Endo-lysosomal dysfunction in neurodegenerative diseases: opinion on current progress and future direction in the use of exosomes as biomarkers.

Over the past two decades, increased research has highlighted the connection between endosomal trafficking defects and neurodegeneration. The endo-lysosomal network is an important, complex cellular system specialized in the transport of proteins, lipids, and other metabolites, essential for cell homeostasis. Disruption of this pathway is linked to a wide range of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease and frontotemporal dementia. Furthermore, there is strong evidence that defects in this pathway create opportunities for diagnostic and therapeutic intervention. In this Opinion piece, we concisely address the role of endo-lysosomal dysfunction in five neurodegenerative diseases and discuss how future research can investigate this intracellular pathway, including extracellular vesicles with a specific focus on exosomes for the identification of novel disease biomarkers. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Cerebrovascular disease drives Alzheimer plasma biomarker concentrations in adults with Down syndrome.

Importance: By age 40 years over 90% of adults with Down syndrome (DS) have Alzheimer's disease (AD) pathology and most progress to dementia. Despite having few systemic vascular risk factors, individuals with DS have elevated cerebrovascular disease (CVD) markers that track with the clinical progression of AD, suggesting a role for CVD that is hypothesized to be mediated by inflammatory factors. Objective: To examine the pathways through which small vessel CVD contributes to AD-related pathophysiology and neurodegeneration in adults with DS. Design: Cross sectional analysis of neuroimaging, plasma, and clinical data. Setting: Participants were enrolled in Alzheimer's Biomarker Consortium - Down Syndrome (ABC-DS), a multisite study of AD in adults with DS. Participants: One hundred eighty-five participants (mean [SD] age=45.2 [9.3] years) with available MRI and plasma biomarker data were included. White matter hyperintensity (WMH) volumes were derived from T2-weighted FLAIR MRI scans and plasma biomarker concentrations of amyloid beta (Aß42/Aß40), phosphorylated tau (p-tau217), astrocytosis (glial fibrillary acidic protein, GFAP), and neurodegeneration (neurofilament light chain, NfL) were measured with ultrasensitive immunoassays. Main Outcomes and Measures: We examined the bivariate relationships of WMH, Aß42/Aß40, p-tau217, and GFAP with age-residualized NfL across AD diagnostic groups. A series of mediation and path analyses examined causal pathways linking WMH and AD pathophysiology to promote neurodegeneration in the total sample and groups stratified by clinical diagnosis. Results: There was a direct and indirect bidirectional effect through GFAP of WMH on p-tau217 concentration, which was associated with NfL concentration in the entire sample. Among cognitively stable participants, WMH was directly and indirectly, through GFAP, associated with p-tau217 concentration, and in those with MCI, there was a direct effect of WMH on p-tau217 and NfL concentrations. There were no associations of WMH with biomarker concentrations among those diagnosed with dementia. Conclusions and Relevance: The findings suggest that among individuals with DS, CVD promotes neurodegeneration by increasing astrocytosis and tau pathophysiology in the presymptomatic phases of AD. This work joins an emerging literature that implicates CVD and its interface with neuroinflammation as a core pathological feature of AD in adults with DS.

Juvenile CLN3 disease is a lysosomal cholesterol storage disorder: similarities with Niemann-Pick type C disease.

BACKGROUND: The most common form of neuronal ceroid lipofuscinosis (NCL) is juvenile CLN3 disease (JNCL), a currently incurable neurodegenerative disorder caused by mutations in the CLN3 gene. Based on our previous work and on the premise that CLN3 affects the trafficking of the cation-independent mannose-6 phosphate receptor and its ligand NPC2, we hypothesised that dysfunction of CLN3 leads to the aberrant accumulation of cholesterol in the late endosomes/lysosomes (LE/Lys) of JNCL patients' brains. METHODS: An immunopurification strategy was used to isolate intact LE/Lys from frozen autopsy brain samples. LE/Lys isolated from samples of JNCL patients were compared with age-matched unaffected controls and Niemann-Pick Type C (NPC) disease patients. Indeed, mutations in NPC1 or NPC2 result in the accumulation of cholesterol in LE/Lys of NPC disease samples, thus providing a positive control. The lipid and protein content of LE/Lys was then analysed using lipidomics and proteomics, respectively. FINDINGS: Lipid and protein profiles of LE/Lys isolated from JNCL patients were profoundly altered compared to controls. Importantly, cholesterol accumulated in LE/Lys of JNCL samples to a comparable extent than in NPC samples. Lipid profiles of LE/Lys were similar in JNCL and NPC patients, except for levels of bis(monoacylglycero)phosphate (BMP). Protein profiles detected in LE/Lys of JNCL and NPC patients appeared identical, except for levels of NPC1. INTERPRETATION: Our results support that JNCL is a lysosomal cholesterol storage disorder. Our findings also support that JNCL and NPC disease share pathogenic pathways leading to aberrant lysosomal accumulation of lipids and proteins, and thus suggest that the treatments available for NPC disease may be beneficial to JNCL patients. This work opens new avenues for further mechanistic studies in model systems of JNCL and possible therapeutic interventions for this disorder. FUNDING: San Francisco Foundation.

Genetic variation and cryptic lineage among the sergestid shrimp Acetes americanus (Decapoda).

The taxonomic status of the sergestid shrimp, Acetes americanus, has been questioned for several decades. No specific study has been performed thus far to resolve the incongruences. This species has a wide geographical range in the western Atlantic and is represented by two formally accepted subspecies: Acetes americanus carolinae, distributed in North America, and Acetes americanus americanus, present in South America. However, there are regions where the coexistence of both subspecies has been reported, such as Central America. This study aimed to genetically compare specimens of A. a. americanus collected in South America with A. a. carolinae sampled in North America to check for possible differences and the existence of more than one subspecies of A. americanus on the Brazilian coast. Based on the sequences of two informative markers, the cytochrome oxidase I region (COI) and 16S rRNA, phylogenetic reconstruction demonstrated well-defined clades with high support values, reinforcing the idea that A. a. americanus is genetically different from A. a. carolinae. Our hypothesis was corroborated as the specimens collected in Brazil were divided into two distinct lineages: the first composed of A. a. americanus sensu stricto (Brazil 1) and the second by Acetes americanus (Brazil 2). The three groups evidenced in the haplotype network were the same as those observed in the phylogenetic tree. The morphometric character (height/length of the thelycum) was effective in distinguishing A. a. Brazil 1 from A. a. carolinae. However, more detailed and conclusive studies comprising other characteristics to propose and describe a possible new entity are necessary. To the best of our knowledge, for the first time, the results of this study provide some insights into the taxonomic status of the sergestid shrimp A. americanus in the western Atlantic.

Ergonomic assessment of postal workers' pain symptoms and musculoskeletal risks related to parcel processing activity for delivery.

The parcel delivery activity is carried out all over the world and workers in this sector have suffered from musculoskeletal disorders (MSDs) due to the strong demand for work generated by the recent increase in e-commerce. This study aimed to evaluate postal workers' pain symptoms, movements and identify MSDs risks related to the parcel processing activity for delivery, proposing preventive measures. A sample of thirty-two workers was evaluated with the application of sociodemographic and Nordic questionnaires and electrical bioimpedance. The motion capture sensors were used to evaluate right/left shoulder joints, segment C7-T1 (Cervical) and segment L5-S1 (Lumbar) of three postal workers (percentiles of anthropometric data: 5, 50, and 95) during four real work activities that are part of the parcel processing. The analyzed workers presented musculoskeletal complaints in practically all body regions, with a greater prevalence in shoulders, hands, lower back, and knees. According to the Body Mass Index (BMI), they were on average overweight (27.8 ± 3.7 kg/m2). In the movement analysis, we identified risks related to cervical protrusion, anterior trunk flexion, and shoulder flexion, in addition to repetitive movements. In some activities, the higher stature showed an increase in lumbar and cervical anterior flexion. The set of evaluations showed that the activity of processing orders for delivery offers musculoskeletal risks. We identify that ergonomic adaptations are necessary to adapt the heights of the work environment to the statures of the postal workers. Relevance to industry: The activity of processing orders for delivery is carried out practically all over the world generating jobs and income for its employees. Nonetheless, there are still situations of ergonomic disadvantage that can generate musculoskeletal risks. The findings elucidate ergonomic risks and provide useful information for future ergonomic interventions in the postal/delivery workplace environment.

The neuronal retromer can regulate both neuronal and microglial phenotypes of Alzheimer's disease.

Disruption of retromer-dependent endosomal trafficking is considered pathogenic in late-onset Alzheimer's disease (AD). Here, to investigate this disruption in the intact brain, we turn to a genetic mouse model where the retromer core protein VPS35 is depleted in hippocampal neurons, and then we replete VPS35 using an optimized viral vector protocol. The VPS35 depletion-repletion studies strengthen the causal link between the neuronal retromer and AD-associated neuronal phenotypes, including the acceleration of amyloid precursor protein cleavage and the loss of synaptic glutamate receptors. Moreover, the studies show that the neuronal retromer can regulate a distinct, dystrophic, microglia morphology, phenotypic of hippocampal microglia in AD. Finally, the neuronal and, in part, the microglia responses to VPS35 depletion were found to occur independent of tau. Showing that the neuronal retromer can regulate AD-associated pathologies in two of AD's principal cell types strengthens the link, and clarifies the mechanism, between endosomal trafficking and late-onset sporadic AD.

Alzheimer's vulnerable brain region relies on a distinct retromer core dedicated to endosomal recycling.

Whether and how the pathogenic disruptions in endosomal trafficking observed in Alzheimer's disease (AD) are linked to its anatomical vulnerability remain unknown. Here, we began addressing these questions by showing that neurons are enriched with a second retromer core, organized around VPS26b, differentially dedicated to endosomal recycling. Next, by imaging mouse models, we show that the trans-entorhinal cortex, a region most vulnerable to AD, is most susceptible to VPS26b depletion-a finding validated by electrophysiology, immunocytochemistry, and behavior. VPS26b was then found enriched in the trans-entorhinal cortex of human brains, where both VPS26b and the retromer-related receptor SORL1 were found deficient in AD. Finally, by regulating glutamate receptor and SORL1 recycling, we show that VPS26b can mediate regionally selective synaptic dysfunction and SORL1 deficiency. Together with the trans-entorhinal's unique network properties, hypothesized to impose a heavy demand on endosomal recycling, these results suggest a general mechanism that can explain AD's regional vulnerability.

Tau and other proteins found in Alzheimer's disease spinal fluid are linked to retromer-mediated endosomal traffic in mice and humans.

Endosomal trafficking has emerged as a defective biological pathway in Alzheimer's disease (AD), and the pathway is a source of cerebrospinal fluid (CSF) protein accumulation. Nevertheless, the identity of the CSF proteins that accumulate in the setting of defects in AD's endosomal trafficking pathway remains unknown. Here, we performed a CSF proteomic screen in mice with a neuronal-selective knockout of the core of the retromer complex VPS35, a master conductor of endosomal traffic that has been implicated in AD. We then validated three of the most relevant proteomic findings: the amino terminus of the transmembrane proteins APLP1 and CHL1, and the mid-domain of tau, which is known to be unconventionally secreted and elevated in AD. In patients with AD dementia, the concentration of amino-terminal APLP1 and CHL1 in the CSF correlated with tau and phosphorylated tau. Similar results were observed in healthy controls, where both proteins correlated with tau and phosphorylated tau and were elevated in about 70% of patients in the prodromal stages of AD. Collectively, the mouse-to-human studies suggest that retromer-dependent endosomal trafficking can regulate tau, APLP1, and CHL1 CSF concentration, informing on how AD's trafficking pathway might contribute to disease spread and how to identify its trafficking impairments in vivo.

Cerebrovascular disease promotes tau pathology in Alzheimer's disease.

Small vessel cerebrovascular disease, visualized as white matter hyperintensities on T2-weighted magnetic resonance imaging, 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 ischaemic injury. Three hundred ninety-one participants from the Alzheimer's Disease Neuroimaging Initiative (74.5 ± 7.1 years of age) were included in this cross-sectional analysis. Participants had measurements of plasma total-tau, cerebrospinal fluid 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 Alzheimer's Disease Neuroimaging Initiative 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 mouse model, aged mice that received transient middle cerebral artery occlusion, but not sham surgery, had increased plasma and cerebrospinal fluid 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.

Neuronal lysosomal dysfunction releases exosomes harboring APP C-terminal fragments and unique lipid signatures.

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.

Growth, longevity, and juvenile recruitment of the white shrimpLitopenaeus schmitti (Burkenroad, 1936) (Decapoda: Penaeoidea) insoutheastern Brazil / Crescimento, longevidade e recrutamento juvenil do camarão-branco Litopenaeus schmitti (Burkenroad, 1936) (Decapoda: Penaeoidea) na região Sudeste do Brasil

Litopenaeus schmitti is one of the most commercially exploited penaeids on the southeastern and southern Atlantic coast of Brazil. Information about juvenile recruitment and growth patterns of individuals is important for management programs. The present study estimated the growth parameters, longevity, and recruitment of L. schmitti in the region of Ubatuba, São Paulo State. The growth parameters of individuals were estimated by the Bertalanffy growth model, and longevity was estimated with the inverted Bertalanffy equation. The growth parameters were, for females and males respectively: CL∞ = 53.10 mm and 43.23 mm, k = 1.82 year-1 and 2.19 years-1, t0= 1.10 and 0.69. Longevity was calculated to be 2.27 years for females and 2.10 years for males. Juvenile recruitment occurred seasonally from December to April during the first year of sampling and from November to May in the second year. Although the large peak in juveniles seen in March 2007 in the estuarine regions coincided with the closed season, the reopening of the fishery in June may promote the capture of large numbers of newly grown adults who have not yet reproduced.

Litopenaeus schmitti é um dos peneídeos mais explorados comercialmente na costa do Atlântico Sudeste e Sul do Brasil. Desta forma, informações acerca do recrutamento juvenil e dos padrões de crescimento dos indivíduos são importantes para futuros programas de manejo. O presente estudo teve como objetivo estimar os parâmetros de crescimento, longevidade e recrutamento de L. schmitti na região de Ubatuba, Estado de São Paulo. Tais parâmetros foram estimados pelo modelo de crescimento de von Bertalanffy e a longevidade foi estimada por meio da equação inversa de von Bertalanffy. Os parâmetros de crescimento estimados para fêmeas e machos foram, respectivamente: CC∞ = 53,10 mm e 43,23 mm, k = 1,82 anos-1 e 2,19 anos-1, t0 = 1,10 e 0,69, e longevidade de 2,27 e 2,10 anos. O recrutamento juvenil ocorreu sazonalmente entre dezembro e abril no primeiro ano de amostragem e de novembro a maio no segundo ano. Embora o principal pico de juvenis, na região estuarina em março/2007, tenha coincidido com o período de fechamento da pesca, a reabertura da pesca em junho, possivelmente, promove a captura de grandes quantidades de recém-adultos, que ainda não participaram da primeira reprodução.

Lifespan and population dynamics of the endemic South American shrimp Artemesia longinaris (Crustacea: Penaeidae) in southeastern Brazil.

The present study investigated the growth, longevity and reproductive dynamics of Artemesia longinaris in the southeastern coast of Brazil over a two-year period. Monthly collections were conducted in Ubatuba and Caraguatatuba using a shrimp fishing boat equipped with "double-rig" nets. Each region was divided into 7 sampling stations up to 35 m deep. Size frequency distributions, growth, longevity, sex ratio, and abundance of individuals in each demographic class, were compared. The relationship between abiotic factors and abundance of each demographic class was assessed using a Canonical Correlation Analysis. A total of 64,641 individuals were collected (6,928 measured) with an estimated longevity of 1.30 (Ubatuba) and 1.14 (Caraguatatuba) years for females and 1.03 years for males in both regions. There was a statistically significant bias in sex ratio toward females (Chi-squared test, p < 0.05) in both regions. The Canonical Correlation Analysis resulted in a canonical correlation coefficient of 0.31 (p = 0.00002). Salinity and temperature showed high correlation mainly with the presence of reproductive females. In general, this demographic class was most common in conditions of low temperature and high salinity. These findings, as well as other studies carried out in colder regions with the same species, are consistent with classical latitudinal paradigm.

Efficient inhibition of infectious prions multiplication and release by targeting the exosomal pathway.

Exosomes are secreted membrane vesicles of endosomal origin present in biological fluids. Exosomes may serve as shuttles for amyloidogenic proteins, notably infectious prions, and may participate in their spreading in vivo. To explore the significance of the exosome pathway on prion infectivity and release, we investigated the role of the endosomal sorting complex required for transport (ESCRT) machinery and the need for ceramide, both involved in exosome biogenesis. Silencing of HRS-ESCRT-0 subunit drastically impairs the formation of cellular infectious prion due to an altered trafficking of cholesterol. Depletion of Tsg101-ESCRT-I subunit or impairment of the production of ceramide significantly strongly decreases infectious prion release. Together, our data reveal that ESCRT-dependent and -independent pathways can concomitantly regulate the exosomal secretion of infectious prion, showing that both pathways operate for the exosomal trafficking of a particular cargo. These data open up a new avenue to regulate prion release and propagation.

Annual, seasonal and spatial abundance of the seabob shrimp Xiphopenaeus kroyeri (Decapoda, Penaeidae) off the Southeastern coast of Brazil.

We investigated the influence of environmental factors in spatial and temporal distribution of the seabob shrimp Xiphopenaeus kroyeri in Santos Bay and São Vicente Estuary, state of São Paulo, Brazil. Monthly samples were obtained, from May 2008 through April 2010, from four locations in the estuary and four in the bay. No individual was collected in the estuary and this was attributed to the low salinity means recorded in this environment. We collected 109,153 individuals in the bay and there was no difference in abundance between the two years comprised by the study period. The similarity in spatial distribution can be related to sediment grain size that in all sampling locations showed great amount of very fine sand. The largest amount of reproductive females was obtained in early 2010, when temperature was high, and this could have increased the juvenile recruitment in April 2010. According to our results, the distribution of X. kroyeri in the study area is influenced by temperature, which is related to reproduction, and salinity, limiting the entrance of individuals in the estuarine region.

Pharmacological chaperones stabilize retromer to limit APP processing.

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.

Population structure, sex ratio and growth of the seabob shrimp Xiphopenaeuskroyeri (Decapoda, Penaeidae) from coastal waters of southern Brazil.

This study evaluated the growth and population structure of Xiphopenaeuskroyeri in Babitonga Bay, southern Brazil. Monthly trawls were conducted from July 2010 through June 2011, using a shrimp boat outfitted with double-rig nets, at depths from 5 to 17 m. Differences from the expected 0.5 sex ratio were determined by applying a Binomial test. A von Bertalanffy growth model was used to estimate the individual growth, and longevity was calculated using its inverted formula. A total of 4,007 individuals were measured, including 1,106 juveniles (sexually immature) and 2,901 adults. Females predominated in the larger size classes. Males and females showed asymptotic lengths of 27.7 mm and 31.4 mm, growth constants of 0.0086 and 0.0070 per day, and longevities of 538 and 661 days, respectively. The predominance of females in larger size classes is the general rule in species of Penaeidae. The paradigm of latitudinal-effect does not appear to apply to seabob shrimp on the southern Brazilian coast, perhaps because of the small proportion of larger individuals, the occurrence of cryptic species, or the intense fishing pressure in this region. The longevity values are within the general range for species of Penaeidae. The higher estimates for longevity in populations at lower latitudes may have occurred because of the growth constants observed at these locations, resulting in overestimation of this parameter.

Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system.

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

Small misfolded Tau species are internalized via bulk endocytosis and anterogradely and retrogradely transported in neurons.

The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845-12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909-913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis.

Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for 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.

The ocular albinism type 1 (OA1) GPCR is ubiquitinated and its traffic requires endosomal sorting complex responsible for transport (ESCRT) function.

The function of signaling receptors is tightly controlled by their intracellular trafficking. One major regulatory mechanism within the endo-lysosomal system required for receptor localization and down-regulation is protein modification by ubiquitination and downstream interactions with the endosomal sorting complex responsible for transport (ESCRT) machinery. Whether and how these mechanisms operate to regulate endosomal sorting of mammalian G protein-coupled receptors (GPCRs) remains unclear. Here, we explore the involvement of ubiquitin and ESCRTs in the trafficking of OA1, a pigment cell-specific GPCR, target of mutations in Ocular Albinism type 1, which localizes intracellularly to melanosomes to regulate their biogenesis. Using biochemical and morphological methods in combination with overexpression and inactivation approaches we show that OA1 is ubiquitinated and that its intracellular sorting and down-regulation requires functional ESCRT components. Depletion or overexpression of subunits of ESCRT-0, -I, and -III markedly inhibits OA1 degradation with concomitant retention within the modified endosomal system. Our data further show that OA1 ubiquitination is uniquely required for targeting to the intralumenal vesicles of multivesicular endosomes, thereby regulating the balance between down-regulation and delivery to melanosomes. This study highlights the role of ubiquitination and the ESCRT machinery in the intracellular trafficking of mammalian GPCRs and has implications for the physiopathology of ocular albinism type 1.