibiting the permeability of the mitochondrial outer membrane, release of cytochrome c from mitochondria to cytoplasm and the activation of Capase3. Conversely, knockdown of REST exacerbates morphological damage and inhibits the expression of FOXO1. Additionally, by overexpression or knockdown of LRP6, we further show that LRP6-mediated Wnt-β-catenin signaling partly regulates the expression of REST. Collectively, we demonstrate for the first time novel neuroprotective function of REST in prion diseases and hypothesise that the LRP6-Wnt-β-catenin/REST signaling plays critical and collaborative roles in neuroprotection. This signaling of neuronal survival regulation could be explored as a viable therapeutic target for prion diseases and associated neurodegenerative diseases.

HDAC6 alleviates prion peptide-mediated neuronal death via modulating PI3K-Akt-mTOR pathway

Histone deacetylase 6 (HDAC6) controls several major cellular responses to stress that play a role in neurodegenerative diseases, including aggresome formation, autophagy, and apoptosis. However, the specific role of HDAC6 in prion diseases is not known. In this study, we examined the relationship between HDAC6 and cellular response to the neurotoxic synthetic prion protein fragment PrP106-126. We determined that exposure of cerebral cortical neurons to this fragment alters the expression and localization of HDAC6. Suppression of HDAC6 activity or knockdown of HDAC6 expression exacerbates the neuronal cell death induced by PrP106-126, but that overexpression of HDAC6 alleviates PrP106-126-induced neuronal death. We also found that this protective effect of HDAC6 involves the activation of autophagy and modulation of PI3K-Akt-mammalian target of rapamycin (mTOR) signaling. Overexpression of HDAC6 in neurons-induced autophagy correlated with a reduction in phosphorylated mTOR and phosphorylated p70S6K in response to PrP106-126 stimulation, conversely, HDAC6 deficiency interfered with autophagy and increased phosphorylated mTOR and phosphorylated 70S6K. In addition, HDAC6 also appears to modulate the phosphorylation of Akt; overexpression of HDAC6 increased the phosphorylated Akt, but HDAC6 deficiency resulted in further reduction of phosphorylated Akt. Overall, we demonstrate that HDAC6 protects neurons from toxicity of prion peptide, and that this protection occurs at through the regulation of the PI3k-Akt-mTOR axis.

Death Receptor 6 and Caspase-6 Regulate Prion Peptide-Induced Axonal Degeneration in Rat Spinal Neurons

Axonal degeneration is a hallmark of many neurodegenerative disorders including transmissible spongiform encephalopathies (TSE). However, the full complement of axonal degeneration triggers is not fully understood. In an in vitro prion model, we observed that treatment of rat spinal neurons with the prion peptide, PrP106-126, activated death receptor 6 (DR6, also known as TNFRSF21), caspase-6, caspase-3, and induced axonal degeneration. Knockdown of DR6 by siRNA blocked caspase-6 and caspase-3 activation and axonal degeneration. We also found that cleaved caspase-3 is only enriched in cell bodies, but cleaved caspase-6 is expressed in both cell bodies and axons. Axonal degeneration was prevented by preincubation of neurons with a caspase-6 inhibitor or siRNA of caspase-6. Our findings suggest that both DR6 and caspase-6 play important roles in axonal degeneration and caspase-6 acts downstream of DR6. We also observed that nicotinamide nucleotide adenylyltransferase 1 protein (Nmnat1), which had been reported to protect neurons from degeneration, alleviated axonal degeneration without blocking caspase-6 activation, suggesting that Nmnat acts downstream or parallel to caspase-6 activation. Our results indicate that PrP106-126 triggered axonal degeneration of the spinal cord neurons, DR6 is a key regulator of axonal degeneration, and the signaling pathway of DR6/caspase-6 mediates axonal degeneration induced by the prion fragment. Our findings raise the hope of targeting the DR6 as a potential therapeutic strategy in prion-related neurodegenerative diseases.

NRSF: an Angel or a Devil in Neurogenesis and Neurological Diseases

The neuron-restrictive silencer factor (NRSF) a transcriptional regulator that function as a hub that coordinately regulates multiple aspects of neurogenesis, orchestrates neural differentiation, and preserves the unique neural phenotype. NRSF also acts as an oncogene in neural tumorigenesis, although its effect differs depending on the cell type and tissues. Intriguingly, far more than above functions, potential roles for NRSF and its target genes have also been implicated in the pathogenesis and therapeutic mechanism of neurodegenerative diseases. NRSF acts as a flexible and complicated regulator in nervous system, from transcriptional repressor to activator or modulator, and plays a part in neuronal survival or neuronal death. Here, we present the mechanisms proposed to account for the multiple roles of NRSF in neurogenesis and neurological diseases and discuss the therapeutic perspective of recent advances. The mechanisms underlying this duality of NRSF are helpful to understanding the physiological and pathological conditions of neurons and provide new therapeutic approaches to neurological disorders and diseases.

Overexpression of BAT3 Alleviates Prion Protein Fragment PrP106-126-Induced Neuronal Apoptosis

Prion diseases are a group of infectious neurodegenerative diseases characterized by neuronal death and degeneration. Human leukocyte antigen‐B‐associated transcript 3 (BAT3) is an important apoptosis regulator. We therefore investigated the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106‐126‐induced apoptosis.

Molecular mechanisms of neurodegeneration mediated by dysfunctional subcellular organelles in transmissible spongiform encephalopathies

Transmissible spongiform encephalopathies refer to a group of infectious neurodegenerative diseases with an entirely novel mechanism of transmission and pathophysiology including synaptic damage, dendritic atrophy, vacuolization, and microglial activation. Extensive neuronal loss is the main cause of chronic brain deterioration and fatal outcome of prion diseases. As the final outcome of pathological alterations, neuronal death is a prominent feature of all prion diseases. The mechanisms responsible for prion diseases are not well understood. A more comprehensive understanding of the molecular basis of neuronal damage is essential for the development of an effective therapy for transmissible spongiform encephalopathies and other neurodegenerative diseases sharing similar features. Here, we review the molecular mechanisms of mitochondrial dysfunction and endoplasmic reticulum stress-mediated neuronal death, which play crucial roles in the pathogenisis of prion diseases.

Lithium alleviates neurotoxic prion peptide-induced synaptic damage and neuronal death partially by the upregulation of nuclear target REST and the restoration of Wnt signaling

&NA; Prion diseases are a group of infectious neurodegenerative diseases characterized by multiple neuropathological hallmarks, including accumulation of PrPSc, synaptic damage, and neuronal death. We previously reported that the repressor element 1‐silencing transcription factor (REST), a novel neuroprotective marker in neurodegeneration, protects neurons against neurotoxic peptide (PrP106‐126)‐induced neurotoxicity, but fails to maintain survival following prolonged exposure to PrP106‐126. Because Wnt signaling partially induces REST and is activated by lithium, we investigated the effects of lithium on REST in prion diseases. Lithium restores nuclear expression of REST, which is essential for regulating survival proteins. Lithium also mimics neuroprotective functions when REST is blocked, and these beneficial effects are additive with REST overexpression under physiological conditions. Reciprocally, under PrP106‐126‐stimulated pathological conditions, REST plays a critical role in the neuroprotective mechanisms of lithium treatment. Although lithium recovers Wnt signaling by inhibiting glycogen synthase kinase‐3&bgr; and stabilizing &bgr;‐catenin, restores survival associated proteins after exposure to PrP106‐126 in primary cortical neurons. Knockdown of REST significantly suppresses the neuroprotective function of lithium. Conversely, overexpression of REST partially recovers its actions. Notably, lithium directly alleviates PrP106‐126‐induced synaptic damage and neuronal cell death by preventing changes in presynaptic and postsynaptic marker proteins and promoting survival pathways also partially via the expression of REST. Our results suggest that REST acts as a novel and important nuclear target for lithium. We hypothesize that PrP106‐126‐stimulated neurotoxicity induces Wnt signaling dysfunction and lithium mimics this signaling cascade, suggesting that lithium should be considered as a potential therapeutic agent against prion diseases. Graphical abstract Schematic signaling pathways for lithium acts as a neuroprotective reagent in PrP106‐126‐stimulated primary neurons. Mito, mitochondria. Symbol, phosphorylation. →: Direct stimulatory modification, Symbol: Direct inhibitory modification. Symbol. No caption available. Symbol. No caption available. Figure. No caption available. HighlightsREST acts as a novel neuroprotective nuclear target for lithium.Lithium mimics neuroprotective functions when REST is blocked.Lithium protects primary neurons from PrP106‐126‐induced synaptic damage partially via REST.Lithium protects against PrP106‐126‐induced neurotoxicity partially via REST.

Downregulation of the Repressor Element 1-Silencing Transcription Factor (REST) Is Associated with Akt-mTOR and Wnt-β-Catenin Signaling in Prion Diseases Models

Prion diseases are a group of infectious diseases characterized by multiple neuropathological changes, yet the mechanisms that preserve function and protect against prion-associated neurodegeneration are still unclear. We previously reported that the repressor element 1-silencing transcription factor (REST) alleviates neurotoxic prion peptide (PrP106-126)-induced toxicity in primary neurons. Here we confirmed the findings of the in vitro model in 263K infected hamsters, an in vivo model of prion diseases and further showed the relationships between REST and related signaling pathways. REST was depleted from the nucleus in prion infected brains and taken up by autophagosomes in the cytoplasm, co-localizing with LC3-II. Importantly, downregulation of the Akt–mTOR and at least partially inactivation of LRP6-Wnt-β-catenin signaling pathways correlated with the decreased levels of REST in vivo in the brain of 263K-infected hamsters and in vitro in PrP106-126-treated primary neurons. Overexpression of REST in primary cortical neurons alleviated PrP106-126 peptide-induced neuronal oxidative stress, mitochondrial damage and partly inhibition of the LRP6-Wnt-β-catenin and Akt–mTOR signaling. Based on our findings, a model of REST-mediated neuroprotection in prion infected animals is proposed, with Akt–mTOR and Wnt-β-catenin signaling as the key pathways. REST-mediated neuronal survival signaling could be explored as a viable therapeutic target for prion diseases and related neurodegenerative diseases.

Parkin Overexpression Ameliorates PrP106–126-Induced Neurotoxicity via Enhanced Autophagy in N2a Cells

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrPSc). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson’s disease and Alzheimer’s disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106–126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106–126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106–126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106–126 toxicity, which is a novel potential therapeutic target for treating prion diseases.

Mycobacterium bovis Induces Endoplasmic Reticulum Stress Mediated-Apoptosis by Activating IRF3 in a Murine Macrophage Cell Line

Mycobacterium bovis (M. bovis) is highly adapted to macrophages and has developed multiple mechanisms to resist intracellular assaults. However, the host cells in turn deploy a multipronged defense mechanism to control bacterial infection. Endoplasmic reticulum (ER) stress-mediated apoptosis is one such primary defense mechanism. However, the role of interferon regulatory factor 3 (IRF3) between ER stress and apoptosis during M. bovis infection is unknown. Here, we demonstrate that M. bovis effectively induced apoptosis in murine macrophages. Caspase-12, caspase-9, and caspase-3 were activated over a 48 h infection period. The splicing of XBP-1 mRNA and the level of phosphorylation of eIF2α, indicators of ER stress, significantly increased at early time points after M. bovis infection. The expansion of the ER compartment, a morphological hallmark of ER stress, was observed at 6 h. Pre-treatment of Raw 264.7 cells with 4-PBA (an ER stress-inhibitor) reduced the activation of the ER stress indicators, caspase activation and its downstream poly (ADP-ribose) polymerase (PARP) cleavage, phosphorylation of TBK1 and IRF3 and cytoplasmic co-localization of STING and TBK1. M. bovis infection led to the interaction of activated IRF3 and cytoplasmic Bax leading to mitochondrial damage. Role of IRF3 in apoptosis was further confirmed by blocking this molecule with BX-795 that showed significant reduction expression of caspase-8 and caspase-3. Intracellular survival of M. bovis increased in response to 4-PBA and BX-795. These findings indicate that STING-TBK1-IRF3 pathway mediates a crosstalk between ER stress and apoptosis during M. bovis infection, which can effectively control intracellular bacteria.