Endosomal-Lysosomal Processing of Neurodegeneration-Associated Proteins in Astrocytes

Most common neurodegenerative diseases (NDs) are characterized by deposition of protein aggregates that are resulted from misfolding, dysregulated trafficking, and compromised proteolytic degradation. These proteins exert cellular toxicity to a broad range of brain cells and are found in both neurons and glia. Extracellular monomeric and oligomeric ND-associated proteins are taken up by astrocytes, the most abundant glial cell in the brain. Internalization, intracellular trafficking, processing, and disposal of these proteins are executed by the endosomal-lysosomal system of astrocytes. Endosomal-lysosomal organelles thus mediate the cellular impact and metabolic fate of these toxic protein species. Given the indispensable role of astrocytes in brain metabolic homeostasis, the endosomal-lysosomal processing of these proteins plays a fundamental role in altering the trajectory of neurodegeneration. This review aims at summarizing the mounting evidence that has established the essential role of astrocytic endosomal-lysosomal organelles in the processing of amyloid precursor proteins, Apolipoprotein E (ApoE), tau, alpha synuclein, and huntingtin, which are associated with NDs such as Alzheimer’s, Parkinson’s, and Huntington diseases.

Size-dependent protein segregation creates a spatial switch for Notch and APP signaling

Aberrant cleavage of Notch and amyloid precursor proteins (APPs) by γ-secretase is implicated in numerous diseases, but how cleavage is regulated in space and time is unclear. Here, we report that cadherin-based adherens junctions (cadAJs) are sites of high cell-surface γ-secretase activity, while simultaneously excluding these γ-secretase substrates by a size-dependent mechanism, prohibiting enzyme-substrate interactions. Upon activation, Notch and APP undergo drastic spatial rearrangements to cadAJs, concentrating them with γ-secretase, wherein they are further processed for downstream signaling. Spatial mutation by decreasing (or increasing) the size of Notch extracellular domain promotes (or inhibits) signaling, respectively. Dysregulation of this spatial switch also promotes formation of more amyloidogenic Aβ. Therefore, cadAJs creates distinct biochemical compartments regulating signaling events involving γ-secretase and prevent pathogenic activation of its substrates.

Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

Substrate interaction inhibits γ-secretase production of amyloid-β peptides

A novel compound C1 interacts with C-terminal juxtamembrane lysines of amyloid precursor proteins and inhibits γ-secretase production of Aβ.

Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot

Nicastrin (NCSTN) is a transmembrane glycoprotein that is part of the gamma-secretase complex. Gamma-secretase is a protease complex that cleaves type-I single-pass transmembrane proteins. There are many potential substrates for this complex, including NOTCH receptors and amyloid precursor proteins (APP). There are a number of commercial antibodies to nicastrin, but they do not agree on expected peptide size. We confirmed the specificity of a C-terminal binding rabbit anti-human antibody from Sigma-Aldrich (#N1660) using wildtype HEK293 cells and HEK293 cells deleted for nicastrin. The wildtype cells showed a prominent band at approximately 110 kDa. We confirmed this larger than expected sized was due to glycosylation by treating the lysate with peptide-N-glycosidase F (PNGase F), which reduced the band to less than 75 kDa. These data suggest that this polyclonal is specific for nicastrin and can detect endogenous levels of protein.

Potential Roles of Exosomal MicroRNAs as Diagnostic Biomarkers and Therapeutic Application in Alzheimer’s Disease

Exosomes are bilipid layer-enclosed vesicles derived from endosomes and are released from neural cells. They contain a diversity of proteins, mRNAs, and microRNAs (miRNAs) that are delivered to neighboring cells and/or are transported to distant sites. miRNAs released from exosomes appear to be associated with multiple neurodegenerative conditions linking to Alzheimer’s disease (AD) which is marked by hyperphosphorylated tau proteins and accumulation of Aβ plaques. Exciting findings reveal that miRNAs released from exosomes modulate the expression and function of amyloid precursor proteins (APP) and tau proteins. These open up the possibility that dysfunctional exosomal miRNAs may influence AD progression. In addition, it has been confirmed that the interaction between miRNAs released by exosomes and Toll-like receptors (TLR) initiates inflammation. In exosome support-deprived neurons, exosomal miRNAs may regulate neuroplasticity to relieve neurological damage. In this review, we summarize the literature on the function of exosomal miRNAs in AD pathology, the potential of these miRNAs as diagnostic biomarkers in AD, and the use of exosomes in the delivery of miRNAs which may lead to major advances in the field of macromolecular drug delivery.