Author ORCID Identifier

https://orcid.org/0000-0002-8414-4348

Date Available

12-4-2020

Year of Publication

2020

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Physiology

Advisor

Dr. Erhard Bieberich

Abstract

Alzheimer’s disease (AD) is an age-dependent, progressive, neurodegenerative disorder that is characterized clinically by the impairment of cognitive functions concomitant with behavioral and personality changes. AD is associated with distinct pathological hallmarks, namely, intracellular neurofibrillary tangles comprised of hyperphosphorylated tau protein, extracellular amyloid beta (Aβ) plaques, and marked brain atrophy. Besides their main role as the core component of amyloid plaques, oligomeric Aβ have been shown to be neurotoxic. The exact mechanism of Aβ neurotoxicity is yet to be elucidated.

Recently, a pathogenic function of small extracellular vesicles- also known as exosomes- has been proposed, suggesting that exosomes can transfer pathogens between cells. One such pathogen that exploits this pathway is Aβ in Alzheimer’s disease, however, it is not known yet whether this Aβ/exosomes association would affect the neuronal toxicity of Aβ.

Exosomes are nano-sized lipid vesicles that are formed by inward budding of late endosomes to form multi vesicular bodies (MVB) which fuse to the plasma membrane and release exosomes to the extracellular space. Exosomes serve as a means of intercellular communication due to their ability in carrying cargoes including microRNA (miRNA), messenger RNA (mRNA), proteins, and other biomolecules. There are several established pathways for exosomes biogenesis, one of which is triggered by the sphingolipid ceramide. Ceramide is a key molecule in sphingolipids metabolism and it is involved in several cellular processes such as proliferation, senescence and apoptosis. It has also been reported that ceramide levels are elevated in AD patients brain specimens.

Exploiting the fact that exosomes can cross the blood brain barrier we therefore used serum derived exosomes to study the biophysical and biochemical characteristics of Alzheimer’s disease mouse model (5xFAD) and AD patients’ exosomes compared to wild type and healthy individuals. We found that serum from 5xFAD mice and AD patients contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide, particularly C16:0, C18:0, C20:0, 22:0, C24:0, and C24:1 ceramide species. This subpopulation (termed astrosomes) was shown to associated with Aβ and are prone to aggregation as confirmed by nanoparticle tracking and cluster analyses. To study the functional characteristics of these Aβ-associated astrosomes, we used Neuro-2a (N2a) cells, human iPS cell-derived neurons, and mouse primary cultured neurons as in vitro tissue culture models. When taken up by neurons, Aβ-associated astrosomes were specifically transported to mitochondria where they induced mitochondria clustering, evident by elevation of expression of the fission protein dynamin related protein1 (Drp1). Aβ-associated astrosomes, but not wild type or healthy control human exosomes, mediated binding of Aβ to voltage-dependent anion channel 1 (VDAC1), a gate keeper protein in the outer mitochondrial membrane that is involved in regulating passage of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis. This Aβ/VDAC1 interaction leads to caspase activation and subsequently apoptosis. Interestingly, removing the ceramide-enriched astrosomes from the exosome pool using lipid-mediated affinity chromatography (LIMAC) mitigated that toxic effect on neurons. These results were replicated using brain derived exosomes.

To investigate the in vivo significance of our in vitro results, we stereotaxically injected wild type mice (two weeks old) with 5xFAD or wild type brain derived exosomes (nine months old). We found that within two days, the injected exosomes were specifically taken up by neurons and transported to mitochondria. Consistent with our in vitro data using Aβ-associated astrosomes, the exosomes isolated from 5xFAD brain, but not those from wild type brain, induced complex formation of Aβ with VDAC1 and activation of caspase 3.

To test that our observations hold true in physiological conditions, we generated a novel astrosome reporter mouse model. This was accomplished by crossing of Aldh1l1-Cre/ERT with floxed CD63-GFP and 5xFAD mice (5XFAD xAldh1l1-Cre/ERTxCD63-GFPfl/fl) which allows us to track astrosome uptake and their subsequent effects. As seen with the injected exosomes, we found that endogenous GFP-labeled astrosomes are taken up by neurons where they shuttle Aβ and induce mitotoxicity.

In conclusion, our data show that association of Aβ to astrosomes in critical for Aβ neurotoxicity. Therefore, we discovered a novel mechanism by which Aβ induces AD neuropathology as well as potential pharmacological target.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.474

Funding Information

This study was (in part) supported by grants from the National Institutes of Health (R01AG034389, R01NS095215, and R01AG064234) in 2018-2019 and the Department of Veteran Affairs (I01BX003643) in 2019.

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