Cystatin C prevents neuronal loss and behavioral deficits via the endosomal pathway in a mouse model of down syndrome
Cystatin C (CysC) plays diverse protective roles under conditions of neuronal challenge. We investigated whether CysC protects from trisomy-induced pathologies in a mouse model of Down syndrome (DS), the most common cause of developmental cognitive and behavioral impairments in humans. We have previously shown that the segmental trisomy mouse model, Ts[Rb(12.1716)]2Cje (Ts2) has DS-like neuronal and behavioral deficiencies. The current study reveals that transgene-mediated low levels of human CysC overexpression has a preventive effect on numerous neuropathologies in the brains of Ts2 mice, including reducing early and late endosome enlargement in cortical neurons and decreasing loss of basal forebrain cholinergic neurons (BFCNs). Consistent with these cellular benefits, behavioral dysfunctions were also prevented, including deficits in nesting behavior and spatial memory. We determined that the CysC-induced neuroprotective mechanism involves activation of the phosphotidylinositol kinase (PI3K)/AKT pathway. Activating this pathway leads to enhanced clearance of accumulated endosomal substrates, protecting cells from DS-mediated dysfunctions in the endosomal system and, for BFCNs, from neurodegeneration. Our findings suggest that modulation of the PI3/AKT pathway offers novel therapeutic interventions for patients with DS.
Intracellular metalloprotease activity controls intraneuronal AÎ² aggregation and limits secretion of AÎ² via exosomes
Accumulating evidence suggests that the abnormal aggregation of amyloid-Î² (Î‘Î²) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where AÎ² is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced AÎ². In this study, we determined whether disruption of AÎ² catabolism could trigger AÎ² aggregation within neurons and increase the amount of AÎ² associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular AÎ² levels and promoted the intracellular formation of AÎ² oligomers, a process that did not require internalization of secreted AÎ². In vivo, the accumulation of intraneuronal AÎ² aggregates was accompanied by increased levels of both extracellular and exosome-associated AÎ², including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of AÎ² degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal AÎ² aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., PÃ©rez-GonzÃ¡lez, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal AÎ² aggregation and limits secretion of AÎ² via exosomes.
Human apolipoprotein E genotype differentially affects olfactory behavior and sensory physiology in mice [Meeting Abstract]
Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE e4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the e2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, e3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. The hyper-excitability of ApoE4 mice may contribute to the impairment of odor habituation memory, while the hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate the potential process by which ApoE affects the olfactory system at early stages, prior to the development of AD
Human apolipoprotein E genotype differentially affects olfactory behavior and sensory physiology in mice
Apolipoprotein E (ApoE) is an important lipid carrier in both the periphery and the brain. The ApoE Îµ4 allele (ApoE4) is the single most important genetic risk-factor for Alzheimer's disease (AD) while the Îµ 2 allele (ApoE2) is associated with a lower risk of AD-related neurodegeneration compared to the most common variant, Îµ 3 (ApoE3). ApoE genotype affects a variety of neural circuits; however, the olfactory system appears to provide early biomarkers of ApoE genotype effects. Here, we directly compared olfactory behavior and olfactory system physiology across all three ApoE genotypes in 6-month- and 12-month-old mice with targeted replacement for the human ApoE2, ApoE3, or ApoE4 genes. Odor investigation and habituation were assessed, along with, olfactory bulb and piriform cortical local field potential activity. The results demonstrate that while initial odor investigation was unaffected by ApoE genotype, odor habituation was impaired in E4 relative to E2 mice, with E3 mice intermediate in function. There was also significant deterioration of odor habituation from 6 to 12 months of age regardless of the ApoE genotype. Olfactory system excitability and odor responsiveness were similarly determined by ApoE genotype, with an ApoE4 > ApoE3 > ApoE2 excitability ranking. Although motivated behavior is influenced by many processes, hyper-excitability of ApoE4 mice may contribute to impaired odor habituation, while hypo-excitability of ApoE2 mice may contribute to its protective effects. Given that these ApoE mice do not have AD pathology, our results demonstrate how ApoE affects the olfactory system at early stages, prior to the development of AD.
Correction: MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease [Correction]
[This corrects the article DOI: 10.1371/journal.ppat.1005032.].
Processing of the amyloid precursor protein in the exosomal pathway: Propagation of Alzheimer's disease pathology [Meeting Abstract]
Background: The main component of the amyloid deposited in the brain of Alzheimer's disease patients is beta-amyloid (Abeta), a proteolytic product of the amyloid beta precursor protein (APP). Mature APP undergoes proteolytic cleavage by alpha- and beta-secretases to produce C-terminal fragments (APP-CTFs). beta-APP-CTF is a neurotoxic protein that is also the source of Abeta following cleavage by gamma-secretase. It was previously shown that amyloidogenic APP processing mainly occurs in endosomes and that exosomes contain APP, APP-CTFs, a minute fraction of Abeta, and the secretases involved in APP metabolism, but the exosomal contribution to amyloid pathology remains unknown. We have investigated whether APP processing occurs in the exosomal pathway. Methods: Exosomes were isolated from postmortem human and mouse brains, and from the culture media of human fibroblasts and of the neuroblastoma cell line SH-SY5Y. The content of APP, APP metabolites and APP secretases in exosomes was analysed by Western blot and compared with the content in the brain or cell homogenates. Results: We found that exosomes isolated from human and mouse brains as well as exosomes secreted by cells in vitro are enriched in APP-CTFs. All three APP secretases were detected in the exosome preparations and interestingly, beta-secretase 1 (BACE1) and the mature form of the -secretase ADAM10 were also enriched in exosomes, whereas the gamma-secretase subunit Nicastrin was not. Our data also show that exosomal beta- and alpha- secretases are active, based on the observation of continuous generation of APP-CTFs in isolated exosomes. Summary/Conclusion: Our data show that APP processing continues in exosomes following their release into the extracellular space from the endosomal multivesicular bodies, implicating exosomes as carriers and generation sites of the neurotoxic beta-APP-CTF and an extracellular source of Abeta. Given the stability of exosomes, this may propagate amyloid pathogenicity throughout the brain
Interrelationships between endosomal pathology and exosomal generation and release in neurodegenerative disorders [Meeting Abstract]
Background: Dysfunction of the neuronal endosomal pathway is a characteristic of down syndrome (DS) and Alzheimer's disease (AD) and of carriers of the AD-risk apolipoprotein E 4 allele (APOE4). We hypothesized that the efficient release of endosomal material via exosomes into the extracellular space, as observed in the brains of DS patients and a mouse model of the disease and by DS fibroblasts, is necessary for a neuron to prevent accumulation of endosomal contents. Conversely, APOE4-driven downregulation of exosome release in the brains of APOE4 human carriers and APOE4 targeted-replacement mice appears to contribute to endosomal pathology. We investigated in vitro the interrelationship between the endosomal and exosomal pathways. Methods: Fibroblasts from DS patients and age-matched controls were transfected with CD63 siRNA or negative control siRNA. Level of exosomal secretion was studied by western blot analysis, and number and area of endosomes by immunohistochemistry. Results: Knockdown of the tetraspanin CD63, a regulator of exosome biogenesis, diminished exosome release by DS fibroblasts but not by control cells. CD63 knockdown did not affect endosomal morphology in control cells, but the number and total area occupied by endosomes was greater in DS fibroblasts in which CD63 expression was reduced. Summary/Conclusion: In neurodegenerative disorders with endosomallysosomal dysfunction, exosome secretion serves as a disposal mechanism for potentially toxic materials that are abnormally accumulated in endosomal compartments. Conversely, APOE4-driven downregulation of brain exosome biosynthesis and release contributes to endosomal pathology. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging likely contributes to neuron degeneration and our findings argue that exosome production plays a central role maintaining homeostatic function of the endosomal-lysosomal system
Apolipoprotein E4 compromises brain exosome production and secretion [Meeting Abstract]
Background: The apolipoprotein E (APOE) gene codes for the brain's primary cholesterol carrier protein. In both humans and humanized APOE mice the Alzheimer's disease-risk APOE 4 allele (APOE4) alters the number and size of neuronal endosomes, a pathology common to several neurodegenerative disorders, including Alzheimer's disease. Given that exosomes derive from the endosomal system, we investigated the impact of APOE4 on brain-derived exosomes. Methods: Extracellular vesicles (EV) were isolated from brain tissue of neuropathologically normal humans and of APOE targeted-replacement mice at 6, 12 and 18 months of age. Antibodies against TSG101 and ALIX were used to identify the exosome population within these samples. Protein, mRNA and lipid analyses were performed on both EV and whole-brain samples. Results: We found lower exosome levels in the brains of neuropathologically normal human APOE4 carriers compared to individuals homozygous for the risk-neutral 3 allele (APOE3). In APOE4 compared with APOE3 mice, brain exosome levels were lower in an age-dependent manner: lower levels were observed at 12 and 18 but not at 6 months of age. Protein and mRNA expressions of the exosome pathway regulators TSG101 and Rab35 were also lower in APOE4 compared with APOE3 mouse brains at 12 months of age, arguing for decreased exosome biosynthesis and secretion, respectively, from the endosomal pathway. Cholesterol and ganglioside levels were higher in brain exosomes isolated from 12-month-old APOE4 compared with APOE3 mice. Summary/Conclusion: Our findings show an APOE4-driven downregulation of brain exosome biosynthesis and release that is associated with altered lipid homeostasis. Failure to maintain proper functioning of the interdependent endosomal-exosomal pathways during aging, which is essential for diverse homeostatic and catabolic cellular processes, is likely to contribute to neuronal vulnerability in neurodegenerative disorders, including Alzheimer's disease
Enhanced exosome secretion in Down syndrome brain - a protective mechanism to alleviate neuronal endosomal abnormalities
A dysfunctional endosomal pathway and abnormally enlarged early endosomes in neurons are an early characteristic of Down syndrome (DS) and Alzheimer's disease (AD). We have hypothesized that endosomal material can be released by endosomal multivesicular bodies (MVBs) into the extracellular space via exosomes to relieve neurons of accumulated endosomal contents when endosomal pathway function is compromised. Supporting this, we found that exosome secretion is enhanced in the brains of DS patients and a mouse model of the disease, and by DS fibroblasts. Furthermore, increased levels of the tetraspanin CD63, a regulator of exosome biogenesis, were observed in DS brains. Importantly, CD63 knockdown diminished exosome release and worsened endosomal pathology in DS fibroblasts. Taken together, these data suggest that increased CD63 expression enhances exosome release as an endogenous mechanism mitigating endosomal abnormalities in DS. Thus, the upregulation of exosome release represents a potential therapeutic goal for neurodegenerative disorders with endosomal pathology.
Lysosomal dysfunction in the brain of a mouse model with intraneuronal accumulation of carboxyl terminal fragments of the amyloid precursor protein
Recent data suggest that intraneuronal accumulation of metabolites of the amyloid-beta-precursor protein (APP) is neurotoxic. We observed that transgenic mice overexpressing in neurons a human APP gene harboring the APPE693Q (Dutch) mutation have intraneuronal lysosomal accumulation of APP carboxylterminal fragments (APP-CTFs) and oligomeric amyloid beta (oAbeta) but no histological evidence of amyloid deposition. Morphometric quantification using the lysosomal marker protein 2 (LAMP-2) immunolabeling showed higher neuronal lysosomal counts in brain of 12-months-old APPE693Q as compared with age-matched non-transgenic littermates, and western blots showed increased lysosomal proteins including LAMP-2, cathepsin D and LC3. At 24 months of age, these mice also exhibited an accumulation of alpha-synuclein in the brain, along with increased conversion of LC3-I to LC3-II, an autophagosomal/autolysosomal marker. In addition to lysosomal changes at 12 months of age, these mice developed cholinergic neuronal loss in the basal forebrain, GABAergic neuronal loss in the cortex, hippocampus and basal forebrain and gliosis and microgliosis in the hippocampus. These findings suggest a role for the intraneuronal accumulation of oAbeta and APP-CTFs and resultant lysosomal pathology at early stages of Alzheimer's disease-related pathology.Molecular Psychiatry advance online publication, 25 October 2016; doi:10.1038/mp.2016.189.