ABSTRACT
The principal signals that drive memory and cognitive impairment in Alzheimer's disease (AD) remain elusive. Here, we revealed brain-wide cellular reactions to type I interferon (IFN-I), an innate immune cytokine aberrantly elicited by amyloid ß plaques, and examined their role in cognition and neuropathology relevant to AD in a murine amyloidosis model. Using a fate-mapping reporter system to track cellular responses to IFN-I, we detected robust, Aß-pathology-dependent IFN-I activation in microglia and other cell types. Long-term blockade of IFN-I receptor (IFNAR) rescued both memory and synaptic deficits and resulted in reduced microgliosis, inflammation, and neuritic pathology. Microglia-specific Ifnar1 deletion attenuated the loss of post-synaptic terminals by selective engulfment, whereas neural Ifnar1 deletion restored pre-synaptic terminals and decreased plaque accumulation. Overall, IFN-I signaling represents a critical module within the neuroinflammatory network of AD and prompts concerted cellular states that are detrimental to memory and cognition.
Subject(s)
Alzheimer Disease , Interferon Type I , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Disease Models, Animal , Immunity, Innate , Interferon Type I/metabolism , Memory Disorders/metabolism , Mice , Mice, Transgenic , Microglia/metabolism , Plaque, Amyloid/metabolismABSTRACT
Impaired neurogenesis in the adult hippocampus has been implicated in AD pathogenesis. Here we reveal that the APP plays an important role in the neural progenitor proliferation and newborn neuron maturation in the mouse dentate gyrus. APP controls adult neurogenesis through a non cell-autonomous mechanism by GABAergic neurons, as selective deletion of GABAergic, but not glutamatergic, APP disrupts adult hippocampal neurogenesis. APP, highly expressed in the majority of GABAergic neurons in the dentate gyrus, enhances the inhibitory tone to granule cells. By regulating both tonic and phasic GABAergic inputs to dentate granule cells, APP maintains excitatory-inhibitory balance and preserves cognitive functions. Our studies uncover an indispensable role of APP in the GABAergic system for controlling adult hippocampal neurogenesis, and our findings indicate that APP dysfunction may contribute to impaired neurogenesis and cognitive decline associated with AD.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , GABAergic Neurons/physiology , Hippocampus/cytology , Interneurons/physiology , Neurogenesis/physiology , Age Factors , Amyloid beta-Protein Precursor/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Cells, Cultured , Excitatory Amino Acid Antagonists/pharmacology , Female , Male , Membrane Potentials/drug effects , Membrane Potentials/genetics , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Tissue Proteins/deficiency , Nestin/genetics , Nestin/metabolism , Neurogenesis/genetics , Sodium Channel Blockers/pharmacology , Synaptic Transmission/genetics , Tetrodotoxin/pharmacology , Vesicular Inhibitory Amino Acid Transport Proteins/deficiencyABSTRACT
Correct development of the cerebellum requires coordinated sonic hedgehog (Shh) signaling from Purkinje to granule cells. How Shh expression is regulated in Purkinje cells is poorly understood. Using a novel tyrosinase minigene-tagged Sleeping Beauty transposon-mediated mutagenesis, which allows for coat color-based genotyping, we created mice in which the Ski/Sno family transcriptional co-repressor 2 (Skor2) gene is deleted. Loss of Skor2 leads to defective Purkinje cell development, a severe reduction of granule cell proliferation and a malformed cerebellum. Skor2 is specifically expressed in Purkinje cells in the brain, where it is required for proper expression of Shh. Skor2 overexpression suppresses BMP signaling in an HDAC-dependent manner and stimulates Shh promoter activity, suggesting that Skor2 represses BMP signaling to activate Shh expression. Our study identifies an essential function for Skor2 as a novel transcriptional regulator in Purkinje cells that acts upstream of Shh during cerebellum development.
Subject(s)
Cerebellum/growth & development , Cerebellum/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Animals , Bone Morphogenetic Proteins/metabolism , Cell Differentiation , Cerebellum/abnormalities , Gene Expression Regulation, Developmental , Genotype , Hair Color/genetics , Histone Deacetylases/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Monophenol Monooxygenase/genetics , Mutagenesis, Insertional , Proto-Oncogene Proteins/deficiency , Purkinje Cells/cytology , Purkinje Cells/metabolism , Repressor Proteins/deficiency , Signal Transduction , Transforming Growth Factor beta/metabolism , Transposases/geneticsABSTRACT
Transcription factor EB (TFEB) mediates gene expression through binding to the coordinated lysosome expression and regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase), which are essential for lysosome acidification. Single-nucleus RNA sequencing of wild-type and PS19 (Tau) transgenic mice expressing the P301S mutant tau identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and were locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.
Subject(s)
Tauopathies , Vacuolar Proton-Translocating ATPases , Animals , Mice , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Lysosomes/metabolism , Mice, Transgenic , Microglia/metabolism , Signal Transduction/physiology , Tauopathies/metabolism , Vacuolar Proton-Translocating ATPases/geneticsABSTRACT
Amyloidogenic processing of the amyloid precursor protein (APP) generates a large secreted ectodomain fragment (APPsß), ß-amyloid (Aß) peptides, and an APP intracellular domain (AICD). Whereas Aß is viewed as critical for Alzheimer's disease pathogenesis, the role of other APP processing products remains enigmatic. Of interest, the AICD has been implicated in transcriptional regulation, and N-terminal cleavage of APPsß has been suggested to produce an active fragment that may mediate axonal pruning and neuronal cell death. We previously reported that mice deficient in APP and APP-like protein 2 (APLP2) exhibit early postnatal lethality and neuromuscular synapse defects, whereas mice with neuronal conditional deletion of APP and APLP2 are viable. Using transcriptional profiling, we now identify transthyretin (TTR) and Klotho as APP/APLP2-dependent genes whose expression is decreased in loss-of-function states but increased in gain-of-function states. Significantly, by creating an APP knockin allele that expresses only APPsß protein, we demonstrate that APPsß is not normally cleaved in vivo and is fully capable of mediating the APP-dependent regulation of TTR and Klotho gene expression. Despite being an active regulator of gene expression, APPsß did not rescue the lethality and neuromuscular synapse defects of APP and APLP2 double-KO animals. Our studies identify TTR and Klotho as physiological targets of APP that are regulated by soluble APPsß independent of developmental APP functions. This unexpected APP-mediated signaling pathway may play an important role in maintaining TTR and Klotho levels and their respective functions in Aß sequestration and aging.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Gene Expression Regulation , Glucuronidase/metabolism , Nerve Tissue Proteins/metabolism , Prealbumin/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Cells, Cultured , Gene Expression Profiling , Genotype , Glucuronidase/genetics , Humans , Klotho Proteins , Mice , Mice, Knockout , Microarray Analysis , Nerve Tissue Proteins/genetics , Neurons/cytology , Neurons/physiology , Prealbumin/metabolismABSTRACT
Synapses are crucial structures that mediate signal transmission between neurons in complex neural circuits and display considerable morphological and electrophysiological heterogeneity. So far we still lack a high-throughput method to profile the molecular heterogeneity among individual synapses. In the present study, we develop a droplet-based single-cell (sc) total-RNA-sequencing platform, called Multiple-Annealing-and-Tailing-based Quantitative scRNA-seq in Droplets, for transcriptome profiling of individual neurites, primarily composed of synaptosomes. In the synaptosome transcriptome, or 'synaptome', profiling of both mouse and human brain samples, we detect subclusters among synaptosomes that are associated with neuronal subtypes and characterize the landscape of transcript splicing that occurs within synapses. We extend synaptome profiling to synaptopathy in an Alzheimer's disease (AD) mouse model and discover AD-associated synaptic gene expression changes that cannot be detected by single-nucleus transcriptome profiling. Overall, our results show that this platform provides a high-throughput, single-synaptosome transcriptome profiling tool that will facilitate future discoveries in neuroscience.
Subject(s)
Alzheimer Disease , Synapses , Humans , Mice , Animals , Synapses/genetics , Synapses/metabolism , Gene Expression Profiling/methods , Synaptosomes/metabolism , Transcriptome/genetics , Alzheimer Disease/genetics , Single-Cell Analysis/methods , Sequence Analysis, RNA/methodsABSTRACT
Transcription factor EB (TFEB) mediates gene expression through binding to the Coordinated Lysosome Expression And Regulation (CLEAR) sequence. TFEB targets include subunits of the vacuolar ATPase (v-ATPase) essential for lysosome acidification. Single nucleus RNA-sequencing (snRNA-seq) of wild-type and PS19 (Tau) transgenic mice identified three unique microglia subclusters in Tau mice that were associated with heightened lysosome and immune pathway genes. To explore the lysosome-immune relationship, we specifically disrupted the TFEB-v-ATPase signaling by creating a knock-in mouse line in which the CLEAR sequence of one of the v-ATPase subunits, Atp6v1h, was mutated. We show that the CLEAR mutant exhibited a muted response to TFEB, resulting in impaired lysosomal acidification and activity. Crossing the CLEAR mutant with Tau mice led to higher tau pathology but diminished microglia response. These microglia were enriched in a subcluster low in mTOR and HIF-1 pathways and was locked in a homeostatic state. Our studies demonstrate a physiological function of TFEB-v-ATPase signaling in maintaining lysosomal homoeostasis and a critical role of the lysosome in mounting a microglia and immune response in tauopathy and Alzheimer's disease.
ABSTRACT
Background: Hepatocellular carcinoma is one of the malignant tumors with the highest incidence in the world. According to the latest statistics of the National Cancer Center, the incidence of liver cancer ranks fifth in malignant tumors and its mortality rate ranks second in China, which seriously threatens people' s life and health. Aim: To investigate the value of CT perfusion parameters and apparent diffusion coefficient (ADC) of magnetic resonance imaging (MRI) diffusion weighted imaging (DWI) in the diagnosis of hepatocellular carcinoma. Methods: 43 patients with hepatocellular carcinoma and 40 patients with hepatic hemangioma treated in our hospital from August 2018 to August 2021 were selected for CT perfusion imaging and MRI examination. Results: The liver blood flow (BF), liver blood volume (BV), and hepatic artery perfusion (HAP) in the hepatocellular carcinoma group were (267.38 ± 35.59) ml/(min·100 g), (30.20 ± 8.82) ml/100 g, and (0.64 ± 0.10) ml/(min·ml), respectively, which were significantly higher than those in the hepatic hemangioma group (p < 0.05). The ADC value of hepatocellular carcinoma DWI sequence was (1.20 ± 0.17) ×10-3 mm2, which was significantly lower than that of hepatic hemangioma (p < 0.05). The area under ROC curve of BF, BV, HAP, and ADC values for hepatocellular carcinoma was 0.860, 0.754, 0.804, and 0.890, respectively. The area under ROC curve of the four groups was compared (p > 0.05). Conclusion: CT perfusion parameters BF, BV, HAP, and DWI sequence ADC values have certain application value in the diagnosis of hepatocellular carcinoma, and there is no significant difference between the diagnostic value of each parameter.
Subject(s)
Carcinoma, Hepatocellular , Hemangioma , Liver Neoplasms , Carcinoma, Hepatocellular/diagnostic imaging , Diagnosis, Differential , Diffusion Magnetic Resonance Imaging/methods , Hemangioma/diagnostic imaging , Hemangioma/pathology , Humans , Liver Neoplasms/pathology , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy , Perfusion , ROC Curve , Tomography, X-Ray ComputedABSTRACT
Mutations in the amyloid precursor protein (APP) cause early-onset Alzheimer's disease (AD), but the only genetic risk factor for late-onset AD is the varepsilon4 allele of apolipoprotein E (apoE), a major cholesterol carrier. Using Cre-lox conditional knockout mice, we demonstrate that lipoprotein receptor LRP1 expression regulates apoE and cholesterol levels within the CNS. We also found that deletion of APP and its homolog APLP2, or components of the gamma-secretase complex, significantly enhanced the expression and function of LRP1, which was reversed by forced expression of the APP intracellular domain (AICD). We further show that AICD, together with Fe65 and Tip60, interacts with the LRP1 promoter and suppresses its transcription. Together, our findings support that the gamma-secretase cleavage of APP plays a central role in regulating apoE and cholesterol metabolism in the CNS via LRP1 and establish a biological linkage between APP and apoE, the two major genetic determinants of AD.
Subject(s)
Amyloid beta-Protein Precursor/physiology , Apolipoproteins E/metabolism , Brain/metabolism , Cholesterol/metabolism , Gene Expression Regulation, Developmental/physiology , Receptors, LDL/physiology , Tumor Suppressor Proteins/physiology , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/deficiency , Animals , Animals, Newborn , Cell Line, Transformed , Chromatin Immunoprecipitation , Cricetinae , Cytidine Deaminase/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Gene Expression Regulation, Developmental/genetics , Histone Acetyltransferases/pharmacology , Humans , Low Density Lipoprotein Receptor-Related Protein-1 , Lysine Acetyltransferase 5 , Mice , Mice, Knockout , Nerve Tissue Proteins/pharmacology , Nuclear Proteins/pharmacology , RNA, Messenger/biosynthesis , Receptors, LDL/deficiency , Reverse Transcriptase Polymerase Chain Reaction/methods , Trans-Activators , Transfection/methods , Tumor Suppressor Proteins/deficiencyABSTRACT
Proteolytic processing of the amyloid precursor protein (APP) generates large soluble APP derivatives, ß-amyloid (Aß) peptides, and APP intracellular domain. Expression of the extracellular sequences of APP or its Caenorhabditis elegans counterpart has been shown to be sufficient in partially rescuing the CNS phenotypes of the APP-deficient mice and the lethality of the apl-1 null C. elegans, respectively, leaving open the question as what is the role of the highly conserved APP intracellular domain? To address this question, we created an APP knock-in allele in which the mouse Aß sequence was replaced by the human Aß. A frameshift mutation was introduced that replaced the last 39 residues of the APP sequence. We demonstrate that the C-terminal mutation does not overtly affect APP processing and amyloid pathology. In contrast, crossing the mutant allele with APP-like protein 2 (APLP2)-null mice results in similar neuromuscular synapse defects and early postnatal lethality as compared with mice doubly deficient in APP and APLP2, demonstrating an indispensable role of the APP C-terminal domain in these development activities. Our results establish an essential function of the conserved APP intracellular domain in developmental regulation, and this activity can be genetically uncoupled from APP processing and Aß pathogenesis.
Subject(s)
Alleles , Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Neuromuscular Junction/metabolism , Receptors, Cell Surface/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Disease Models, Animal , Frameshift Mutation , Gene Knock-In Techniques , Humans , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Neuromuscular Junction/genetics , Neuromuscular Junction/pathology , Protease Nexins , Protein Structure, Tertiary , Receptors, Cell Surface/geneticsABSTRACT
BACKGROUND: Genome-wide association studies have established clusterin (CLU) as a genetic modifier for late-onset Alzheimer's disease (AD). Both protective and risk alleles have been identified which may be associated with its expression levels. However, the physiological function of clusterin in the central nervous system remains largely unknown. METHODS: We examined Clu expression in mouse brains by immunohistochemistry and high-resolution imaging. We performed electrophysiological recordings and morphological analysis of dendritic spines in wild-type and Clu knockout mice. We tested synaptic function of astrocytic Clu using neuron-glia co-cultures and by AAV-mediated astroglial Clu expression in vivo. Finally, we investigated the role of astrocytic Clu on synaptic properties and amyloid pathology in 5xFAD transgenic mouse model of AD. RESULTS: We show that astrocyte secreted Clu co-localizes with presynaptic puncta of excitatory neurons. Loss of Clu led to impaired presynaptic function and reduced spine density in vivo. Neurons co-cultured with Clu-overexpressing astrocytes or treated with conditioned media from HEK293 cells transfected with Clu displayed enhanced excitatory neurotransmission. AAV-mediated astroglial Clu expression promoted excitatory neurotransmission in wild-type mice and rescued synaptic deficits in Clu knockout mice. Overexpression of Clu in the astrocytes of 5xFAD mice led to reduced Aß pathology and fully rescued the synaptic deficits. CONCLUSION: We identify Clu as an astrocyte-derived synaptogenic and anti-amyloid factor; the combination of these activities may influence the progression of late-onset AD.
Subject(s)
Alzheimer Disease/metabolism , Astrocytes/metabolism , Clusterin/metabolism , Neurons/metabolism , Synaptic Transmission/physiology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Clusterin/genetics , Disease Models, Animal , Genome-Wide Association Study , Humans , Mice, Transgenic , Neuropathology/methods , Synaptic Transmission/geneticsABSTRACT
Amyloid precursor protein (APP) has been strongly implicated in the pathogenesis of Alzheimer's disease (AD). Although impaired synaptic function is believed to be an early and causative event in AD, how APP physiologically regulates synaptic properties remains poorly understood. Here, we report a critical role for APP in the regulation of L-type calcium channels (LTCC) in GABAergic inhibitory neurons in striatum and hippocampus. APP deletion in mice leads to an increase in the levels of Ca(v)1.2, the pore-forming subunit of LTCCs, and subsequent increases in GABAergic calcium currents (I(Ca(2+))) that can be reversed by reintroduction of APP. Upregulated levels of Ca(v)1.2 result in reduced GABAergic paired-pulse inhibition and increased GABAergic post-tetanic potentiation in both striatal and hippocampal neurons, indicating that APP modulates synaptic properties of GABAergic neurons by regulating Ca(v)1.2. Furthermore, APP physically interacts with Ca(v)1.2, suggesting a mechanism in which loss of APP leads to an inappropriate accumulation and aberrant activity of Ca(v)1.2. These results provide a direct link between APP and calcium signaling and might help explain how altered APP regulation leads to changes in synaptic function that occur with AD.
Subject(s)
Amyloid beta-Protein Precursor/physiology , Calcium Channels, L-Type/physiology , Neuronal Plasticity/physiology , gamma-Aminobutyric Acid/physiology , Animals , Animals, Newborn , Cells, Cultured , Long-Term Potentiation/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Time FactorsABSTRACT
A critical role of the amyloid precursor protein (APP) in Alzheimer's disease (AD) pathogenesis has been well established. However, the physiological function of APP remains elusive and much debated. We reported previously that the APP family of proteins is essential in mediating the developing neuromuscular synapse. In the current study, we created a conditional allele of APP and deleted APP in presynaptic motor neuron or postsynaptic muscle. Crossing these alleles onto the APP-like protein 2-null background reveals that, unexpectedly, inactivating APP in either compartment results in neuromuscular synapse defects similar to the germline deletion and that postsynaptic APP is obligatory for presynaptic targeting of the high-affinity choline transporter and synaptic transmission. Using a HEK293 and primary hippocampus mixed-culture assay, we report that expression of APP in HEK293 cells potently promotes synaptogenesis in contacting axons. This activity is dependent on neuronal APP and requires both the extracellular and intracellular domains; the latter forms a complex with Mint1 and Cask and is replaceable by the corresponding SynCAM (synaptic cell adhesion molecule) sequences. These in vitro and in vivo studies identify APP as a novel synaptic adhesion molecule. We postulate that transsynaptic APP interaction modulates its synaptic function and that perturbed APP synaptic adhesion activity may contribute to synaptic dysfunction and AD pathogenesis.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Central Nervous System/physiology , Peripheral Nervous System/metabolism , Presynaptic Terminals/metabolism , Synapses/metabolism , Amyloid beta-Protein Precursor/physiology , Animals , Cell Communication/physiology , Cell Line , Cells, Cultured , Central Nervous System/embryology , Central Nervous System/metabolism , Coculture Techniques , Female , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neurogenesis/physiology , Peripheral Nervous System/embryology , Pregnancy , Presynaptic Terminals/physiology , Synapses/physiology , Synaptic Potentials/physiology , Synaptic Transmission/physiologyABSTRACT
FE65 is expressed predominantly in the brain and interacts with the C-terminal domain of beta-amyloid precursor protein (APP). We examined hippocampus-dependent memory and in vivo long-term potentiation (LTP) at the CA1 synapses with isoform-specific FE65 knockout (p97FE65(-/-)) mice. When examined using the Morris water maze, p97FE65(-/-) mice were impaired for the hidden platform task but showed normal performance in the probe test. To further discriminate the role of FE65 in acquisition and memory consolidation, we examined p97FE65(-/-) mice with temporal dissociative passive avoidance (TDPA) and contextual fear conditioning (CFC). p97FE65(-/-) mice showed impaired short-term memory for both TDPA and CFC when tested 10 min after training. After multiple TDPA training sessions, the crossover latency of some p97FE65(-/-) mice reached the cutoff value, but it significantly decayed in 8 d. At the Schaffer collateral-CA1 synapses, p97FE65(-/-) mice showed defective early-phase LTP (E-LTP). These results demonstrate novel roles of FE65 in synaptic plasticity, acquisition, and retention for certain forms of memory formation.
Subject(s)
Avoidance Learning/physiology , Hippocampus/physiology , Long-Term Potentiation/physiology , Adaptation, Physiological/drug effects , Adaptation, Physiological/genetics , Analysis of Variance , Animals , Avoidance Learning/drug effects , Behavior, Animal/drug effects , Behavior, Animal/physiology , Biophysics , Conditioning, Classical/drug effects , Conditioning, Classical/physiology , Electric Stimulation/methods , Fear/drug effects , Fear/physiology , Hippocampus/drug effects , In Vitro Techniques , Learning Disabilities/drug therapy , Learning Disabilities/genetics , Long-Term Potentiation/drug effects , Maze Learning/drug effects , Maze Learning/physiology , Memory Disorders/drug therapy , Memory Disorders/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation/genetics , Nerve Tissue Proteins/deficiency , Nerve Tissue Proteins/genetics , Nuclear Proteins/deficiency , Nuclear Proteins/genetics , Phosphodiesterase Inhibitors/pharmacology , Phosphodiesterase Inhibitors/therapeutic use , RNA, Small Interfering/pharmacology , Rolipram/pharmacology , Rolipram/therapeutic use , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Accumulation of ß-amyloid (Aß) could induce neurotoxicity in Alzheimer's disease (AD). microRNA (miR)-34a-5p and miR-125b-5p have been reported to be aberrantly expressed in AD patients. However, the roles and mechanisms of these two miRNAs in AD remain poorly understood. METHODS: Serum samples of 27 AD patients were collected. Primary mouse cortical neurons (MCN) and Neuro2a (N2a) cells were incubated with Aß. The expression levels of miR-34a-5p, miR-125b-5p and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) were detected by quantitative real-time polymerase chain reaction and western blot. The effect of miRNAs or epigallocatechin-3-gallate (EGCG) on Aß-induced neurotoxicity was investigated by cell viability, Caspase 3 activity, apoptosis and intracellular ROS production. The interaction between BACE1 and miR-34a-5p or miR-125b-5p was analyzed by luciferase reporter assay. RESULTS: miR-34a-5p and miR-125b-5p levels were decreased and BACE1 mRNA expression was increased in AD patients and Aß-treated MCN and N2a cells. Addition of miR-34a-5p or miR-125b-5p attenuated Aß-induced apoptosis and oxidative stress. BACE1 acted as a target of miR-34a-5p and miR-125b-5p and its restoration weakened the effect of miR-34a-5p or miR-125b-5p on Aß-induced neurotoxicity. Moreover, EGCG could mitigate Aß-induced neurotoxicity, which might be associated with miR-34a-5p and miR-125b-5p. CONCLUSION: miR-34a-5p and miR-125b-5p inhibited Aß-induced neurotoxicity by decreasing apoptosis and oxidative stress via targeting BACE1, providing novel targets for treatment of AD.
Subject(s)
Amyloid Precursor Protein Secretases , MicroRNAs , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Peptides/toxicity , Animals , Aspartic Acid Endopeptidases/genetics , Cell Line, Tumor , Humans , Mice , MicroRNAs/geneticsABSTRACT
Type I interferon (IFN) is a key cytokine that curbs viral infection and cell malignancy. Previously, we demonstrated a potent IFN immunogenicity of nucleic acid-containing (NA-containing) amyloid fibrils in the periphery. Here, we investigated whether IFN is associated with ß-amyloidosis inside the brain and contributes to neuropathology. An IFN-stimulated gene (ISG) signature was detected in the brains of multiple murine Alzheimer disease (AD) models, a phenomenon also observed in WT mouse brain challenged with generic NA-containing amyloid fibrils. In vitro, microglia innately responded to NA-containing amyloid fibrils. In AD models, activated ISG-expressing microglia exclusively surrounded NA+ amyloid ß plaques, which accumulated in an age-dependent manner. Brain administration of rIFN-ß resulted in microglial activation and complement C3-dependent synapse elimination in vivo. Conversely, selective IFN receptor blockade effectively diminished the ongoing microgliosis and synapse loss in AD models. Moreover, we detected activated ISG-expressing microglia enveloping NA-containing neuritic plaques in postmortem brains of patients with AD. Gene expression interrogation revealed that IFN pathway was grossly upregulated in clinical AD and significantly correlated with disease severity and complement activation. Therefore, IFN constitutes a pivotal element within the neuroinflammatory network of AD and critically contributes to neuropathogenic processes.
Subject(s)
Alzheimer Disease/immunology , Amyloid/immunology , Interferon-beta/immunology , Synapses/immunology , Alzheimer Disease/chemically induced , Alzheimer Disease/pathology , Animals , Complement C3/immunology , Disease Models, Animal , Humans , Inflammation/chemically induced , Inflammation/immunology , Inflammation/pathology , Interferon-beta/adverse effects , Interferon-beta/pharmacology , Mice , Microglia/immunology , Microglia/pathology , Synapses/pathology , Up-Regulation/drug effects , Up-Regulation/immunologyABSTRACT
Nonneuronal cell types in the CNS are increasingly implicated as critical players in brain health and disease. While gene expression profiling of bulk brain tissue is routinely used to examine alterations in the brain under various conditions, it does not capture changes that occur within single cell types or allow interrogation of crosstalk among cell types. To this end, we have developed a concurrent brain cell type acquisition (CoBrA) methodology, enabling the isolation and profiling of microglia, astrocytes, endothelia, and oligodendrocytes from a single adult mouse forebrain. By identifying and validating anti-ACSA-2 and anti-CD49a antibodies as cell surface markers for astrocytes and vascular endothelial cells, respectively, and using established antibodies to isolate microglia and oligodendrocytes, we document that these 4 major cell types are isolated with high purity and RNA quality. We validated our procedure by performing acute peripheral LPS challenge, while highlighting the underappreciated changes occurring in astrocytes and vascular endothelia in addition to microglia. Furthermore, we assessed cell type-specific gene expression changes in response to amyloid pathology in a mouse model of Alzheimer's disease. Our CoBrA methodology can be readily implemented to interrogate multiple CNS cell types in any mouse model at any age.
Subject(s)
Alzheimer Disease/pathology , Brain/pathology , Inflammation/pathology , Alzheimer Disease/genetics , Animals , Astrocytes/metabolism , Astrocytes/pathology , Brain/metabolism , Disease Models, Animal , Endothelial Cells/metabolism , Endothelial Cells/pathology , Female , Gene Expression , Gene Expression Profiling , Inflammation/genetics , Integrin alpha1 , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Microglia/metabolism , Microglia/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathologyABSTRACT
The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and ß-amyloid production play a central role in Alzheimer's disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation.
Subject(s)
Amyloid beta-Protein Precursor/metabolism , Axon Guidance/genetics , Cerebral Cortex/cytology , Intercellular Signaling Peptides and Proteins/metabolism , Nerve Tissue Proteins/metabolism , Neurons/cytology , Amyloid beta-Protein Precursor/genetics , Animals , Animals, Newborn , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cerebral Cortex/growth & development , Cerebral Cortex/metabolism , Embryo, Mammalian , HEK293 Cells , Humans , Intercellular Signaling Peptides and Proteins/genetics , Mice , Mice, Inbred C57BL , Nerve Net/physiology , Nerve Tissue Proteins/genetics , Neurons/physiology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA Interference/physiology , Receptors, Immunologic/metabolism , p21-Activated Kinases/metabolism , rac1 GTP-Binding Protein/metabolism , Roundabout ProteinsABSTRACT
Accumulating evidence implicates impairment of the autophagy-lysosome pathway in Alzheimer's disease (AD). Recently discovered, transcription factor EB (TFEB) is a molecule shown to play central roles in cellular degradative processes. Here we investigate the role of TFEB in AD mouse models. In this study, we demonstrate that TFEB effectively reduces neurofibrillary tangle pathology and rescues behavioral and synaptic deficits and neurodegeneration in the rTg4510 mouse model of tauopathy with no detectable adverse effects when expressed in wild-type mice. TFEB specifically targets hyperphosphorylated and misfolded Tau species present in both soluble and aggregated fractions while leaving normal Tau intact. We provide in vitro evidence that this effect requires lysosomal activity and we identify phosphatase and tensin homolog (PTEN) as a direct target of TFEB that is required for TFEB-dependent aberrant Tau clearance. The specificity and efficacy of TFEB in mediating the clearance of toxic Tau species makes it an attractive therapeutic target for treating diseases of tauopathy including AD.
Subject(s)
Alzheimer Disease/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/physiology , Nerve Degeneration/genetics , Neurofibrillary Tangles/genetics , Tauopathies/genetics , tau Proteins/metabolism , Alzheimer Disease/pathology , Animals , Autophagy , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Gene Expression Regulation , Lysosomes/physiology , Mice, Inbred C57BL , Mice, Transgenic , Nerve Degeneration/pathology , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Tauopathies/pathologyABSTRACT
The key pathological features of Alzheimer's disease include synaptic dysfunction, profound changes in the cholinergic system, and deposition of beta-amyloid peptides generated by proteolytic processing of the amyloid-beta precursor protein (APP). However, the pathways linking APP with synaptic activity and cholinergic neuronal function are poorly understood. We report here that APP is essential in regulating the presynaptic expression and activity of the high-affinity choline transporter (CHT), a molecule that mediates the rate-limiting step of cholinergic synaptic transmission in both the neuromuscular junction and central cholinergic neurons. Loss of APP leads to aberrant localization of CHT at the neuromuscular synapses and reduced CHT activity at cholinergic projections. At the cellular level, we show that APP and CHT can be found in Rab5-positive endosomal compartments and that APP affects CHT endocytosis. Furthermore, we demonstrate that APP interacts with CHT through the C-terminal domain, providing support for a specific and direct regulation of CHT by APP through protein-protein interactions. These results identify a physiological activity of APP in cholinergic neurons, and our data indicate that deregulation of APP function may contribute to cholinergic impairment and AD pathogenesis.