Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer’s disease (AD) has facilitated advancement of the ...amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1 . U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.
Presynaptic synthesis of acetylcholine (ACh) requires a steady supply of choline, acquired by a plasma membrane, hemicholinium-3-sensitive (HC-3) choline transporter (CHT). A significant fraction of ...synaptic choline is recovered from ACh hydrolyzed by acetylcholinesterase (AChE) after vesicular release. Although antecedent neuronal activity is known to dictate presynaptic CHT activity, the mechanisms supporting this regulation are unknown. We observe an exclusive localization of CHT to cholinergic neurons and demonstrate that the majority of CHTs reside on small vesicles within cholinergic presynaptic terminals in the rat and mouse brain. Furthermore, immunoisolation of presynaptic vesicles with multiple antibodies reveals that CHT-positive vesicles carry the vesicular acetylcholine transporter (VAChT) and synaptic vesicle markers such as synaptophysin and Rab3A and also contain acetylcholine. Depolarization of synaptosomes evokes a Ca2+-dependent botulinum neurotoxin C-sensitive increase in the Vmax for HC-3-sensitive choline uptake that is accompanied by an increase in the density of CHTs in the synaptic plasma membrane. Our study leads to the novel hypothesis that CHTs reside on a subpopulation of synaptic vesicles in cholinergic terminals that can transit to the plasma membrane in response to neuronal activity to couple levels of choline re-uptake to the rate of ACh release.
The redox-sensitive transcription factor NF-κB mediates the expression of genes involved in inflammation and cell survival. Thioredoxin reductase-1 (TR1) and its substrate thioredoxin-1 act together ...to reduce oxidized cysteine residues within the DNA-binding domain of NF-κB and promote maximal DNA-binding activity in vitro. It is not clear, however, if NF-κB is regulated via this mechanism within living cells. The purpose of this study was to determine the mechanism of NF-κB modulation by TR1 in cells stimulated with the inflammatory cytokine tumor necrosis factor-α (TNF). In both control cells and cells depleted of TR1 activity through chemical inhibition or siRNA knockdown, TNF stimulation resulted in degradation of the cytoplasmic NF-κB inhibitor IκB-α and translocation of NF-κB to the nucleus. Similarly, the DNA-binding activity and redox state of NF-κB were unaffected by TR1 depletion. In contrast, NF-κB-mediated gene expression was markedly inhibited in cells lacking TR1 activity, suggesting that the transactivation potential of NF-κB is sensitive to changes in TR1 activity. Consistent with this concept, phosphorylation of the transactivation domain of NF-κB was inhibited in the presence of curcumin. Surprisingly, another TR1 inhibitor, 1-chloro-2,4-dinitrobenzene, had no effect, and siRNA knockdown of TR1 actually increased phosphorylation at this site. These results demonstrate that TR1 activity controls the transactivation potential of NF-κB and that more than one mechanism may mediate this effect.
Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate ...that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis.
► Components of the A20 ubiquitin-editing complex are present in the human brain. ► A20 complex components are mainly expressed in neurons. ► A20 complex components are differentially expressed in ...various regions of the human brain.
Activation of innate and adaptive immune responses is tightly regulated, as insufficient activation could result in defective clearance of pathogens, while excessive activation might lead to lethal systemic inflammation or autoimmunity. A20 functions as a negative regulator of innate and adaptive immunity by inhibiting NF-κB activation. A20 mediates its inhibitory function in a complex with other proteins including RNF11 and Itch, both E3 ubiquitin ligases and TAX1BP1, an adaptor protein. Since NF-κB has been strongly implicated in various neuronal functions, we predict that its inhibitor, the A20 complex, is also present in the nervous system. In efforts to better understand the role of A20 complex and NF-κB signaling pathway, we determined regional distribution of A20 mRNA as well as protein expression levels and distribution of RNF11, TAX1BP1 and Itch, in different brain regions. The distribution of TRAF6 was also investigated since TRAF6, also an E3 ligase, has an important role in NF-κB signaling pathway. Our investigations, for the first time, describe and demonstrate that the essential components of the A20 ubiquitin-editing complex are present and mainly expressed in neurons. The A20 complex components are also differentially expressed throughout the human brain. This study provides useful information about region specific expression of the A20 complex components that will be invaluable while determining the role of NF-κB signaling pathway in neuronal development and degeneration.
Detergent-insoluble protein accumulation and aggregation in the brain is one of the pathological hallmarks of neurodegenerative diseases. Here, we describe the identification of septin 11 (SEPT11), ...an enriched component of detergent-resistant fractions in frontotemporal lobar degeneration with ubiquitin-immunoreactive inclusions (FTLD-U), using large-scale unbiased proteomics approaches.
We developed and applied orthogonal quantitative proteomic strategies for the unbiased identification of disease-associated proteins in FTLD-U. Using these approaches, we proteomically profiled detergent-insoluble protein extracts prepared from frontal cortex of FTLD-U cases, unaffected controls, or neurologic controls (i.e. Alzheimer's disease; AD). Among the proteins altered specifically in FTLD-U, we identified TAR DNA binding protein-43 (TDP-43), a known component of ubiquitinated inclusions. Moreover, we identified additional proteins enriched in detergent-resistant fractions in FTLD-U, and characterized one of them, SEPT11, in detail. Using independent highly sensitive targeted proteomics approaches, we confirmed the enrichment of SEPT11 in FTLD-U extracts. We further showed that SEPT11 is proteolytically cleaved into N-terminal fragments and, in addition to its prominent glial localization in normal brain, accumulates in thread-like pathology in affected cortex of FTLD-U patients.
The proteomic discovery of insoluble SEPT11 accumulation in FTLD-U, along with novel pathological associations, highlights a role for this cytoskeleton-associated protein in the pathogenesis of this complex disorder.
Several genes have been implicated in the pathogenesis of early-onset familial Alzheimer's disease. A majority of the autosomal dominant cases are linked to recently identified mutations in the ...presenilin-1 gene on chromosome 14. The native presenilin-1 protein in primates has not been well characterized, and its precise localization is unknown. We have studied the native presenilin-1 protein in monkey brain and peripheral tissues by using a monoclonal antibody specific for the N-terminal domain of human presenilin-1. Western blots detect polypeptide species of approximately 49 and approximately 32 kDa from COS-7 and PC12 cells transfected with full-length human presenilin-1 cDNA and from in vitro translations of the normal human presenilin-1 mRNA. A 32 kDa polypeptide is detected in monkey peripheral tissues, with the highest expression in testis and lung. In all brain regions the 32 kDa band is the predominant form of presenilin-1, and it is found in particulate subfractions. Light microscopic immunocytochemistry reveals presenilin-1 staining in all brain regions, with the strongest labeling in neurons and neuropil. In addition, weaker immunoreactivity is also present in glia and blood vessels. Neuronal staining shows significant variability, with particularly intense labeling of certain cell types, including large neocortical and hippocampal pyramidal neurons, magnocellular basal forebrain neurons, brainstem motoneurons, and some populations of interneurons. By electron microscopic immunocytochemistry, highly selective presenilin-1 staining is seen on the cytoplasmic surfaces of membranous organelles, which suggest localization to the endoplasmic reticulum-Golgi intermediate compartment, a subdomain of the endoplasmic reticulum, and some coated transport vesicles.
The plasma membrane dopamine transporter (DAT) is considered to be a reliable marker of presynaptic dopaminergic terminal loss. Previous in vivo imaging and postmortem binding studies have detected a ...loss in striatal DAT binding in Parkinson's diseased (PD) brain; however, these techniques have poor spatial resolution and may suffer from nonspecific binding of some ligands. In this study, we use novel highly specific monoclonal antibodies to distinct epitopes of human DAT to quantify and localize the protein. Western blot analysis revealed marked reductions in DAT immunoreactivity in putamen, caudate, and nucleus accumbens of PD brain compared with control cases, and the reductions were significantly correlated to disease duration. Immunohistochemistry revealed DAT‐immunoreactive fibers and puncta that were dense throughout the striatum of control brains but that were drastically reduced in putamen of PD brains. Caudate from PD brains showed a significant degree of sparing along the border of the ventricle, and the nucleus accumbens was relatively preserved. An unexpected finding was that discrete islands of DAT immunoreactivity were preserved within the matrix of PD putamen. Thus, immunological analysis of DAT protein provides novel and sensitive means for localizing and quantifying DAT protein in PD and other neurological disorders involving dopaminergic systems.
The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntingtin. We used polyclonal and ...monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cell lines from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.
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