alzheimer 's disease ( ad ) is defined by the appearance of pathological hallmarks within specific areas of the brain , including amyloid plaques , composed of extracellular amyloid beta ( a ) peptide   and neurofibrillary tangles ( nfts ) , composed of intracellular aggregates of the microtubule - associated protein tau . definitive diagnosis of ad is made at autopsy on the basis of these defining lesions , which have a well - documented but incompletely understood connection to disease . although it has been debated whether these lesions are mediators of disease , they have clear correlation to disease progression and are useful as clues to understanding the pathological processes accompanying neurodegeneration . additional hallmarks of ad include granulovacuolar degeneration ( gvd ) , characterized morphologically as intracellular double membrane - bound organelles harboring a dense core and extracellular tau detectable in the cerebrospinal fluid ( csf ) [ 3 , 4 ] . although the molecular mechanisms for individual pathological lesions have been proposed [ 57 ] , a unifying hypothesis that rationalizes the appearance of all hallmarks of disease has been elusive . here we review recent studies involving the intracellular vesicular pathways and their connection to the hallmark lesions of ad . we propose that vesicle trafficking dysfunction , and more specifically a failure in lysosomal fusion , may be the nidus of both defining lesions of ad as well as of gvd and extracellular tau , thus providing a unifying hypothesis for disease pathology . intracellular vesicle trafficking pathways form an interconnected , dynamic system for transfer of cellular constituents within the cell and between intracellular and extracellular compartments   ( figure 1 ) . for example , the epidermal growth factor receptor is endocytosed and incorporated into early endosomes after binding its ligand and becoming ubiquitylated [ 9 , 10 ] . endocytosed material can then be budded off into recycling endosomes for exocytic release or incorporated into intralumenal vesicles of the multivesicular body ( mvb ) by the sequential activity of the endosomal sorting complexes required for transport ( escrts ) [ 1113 ] . intralumenal vesicles and their contents may also be released as exosomes by fusion of the outer membrane of the mvb with the plasma membrane [ 14 , 15 ] . the endocytic pathway merges with the macroautophagic ( hereafter referred to as simply autophagic ) pathway at either the early endosome or mvb stage . in the autophagic pathway , target proteins are polyubiquitylated , fostering the binding of p62 , the autophagic protein microtubule - associated protein 1 light chain 3 ( lc3 ) , and the assembly of the autophagic membrane around the ubiquitylated cargo to form the autophagosome . the autophagosome can then fuse with the lysosome directly or with the mvb to form the amphisome , a slightly acidic hybrid organelle . the amphisome also can fuse with the lysosome to form the autolysosome , a caustic organelle that degrades the enclosed proteins and organelles [ 19 , 20 ] . the dynamic nature of this system is difficult to capture in fixed postmortem tissue ; however , there are several lines of evidence suggesting that changes in pathway flux are among the earliest pathologies observed in ad , preceding clinical symptoms of ad , intracellular nft formation , and extracellular amyloid deposition [ 21 , 22 ] . first , expression profiling during the progression of ad has revealed significant upregulation of effector genes of the early endosome ( including rab4 and rab5 ) , the late endosome ( rab7 ) , and the exocytic pathway ( rab27 ) [ 2325 ] . for example , overexpression of rab5 causes enlarged endosomes , one of the earliest pathological alterations observed in ad . rab7 upregulation is found in vulnerable hippocampal basal forebrain regions but not in relatively spared striatum and cerebellum in mild cognitive impairment and ad [ 24 , 27 ] . second , the accumulation of a and tau protein and the appearance of gvd bodies correlates with changes in trafficking pathways . full - length amyloid precursor protein ( app ) is a transmembrane protein with an incompletely understood function . a , the secreted peptide that aggregates to form senile plaques , is cleaved from app by sequential activities of - and -secretase enzymes . -secretase activity is catalyzed by multiple enzymes including bace1 , which contributes strongly to a peptide production , bace2 , which is a bace1 structural homolog , and lysosomal enzyme cathepsin b [ 29 , 30 ] . following - cleavage , -secretase - mediated cleavage takes place within the transmembrane domain , yielding primarily 40 ( a 
 40 ) or 42 ( a 
 42 ) amino acid peptides . a 
 40 is continuously and abundantly produced in both healthy and ad - affected brain , whereas a 
 42 is produced at lower levels in healthy individuals but is increased by familial ad ( fad- ) causing mutations . in general , csf a 
 42 , but not a 
 40 , can serve as a surrogate biomarker for a deposition in the brain [ 4 , 32 ] . observations in both human tissue and cell culture implicate the endocytic pathway in a production [ 22 , 3337 ] . ad - related endocytic dysfunction coincides with the detection of a within endosomal compartments and autophagic vacuoles that collect within dystrophic neurites with the initial rise in soluble a peptides [ 35 , 36 , 38 ] . the presence of a in early endosomes also is consistent with the colocalization of app and bace1 within the same early endocytic compartments [ 3942 ] and the degradation of bace within the endosomal - lysosomal system . one study suggests that internalized a can aggregate within the cell and disrupt the vesicular membrane , thus contributing to its pathologic effect . intracellular trafficking of proteins involves a series of cytosolic factors , some of which are implicated in the regulation of app trafficking and a generation . for example , rab6 , a protein implicated in membrane budding and clathrin , which mediates the internalization of app from the cell surface , affect app processing [ 45 , 46 ] . altogether , these data support a relationship between endocytic pathway dysfunction and the amyloidogenic processing of app . rare disease - causing mutations have been discovered within the app gene that result in increased a production , a42  :  a40 ratio , or a aggregation rate . more common , although still rare , are autosomal dominant mutations within the psen1 gene encoding presenilin 1(ps1 ) , a transmembrane protein that acts as the catalytic subunit of the -secretase complex [ 48 , 49 ] . this complex is located in the endoplasmic reticulum , transgolgi network , and endocytic compartments [ 50 , 51 ] . the precise mechanism through which ps1 mutations drive ad is unclear , but they can cause aberrant processing of app leading to increased a42  :  a40 ratio [ 52 , 53 ] . further studies have revealed that familial ad - linked ps1 mutations significantly reduce budding from the endoplasmic reticulum and golgi , thereby decreasing delivery of app to the cell surface . this suggests that familial ad - linked ps1 variants increase a production by decreasing intracellular transport of app , thus prolonging the availability of app for cleavage by - and -secretases within the golgi . ps1 may regulate protein trafficking through its interaction with several cytosolic factors involved in the regulation of vesicular transport , such as rab11 , rab6 , and rab regulators [ 5557 ] . other data have ascribed the pathogenic effects of ps1 mutations to its role in facilitating maturation and targeting of a subunit of the vacuolar atpase to the lysosome , which is essential for lysosomal acidification , protease activation , and degradation of lysosomal substrates . this supports the impaired lysosomal fusion hypothesis , in that bafilomycin a , a vacuolar atpase inhibitor , is known to impair fusion of the lysosome with other membrane - bound vesicles . however , recently the role of ps1 in the maturation of the vacuolar atpase has been challenged , instead attributing lysosomal dysfunction in ps1-mutant cells to changes in gene expression associated with lysosome biogenesis   or alteration in lysosomal calcium storage and release . calcium storage and release have been proposed to regulate vesicular fusion , and in fact , is dependent on protonmotive force . therefore differentiating between these potential mechanisms it will be important to determine the actual role of ps1 in the adult brain in order to parse these differences . because a is both generated and degraded via the endocytic and autophagic pathways , impaired lysosomal clearance could mediate ps1-dependent increases in a concentration . the identification of genetic mutations that cause early - onset ad has lead to a greater understanding of the molecular mechanisms of disease ; however , these mutations account for only a small fraction of cases . rather , the majority of cases are sporadic with multiple susceptibility genes contributing incremental risk of developing disease [ 64 , 65 ] . the role of each in ad pathogenesis will require further investigation ; however , several of these susceptibility genes have known functions within or adjacent to the endocytic pathway . the strongest risk locus identified thus far is the gene that encodes apolipoprotein e ( apoe ) , a protein component of lipoprotein particles that bind to cell surface receptors with function in lipid transport , a trafficking , synaptic function , immune regulation , and intracellular signaling [ 66 , 67 ] . a second risk - conferring gene is sorl1 , a receptor that participates in trafficking vesicles from the cell surface to the golgi - endoplasmic reticulum . other new risk genes , including bin1   and picalm , are involved in clathrin - mediated endocytosis . clusterin , also known as apolipoprotein j , has also been identified to increase risk of ad [ 64 , 65 ] and is hypothesized to act as an extracellular chaperone that influences a-aggregation and receptor - mediated a clearance by endocytosis [ 70 , 71 ] . although each of these genes confers only incremental risk , together they highlight the intracellular vesicle trafficking in the molecular pathogenesis of sporadic ad . the appearance of tau and phospho - tau ( p - tau ) in csf has been assumed to result from the passive release of tau from dying neurons . however , recently it was shown that tau may be actively secreted into the extracellular compartment , where it is positioned to participate in the transmission of neurofibrillary pathology [ 72 , 73 ] . exosome - mediated release is considered a common , yet unconventional , mechanism responsible for the secretion of other aggregation - prone proteins including -synuclein , prion protein , a , and tau . secreted tau associates with both typical exosomal proteins , such as alix and with proteins involved in tau misprocessing and ad pathogenesis , such as a , presenilins , and fyn kinase . somewhat surprisingly , the greatest amount of exosomal tau was found in early ad , with the greatest being in braak stage 3 , progressively less so in later stages of ad when neuronal death is rampant and absent from non - ad controls diagnosed with nontauopathic dementias . because there is relatively little neurofibrillary degeneration at this early stage , the elevation of p - tau in ad csf is not due to passive , nonspecific tau release consequent to neuronal death . .    a lysosomal fusion defect may account for the disease - related detection of tau in the csf . the p - tau that accumulates where dendritic microtubules are being lost in dendrites is largely vesicle associated , and some of these vesicles are amphisome like , harboring both endocytic ( fyn ) and autophagic ( lc3 ) markers . fyn activation during signal transduction typically causes the oligomerization and endocytosis of downstream elements   and targets at least some oligomerized targets of fyn phosphorylation to exosomes [ 81 , 82 ] . although in normal autophagy , lc3 is rarely exocytosed , the inability of the autophagic vesicle to fuse with the lysosome may alternatively shuttle vesicles towards the exosomal pathway ( figure 1 ) . this exosomal secretion of tau corroborates the recent suggestion that interneuronal transfer of tau may be an important aspect of pathogenesis and account for the stereotypic neurofibrillary lesion progression [ 8488 ] . furthermore , this is consistent with recent evidence gathered from the lysosomal storage disorder , niemann - pick type c disease , in which exosomal release of cholesterol may serve as a cellular mechanism to partially bypass the traffic block that results in its toxic accumulation within the lysosome . altogether , although tau lacks the usual features of secreted proteins , such as an n - terminal hydrophobic leader  sequence and lipidation sites , data suggests that tau is secreted , though it may be low in healthy neurons such as when the lysosomal system is functioning normally . failure of the lysosome to efficiently fuse with membrane - bound organelles and degrade encaptured proteins of the endocytic and autophagic pathways may also affect the intracellular concentrations of tau . consistent with lysosome - mediated effects on tau levels , soluble tau is a substrate for lysosomal proteases such as cathepsin d in vitro and in cultured cells [ 90 , 91 ] , and inhibition of lysosome function with chloroquine can increase bulk tau levels . moreover , tau immunoreactivity associates with lysosomal protease cathepsin b in ad brain   and neuronal lysosomes in sections of both ad and control brain . because tau concentration modulates tau aggregation at both nucleation and extension steps [ 95 , 96 ] , it is a direct modulator of neurofibrillary lesion formation . first among these is autophagy , which can mediate turnover of tau phosphorylated on kxgs motifs in the microtubule repeat region [ 97 , 98 ] . evidence suggests that p - tau is less susceptible to proteasomal degradation mediated by hsp90 . therefore , under these conditions , failure of lysosome degradation could lead to elevated p - tau levels . second , tau contains two sequence motifs for chaperone - mediated autophagy , a pathway that relies on hsc70 to target substrates directly to the lysosome . in a cellular model of tauopathy , the chaperone - mediated autophagy machinery and associated recognition motifs on tau protein enable the generation of tau fragments by lysosomal proteases . these fragments have a higher propensity to form -sheet aggregates that , once nucleated , can seed the aggregation of full length tau . because these fragments are generated via chaperone - mediated autophagy , they can be produced even if lysosomal fusion stalls so long as lysosomal proteases remain functional . overall , the failure of the lysosomal pathway to efficiently clear intracellular tau may foster its pathological aggregation not only by increasing cytosolic concentrations but by generating aggregation - nucleating fragments as well . endocytic abnormalities may be involved in non - ad tauopathies , independent from a deposition . tau is redistributed to microtubule - poor regions of the cell when it is present in excess of available microtubule - binding capacity , which can result from overexpression , mutation , or posttranslational modifications ( ptms ) that limit tau - microtubule binding [ 79 , 101 ] . since tau has been shown to modulate the activity of microtubule - associated motor proteins that mediate dendritic transport [ 102 , 103 ] , it is possible that toxicity resulting from tau accumulation at localized dendritic loci may have relevance to pathogenesis of non - ad tauopathies . this toxic , vesicle - associated tau accumulates selectively in microtubule - poor segments containing organized microtubule bundles , suggesting that its accumulation is both the cause and consequence of localized microtubule destabilization . furthermore , neuropathological examination of niemann - pick - type c disease has revealed tau - laden nfts without accompanying amyloid pathology . although the pathogenesis of the nfts in niemann - pick - type c disease is not clear , the presence of dystrophic axonal swellings suggest cytoskeletal abnormalities . this indicates that under certain conditions , lysosomal dysfunction alone can ultimately lead to nft formation in the absence of a deposits . the mechanisms regulating tau secretion through the intracellular vesicular trafficking pathways are unknown but may involve those also responsible for modulating pathological and normal tau biology , including alternative splicing and ptms . tau protein is encoded by a single gene comprising 16 exons [ 105 , 106 ] . exons 2 , 3 , and 10 undergo alternative splicing ; however , exon 3 is expressed only in the presence of exon 2 , thus yielding 6 isoforms . depending on the presence or absence of the protein sequence encoded by exon 10 ( e10 ) , tau isoforms are called 4r ( with e10 ) or 3r ( without e10 ) , referring to the number of imperfect microtubule - binding repeats . similarly , tau isoforms are called 0n ( without n - terminal inserts ) , 1n ( with one n - terminal insert , encoded by exon 2 ) , or 2n ( with two n - terminal inserts , encoded by exons 2 and 3 ) . e10 has received relatively more attention than either n - terminal inserts ( e2 and e3 ) due to its role in microtubule binding and self - aggregation . however , recent evidence suggests that while extracellular secretion of tau requires the presence of an unknown element in the n - terminal domain , e2 specifically inhibits this secretion . this agrees with known functions of the n - terminus of tau to mediate association with the plasma membrane and perimembranous structures .    in conjunction with the inclusion or exclusion of e10 , the affinity of tau for microtubules is regulated by its phosphorylation at sites in and around the microtubule - binding repeat region , with certain sites having more acute affects on the protein than others [ 108110 ] . exosomal fractions of conditioned media of human neuroblastoma cells are enriched in p - tau species associated with neurodegeneration . quantitative analysis has identified four epitopes within the proline - rich domain of tau that are most strongly enriched in secreted tau compared with intracellular tau . these epitopes include at270 , at8 , at100 , and at180 , which correspond to phosphorylation at thr-181 , ser-198 and ser-202 , ser-210 and thr-212 , and thr-231 , respectively . significantly , epitope at270 ( corresponding to phosphorylated thr-181 ) is the epitope most highly enriched in secreted tau and is also an established biomarker for csf - based diagnostics for early - stage ad [ 77 , 111 ] . the potential role of phosphorylation as a regulator of tau secretion is corroborated by csf biomarker data gathered from patients with acute brain injury , which at the cellular level involves axonal injury . importantly , total tau , but not p - tau , increases following acute brain injury , suggesting that while total tau may be released as a result of cellular damage and death , ptms may actively regulate tau secretion . as many as 30 different phosphorylation sites have been identified on tau , and although phosphorylation is the most comprehensively studied of the tau ptms , filamentous tau is known to be extensively modified by several ptms , including lysine - directed ubiquitylation . we and others have identified at least three sites of ubiquitylation on tau [ 115117 ] . although most well known for its role in proteasomal degradation of proteins , ubiquitin is also the best known signal for endocytic sorting . as a general rule , targeting to the proteasome requires attachment of a chain of at least four ubiquitins , whereas targeting to the endocytic pathway requires only a single ubiquitin or a short chain of two or three ubiquitins . these ubiquitins are recognized by the escrt machinery , which sort the ubiquitylated cargo and direct it towards its destined pathway [ 12 , 13 ] . in addition to the influence that ptms exert on tau function individually , it is thought that various modifications may cooperate and compete in a coordinated fashion . for example , recently two modifications have been identified that are also lysine directed : acetylation [ 120 , 121 ] and methylation . because these modifications both directly compete with ubiquitin for lysine site occupancy , there is potential for acetylation and methylation to directly affect the rate of turnover of tau protein and its motility through the endocytic pathway . furthermore , in other biochemical pathways , such as histone - regulated processes , there is precedent for ptms , including acetylation and methylation , to indirectly affect neighboring site modification , such as phosphorylation , though the potential for this type of modification crosstalk to be occurring in tau protein and its effect on tau metabolism has yet to be examined . gvd body load increases with disease severity and episodic memory decline [ 3 , 123 , 124 ] . gvd body ultrastructure has been extensively studied , revealing an electron - dense core with coarse or vesicular morphology surrounded by a double - layered membrane . because of the two - layered membrane , it has long been hypothesized that these lesions are of autophagic origin . recent work by us and others has implicated the endocytic pathway in the formation of gvd bodies due to strong immunoreactivity of chmp2b , a component of escrt - iii , in the gvd body core [ 126 , 127 ] . this work also suggests a failure of the mvb to fuse with the lysosome , which could account for an accumulation of autophagic intermediates   and an increased size and density of the mvb . we hypothesize that consequently , flux is routed to the autophagic pathway , which results in the accumulation of abnormally large amphisome - like intermediates owing to the same lysosomal fusion defect combined with mtor - mediated suppression of phagophore formation [ 129131 ] . the genetic increase in rab7 in ad [ 23 , 25 ] is somewhat inconsistent with this hypothetical lysosomal fusion failure , though it is possible that this upregulation is acting in an ineffective compensatory manner . however , it is interesting that late endosome marker rab24 , but not secretory protein rab27 , is upregulated in ca1 pyramidal neurons [ 23 , 25 ] considering that gvd most greatly affects the ca1 region of the hippocampus . this may reflect alternative responses to the disease process with one cell type opting for increased secretion and another for increased degradation to remove the toxic protein . we propose that lysosome fusion dysfunction is a candidate nidus for the major pathological hallmarks of ad including both defining lesions of ad , gvd bodies , as well as the presence of tau in the csf . although the origin of fusion dysfunction is not yet fully understood , it most likely results from the convergence of multiple factors , like the disease it manifests . the involvement of lysosomal dysfunction as an underlying and unifying hypothesis for ad pathology may change the overall view of ad pathogenesis . it provides novel insights into disease - associated mechanisms of protein misprocessing and potentially new modes of disease progression . there is also potential clinical importance of tau secretion biomarkers for csf - based diagnostics and for the direction of future disease - modifying therapeutics .
alzheimer 's disease is characterized pathologically by extracellular senile plaques , intracellular neurofibrillary tangles , and granulovacuolar degeneration . 
 it has been debated whether these hallmark lesions are markers or mediators of disease progression , and numerous paradigms have been proposed to explain the appearance of each lesion individually . 
 however , the unfaltering predictability of these lesions suggests a single pathological nidus central to disease onset and progression . 
 one of the earliest pathologies observed in alzheimer 's disease is endocytic dysfunction . here 
 we review the recent literature of endocytic dysfunction with particular focus on disrupted lysosomal fusion and propose it as a unifying hypothesis for the three most - studied lesions of alzheimer 's disease .