Filter Trap Assay Research Articles

Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3.

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases.

Trehalose prevents the formation of aggregates of mutant ataxin-3 and reduces soluble ataxin-3 protein levels in an SCA3 cell model

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by mutant ataxin-3 with an abnormally expanded polyQ tract and is the most common dominantly inherited ataxia worldwide. There are no suitable therapeutic options for this disease. Autophagy, a defense mechanism against the toxic effects of aggregation-prone misfolded proteins, has been shown to have beneficial effects on neurodegenerative diseases. Thus, trehalose, which is an autophagy inducer, may have beneficial effects on SCA3. In the present study, we examined the effects of trehalose on an SCA3 cell model. After trehalose treatment, aggregate formation, soluble ataxin-3 protein levels and cell viability were evaluated in HEK293T cells overexpressing ataxin-3-15Q or ataxin-3-77Q. We also explored the mechanism by which trehalose affects autophagy and stress pathways. A filter trap assay showed that trehalose decreased the number of aggregates formed by mutant ataxin-3 containing an expanded polyQ tract. Western blot and Cell Counting Kit-8 (CCK-8) results demonstrated that trehalose also reduced the ataxin-3 protein levels and was safe for ataxin-3-expressing cells, respectively. Western blot and total antioxidant capacity assays suggested that trehalose had great therapeutic potential for treating SCA3, likely through its antioxidant activity. Our data indicate that trehalose plays a neuroprotective role in SCA3 by inhibiting the aggregation and reducing the protein level of ataxin-3, which is also known to protect against oxidative stress. These findings provide a new insight into the possibility of treating SCA3 with trehalose and highlight the importance of inducing autophagy in SCA3.

An optimized filter trap assay for detecting recombinant authentic tau fibrils.

The development and optimization of the Filter Trap Assay (FTA) for the detection of authentic tau fibrils in vitro mark a pivotal advancement in the realm of tauopathy research, particularly by addressing the limitations of using polyanion-induced tau fibrils, which structurally differ from those isolated from tauopathy patients. Recently it has been shown that truncated tau fragment (297-391), also termed dGAE, can form authentic tau fibrils in the absence of polyanions. This study introduces a refined protocol that reliably detects authentic tau fibrils in a physiologically relevant framework, utilizing nitrocellulose membranes to achieve heightened sensitivity. Our investigation highlights the superior efficacy of sarkosyl, an anionic surfactant traditionally used to prepare protein lysates from brains and cultured neurons, in preserving the aggregated state of tau dGAE fibrils in vitro, underscoring its potential for further exploratory studies. By offering a user-friendly and economically feasible approach, this technique enables a broad range of laboratories to measure the presence of authentic tau fibrils. This methodological enhancement propels our understanding of tauopathies forward and bridges the gap between basic research and advanced structural analyses, enriching the scientific community's methodologies for studying neurodegenerative disorders.

Hypochlorite-induced oxidation promotes aggregation and reduces toxicity of amyloid beta 1-42

Exacerbated hypochlorite (OCl−) production is linked to neurodegenerative processes, but there is growing evidence that lower levels of hypochlorite activity are important to protein homeostasis. In this study we characterise the effects of hypochlorite on the aggregation and toxicity of amyloid beta peptide 1–42 (Aβ1-42), a major component of amyloid plaques that form in the brain in Alzheimer's disease. Our results demonstrate that treatment with hypochlorite promotes the formation of Aβ1-42 assemblies ≥100 kDa that have reduced surface exposed hydrophobicity compared to the untreated peptide. This effect is the result of the oxidation of Aβ1-42 at a single site as determined by mass spectrometry analysis. Although treatment with hypochlorite promotes the aggregation of Aβ1-42, the solubility of the peptide is enhanced and amyloid fibril formation is inhibited as assessed by filter trap assay, thioflavin T assay and transmission electron microscopy. The results of in vitro assays using SH-SY5Y neuroblastoma cells show that pre-treatment of Aβ1-42 with a sub-stoichiometric amount of hypochlorite substantially reduces its toxicity. The results of flow cytometry analysis and internalisation assays indicate that hypochlorite-induced modification of Aβ1-42 reduces its toxicity via at least two-distinct mechanism, reducing the total binding of Aβ1-42 to the surface of cells and facilitating the cell surface clearance of Aβ1-42 to lysosomes. Our data is consistent with a model in which tightly regulated production of hypochlorite in the brain is protective against Aβ-induced toxicity.

Degradation of neurodegenerative disease-associated TDP-43 aggregates and oligomers via a proteolysis-targeting chimera

BackgroundAmyotrophic lateral sclerosis (ALS) associated with TAR DNA-binding protein 43 (TDP-43) aggregation has been considered as a lethal and progressive motor neuron disease. Recent studies have shown that both C-terminal TDP-43 (C-TDP-43) aggregates and oligomers were neurotoxic and pathologic agents in ALS and frontotemporal lobar degeneration (FTLD). However, misfolding protein has long been considered as an undruggable target by applying conventional inhibitors, agonists, or antagonists. To provide this unmet medical need, we aim to degrade these misfolding proteins by designing a series of proteolysis targeting chimeras (PROTACs) against C-TDP-43.MethodsBy applying filter trap assay, western blotting, and microscopy imaging, the degradation efficiency of C-TDP-43 aggregates was studied in Neuro-2a cells overexpressing eGFP-C-TDP-43 or mCherry-C-TDP-43. The cell viability was characterized by alarmarBlue assay. The beneficial and disaggregating effects of TDP-43 PROTAC were examined with the YFP-C-TDP-43 transgenic C. elegans by motility assay and confocal microscopy. The impact of TDP-43 PROTAC on C-TDP-43 oligomeric intermediates was monitored by fluorescence lifetime imaging microscopy and size exclusion chromatography in the Neuro-2a cells co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.ResultsFour PROTACs with different linker lengths were synthesized and characterized. Among these chimeras, PROTAC 2 decreased C-TDP-43 aggregates and relieved C-TDP-43-induced cytotoxicity in Neuro-2a cells without affecting endogenous TDP-43. We showed that PROTAC 2 bound to C-TDP-43 aggregates and E3 ligase to initiate ubiquitination and proteolytic degradation. By applying advanced microscopy, it was further shown that PROTAC 2 decreased the compactness and population of C-TDP-43 oligomers. In addition to cellular model, PROTAC 2 also improved the motility of transgenic C. elegans by reducing the C-TDP-43 aggregates in the nervous system.ConclusionsOur study demonstrated the dual-targeting capacity of the newly-designed PROTAC 2 against both C-TDP-43 aggregates and oligomers to reduce their neurotoxicity, which shed light on the potential drug development for ALS as well as other neurodegenerative diseases.

Modulation of synaptic plasticity, motor unit physiology, and TDP-43 pathology by CHCHD10

Mutations in CHCHD10, a gene coding for a mitochondrial intermembrane space protein, are associated with Frontotemporal dementia (FTD)-Amyotrophic lateral sclerosis (ALS) spectrum disorders, which are pathologically characterized by cytoplasmic inclusions containing TDP-43. FTD/ALS-linked CHCHD10 mutations and TDP-43 inclusions similarly induce mitochondrial defects in respiration, fusion/fission, mtDNA stability, and cristae structure, while sizeable amounts of cytoplasmic TDP-43 aggregates are found in mitochondria. However, the mechanistic link between CHCHD10 and TDP-43 pathogenesis remains unclear. In this study, we present immunohistochemical and biochemical evidence demonstrating that insoluble CHCHD10 aggregates accumulate and colocalize with phospho-TDP-43 inclusions in brains of FTLD-TDP and AD patients, and that insoluble CHCHD10 levels tightly correlate with insoluble TDP-43 levels in control and FTLD-TDP brains. In an experimental exploration of this pathological phenotype, transgenic mice neuronally expressing FTD/ALS-linked CHCHD10R15L or CHCHDS59L mutations but not CHCHD10WT transgenic mice exhibit significantly increased CHCHD10 aggregation and phospho-TDP-43 pathology, which often colocalize within the same inclusions. Such pathologies are reflected in poor functional outcomes in long-term synaptic plasticity, motor unit physiology, and behavior in CHCHD10R15L and CHCHDS59L transgenic mice. In contrast, expression of CHCHD10WT in hTDP-43 transgenic mice (TAR4;CHCHD10WT) significantly mitigates phospho-TDP-43 pathology and rescues TDP-43-induced impairments in synaptic integrity and long-term synaptic plasticity. In isolated mitochondria, the S59L mutation induces the aggregation of resident CHCHD10S59L protein as well as the aggregation and slower turnover of recombinant TDP-43 imported into mitochondria. Likewise, in an in vitro cell-free system, the S59L mutation induces the aggregation of CHCHD10S59L protein while simultaneously enhancing the aggregation of recombinant TDP-43, as evidenced by filter trap assays and atomic force microscopy. In contrast, recombinant CHCHD10WT inhibits the growth of TDP-43 aggregates. These results in human brains, transgenic mice, and in vitro systems substantiate the role of wild type and mutant CHCHD10 in modulating mitochondrial CHCHD10 and TDP-43 pathogenesis together with associated phenotypes in long-term synaptic plasticity and motor unit physiology in mice and humans.

Pathogenic variants of Valosin-containing protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.

Mutations in the valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone and frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage. By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. With the use of western blotting, real-time quantitative polymerase chain reaction (RT-qPCR), immunofluorescence and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization and their impact on autophagy. We found that VCP mutants induce the formation of aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterised by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP1LC3B levels inducing autophagy. Moreover, we found that wild type VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies. Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

Novel SOD1 monoclonal antibodies against the electrostatic loop preferentially detect misfolded SOD1 aggregates

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that leads to motor neuron degeneration and paralysis. Superoxide dismutase (SOD1) mutations are the second most common cause of familial ALS and are responsible for up to 20 % of familial ALS cases. In ALS patients, SOD1 can form toxic misfolded aggregates that deposit in the brain and spinal cord. To better detect SOD1 aggregates and expand the repertoire of conformational SOD1 antibodies, SOD1 monoclonal antibodies were generated by immunizing SOD1 knockout mice with an SOD1 fragment consisting of amino acids 129–146, which make up part of the electrostatic loop. A series of hybridomas secreting antibodies were screened and five different SOD1 monoclonal antibodies (2C10, 2F8, 4B11, 5H5, and 5A10) were found to preferentially detect denatured or aggregated SOD1 by enzyme-linked immunosorbent assay (ELISA), filter trap assay, and immunohistochemical analysis in SOD1 mouse models. The staining with these antibodies was compared to Campbell-Switzer argyrophilic reactivity of pathological inclusions. These new conformational selective SOD1 antibodies will be useful for clinical diagnosis of SOD1 ALS and potentially for passive immunotherapy.

Co-chaperones DNAJA1 and DNAJB6 are critical for regulation of polyglutamine aggregation

Huntington’s disease (HD) is caused by CAG repeat expansion in the huntingtin gene. The expanded polyglutamine (polyQ) repeat of the encoded protein leads to protein misfolding and aggregation, resulting in increased neuronal cell death. DNAJ co-chaperones play a crucial role in transferring misfolded/unfolded proteins to HSP70 chaperones, which play an essential role for protein folding. Here, we investigated the effect of knock out (KO) of three individual DNAJ genes in HEK293 cells expressing polyglutamine74exon1 huntingtin (polyQ74htt). Flourescence microscopy analysis revealed that KO of DNAJB6 resulted in a 5-fold increase in polyQ74htt aggregation and that DNAJA1 KO resulted in a 4-fold decrease of polyQ74htt aggregation. KO of DNAJB1 did not change the propensity of polyQ74htt to aggregate in cells. These findings where confirmed both by fluorescence microscopy analysis and filter trap assay (FTA). DNAJB6 KO cells displayed an increased rate of cell death as assessed by trypan blue exclusion and propidium iodide (PI) uptake assays. These results demonstrate that the DNAJ proteins DNAJA1 and DNAJB6 can modulate polyQ aggregation in opposite manners, and thus that fine-tuning the cellular levels of DNAJ proteins is critical for suppression of polyQ aggregation and cell survival.

FAIM Opposes Aggregation of Mutant SOD1 That Typifies Some Forms of Familial Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative illness that is unremittingly fatal and for which no effective treatment exists. All forms of ALS are characterized by protein aggregation. In familial forms of ALS, specific and heritable aggregation-prone proteins have been identified, such as mutant superoxide dismutase (SOD1). It has been suggested that a factor capable of preventing mutant SOD1 protein aggregation and/or disassembling mutant SOD1 protein aggregates would ameliorate SOD1-associated forms of familial ALS. Here we identify Fas Apoptosis Inhibitory Molecule (FAIM), a highly evolutionarily conserved 20 kDa protein, as an agent with this activity. We show FAIM counteracts intracellular accumulation of mutant SOD1 protein aggregates, which is increased in the absence of FAIM, as determined by pulse-shape analysis and filter trap assays. In a cell-free system, FAIM inhibits aggregation of mutant SOD1, and further disassembles and solubilizes established mutant SOD1 protein aggregates, as determined by thioflavin T (ThT), filter trap, and sedimentation assays. In sum, we report here a previously unknown activity of FAIM that opposes ALS disease-related protein aggregation and promotes proteostasis of an aggregation-prone ALS protein.

Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples.

Optimizing filter trap assay for the detection of aggregated alpha-synuclein in brain samples.

Mutations in the J domain of DNAJB6 cause dominant distal myopathy

Eight patients from five families with undiagnosed dominant distal myopathy underwent clinical, neurophysiological and muscle biopsy examinations. Molecular genetic studies were performed using targeted sequencing of all known myopathy genes followed by segregation of the identified mutations in the affected families using Sanger sequencing. Two novel mutations in DNAJB6 J domain, c.149C>T (p.A50V) and c.161A>C (p.E54A), were identified as the cause of disease. The muscle involvement with p.A50V was distal calf-predominant, and the p.E54A was more proximo-distal. Histological findings were similar to those previously reported in DNAJB6 myopathy. In line with reported pathogenic mutations in the glycine/phenylalanine (G/F) domain of DNAJB6, both the novel mutations showed reduced anti-aggregation capacity by filter trap assay and TDP-43 disaggregation assays. Modeling of the protein showed close proximity of the mutated residues with the G/F domain. Myopathy-causing mutations in DNAJB6 are not only located in the G/F domain, but also in the J domain. The identified mutations in the J domain cause dominant distal and proximo-distal myopathy, confirming that mutations in DNAJB6 should be considered in distal myopathy cases.

LGMD AUTOSOMAL RESSESSIVE AND DOMINANT: P.108Functional characterization of DNAJB6 J-domain mutations

Limb-girdle muscular dystrophy type 1D (LGMD1D) is caused by dominant mutations in DNAJB6. This ubiquitously expressed co-chaperone interacts with the HSPA (Hsp70) chaperones and promotes their function, and also acts as a powerful suppressor or protein aggregation. Until recently, all of the known LGMD1D-causing mutations in DNAJB6 were located relatively close to each other within the glycine/phenylalanine-rich (GF) domain of the protein. This domain, whose function remains poorly understood, links the HSPA-interacting J domain and the substrate-binding C-terminal domain. The mutations impair the anti-aggregation activity of DNAJB6 and confer a dominant toxic effect to the cytoplasmic isoform DNAJB6 isoform (DNAJB6b). Some GF-domain mutations have also been shown to interfere with the turnover of DNAJB6; however, not all mutations have been characterized in this respect. The first LGMD1D patients with missense variants in the J domain of DNAJB6 have now been identified. To evaluate their pathogenicity and to gain further insight into the molecular pathomechanisms of LGMD1D, we performed functional studies on two putative disease-causing J-domain variants, p.Ala50Val and p.Glu54Ala. The nearby, presumably harmless variant p.Ser57Leu served as control. In filter trap assay, DNAJB6b constructs with disease-associated variants showed impaired anti-aggregation activity towards polyQ-huntingtin, supporting the causative role of the variants and highlighting the importance of the anti-aggregation defect in the pathogenesis of LGMD1D. Protein turnover studies indicated a reduced turnover rate for p.Ala50Val but not for p.Glu54Ala, suggesting that impaired turnover may not be a universal feature of pathogenic DNAJB6 variants.

FYCO1 mediates clearance of α-synuclein aggregates through a Rab7-dependent mechanism.

Parkinson's disease can be caused by mutations in the α-synuclein gene and is characterized by aggregates of α-synuclein protein. We have previously shown that over-expression of the small GTPase Rab7 can induce clearance of α-synuclein aggregates. In this study, we investigate which Rab7 effectors mediate this effect. To model Parkinson's disease, we expressed the pathogenic A53T mutant of α-synuclein in HEK293T cells and Drosophila melanogaster. We tested the Rab7 effectors FYVE and coiled-coil domain-containing protein 1 (FYCO1) and Rab-interacting lysosomal protein (RILP). FYCO1-EGFP-decorated vesicles containing α-synuclein. RILP-EGFP also decorated vesicular structures, but they did not contain α-synuclein. FYCO1 over-expression reduced the number of cells with α-synuclein aggregates, defined as visible particles of EGFP-tagged α-synuclein, whereas RILP did not. FYCO1 but not RILP reduced the amount of α-synuclein protein as assayed by western blot, increased the disappearance of α-synuclein aggregates in time-lapse microscopy and decreased α-synuclein-induced toxicity assayed by the Trypan blue assay. siRNA-mediated knockdown of FYCO1 but not RILP reduced Rab7-induced aggregate clearance. Collectively, these findings indicate that FYCO1 and not RILP mediates Rab7-induced aggregate clearance. The effect of FYCO1 on aggregate clearance was blocked by dominant negative Rab7 indicating that FYCO1 requires active Rab7 to function. Electron microscopic analysis and insertion of lysosomal membranes into the plasma membrane indicate that FYCO1 could lead to secretion of α-synuclein aggregates. Extracellular α-synuclein as assayed by ELISA was, however, not increased with FYCO1. Coexpression of FYCO1 in the fly model decreased α-synuclein aggregates as shown by the filter trap assay and rescued the locomotor deficit resulting from neuronal A53T-α-synuclein expression. This latter finding confirms that a pathway involving Rab7 and FYCO1 stimulates degradation of α-synuclein and could be beneficial in patients with Parkinson's disease. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species.

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution. For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

Measurement of Chaperone-Mediated Effects on Polyglutamine Protein Aggregation by the Filter Trap Assay.

The formation of aggregates by polyglutamine-containing (polyQ) proteins in neurons is a key to the pathogenesis of several progressive neurodegenerative diseases such as Huntington's disease (HD) spinocerebellar ataxias (SCAs), and spinal and bulbar muscular atrophy (SBMA). In order to study whether the members of the heat shock protein (HSP) families, by virtue of their molecular chaperone activity, can inhibit the formation of polyQ aggregates, we developed a cell culture model expressing the GFP tagged fragment of exon1 of the huntingtin gene with an expanded polyQ chain and tetracycline inducible chaperones. Expression of mutated Huntington's protein leads to the formation of 2% SDS insoluble high molecular weight polyQ aggregates that are retarded on a cellulose acetate membrane in the so-called filter trap assay (FTA). This chapter explains in detail the protocols of the FTA and how it can be a useful tool to study the effect of HSPs or their functional mutants on aggregation of polyglutamine proteins. Moreover, the assay is useful to investigate how externally added polyQ peptides can act as nucleation seeds for internally expressed polyQ proteins.

THE ROLE OF NUB1 IN THE CLEARANCE OF TAU AGGREGATES IN ALZHEIMER'S DISEASE

BACKGROUND: Intraneuronal aggregations of abnormal hyperphosphorylated tau are a hallmark of Alzheimer’s disease. Previous data revealed that NEDD8 ultimate buster 1 (NUB1) reduces the aggregation and phosphorylation of tau in an in vitro model. NUB1 is a ubiquitin-like (UBL)/ ubiquitin-associated (UBA) protein that targets the down regulation of ubiquitin-like modifiers (NEDD8, FAT10), and aggregation-prone proteins leading to neurodegenerative disease (synphilin-1, huntingtin). The aim of the study is to understand how NUB1 affects tau aggregation and phosphorylation. METHODS: A GFP-tau inducible neuroblastoma cell line was generated and treated with proteasome inhibitor to induce the formation of aggresomes. GFP-tau cells were transduced with NUB1 lentiviral vectors both in the absence and presence of proteasome inhibitor, followed by western blotting, filter trap assays, immunoprecipitation and immunofluorescence. RESULTS: Inhibition of the proteasome induced the formation of GFP-tau aggresomes that were positive for phospho-tau (p-tau) markers (AT8, pS396). Filter trap assays revealed that NUB1 significantly decreased the ratio of p-tau to tau in the total cellular fraction (both soluble and insoluble), as well as in only the detergent insoluble fraction, both with and without proteasome inhibition. However, the ratio of tau and p-tau in the insoluble fraction to that in the total fraction revealed that the NUB1-mediated decrease in the levels of detergent insoluble tau and p-tau exceeded that of the overall decline in total levels, suggesting NUB1 can target aggregation-prone tau and p-tau. This effect of NUB1 was more pronounced for p-tau following proteasome inhibition. Blocking the proteasome induced the upregulation of p62 and increased the ratio of LC3B-II to LC3B-I. NUB1 induced further upregulation of autophagy following proteasome inhibition as seen by an increase in the LC3B-II/LC3B-I ratio and changes in LC3B positive puncta in cells. Finally, GFP-tau aggresomes were positive for p62 and NUB1 overexpression promoted the interaction between GFP-tau and p62. CONCLUSIONS: These data suggest that NUB1 may play a role in the clearance of aggregation-prone tau and p-tau following inhibition of the proteasome via autophagy.

A novel DNAJB6 mutation causes dominantly inherited distal‐onset myopathy and compromises DNAJB6 function

Mutations in the DNAJB6 gene have been identified as a rare cause of dominantly inherited limb-girdle muscular dystrophy or distal-onset myopathy. Exome sequencing was performed to investigate a Taiwanese family with a dominantly inherited distal-onset myopathy. Functional effects of the causal mutation were investigated in vitro. Exome sequencing of the two affected individuals in this family identified a heterozygous mutation, c.287C>T (p.Pro96Leu) in the DNAJB6 gene, which co-segregated with the myopathy within all 12 family members. Notably, this mutation is novel and localizes within the glycine and phenylalanine-rich (G/F) domain and alters an amino acid residue previously reported with a different mutation. Furthermore, immunofluorescence analyses and filter trap assay demonstrated that the c.287C>T (p.Pro96Leu) mutation possessed a dominant negative effect on the anti-aggregation function of DNAJB6 protein. This study expands the molecular spectrum of DNAJB6 mutations and also emphasizes the pathogenic role of DNAJB6 dysfunction in distal-onset myopathy.

Taxifolin inhibits amyloid-\u03b2 oligomer formation and fully restores vascular integrity and memory in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) induces various forms of cerebral infarcts and hemorrhages from vascular amyloid-β accumulation, resulting in acceleration of cognitive impairment, which is currently untreatable. Soluble amyloid-β protein likely impairs cerebrovascular integrity as well as cognitive function in early stage Alzheimer’s disease. Taxifolin, a flavonol with strong anti-oxidative and anti-glycation activities, has been reported to disassemble amyloid-β in vitro but the in vivo relevance remains unknown. Here, we investigated whether taxifolin has therapeutic potential in attenuating CAA, hypothesizing that inhibiting amyloid-β assembly may facilitate its clearance through several elimination pathways. Vehicle- or taxifolin-treated Tg-SwDI mice (commonly used to model CAA) were used in this investigation. Cognitive and cerebrovascular function, as well as the solubility and oligomerization of brain amyloid-β proteins, were investigated. Spatial reference memory was assessed by water maze test. Cerebral blood flow was measured with laser speckle flowmetry and cerebrovascular reactivity evaluated by monitoring cerebral blood flow changes in response to hypercapnia. Significantly reduced cerebrovascular pan-amyloid-β and amyloid-β1-40 accumulation was found in taxifolin-treated Tg-SwDI mice compared to vehicle-treated counterparts (n = 5). Spatial reference memory was severely impaired in vehicle-treated Tg-SwDI mice but normalized after taxifolin treatment, with scoring similar to wild type mice (n = 10–17). Furthermore, taxifolin completely restored decreased cerebral blood flow and cerebrovascular reactivity in Tg-SwDI mice (n = 4–6). An in vitro thioflavin-T assay showed taxifolin treatment resulted in efficient inhibition of amyloid-β1-40 assembly. In addition, a filter trap assay and ELISA showed Tg-SwDI mouse brain homogenates exhibited significantly reduced levels of amyloid-β oligomers in vivo after taxifolin treatment (n = 4–5), suggesting the effects of taxifolin on CAA are attributable to the inhibition of amyloid-β oligomer formation. In conclusion, taxifolin prevents amyloid-β oligomer assembly and fully sustains cognitive and cerebrovascular function in a CAA model mice. Taxifolin thus appears a promising therapeutic approach for CAA.

NIP-SNAP-1 and -2 mitochondrial proteins are maintained by heat shock protein 60

NIP-SNAP-1 and -2 are ubiquitous proteins thought to be associated with maintenance of mitochondrial function, neuronal transmission, and autophagy. However, their physiological functions remain largely unknown. To elucidate their functional importance, we screened for proteins that interact with NIP-SNAP-1 and -2, resulting in identification of HSP60 and P62/SQSTM1 as binding proteins. NIP-SNAP-1 and -2 localized in the mitochondrial inner membrane space, whereas HSP60 localized in the matrix. Native gel electrophoresis and filter trap assays revealed that human HSP60 prevented aggregation of newly synthesized NIP-SNAP-2 in an in vitro translation system. Moreover, expression levels of NIP-SNAP-1 and -2 in cells were decreased by knockdown of HSP60, but not HSP10. These findings indicate that HSP60 promotes folding and maintains the stability of NIP-SNAP-1 and -2.