Exosomes are membrane vesicles that are released by cells upon fusion of multivesicular bodies with the plasma membrane. Their molecular composition reflects their origin in endosomes as intraluminal vesicles. In addition to a common set of membrane and cytosolic molecules, exosomes harbor unique subsets of proteins linked to cell type-associated functions. Exosome secretion participates in the eradication of obsolete proteins but several findings, essentially in the immune system, indicate that exosomes constitute a potential mode of intercellular communication. Release of exosomes by tumor cells and their implication in the propagation of unconventional pathogens such as prions suggests their participation in pathological situations. These findings open up new therapeutic and diagnostic strategies.

Summary Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for numerous physiological processes including lysosome-related organelle (LRO) biogenesis. PMEL – a component of melanocyte LROs (melanosomes) – is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.

Background: Exogenous, misfolded Tau can be internalized, but details of the mechanism are unknown. Results: Small misfolded Tau species are internalized through endocytosis, anterogradely and retrogradely transported. Conclusion: Tau uptake is dependent on conformation and size of aggregates, and regulated through endocytosis. Significance: Understanding the mechanism by which pathological Tau is internalized provides a foundation for therapeutic approaches targeting uptake and propagation of tauopathy.

Retromer is a multiprotein complex that trafficks cargo out of endosomes. The neuronal retromer traffics the amyloid-precursor protein (APP) away from endosomes, a site where APP is cleaved into pathogenic fragments in Alzheimer’s disease. Here we determined whether pharmacological chaperones can enhance retromer stability and function. First, we relied on the crystal structures of retromer proteins to help identify the ‘weak link’ of the complex and to complete an in silico screen of small molecules predicted to enhance retromer stability. Among the hits, an in vitro assay identified one molecule that stabilized retromer against thermal denaturation. Second, we turned to cultured hippocampal neurons, showing that this small molecule increases the levels of retromer proteins, shifts APP away from the endosome, and decreases the pathogenic processing of APP. These findings show that pharmacological chaperones can enhance the function of a multiprotein complex and may have potential therapeutic implications for neurodegenerative diseases.

Defects in endosomal sorting have been implicated in Alzheimer’s disease (AD). Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that PI3P is selectively deficient in brain tissue from humans with AD and AD mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein (APP) in these organelles and reduces APP sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT pathway, including the PI3P effector Hrs and Tsg101. APP is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of APP to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing PI3P deficiency as a contributing factor in AD, these results clarify the mechanisms of APP trafficking through the endosomal system in normal and pathological states.

Defects in endolysosomal and autophagic functions are increasingly viewed as key pathological features of neurodegenerative disorders. A master regulator of these functions is phosphatidylinositol-3-phosphate (PI3P), a phospholipid synthesized primarily by class III PI 3-kinase Vps34. Here we report that disruption of neuronal Vps34 function in vitro and in vivo impairs autophagy, lysosomal degradation as well as lipid metabolism, causing endolysosomal membrane damage. PI3P deficiency also promotes secretion of unique exosomes enriched for undigested lysosomal substrates, including amyloid precursor protein C-terminal fragments (APP-CTFs), specific sphingolipids, and the phospholipid bis(monoacylglycero)phosphate (BMP), which normally resides in the internal vesicles of endolysosomes. Secretion of these exosomes requires neutral sphingomyelinase 2 and sphingolipid synthesis. Our results reveal a homeostatic response counteracting lysosomal dysfunction via secretion of atypical exosomes eliminating lysosomal waste and define exosomal APP-CTFs and BMP as candidate biomarkers for endolysosomal dysfunction associated with neurodegenerative disorders.

Background information. TSEs (transmissible spongiform encephalopathies) are neurodegenerative disorders affecting humans and animals. PrPSc, a conformationally altered isoform of the normal prion protein (PrPC), is thought to be the pathogenic agent. However, the biochemical composition of the prion agent is still matter of debate. The potential transmission risk of the prion agent through biological fluids has been shown, but the development of competitive diagnostic tests and treatment for TSEs requires a more comprehensive knowledge of the agent and the cellular mechanisms by which it is disseminated. With this aim, we initiated characterization of the prion agent and the pathways by which it can be propagated using the cellular model system neuroblastoma (N2a). Results. The present study shows that N2a cells infected with scrapie release the prion agent into the cell culture medium in association with exosome‐like structures and viral particles of endogenous origin. We found that both prion proteins and scrapie infectivity are mainly associated with exosome‐like structures that contain viral envelope glycoprotein and nucleic acids, such as RNAs. Conclusions. The dissemination of prions in N2a cell culture is mediated through the exosomal pathway.

Small vessel cerebrovascular disease, visualized as white matter hyperintensities on T2-weighted MRI, contributes to the clinical presentation of Alzheimer’s disease. However, the extent to which cerebrovascular disease represents an independent pathognomonic feature of Alzheimer's disease or directly promotes Alzheimer’s pathology is unclear. The purpose of this study was to examine the association between white matter hyperintensities and plasma levels of tau and to determine if white matter hyperintensities and tau levels interact to predict Alzheimer’s disease diagnosis. To confirm that cerebrovascular disease promotes tau pathology, we examined tau fluid biomarker concentrations and pathology in a mouse model of ischemic injury. Three hundred ninety-one participants from the Alzheimer’s Disease Neuroimaging Initiative (ADNI; 74.5 ± 7.1 years of age) were included in this cross-sectional analysis. Participants had measurements of plasma total-tau, CSF beta amyloid, and white matter hyperintensities, and were diagnosed clinically as Alzheimer’s disease (n = 97), mild cognitive impairment (n = 186), or cognitively normal control (n = 108). We tested the relationship between plasma tau concentration and white matter hyperintensity volume across diagnostic groups. We also examined the extent to which white matter hyperintensity volume, plasma tau, amyloid positivity status, and the interaction between white matter hyperintensities and plasma tau correctly classifies diagnostic category. Increased white matter hyperintensity volume was associated with higher plasma tau concentration, particularly among those diagnosed clinically with Alzheimer’s disease. Presence of brain amyloid and the interaction between plasma tau and white matter hyperintensity volume distinguished Alzheimer’s disease and mild cognitive impairment participants from controls with 77.6% and 63.3% accuracy, respectively. In 63 ADNI participants who came to autopsy (82.33 ± 7.18 age at death) we found that higher degrees of arteriosclerosis were associated with higher Braak staging, indicating a positive relationship between cerebrovascular disease and neurofibrillary pathology. In a transient middle cerebral artery occlusion (tMCAo) mouse model, aged mice that received tMCAo, but not sham surgery, had increased plasma and CSF tau concentrations, induced myelin loss, and hyperphosphorylated tau pathology in the ipsilateral hippocampus and cerebral hemisphere. These findings demonstrate a relationship between cerebrovascular disease, operationalized as white matter hyperintensities, and tau levels, indexed in the plasma, suggesting that hypoperfusive injury promotes tau pathology. This potential causal association is supported by the demonstration that transient cerebral artery occlusion induces white matter damage, increases biofluidic markers of tau, and promotes cerebral tau hyperphosphorylation in older adult mice.