Arginine methylation next to the PY‐NLS modulates Transportin binding and nuclear import of FUS

Fused in sarcoma (FUS) is a nuclear protein that carries a proline‐tyrosine nuclear localization signal (PY‐NLS) and is imported into the nucleus via Transportin (TRN). Defects in nuclear import of FUS have been implicated in neurodegeneration, since mutations in the PY‐NLS of FUS cause amyotrophic lateral sclerosis (ALS). Moreover, FUS is deposited in the cytosol in a subset of frontotemporal lobar degeneration (FTLD) patients. Here, we show that arginine methylation modulates nuclear import of FUS via a novel TRN‐binding epitope. Chemical or genetic inhibition of arginine methylation restores TRN‐mediated nuclear import of ALS‐associated FUS mutants. The unmethylated arginine–glycine–glycine domain preceding the PY‐NLS interacts with TRN and arginine methylation in this domain reduces TRN binding. Inclusions in ALS‐FUS patients contain methylated FUS, while inclusions in FTLD‐FUS patients are not methylated. Together with recent findings that FUS co‐aggregates with two related proteins of the FET family and TRN in FTLD‐FUS but not in ALS‐FUS, our study provides evidence that these two diseases may be initiated by distinct pathomechanisms and implicates alterations in arginine methylation in pathogenesis.     

Loss of fused in sarcoma (FUS) promotes pathological Tau splicing

A subset of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients present pathological redistribution and aggregation of the nuclear protein fused in sarcoma (FUS) in the cytoplasm. Although FUS associates with the spliceosomal complex, no endogenous neuronal splicing targets have been identified. Here we identify Tau mRNA as a physiological splicing target of FUS. In mouse brain, FUS directly binds to Tau pre-mRNA, and knockdown of FUS in hippocampal neurons leads to preferential inclusion of Tau exons 3 and 10. FUS knockdown causes significant growth cone enlargement and disorganization reminiscent of Tau loss of function. These findings suggest that disturbed cytoskeletal function and enhanced expression of the neurodegeneration-associated Tau exon 10 might contribute to FTLD/ALS with FUS inclusions.     

Phosphorylation of the FUS low‐complexity domain disrupts phase separation,aggregation, and toxicity

Neuronal inclusions of aggregated RNA‐binding protein fused in sarcoma (FUS) are hallmarks of ALS and frontotemporal dementia subtypes. Intriguingly, FUS's nearly uncharged, aggregation‐prone, yeast prion‐like, low sequence‐complexity domain (LC) is known to be targeted for phosphorylation. Here we map in vitro and in‐cell phosphorylation sites across FUS LC. We show that both phosphorylation and phosphomimetic variants reduce its aggregation‐prone/prion‐like character, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Nuclear magnetic resonance spectroscopy demonstrates the intrinsically disordered structure of FUS LC is preserved after phosphorylation; however, transient domain collapse and self‐interaction are reduced by phosphomimetics. Moreover, we show that phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS‐associated cytotoxicity. Hence, post‐translational modification may be a mechanism by which cells control physiological assembly and prevent pathological protein aggregation, suggesting a potential treatment pathway amenable to pharmacologic modulation.     

Inhibition of TIM‐4 protects against cerebral ischaemia‐reperfusion injury

TIM‐4 plays an important role in ischaemia‐reperfusion injury of liver and kidney; however, the effects of TIM‐4 on cerebral ischaemia‐reperfusion injury (IRI) are unknown. The purpose of the present study was to investigate the potential role of TIM‐4 in experimental brain ischaemia‐reperfusion injury. In this study, cerebral ischaemia reperfusion was induced by transient middle cerebral artery occlusion (MCAO) for 1 hour in C57/BL6 mice. The TIM‐4 expression was detected in vivo or vitro by real‐time quantitative polymerase chain reaction, Western blot and flow cytometric analysis. In vivo, the administration of anti‐TIM‐4 antibodies significantly suppressed apoptosis, inhibited inflammatory cells and enhanced anti‐inflammatory responses. In vitro, activated microglia exhibited reduced cellular proliferation and induced IRI injury when co‐cultured with neurons; these effects were inhibited by anti‐TIM‐4 antibody treatment. Similarly, microglia transfected with TIM‐4 siRNA and stimulated by LPS + IFN‐γ alleviated the TIM‐4‐mediated damage to neurons. Collectively, our data indicate that the inhibition of TIM‐4 can improve the inflammatory response and exerts a protective effect in cerebral ischaemia‐reperfusion injury.     

ALS‐associated fused in sarcoma (FUS) mutations disrupt Transportin‐mediated nuclear import

Mutations in fused in sarcoma (FUS) are a cause of familial amyotrophic lateral sclerosis (fALS). Patients carrying point mutations in the C‐terminus of FUS show neuronal cytoplasmic FUS‐positive inclusions, whereas in healthy controls, FUS is predominantly nuclear. Cytoplasmic FUS inclusions have also been identified in a subset of frontotemporal lobar degeneration (FTLD‐FUS). We show that a non‐classical PY nuclear localization signal (NLS) in the C‐terminus of FUS is necessary for nuclear import. The majority of fALS‐associated mutations occur within the NLS and impair nuclear import to a degree that correlates with the age of disease onset. This presents the first case of disease‐causing mutations within a PY‐NLS. Nuclear import of FUS is dependent on Transportin, and interference with this transport pathway leads to cytoplasmic redistribution and recruitment of FUS into stress granules. Moreover, proteins known to be stress granule markers co‐deposit with inclusions in fALS and FTLD‐FUS patients, implicating stress granule formation in the pathogenesis of these diseases. We propose that two pathological hits, namely nuclear import defects and cellular stress, are involved in the pathogenesis of FUS‐opathies.     

ALS/FTD‐associated FUS activates GSK‐3β to disrupt the VAPB–PTPIP51 interaction and ER–mitochondria associations

Defective FUS metabolism is strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), but the mechanisms linking FUS to disease are not properly understood. However, many of the functions disrupted in ALS/FTD are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. This signalling is facilitated by close physical associations between the two organelles that are mediated by binding of the integral ER protein VAPB to the outer mitochondrial membrane protein PTPIP51, which act as molecular scaffolds to tether the two organelles. Here, we show that FUS disrupts the VAPB–PTPIP51 interaction and ER–mitochondria associations. These disruptions are accompanied by perturbation of Ca²⁺ uptake by mitochondria following its release from ER stores, which is a physiological read‐out of ER–mitochondria contacts. We also demonstrate that mitochondrial ATP production is impaired in FUS‐expressing cells; mitochondrial ATP production is linked to Ca²⁺ levels. Finally, we demonstrate that the FUS‐induced reductions to ER–mitochondria associations and are linked to activation of glycogen synthase kinase‐3β (GSK‐3β), a kinase already strongly associated with ALS/FTD.     

Rapid anti‐depressant‐like effects of ketamine and other candidates: Molecular and cellular mechanisms

Major depressive disorder takes at least 3 weeks for clinical anti‐depressants, such as serotonin selective reuptake inhibitors, to take effect, and only one‐third of patients remit. Ketamine, a kind of anaesthetic, can alleviate symptoms of major depressive disorder patients in a short time and is reported to be effective to treatment‐resistant depression patients. The rapid and strong anti‐depressant‐like effects of ketamine cause wide concern. In addition to ketamine, caloric restriction and sleep deprivation also elicit similar rapid anti‐depressant‐like effects. However, mechanisms about the rapid anti‐depressant‐like effects remain unclear. Elucidating the mechanisms of rapid anti‐depressant effects is the key to finding new therapeutic targets and developing therapeutic patterns. Therefore, in this review we summarize potential molecular and cellular mechanisms of rapid anti‐depressant‐like effects based on the pre‐clinical and clinical evidence, trying to provide new insight into future therapy.     

Retinal cell type‐specific prevention of ischemia‐induced damages by LPS‐TLR4 signaling through microglia

Reprogramming of toll‐like receptor 4 (TLR4) by brief ischemia or lipopolysacharide (LPS) contributes to superintending tolerance against destructive ischemia in brain. However, beneficial roles of TLR4 signaling in ischemic retina are not well known. This study demonstrated that preconditioning with LPS 48 h prior to the retinal ischemia prevents the cellular damage in morphology with hematoxylin and eosin (H&E) staining and functions of retina with electroretinogram (ERG), while post‐ischemia treatment deteriorated it. The preventive effects of LPS preconditioning showed the cell type‐specificity of retinal cells. There was complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. LPS treatment caused the proliferation and migration of retinal microglia and its preconditioning prevented the ischemia‐induced microglial activation. Preventive actions from cell damages following LPS preconditioning prior to retinal ischemia were abolished in TLR4 knock‐out mice, and by pre‐treatments with anti‐TLR4 antibody or minocycline, a microglia inhibitor, which themselves had no effects on the retinal ischemia‐induced damages or microglia activation. Thus, this study revealed that TLR4 mediates the LPS preconditioning‐induced preventive effects through microglial activation in the retinal ischemia model.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

FUS transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration

Fused in Sarcoma (FUS) proteinopathy is a feature of frontotemporal lobar dementia (FTLD), and mutation of the fus gene segregates with FTLD and amyotrophic lateral sclerosis (ALS). To study the consequences of mutation in the fus gene, we created transgenic rats expressing the human fus gene with or without mutation. Overexpression of a mutant (R521C substitution), but not normal, human FUS induced progressive paralysis resembling ALS. Mutant FUS transgenic rats developed progressive paralysis secondary to degeneration of motor axons and displayed a substantial loss of neurons in the cortex and hippocampus. This neuronal loss was accompanied by ubiquitin aggregation and glial reaction. While transgenic rats that overexpressed the wild-type human FUS were asymptomatic at young ages, they showed a deficit in spatial learning and memory and a significant loss of cortical and hippocampal neurons at advanced ages. These results suggest that mutant FUS is more toxic to neurons than normal FUS and that increased expression of normal FUS is sufficient to induce neuron death. Our FUS transgenic rats reproduced some phenotypes of ALS and FTLD and will provide a useful model for mechanistic studies of FUS-related diseases.     

Molecular determinants and genetic modifiers of aggregation and toxicity for the ALS disease protein FUS/TLS

TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

Modeling ALS and FTLD proteinopathies in yeast: An efficient approach for studying protein aggregation and toxicity

In recent years there have been several reports of human neurodegenerative diseases that involve protein misfolding being modeled in the yeast Saccharomyces cerevisiae. This review summarizes recent advances in understanding the specific mechanisms underlying intracellular neuronal pathology during Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), including SOD1, TDP-43 and FUS protein inclusions and the potential of these proteins to be involved in pathogenic prion-like mechanisms. More specifically, we focus on findings from yeast systems that offer tremendous possibilities for screening for genetic and chemical modifiers of disease-related proteotoxicity.Key words: amyotrophic lateral sclerosis, ALS, frontotemporal lobar degeneration, FTLD, yeast, amyloid, prion, FUS, TDP-43, SOD1     

Protective effect of brown Brazilian propolis against acute vaginal lesions caused by herpes simplex virus type 2 in mice: involvement of antioxidant and anti‐inflammatory mechanisms

Propolis has been highlighted for its antioxidant, anti‐inflammatory and antiviral properties. The purpose of this study was to investigate if brown Brazilian hydroalcoholic propolis extract (HPE) protects against vaginal lesions caused by herpes simplex virus type 2 (HSV‐2) in female BALB/c mice. The treatment was divided in 5 days of pre‐treatment with HPE [50 mg·kg^–1, once a day, intragastric (i.g.)], HSV‐2 infection [10 µl of a solution 1 × 10² plaque‐forming unit (PFU·ml^–1 HSV‐2), intravaginal inoculation at day 6] and post‐treatment with HPE (50 mg·kg^–1) for 5 days more. At day 11, the animals were killed, and the in vivo analysis (score of lesions) and ex vivo analysis [haematological and histological evaluation; superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) activities; reactive species (RS), tyrosine nitration levels, non‐protein thiols (NPSH) and ascorbic acid (AA) levels] were carried out. HPE treatment reduced extravaginal lesions and the histological damage caused by HSV‐2 infection in vaginal tissues of animals. HPE was able to decrease RS, tyrosine nitration, AA levels and MPO activity. Also, it protected against the inhibition of CAT activity in vaginal tissues of mice. HPE promoted protective effect on HSV‐2 infected animals by acting on inflammatory and oxidative processes, and this effect probably is caused by its antioxidant and anti‐inflammatory properties. Copyright © 2011 John Wiley & Sons, Ltd.     

Attenuation of AD‐like neuropathology by harnessing peripheral immune cells: local elevation of IL‐10 and MMP‐9

Immunization with an altered myelin‐derived peptide (MOG45D) improves recovery from acute CNS insults, partially via recruitment of monocyte‐derived macrophages that locally display a regulatory activity. Here, we investigated the local alterations in the cellular and molecular immunological milieu associated with attenuation of Alzheimer’s disease‐like pathology following immunotherapy. We found that immunization of amyloid precursor protein/presenilin 1 double‐transgenic mice with MOG45D peptide, loaded on dendritic cells, led to a substantial reduction of parenchymal and perivascular amyloid beta (Aβ)‐plaque burden and soluble Aβ_(1–42) peptide levels as well as reduced astrogliosis and levels of a key glial scar protein (chondroitin sulphate proteoglycan). These changes were associated with a shift in the local innate immune response, manifested by increased Iba1⁺/CD45^high macrophages that engulfed Aβ, reduced pro‐inflammatory (tumor necrosis factor‐α) and increased anti‐inflammatory (interleukin‐10) cytokines, as well as a significant increase in growth factors (IGF‐1 and TGFβ) in the brain. Furthermore, the levels of matrix metalloproteinase‐9, an enzyme shown to degrade Aβ and is associated with glial scar formation, were significantly elevated in the brain following immunization. Altogether, these results indicate that boosting systemic immune cells leads to a local immunomodulation manifested by elevated levels of anti‐inflammatory cytokines and metalloproteinases that contribute to ameliorating Alzheimer’s disease pathology.     

Age‐at‐onset‐dependent effects of sulfur amino acid restriction on markers of growth and stress in male F344 rats

Trade‐offs in life‐history traits are clinically and mechanistically important. Sulfur amino acid restriction (SAAR) extends lifespan. But whether this benefit comes at the cost of other traits including stress resistance and growth is unclear. We investigated the effects of SAAR on growth markers (body weight, IGF1, and IGFBP3) and physiological stresses. Male‐F344 rats were fed control (0.86% Met) and SAAR (0.17% Met) diets starting at 2, 10, and 20 months. Rats were injected with keyhole‐limpet‐hemocyanin (KLH) to measure immune responses (anti‐KLH‐IgM, anti‐KLH‐IgG, and delayed‐type‐hypersensitivity [DTH]). Markers of ER stress (FGF21 and adiponectin), detoxification capacity (glutathione [GSH] concentrations, GSH‐S‐transferase [GST], and cytochrome‐P₄₅₀‐reductase [CPR] activities), and low‐grade inflammation (C‐reactive protein [CRP]) were also determined. SAAR decreased body weight, liver weight, food intake, plasma IGF1, and IGFBP3; the effect size diminished with increasing age‐at‐onset. SAAR increased FGF21 and adiponectin, but stress damage markers GRP78 and Xbp1_s/us were unchanged, suggesting that ER stress is hormetic. SAAR increased hepatic GST activity despite lower GSH, but CPR activity was unchanged, indicative of enhanced detoxification capacity. Other stress markers were either uncompromised (CRP, anti‐KLH‐IgM, and DTH) or slightly lower (anti‐KLH‐IgG). Increases in stress markers were similar across all ages‐at‐onset, except for adiponectin, which peaked at 2 months. Overall, SAAR did not compromise stress responses and resulted in maximal benefits with young‐onset. In survival studies, median lifespan extension with initiation at 52 weeks was 7 weeks (p = .05); less than the 33.5‐week extension observed in our previous study with 7‐week initiation. Findings support SAAR translational studies and the need to optimize Met dose based on age‐at‐onset.     

Dl‐3n‐butylphthalide improves traumatic brain injury recovery via inhibiting autophagy‐induced blood‐brain barrier disruption and cell apoptosis

Blood‐brain barrier (BBB) disruption and neuronal apoptosis are important pathophysiological processes after traumatic brain injury (TBI). In clinical stroke, Dl‐3n‐butylphthalide (Dl‐NBP) has a neuroprotective effect with anti‐inflammatory, anti‐oxidative, anti‐apoptotic and mitochondrion‐protective functions. However, the effect and molecular mechanism of Dl‐NBP for TBI need to be further investigated. Here, we had used an animal model of TBI and SH‐SY5Y/human brain microvascular endothelial cells to explore it. We found that Dl‐NBP administration exerts a neuroprotective effect in TBI/OGD and BBB disorder, which up‐regulates the expression of tight junction proteins and promotes neuronal survival via inhibiting mitochondrial apoptosis. The expressions of autophagy‐related proteins, including ATG7, Beclin1 and LC3II, were significantly increased after TBI/OGD, and which were reversed by Dl‐NBP treatment both in vivo and in vitro. Moreover, rapamycin treatment had abolished the effect of Dl‐NBP for TBI recovery. Collectively, our current studies indicate that Dl‐NBP treatment improved locomotor functional recovery after TBI by inhibiting the activation of autophagy and consequently blocking the junction protein loss and neuronal apoptosis. Dl‐NBP, as an anti‐inflammatory and anti‐oxidative drug, may act as an effective strategy for TBI recovery.     

Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43)

Cytoplasmic inclusions containing TAR DNA-binding protein of 43 kDa (TDP-43) or Fused in sarcoma (FUS) are a hallmark of amyotrophic lateral sclerosis (ALS) and several subtypes of frontotemporal lobar degeneration (FTLD). FUS-positive inclusions in FTLD and ALS patients are consistently co-labeled with stress granule (SG) marker proteins. Whether TDP-43 inclusions contain SG markers is currently still debated. We determined the requirements for SG recruitment of FUS and TDP-43 and found that cytoplasmic mislocalization is a common prerequisite for SG recruitment of FUS and TDP-43. For FUS, the arginine-glycine-glycine zinc finger domain, which is the protein's main RNA binding domain, is most important for SG recruitment, whereas the glycine-rich domain and RNA recognition motif (RRM) domain have a minor contribution and the glutamine-rich domain is dispensable. For TDP-43, both the RRM1 and the C-terminal glycine-rich domain are required for SG localization. ALS-associated point mutations located in the glycine-rich domain of TDP-43 do not affect SG recruitment. Interestingly, a 25-kDa C-terminal fragment of TDP-43, which is enriched in FTLD/ALS cortical inclusions but not spinal cord inclusions, fails to be recruited into SG. Consistently, inclusions in the cortex of FTLD patients, which are enriched for C-terminal fragments, are not co-labeled with the SG marker poly(A)-binding protein 1 (PABP-1), whereas inclusions in spinal cord, which contain full-length TDP-43, are frequently positive for this marker protein.