Huntington's disease cerebrospinal fluid seeds aggregation of mutant huntingtin.

Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.

Proneurogenic Group II mGluR antagonist improves learning and reduces anxiety in Alzheimer Aß oligomer mouse.

Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aß42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-ß (Aß) oligomers but never develop Aß plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aß-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aß monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aß APP mutation.

Pathogenic amyloid-ß peptide precursor (APP) mutations clustered around position 693 of APP-position 22 of the Aß sequence--are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aß APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aß accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAß) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAß mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aß oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aß peptides form amyloid fibrils, aged E22ΔAß mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aß peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aß42 on E22Δ Aß40 fibrillogenesis. Moreover, E22Δ Aß42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aß aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

Transgenic mice as a model of pre-clinical Alzheimer's disease.

At diagnosis, Alzheimer's disease (AD) brains are extensively burdened with plaques and tangles and display a degree of synaptic failure most likely beyond therapeutic treatment. It is therefore crucial to identify early pathological events in the progression of the disease. While it is not currently feasible to identify and study early, pre-clinical stages of AD, transgenic (Tg) models offer a valuable tool in this regard. Here we investigated cognitive, structural and biochemical CNS alterations occurring in our newly developed McGill-Thyl-APP Tg mice (over-expressing the human amyloid precursor protein with the Swedish and Indiana mutations) prior to extracellular plaque deposition. Pre-plaque, 3-month old Tg mice already displayed cognitive deficits concomitant with reorganization of cortical cholinergic pre-synaptic terminals. Conformational specific antibodies revealed the early appearance of intracellular amyloid ß (Aß)-oligomers and fibrillar oligomers in pyramidal neurons of cerebral cortex and hippocampus. At the same age, the cortical levels of insulin degrading enzyme -a well established Aß-peptidase, were found to be significantly down-regulated. Our results suggest that, in the McGill-Thy1-APP Tg model, functional, structural and biochemical alterations are already present in the CNS at early, pre-plaque stages of the pathology. Accumulation of intraneuronal neurotoxic Aß-oligomers (possibly caused by a failure in the clearance machinery) is likely to be the culprit of such early, pre-plaque pathology. Similar neuronal alterations might occur prior to clinical diagnosis in AD, during a yet undefined 'latent' stage. A better understanding of such pre-clinical AD might yield novel therapeutic targets and or diagnostic tools.

Oxidation of Abeta and plaque biogenesis in Alzheimer's disease and Down syndrome.

The processes involved with beta-amyloid (Abeta) degradation and clearance in human brain are not well understood. We hypothesized that the distribution of oxidatively modified Abeta, as determined by an affinity-purified antibody in the entorhinal and frontal cortices of Alzheimer's disease (AD), Down syndrome (DS), nondemented elderly control cases, and canine brain, would provide insight into the mechanisms of Abeta accumulation. Based upon plaque counts, oxidized Abeta was present within 46-48% of diffuse and primitive plaques and 98% of cored plaques. Dense punctate deposits of oxidized Abeta were distributed throughout the neuropil in AD and DS brains but were also present within controls with mild neuropathology and isolated cognitive impairments. Confocal studies indicate that punctate oxidized Abeta deposits were within activated microglia. Oxidatively modified Abeta may reflect the efforts of microglial cells to take up and degrade Abeta. Oxidative modification of Abeta may be an early event in Abeta pathogenesis and may be important for plaque biogenesis.

Antibody-mediated phagocytosis of the amyloid beta-peptide in microglia is differentially modulated by C1q.

Microglial ingestion of the amyloid beta-peptide (Abeta) has been viewed as a therapeutic target in Alzheimer's disease, in that approaches that enhance clearance of Abeta relative to its production are predicted to result in decreased senile plaque formation, a proposed contributor to neuropathology. In vitro, scavenger receptors mediate ingestion of fibrillar Abeta (fAbeta) by microglia. However, the finding that cerebral amyloid deposition in a transgenic mouse model of Alzheimer's disease was diminished by inoculation with synthetic Abeta has suggested a possible therapeutic role for anti-Abeta Ab-mediated phagocytosis. Microglia also express C1qR(P), a receptor for complement protein C1q, ligation of which in vitro enhances phagocytosis of immune complexes formed with IgG levels below that required for optimal FcR-mediated phagocytosis. The data presented here demonstrate FcR-dependent ingestion of Abeta-anti-Abeta complexes (IgG-fAbeta) by microglia that is a function of the amount of Ab used to form immune complexes. In addition, C1q incorporated into IgG-fAbeta enhanced microglial uptake of these complexes when they contained suboptimal levels of anti-Abeta Ab. Mannose binding lectin and lung surfactant protein A, other ligands of C1qR(P), also enhanced ingestion of suboptimally opsonized IgG-fAbeta, whereas control proteins did not. Our data suggest that C1qR(P)-mediated events may promote efficient ingestion of Abeta at low Ab titers, and this may be beneficial in paradigms that seek to clear amyloid via FcR-mediated mechanisms by minimizing the potential for destructive Ab-induced complement-mediated processes.

Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease.

Cell-culture studies have revealed some of the fundamental features of the interaction of amyloid Abeta with cells and the mechanism of amyloid accumulation and pathogenesis in vitro. A(beta)1-42, the longer isoform of amyloid that is preferentially concentrated in senile plaque (SP) amyloid deposits in Alzheimer's disease (AD), is resistant to degradation and accumulates as insoluble aggregates in late endosomes or lysosomes. Once these aggregates have nucleated inside the cell, they grow by the addition of aberrantly folded APP and amyloidgenic fragments of APP, that would otherwise be degraded, onto the amyloid lattice in a fashion analogous to prion replication. This accumulation of heterogeneous aggregated APP fragments and Abeta appears to mimic the pathophysiologyof dystrophic neurites, where the same spectrum of components has been identified by immunohistochemistry. In the brain, this residue appears to be released into the extracellular space, possibly by a partially apoptotic mechanism that is restricted to the distal compartments of the neuron. Ultimately, this insoluble residue may be further digested to the protease-resistant A(beta)n-42 core, perhaps by microglia, where it accumulates as senile plaques. Thus, the dystrophic neurites are likely to be the source of the immediate precursors of amyloid in the senile plaques. This is the opposite of the commonly held view that extracellular accumulation of amyloid induces dystrophic neurites. Many of the key pathological events of AD may also be directly related to the intracellular accumulation of this insoluble amyloid. The aggregated, intracellular amyloid induces the production of reactive oxygen species (ROS) and lipid peroxidation products and ultimately results in the leakage of the lysosomal membrane. The breakdown of the lysosomal membrane may be a key pathogenic event, leading to the release of heparan sulfate and lysosomal hydrolases into the cytosol. Together, these observations provide the novel view that amyloid deposits and some of the early events of amyloid pathogenesis initiate randomly within single cells in AD. This pathogenic mechanism can explain some of the more enigmatic features of Alzheimer's pathogenesis, like the focal nature of amyloid plaques, the relationship between amyloid, dystrophic neurites and neurofibrillary-tangle pathology, and the miscompartmentalization of extracellular and cytosolic components observed in AD brain.

A rapidly diverging EGF protein regulates species-specific signal transduction in early sea urchin development.

The macromolecules mediating species-specific events during fertilization and early development and their molecular evolution are only beginning to be understood. We screened sea urchin ovary mRNA for species-specific gene products using representational differential analysis to identify unique transcripts in Strongylocentrotus franciscanus that are absent or divergent from a closely related species, S. purpuratus. One of the transcripts identified by this screening process is SfEGF-II, which contains four EGF repeats. SfEGF-II is orthologous to the previously reported genes S. purpuratus SpEGF-II and Anthocidaris crassispina AcEGF-II, encoding exogastrulation-inducing peptides (EGIP). EGF peptides derived from EGIP induce exogastrulation, a classical developmental defect, when added to embryos prior to gastrulation. The first three EGF repeats (EGF1-3) share 50 to 60% identity among the three species, but the fourth repeat (EGF4) is more divergent, displaying only 30% identity. Analysis of the sequence divergence indicates that the EGF-II genes display a relatively high nonsynonymous-to-synonymous ratio, a significant excess of radical compared to conservative amino acid substitutions, and a lack of polymorphism within SfEGF-II, indicating that these genes have been subjected to positive Darwinian selection. Recombinant EGF3 from S. franciscanus induces exogastrulation in both S. franciscanus and S. purpuratus. In contrast, recombinant EGF4 from both S. franciscanus and S. purpuratus induces exogastrula in a species-specific manner. In hybrid embryos, both species of EGF4 induce exogastrulation, suggesting that the receptor for this EGF molecule is expressed from both parental genomes during development. Both EGF3 and EGF4 induce the phosphorylation of membrane proteins of the blastula stage embryos, but EGF4 stimulates phosphorylation of proteins only in membranes prepared from homologous embryos, suggesting that it utilizes a unique pathway involving a species-specific receptor for EGF4. Thus, species-specific events of gastrulation and early development may be controlled by these rapidly diverging EGF molecules, through a novel species-specific signal transduction pathway.

A conformation change in the carboxyl terminus of Alzheimer's Abeta (1-40) accompanies the transition from dimer to fibril as revealed by fluorescence quenching analysis.

Alzheimer's disease is characterized by the presence of insoluble, fibrous deposits composed principally of amyloid beta (Abeta) peptide. A number of studies have provided information on the fibril structure and on the factors affecting fiber formation, but the details of the fibril structure are not known. We used fluorescence quenching to investigate the solvent accessibility and surface charge of the soluble Abeta(1-40) dimer and amyloid fibrils. Analogs of Abeta(1-40) containing a single tryptophan were synthesized by substituting residues at positions 4, 10, 34, and 40 with tryptophan. Quenching measurements in the dimeric state indicate that the amino-terminal analogs (AbetaF4W and AbetaY10W) are accessible to polar quenchers, and the more carboxyl-terminal analog AbetaV34W is less accessible. AbetaV40W, on the other hand, exhibits a low degree of quenching, indicating that this residue is highly shielded from the solvent in the dimeric state. Correcting for the effect of reduced translational and rotational diffusion, fibril formation was associated with a selective increase in solvent exposure of residues 34 and 40, suggesting that a conformation change may take place in the carboxyl-terminal region coincident with the dimer to fibril transition.

Complement component C1q modulates the phagocytosis of Abeta by microglia.

Recent studies showing that microglia internalize the amyloid beta-peptide (Abeta) suggest that these cells have the potential for clearing Abeta deposits in Alzheimer's disease, and mechanisms that regulate the removal of Abeta may therefore be of clinical interest. Previous studies from this laboratory showing that C1q enhances phagocytosis of cellular targets by rat microglia prompted the current investigations characterizing the effects of C1q on microglial phagocytosis of Abeta. Microglia were shown to phagocytose Abeta1-42, in agreement with observations of other investigators. Uptake of Abeta1-42 was observed for concentrations of 5-50 microM, and phagocytosis of peptides containing (14)C or fluorescein (FM) labels was not affected by the interaction of microglia with C1q-coated surfaces. However, inclusion of C1q (125 nM-1.4 microM) in solutions of 50 microM Abeta1-42 inhibited the uptake of (14)C-Abeta1-42 and FM-Abeta1-42, suggesting that C1q blocks the interaction of Abeta with microglia. Uptake of Abeta was partially blocked by the scavenger receptor ligands polyinosinic acid and maleylated BSA. Inhibition of Abeta uptake by C1q may contribute to the accumulation of fibrillar, C1q-containing plaques that occurs in parallel with disease progression. These data suggest that mechanisms which interfere with the binding of C1q to Abeta may be of therapeutic value both through inhibition of the inflammatory events resulting from complement activation and via altered access of Abeta sites necessary for ingestion by microglia.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

Toxicity of pyroglutaminated amyloid beta-peptides 3(pE)-40 and -42 is similar to that of A beta1-40 and -42.

An N-terminal truncated isoform of the amyloid beta-peptide (A beta) that begins with a pyroglutamate (pE) residue at position 3 [A beta3(pE)-42] is the predominant isoform found in senile plaques. Based upon previous in vitro studies regarding A beta N-terminal truncated isoforms, it has been hypothesized that A beta3(pE)-x isoforms may aggregate more rapidly and become more toxic than corresponding Abeta1-x peptides. However, the toxicity and aggregation properties of A beta3(pE)-42 and A beta3(pE)-40 have not previously been examined. After initial solubilization and 1-week preaggregation of each peptide at 37 degrees C and pH 7.4, the toxicity of 5-50 microM A beta3(pE)-42 was similar to that of A beta1-42. Moreover, the toxicity of A beta3(pE)-40 paralleled that induced by A beta1-40 in both 1 day in vitro (DIV) cortical and 7 DIV hippocampal cells. Circular dichroism spectra did not reveal major differences in secondary structure between aged A beta1-42, A beta3(pE)-42, A beta3(pE)-40, and A beta1-40 or freshly solubilized forms of these peptides. Overall, the data indicate that the loss of the two N-terminal amino acids and the cyclization of glutamate at position 3 do not alter the extracellular toxicity of A beta.

Binding of Zn(II), Cu(II), and Fe(II) ions to Alzheimer's A beta peptide studied by fluorescence.

Binding of Zn(II), Cu(II) and Fe(II) ions to A beta1-40, A beta1-42 and a single tryptophan mutant of Abeta 1-40 in solution at pH 7.4 was studied by fluorescent titration. Job plots and fitting of titration curves revealed formation of 1:1 and 1:2 peptide-metal complexes. For dimeric peptides A beta1-40 and A betaF4W the order of metal to peptide affinities is Fe < Cu > Zn, which is in agreement with the Irving-Williams series of complex stability. The affinity of A beta1-42 for Fe increases dramatically upon aggregation: K(D) changes from ca. 100 to ca. 0.2 microM.

Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix.

Most familial early-onset Alzheimer's disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer's disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexes in vivo with actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell-matrix interaction.

Intracellular accumulation of insoluble, newly synthesized abetan-42 in amyloid precursor protein-transfected cells that have been treated with Abeta1-42.

Our early study indicates that intracellular Abeta1-42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Abeta1-42 also leads to the accumulation of newly synthesized intracellular Abeta. Here we describe that newly synthesized Abeta, especially Abetan-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Abeta1-42 treatment. These results suggest that intracellular Abeta may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Abeta1-40, the solid-phase intracellular pathway preferentially produces Abetan-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Abeta also share the same types of Abeta structures that accumulate in the brain of Alzheimer's disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer's disease may arise by this solid-phase pathway.

gamma-secretase cleavage is distinct from endoplasmic reticulum degradation of the transmembrane domain of the amyloid precursor protein.

One of the critical cleavage events that generates Alzheimer's amyloid Abeta peptide occurs within the transmembrane domain (TMD) of the amyloid precursor protein (APP) and is carried out by a poorly understood enzyme activity known as gamma-secretase. To investigate this processing, a probe molecule, H26-57C, was constructed containing the TMD of APP flanked immediately on each side by unique epitope tags. H26-57C-transfected cells secrete a approximately 2.9-kDa fragment, indicating that the lumenal and cytosolic domains of APP are not required for gamma-secretase processing. Pulse-chase experiments indicate that the probe turns over with a half-life of 8 min. No degradation intermediates are detected during the chase period, indicating that TMD turnover is a highly processive mechanism. The protease inhibitors, ALLN and MG132, cause a dramatic (50-fold) increase in the steady-state amount of the probe. All of the inhibitors that prevent degradation of the probe in the rough endoplasmic reticulum increase the amount of the approximately 2.9-kDa fragment that is secreted into the media and also causes a similar increase the secretion of 4 kDa Abeta from APP-transfected cells. These results indicate that the system responsible for the degradation of the probe in the rough endoplasmic reticulum and the intramembrane cleavage by gamma-secretase that produces soluble, secreted Abeta are distinct and opposing processes.

Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis.

Previous studies have implicated the failure to degrade aggregated Abeta1-42 in late endosomes or secondary lysosomes as a mechanism for the accumulation of beta-amyloid in Alzheimer's disease. We examined the consequences of intracellular accumulation of Abeta1-42 on the integrity of the endosomal/lysosomal compartment by monitoring Lucifer Yellow fluorescence and the release of lysosomal hydrolases into the soluble, cytosolic fraction. In control cells, the Lucifer Yellow fluorescence is observed as punctate staining in a perinuclear distribution with no apparent cytoplasmic fluorescence, consistent with its localization in late endosomes or secondary lysosomes. After incubation with Abeta1-42 for 6 hr, a loss of lysosomal membrane impermeability is observed as evidenced by redistribution of the fluorescence to a diffuse, cytoplasmic pattern. The loss of lysosomal membrane impermeability is correlated with Abeta1-42 accumulation, since incubation of the cells with the nonaccumulating isoform of amyloid, Abeta1-40, does not induce leakage. The same results were obtained using the release of soluble lysosomal hydrolases, cathepsin D and beta-hexosaminidase, into the cytosol as an assay for the leakage of lysosomal contents. Together, our results suggest that the loss of lysosomal membrane impermeability may be an early event in Abeta pathogenesis, and provide an explanation for the miscompartmentalization of extracellular and cytoplasmic components observed in Alzheimer's disease (AD). The release of hydrolases may further cause the breakdown of the cytoskeleton and the blebbing of the plasma membrane, and the leakage of heparan sulfate glycosaminoglycans from the lysosome may ultimately promote the assembly of tau into neurofibrillary tangles (NFT).