ABSTRACT
Neuropathological features of Alzheimer's disease include amyloid plaques, neurofibrillary tangles and Lewy bodies, with the former preceding the latter two. However, it is not fully understood how these compound proteinopathies are interconnected. Here, we show that transplantation of amyloid-ß oligomer-activated microglia into the striatum of naïve mice was sufficient to generate all the features of Alzheimer's disease, including widespread tauopathy and synucleinopathy, gliosis, neuroinflammation, synapse loss, neuronal death, and cognitive and motor deficits. These pathological features were eliminated by microglia depletion and anti-inflammatory drug administration. Our results suggest the crucial roles of microglia-driven inflammation in development of mixed pathology. This study provides not only mechanistic insights into amyloid-ß oligomer-triggered proteinopathies but also a novel animal model recapitulating the salient features of Alzheimer's disease.
ABSTRACT
We recently developed a multiplex diagnostic kit, QPLEX™ Alz plus assay kit, which captures amyloid-ß1-40, galectin-3 binding protein, angiotensin-converting enzyme, and periostin simultaneously using microliters of peripheral blood and utilizes an optimized algorithm for screening Alzheimer's disease (AD) by correlating with cerebral amyloid deposition. Owing to the demand for early AD detection, we investigate the potential of our kit for the early clinical diagnosis of AD. A total of 1395 participants were recruited, and their blood samples were analyzed with the QPLEX™ kit. The average of QPLEX™ algorithm values in each group increased gradually in the order of the clinical progression continuum of AD: cognitively normal (0.382 ± 0.150), subjective cognitive decline (0.452 ± 0.130), mild cognitive impairment (0.484 ± 0.129), and AD (0.513 ± 0.136). The algorithm values between each group showed statistically significant differences among groups divided by Mini-Mental State Examination and Clinical Dementia Rating. The QPLEX™ algorithm values could be used to distinguish the clinical continuum of AD or cognitive function. Because blood-based diagnosis is more accessible, convenient, and cost- and time-effective than cerebral spinal fluid or positron emission tomography imaging-based diagnosis, the QPLEX™ kit can potentially be used for health checkups and the early clinical diagnosis of AD.
Subject(s)
Alzheimer Disease , Cognition Disorders , Cognitive Dysfunction , Humans , Alzheimer Disease/metabolism , Neuropsychological Tests , Cognitive Dysfunction/complications , Cognition , Cognition Disorders/etiology , Positron-Emission Tomography , Amyloid beta-Peptides/metabolism , Biomarkers , Disease ProgressionABSTRACT
Phenylketonuria (PKU) is a common genetic metabolic disorder that causes phenylalanine accumulation in the blood. The most serious symptoms are related to the brain, as intellectual disability, seizure, and microcephaly are commonly found in poorly treated PKU patients and the babies of maternal PKU. However, the mechanism of hyperphenylalaninemia on human neurodevelopment is still unclear. Here we utilized human induced pluripotent stem cell (iPSC)-derived cerebral organoids to investigate the neurotoxicity of hyperphenylalaninemia. Cerebral organoids at days 40 or 100 were treated with different concentrations of phenylalanine for 5 days. After phenylalanine treatments, the cerebral organoids displayed alterations in organoid size, induction of apoptosis, and depletion of neural progenitor cells. However, phenylalanine did not have an impact on neurons and glia, including astrocytes, immature oligodendrocytes, and mature oligodendrocytes. Remarkably, a reduction in the thickness of the cortical rosettes and a decrease in myelination at the intermediate zone were inspected with the elevated phenylalanine concentrations. RNA-seq of phenylalanine-treated organoids revealed that gene sets related to apoptosis, p53 signaling pathway, and TNF signaling pathway via NF-kB were enriched in upregulated genes, while those related to cell cycle and amino acid metabolism were enriched in downregulated genes. In addition, there were several microcephaly disease genes, such as ASPM, LMNB1, and CENPE, ranked at the top of the downregulated genes. These findings indicate that phenylalanine exposure may contribute to microcephaly, abnormal cortical expansion, and myelination lesions in the developing human brain.
Subject(s)
Induced Pluripotent Stem Cells , Microcephaly , Phenylketonuria, Maternal , Phenylketonurias , Female , Humans , Microcephaly/genetics , Organoids/pathology , Phenylalanine , Phenylketonurias/diagnosis , PregnancyABSTRACT
BACKGROUND INFORMATION: Aberrant production of amyloid beta (Aß) causes disruption of intracellular calcium homeostasis, a crucial factor in the pathogenesis of Alzheimer's disease. Calcium is required for the fusion and trafficking of vesicles. Previously, we demonstrated that Sec31A, a main component for coat protein complex II (COPII) vesicles at ER exit sites (ERES), is modulated by O-GlcNAcylation. O-GlcNAcylation, a unique and dynamic protein glycosylation process, modulates the formation of COPII vesicles. RESULTS: In this study, we observed that disrupted calcium levels affected the formation of COPII vesicles in ERES through calcium-triggered O-GlcNAcylation of Sec31A. Additionally, we found that Aß impaired ERES through Aß-disturbed calcium homeostasis and O-GlcNAcylation of Sec31A in neuronal cells. Furthermore, we identified that Aß disrupted the ribbon-like structure of Golgi. Golgi fragmentation by Aß was rescued by up-regulation of O-GlcNAcylaion levels using Thiamet G (ThiG), an O-GlcNAcase inhibitor. Additionally, we observed that the Golgi reassembly stacking proteins having a function in Golgi stacking showed attenuation at COPII vesicles following Aß treatment. CONCLUSIONS: This study demonstrated that Aß impaired Sec31A targeting to ERES through altered Sec31A O-GlcNAcylation triggered by disruption of intracellular calcium homeostasis. SIGNIFICANCE: The findings of this study suggested that protection of ERES or Sec31 O-GlcNAcylation may offer a promising novel avenue for development of AD therapeutics.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Apoptosis Regulatory Proteins/metabolism , Calcium-Binding Proteins/metabolism , Calcium/metabolism , Endoplasmic Reticulum/metabolism , Vesicular Transport Proteins/metabolism , COP-Coated Vesicles/metabolism , HeLa Cells , Humans , Protein Processing, Post-Translational , Protein TransportABSTRACT
OBJECTIVE: Cerebral amyloidosis and severe tauopathy in the brain are key pathological features of Alzheimer's disease (AD). Despite a strong influence of the intestinal microbiota on AD, the causal relationship between the gut microbiota and AD pathophysiology is still elusive. DESIGN: Using a recently developed AD-like pathology with amyloid and neurofibrillary tangles (ADLPAPT) transgenic mouse model of AD, which shows amyloid plaques, neurofibrillary tangles and reactive gliosis in their brains along with memory deficits, we examined the impact of the gut microbiota on AD pathogenesis. RESULTS: Composition of the gut microbiota in ADLPAPT mice differed from that of healthy wild-type (WT) mice. Besides, ADLPAPT mice showed a loss of epithelial barrier integrity and chronic intestinal and systemic inflammation. Both frequent transfer and transplantation of the faecal microbiota from WT mice into ADLPAPT mice ameliorated the formation of amyloid ß plaques and neurofibrillary tangles, glial reactivity and cognitive impairment. Additionally, the faecal microbiota transfer reversed abnormalities in the colonic expression of genes related to intestinal macrophage activity and the circulating blood inflammatory monocytes in the ADLPAPT recipient mice. CONCLUSION: These results indicate that microbiota-mediated intestinal and systemic immune aberrations contribute to the pathogenesis of AD in ADLPAPT mice, providing new insights into the relationship between the gut (colonic gene expression, gut permeability), blood (blood immune cell population) and brain (pathology) axis and AD (memory deficits). Thus, restoring gut microbial homeostasis may have beneficial effects on AD treatment.
Subject(s)
Alzheimer Disease/microbiology , Alzheimer Disease/therapy , Fecal Microbiota Transplantation , Gastrointestinal Microbiome/physiology , Alzheimer Disease/metabolism , Animals , Behavior, Animal , Chronic Disease , Disease Models, Animal , Humans , Inflammation/microbiology , Intestines/microbiology , Memory, Short-Term , Mice, Transgenic , Permeability , Plaque, Amyloid/microbiology , Plaque, Amyloid/pathology , Spatial Learning , tau Proteins/analysisABSTRACT
One of the hallmarks of Alzheimer's disease is abnormal deposition of tau proteins in the brain. Although plasma tau has been proposed as a potential biomarker for Alzheimer's disease, a direct link to brain deposition of tau is limited. Here, we estimated the amount of in vivo tau deposition in the brain by PET imaging and measured plasma levels of total tau (t-tau), phosphorylated tau (p-tau, T181) and amyloid-ß1-42. We found significant correlations of plasma p-tau, t-tau, p-tau/amyloid-ß1-42, and t-tau/amyloid-ß1-42 with brain tau deposition in cross-sectional and longitudinal manners. In particular, t-tau/amyloid-ß1-42 in plasma was highly predictive of brain tau deposition, exhibiting 80% sensitivity and 91% specificity. Interestingly, the brain regions where plasma t-tau/amyloid-ß1-42 correlated with brain tau were similar to the typical deposition sites of neurofibrillary tangles in Alzheimer's disease. Furthermore, the longitudinal changes in cerebral amyloid deposition, brain glucose metabolism, and hippocampal volume change were also highly associated with plasma t-tau/amyloid-ß1-42. These results indicate that combination of plasma tau and amyloid-ß1-42 levels might be potential biomarkers for predicting brain tau pathology and neurodegeneration.
Subject(s)
Amyloid beta-Peptides/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Aged , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid/metabolism , Amyloid beta-Peptides/analysis , Amyloid beta-Peptides/blood , Amyloidosis/pathology , Biomarkers/blood , Biomarkers/metabolism , Brain/pathology , Cognitive Dysfunction/metabolism , Cross-Sectional Studies , Female , Humans , Male , Neurodegenerative Diseases/pathology , Neurofibrillary Tangles/pathology , Neuropsychological Tests , Positron-Emission Tomography/methods , Prognosis , tau Proteins/analysis , tau Proteins/bloodABSTRACT
The conventional secretory pathway is indispensable for eukaryotic cells. Newly synthesized membrane and secretory proteins are released from the endoplasmic reticulum (ER) through ER-derived vesicles to their appropriate destination. Vesicle formation is important for steady protein trafficking. O-GlcNAcylation ( O-GlcNAc) is a unique protein glycosylation signature, whose dynamic regulation by O-GlcNAc transferase and O-GlcNAcase occurs exclusively for nuclear and cytoplasmic proteins. Because of this locally limited property, the role of O-GlcNAc in the conventional protein secretory pathway is unknown. We report that Sec31A on COPII vesicles, a specific coat-protein complex for anterograde trafficking in the ER-Golgi network, is O-GlcNAcylated on S964, which accelerates COPII vesicle formation through control of its binding affinity to apoptosis-linked gene 2, a calcium-binding protein. Together, O-GlcNAc on Sec31A regulates conventional secretory vesicle trafficking in the ER-Golgi network. These modifications accelerate COPII vesicle formation and accelerated anterograde transport of vesicles within the ER-Golgi networks.-Cho, H. J., Mook-Jung, I. O-GlcNAcylation regulates endoplasmic reticulum exit sites through Sec31A modification in conventional secretory pathway.
Subject(s)
Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Protein Transport/physiology , Secretory Pathway/physiology , Apoptosis Regulatory Proteins/genetics , Calcium-Binding Proteins/metabolism , Humans , Protein Binding/physiology , Vesicular Transport Proteins/metabolismABSTRACT
The aggregation of amyloid beta (Aß) proteins in senile plaques is a critical event during the development of Alzheimer's disease, and the postmortem detection of Aß-rich proteinaceous deposits through fluorescent staining remains one of the most robust diagnostic tools. In animal models, fluorescence imaging can be employed to follow the progression of the disease, and among the different imaging methods, two-photon microscopy (TPM) has emerged as one of the most powerful. To date, several near-infrared-emissive two-photon dyes with a high affinity for Aß fibrils have been developed, but there has often been a tradeoff between excellent two-photon cross-sections and large fluorescence signal-to-background ratios. In the current work, we introduced a twisted intramolecular charge state (TICT)-based de-excitation pathway, which results in a remarkable fluorescence increase of around 167-fold in the presence of Aß fibrils, while maintaining an excellent two-photon cross section, thereby enabling high-contrast exâ vivo and inâ vivo TPM imaging. Overall, the results suggest that adopting TICT de-excitation in two-photon fluorophores may represent a general method to overcome the tradeoff between probe brightness and signal-to-background ratio.
Subject(s)
Fluorescent Dyes/metabolism , Optical Imaging/methods , Plaque, Amyloid/metabolism , HumansABSTRACT
Glycosylation with O-linked ß-N-acetylglucosamine (O-GlcNAc) is one of the protein glycosylations affecting various intracellular events. However, the role of O-GlcNAcylation in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. Mitochondrial adenosine 5'-triphosphate (ATP) synthase is a multiprotein complex that synthesizes ATP from ADP and Pi. Here, we found that ATP synthase subunit α (ATP5A) was O-GlcNAcylated at Thr432 and ATP5A O-GlcNAcylation was decreased in the brains of AD patients and transgenic mouse model, as well as Aß-treated cells. Indeed, Aß bound to ATP synthase directly and reduced the O-GlcNAcylation of ATP5A by inhibition of direct interaction between ATP5A and mitochondrial O-GlcNAc transferase, resulting in decreased ATP production and ATPase activity. Furthermore, treatment of O-GlcNAcase inhibitor rescued the Aß-induced impairment in ATP production and ATPase activity. These results indicate that Aß-mediated reduction of ATP synthase activity in AD pathology results from direct binding between Aß and ATP synthase and inhibition of O-GlcNAcylation of Thr432 residue on ATP5A.
Subject(s)
Alzheimer Disease/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , N-Acetylglucosaminyltransferases/metabolism , Oxidative Phosphorylation Coupling Factors/metabolism , Acetylglucosamine/metabolism , Adenosine Triphosphate/metabolism , Alzheimer Disease/enzymology , Alzheimer Disease/genetics , Animals , CHO Cells , Cricetulus , Disease Models, Animal , Glycosylation , HeLa Cells , Humans , Mice , Mice, Transgenic , Mitochondria/enzymology , Mitochondria/metabolism , Mitochondrial Proton-Translocating ATPases/genetics , Oxidative Phosphorylation Coupling Factors/genetics , Protein Processing, Post-Translational , beta-N-Acetylhexosaminidases/metabolismABSTRACT
Alzheimer's disease (AD) is the most common form of dementia; however, at the present time there is no disease-modifying drug for AD. There is increasing evidence supporting the role of lipid changes in the process of normal cognitive aging and in the etiology of age-related neurodegenerative diseases. AD is characterized by the presence of intraneuronal protein clusters and extracellular aggregates of ß-amyloid (Aß). Disrupted Aß kinetics may activate intracellular signaling pathways, including tau hyperphosphorylation and proinflammatory pathways. We analyzed and visualized the lipid profiles of mouse brains using MALDI-TOF MS. Direct tissue analysis by MALDI-TOF imaging MS (IMS) can determine the relative abundance and spatial distribution of specific lipids in different tissues. We used 5XFAD mice that almost exclusively generate and rapidly accumulate massive cerebral levels of Aß-42 (1). Our data showed changes in lipid distribution in the mouse frontal cortex, hippocampus, and subiculum, where Aß plaques are first generated in AD. Our results suggest that MALDI-IMS is a powerful tool for analyzing the distribution of various phospholipids and that this application might provide novel insight into the prediction of disease.
Subject(s)
Alzheimer Disease/metabolism , Phospholipids/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Brain/metabolism , Disease Models, Animal , Hippocampus/metabolism , Humans , Mice , Mice, Transgenic , Phospholipids/analysis , Phospholipids/chemistry , Presenilin-1/geneticsABSTRACT
Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid ß (Aß) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain-resident macrophages, are also found surrounding Aß plaques. The study of the brain of AD mouse models revealed that Aß plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aß plaque formation, growth and the mechanisms by which microglia contribute to Aß plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aß plaque formation and their growth in the brain of 5XFAD mice, the Aß-overexpressing AD transgenic mouse model, and performed serial intravital two-photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia-specific GFP-expressing CX3CR1GFP/GFP mice. We found that activated microglia surrounding Aß plaques take up Aß, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aß into the extracellular space, which contributes to Aß plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aß plaques by causing microglial cell death in the brain. GLIA 2016;64:2274-2290.
Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Brain/metabolism , Brain/pathology , Microglia/pathology , Plaque, Amyloid/metabolism , Age Factors , Alzheimer Disease/genetics , Amyloid beta-Peptides/pharmacology , Amyloid beta-Protein Precursor/genetics , Animals , CX3C Chemokine Receptor 1/genetics , CX3C Chemokine Receptor 1/metabolism , Cell Death/genetics , Cell Line , Cells, Cultured , Cytokines/metabolism , Disease Models, Animal , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Mice , Mice, Transgenic , Microglia/drug effects , Mutation/genetics , Nerve Tissue Proteins/metabolism , Plaque, Amyloid/pathology , Presenilin-1/geneticsABSTRACT
Apolipoprotein E (ApoE) polymorphism has been appreciated as a valuable predictor of Alzheimer disease (AD), and the associated ε4 allele has been recognized as an indicator of susceptibility to this disease. However, serum ApoE levels have been a controversial issue in AD, due to the great variability regarding the different target detection methods, ethnicity, and the geographic variations of cohorts. The aim of this study was to validate serum ApoE levels in relation to AD, particularly using two distinct detection methods, liquid chromatography-selected reaction monitoring (SRM) mass spectrometry and microsphere-based fluorescence-activated cell sorting (FACS) analysis, to overcome experimental variations. Also, comparison of serum ApoE levels was performed between the level of protein detection by FACS and peptide level by SRM in both control and AD patients. Results from the two detection methods were cross-confirmed and validated. Both methods produced fairly consistent results, showing a significant decrease of serum ApoE levels in AD patients relative to those of a control cohort (43 control versus 45 AD, p < 0.0001). Significant correlation has been revealed between results from FACS and SRM (p < 0.0001) even though lower serum ApoE concentration values were measured in protein by FACS analysis than in peptide-level detections by SRM. Correlation study suggested that a decrease of the serum ApoE level in AD is related to the mini-mental state exam score in both results from different experimental methods, but it failed to show consistent correlation with age, gender, or clinical dementia rating.
Subject(s)
Alzheimer Disease/blood , Apolipoproteins E/blood , Blood Chemical Analysis/methods , Flow Cytometry/methods , Mass Spectrometry/methods , Aged , Aged, 80 and over , Amino Acid Sequence , Case-Control Studies , Down-Regulation , Female , Humans , Male , Microspheres , Middle Aged , Molecular Sequence Data , Reproducibility of ResultsABSTRACT
Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by ß-amyloid (Aß) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aß and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aß42-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aß-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aß42 oligomers (or aggregates) or the products of αS incubated without Aß42 have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aß and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction.
Subject(s)
Amyloid beta-Peptides/physiology , Cytoplasmic Vesicles/physiology , Membrane Fusion , SNARE Proteins/antagonists & inhibitors , alpha-Synuclein/physiology , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Animals , Exocytosis/genetics , Humans , Membrane Fusion/genetics , PC12 Cells , Protein Binding/genetics , Protein Multimerization/physiology , Rats , SNARE Proteins/metabolism , Synapses/genetics , Synapses/metabolism , Transfection , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
Fluorescence imaging of tissues offer an essential means for studying biological systems. Autofluorescence becomes a serious issue in tissue imaging under excitation at UV-vis wavelengths where biological molecules compete with the fluorophore. To address this critical issue, a novel class of fluorophores that can be excited at â¼900 nm under two-photon excitation conditions and emits in the red wavelength region (≥600 nm) has been disclosed. The new π-extended dipolar dye system shows several advantageous features including minimal autofluorescence in tissue imaging and pronounced solvent-sensitive emission behavior, compared with a widely used two-photon absorbing dye, acedan. As an important application of the new dye system, one of the dyes was developed into a fluorescent probe for amyloid-ß plaques, a key biomarker of Alzheimer's disease. The probe enabled in vivo imaging of amyloid-ß plaques in a disease-model mouse, with negligible background signal. The new dye system has great potential for the development of other types of two-photon fluorescent probes and tags for imaging of tissues with minimal autofluorescence.
Subject(s)
Alzheimer Disease/diagnosis , Amyloid beta-Peptides/analysis , Fluorescent Dyes/chemistry , Optical Imaging , Photons , Animals , Cell Line, Tumor , Cell Survival , Disease Models, Animal , Fluorescent Dyes/chemical synthesis , Humans , Mice , Mice, Transgenic , Molecular StructureABSTRACT
The functional roles of the orphan nuclear receptor, Nurr1, have been extensively studied and well established in the development and survival of midbrain dopamine neurons. As Nurr1 and other NR4A members are widely expressed in the brain in overlapping and distinct manners, it has been an open question whether Nurr1 has important function(s) in other brain areas. Recent studies suggest that up-regulation of Nurr1 expression is critical for cognitive functions and/or long-term memory in forebrain areas including hippocampal formation. Questions remain about the association between Nurr1 expression and Alzheimer's disease (AD) brain pathology. Here, using our newly developed Nurr1-selective antibody, we report that Nurr1 protein is prominently expressed in brain areas with Aß accumulation, that is, the subiculum and the frontal cortex, in the 5XFAD mouse and that Nurr1 is highly co-expressed with Aß at early stages. Furthermore, the number of Nurr1-expressing cells significantly declines in the 5XFAD mouse in an age-dependent manner, accompanied by increased plaque deposition. Thus, our findings suggest that altered expression of Nurr1 is associated with AD progression. Using our newly developed Nurr1-selective antibody, we show that Nurr1 protein is prominently expressed in brain areas accumulating amyloid-beta (Aß) in the transgenic mouse model of Alzheimer's disease (AD) and that Nurr1 is highly co-expressed with Aß at early stages (upper panel). Furthermore, in the AD brain the number of Nurr1-expressing cells significantly declines in an age-dependent manner concomitant with increased Aß accumulation (lower diagram) highlighting a possible Nurr1 involvement in AD pathology.
Subject(s)
Amyloid beta-Peptides/metabolism , Brain/metabolism , Cerebral Cortex/metabolism , Hippocampus/metabolism , Nerve Tissue Proteins/physiology , Nuclear Receptor Subfamily 4, Group A, Member 2/physiology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Sequence , Animals , Antibody Specificity , Cerebral Cortex/pathology , Disease Models, Animal , Disease Progression , Fluorescent Antibody Technique, Direct , Hippocampus/pathology , Immunoenzyme Techniques , Mice , Mice, Transgenic , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/biosynthesis , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/immunology , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
Two DNA/RNA binding proteins, TDP-43 and FUS/TLSU, are involved in RNA processing, and their aberrant mutations induce inherited amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. Wild type TDP-43 and FUS (wtTDP-43 and wtFUS) are mainly localized in the nucleus and biochemically interact with the microRNA processing enzyme Drosha. In this study, we investigated Drosha stability in Neuro 2A cells by gain and loss of function studies of wtTDP-43 and wtFUS and cycloheximide mediated protein degradation assay. We also generated three different phosphomimetic mutants of TDP-43 (S379E, S403/404E and S409/410E) by using a site-directed mutagenesis method and examined Drosha stability to elucidate a correlation between the phosphorylated TDP-43 mutants and Drosha stability. Overexpression of wtTDP-43 and/or wtFUS increased Drosha stability in Neuro 2A cells and double knockdown of wtTDP-43 and wtFUS reduced its stability. However, knockdown of wtTDP-43 or wtFUS did not affect Drosha stability in Neuro 2A cells. Interestingly, a phosphomimetic mutant TDP-43 (S409/410E) significantly reduced Drosha stability via prevention of protein-protein interactions between wtFUS and Drosha, and induced cytotoxicity in Neuro 2A cells. Our findings suggest that TDP-43 and FUS controls Drosha stability in Neuro 2A cells and that a phosphomimetic mutant TDP-43 (S409/410E) which is associated with Drosha instability can induce neuronal toxicity.
Subject(s)
DNA-Binding Proteins/genetics , MicroRNAs/genetics , Neurons/metabolism , Phosphoproteins/genetics , RNA-Binding Protein FUS/genetics , Ribonuclease III/genetics , Animals , Cell Death/genetics , Cell Line, Tumor , Cycloheximide/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Enzyme Stability/genetics , Mice , MicroRNAs/metabolism , Molecular Mimicry , Mutagenesis, Site-Directed , Neurons/drug effects , Neurons/pathology , Phosphoproteins/metabolism , Protein Binding , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Protein FUS/antagonists & inhibitors , RNA-Binding Protein FUS/metabolism , Ribonuclease III/metabolismABSTRACT
µ-Calpain is a calcium-dependent cysteine protease, which is activated by µM concentration of calcium in vitro. Disrupted intracellular calcium homeostasis leads to hyper-activation of µ-calpain. Hyper-activated µ-calpain enhances the accumulation of ß-amyloid peptide by increasing the expression level of ß-secretase (BACE1) and induces hyper-phosphorylation of tau along with the formation of neurofibrillary tangle by mediating p35 cleavage into p25, both of which are the major mechanisms of neurodegeneration in Alzheimer's disease (AD). Hence, inhibition of µ-calpain activity is very important in the treatment and prevention of AD. In this study, conjugated linoleic acid (CLA), an eighteen-carbon unsaturated fatty acid, was discovered as a µ-calpain-specific inhibitor. CLA showed neuroprotective effects against neurotoxins such as H2O2 and Aß1-42 in SH-SY5Y cells, and inhibited Aß oligomerization/fibrillation and Aß-induced Zona Occludens-1 degradation. In addition, CLA decreased the levels of proapoptotic proteins, p35 conversion to p25 and tau phosphorylation. These findings implicate CLA as a new core structure for selective µ-calpain inhibitors with neuroprotective effects. CLA should be further evaluated for its potential use as an AD therapeutic agent.
Subject(s)
Alzheimer Disease/metabolism , Calpain/antagonists & inhibitors , Glycoproteins/pharmacology , Linoleic Acids, Conjugated/pharmacology , Neuroprotective Agents/pharmacology , Amyloid beta-Peptides/metabolism , Cell Line, Tumor , Humans , Hydrogen Peroxide/metabolism , Phosphorylation/drug effects , tau Proteins/metabolismABSTRACT
Huntington's disease (HD) is the most common hereditary neurodegenerative diseases, in which the loss of striatal neuron caused by the aggregation of mutant huntingtin protein (mHtt) is the main pathological feature. Our previous studies have demonstrated that human adipose stem cells (hASC) and its extracts can slow down the progression of HD in vitro and in vivo. hASC are readily accessible adult stem cells, and the cytosolic extracts contain a number of neurotrophic factors. Here, we further explored the role of the hASC extracts in neuronal death and mitochondrial function in HD. Our results showed that the hASC extracts prevent mHtt-induced cell toxicity and cell apoptosis. Moreover, the hASC extracts recovered mHtt-induced mitochondrial oxidative stress and reduced mitochondrial membrane potential. The hASC extracts blocked the interaction between p53 and mHtt, and decreased the endogenous p53 levels at both transcriptional and post-translational levels, resulting in the instability of p53 and increased neuronal survival. Taken together, these findings implicate protective roles of hASC extracts in mHtt-induced mitochondrial apoptosis, providing insights into the molecular mechanism of the hASC in the therapeutic strategy of HD.
Subject(s)
Adipocytes/metabolism , Adult Stem Cells/metabolism , Mitochondria/metabolism , Animals , Apoptosis/physiology , Cell Extracts/pharmacology , Cell Line , Humans , Huntingtin Protein , Huntington Disease/metabolism , Huntington Disease/pathology , Huntington Disease/therapy , Membrane Potential, Mitochondrial , Mice , Multipotent Stem Cells/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neural Stem Cells/metabolism , Neural Stem Cells/pathology , Neurons/metabolism , Neurons/pathology , Oxidative Stress , Tumor Suppressor Protein p53/metabolismABSTRACT
Mitochondrial dysfunction and synaptic damage are important features of Alzheimer's disease (AD) associated with amyloid ß (Aß) and tau. We reported previously that the scaffolding protein RanBP9, which is overall increased in brains of patients with AD and in mutant APP transgenic mice, simultaneously promotes Aß generation and focal adhesion disruption by accelerating the endocytosis of APP and ß1-integrin, respectively. Moreover, RanBP9 induces neurodegeneration in vitro and in vivo and mediates Aß-induced neurotoxicity. Here we show in primary hippocampal neurons that RanBP9 potentiates Aß-induced reactive oxygen species (ROS) overproduction, apoptosis, and calcium deregulation. Analyses of calcium-handling measures demonstrate that RanBP9 selectively delays the clearance of cytosolic Ca(2+) mediated by the mitochondrial calcium uniporter through a process involving the translocation of cofilin into mitochondria and oxidative mechanisms. Further, RanBP9 retards the anterograde axonal transport of mitochondria in primary neurons and decreases synaptic mitochondrial activity in brain. These data indicate that RanBP9, cofilin, and Aß mimic and potentiate each other to produce mitochondrial dysfunction, ROS overproduction, and calcium deregulation, which leads to neurodegenerative changes reminiscent of those seen in AD.
Subject(s)
Actin Depolymerizing Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Calcium Signaling , Cytoskeletal Proteins/metabolism , Mitochondria/metabolism , Nuclear Proteins/metabolism , Actin Depolymerizing Factors/genetics , Adaptor Proteins, Signal Transducing/genetics , Amyloid beta-Peptides/pharmacology , Animals , Apoptosis , Axonal Transport , Calcium/metabolism , Calcium Channels/metabolism , Cytoskeletal Proteins/genetics , Hippocampus/cytology , Membrane Potential, Mitochondrial , Mice , Neurons/drug effects , Neurons/metabolism , Neurons/physiology , Nuclear Proteins/genetics , Peptide Fragments/pharmacology , Reactive Oxygen Species/metabolism , Synapses/metabolismABSTRACT
Alzheimer's disease (AD) is the most common form of dementia caused by neurodegenerative process and is tightly related to amyloid ß (Aß) and neurofibrillary tangles. The lack of early diagnostic biomarker and therapeutic remedy hinders the prevention of increasing population of AD patients every year. In spite of accumulated scientific information, numerous clinical trials for candidate drug targets have failed to be preceded into therapeutic development, therefore, AD-related sufferers including patients and caregivers, are desperate to seek the solution. Also, effective AD intervention is desperately needed to reduce AD-related societal threats to public health. In this review, we summarize various drug targets and strategies in recent preclinical studies and clinical trials for AD therapy: Allopathic treatment, immunotherapy, Aß production/aggregation modulator, tau-targeting therapy and metabolic targeting. Some has already failed in their clinical trials and the others are still in various stages of investigations, both of which give us valuable information for future research in AD therapeutic development.