Autophagy and Tau Protein.

Neurofibrillary tangles, which consist of highly phosphorylated tau protein, and senile plaques (SPs) are pathological hallmarks of Alzheimer's disease (AD). In swollen axons, many autophagic vacuoles are observed around SP in the AD brain. This suggests that autophagy function is disturbed in AD. We used a neuronal cellular model of tauopathy (M1C cells), which harbors wild type tau (4R0N), to assess the effects of the lysosomotrophic agent NH4Cl, and autophagy inhibitors chloroquine and 3 methyladenine (3MA). It was found that chloroquine, NH4Cl and 3MA markedly increased tau accumulation. Thus, autophagy lysosomal system disturbances disturbed the degradation mechanisms of tau protein. Other studies also revealed that tau protein, including aggregated tau, is degraded via the autophagy lysosome system. Phosphorylated and C terminal truncated tau were also reported to disturb autophagy function. As a therapeutic strategy, autophagy upregulation was suggested. Thus far, as autophagy modulators, rapamycin, mTOCR1 inhibitor and its analogues, lithium, metformin, clonidine, curcumin, nicotinamide, bexaroten, and torehalose have been proposed. As a therapeutic strategy, autophagic modulation may be the next target of AD therapeutics.

Clioquinol Decreases Levels of Phosphorylated, Truncated, and Oligomerized Tau Protein.

The neuropathological hallmarks of Alzheimer's disease (AD) are senile plaques (SPs), which are composed of amyloid ß protein (Aß), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 µM CQ had no effect on cell viability; however, 100 µM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

Homocysteine Increases Tau Phosphorylation, Truncation and Oligomerization.

Increased plasma homocysteinemia is considered a risk factor of dementia, including Alzheimer's disease (AD) and vascular dementia. However, the reason elevated plasma homocysteinemia increases the risk of dementia remains unknown. A pathological hallmark of AD is neurofibrillary tangles (NFTs) that consist of pathologically phosphorylated tau proteins. The effect of homocysteine (Hcy) on tau aggregation was explored using human neuroblastoma M1C cells that constitutively express human wild-type tau (4R0N) under the control of a tetracycline off system, primary mouse cultured neurons, and by inducing hyperhomocysteinemia in a mouse model of tauopathy (HHCy mice). A wide range of Hcy concentrations (10-1000 µM) increased total tau and phosphorylated tau protein levels. Hcy activated glycogen synthase kinase 3, and cyclin dependent kinase 5, major tau phosphokinases, and inactivated protein phosphatase 2A, a main tau phosphatase. Hcy exhibited cytotoxic effects associated with enhanced activation of caspase. Truncation of tau in the C-terminus, the cleavage site of caspase 3 (i.e., D421, detected by the TauC3 antibody) was also increased. Total tau, phosphorylated tau, as well as C-terminal cleaved tau were increased in the sarkosyl insoluble tau fraction. Hcy also increased the level of tau oligomers, as indicated by the tau oligomer complex 1 (TOC1) antibody that specifically identifies oligomeric tau species, in the tris insoluble, sarkosyl soluble fraction. The levels of TOC1-positive oligomeric tau were increased in brain lysates from HHCy mice, and treating HHCy mice with S-adenosylmethionine, an intermediate of Hcy, reduced the levels of oligomeric tau to control levels. These observations suggest that Hcy increases the levels of phosphorylated tau as well as truncated tau species via caspase 3 activation, and enhanced tau oligomerization and aggregation.

Histones facilitate α-synuclein aggregation during neuronal apoptosis.

Ample in vitro and in vivo experimental evidence supports the hypothesis that intercellular transmission of α-synuclein (αS) is a mechanism underlying the spread of αS pathology in Parkinson's disease and related disorders. What remains unexplained is where and how initial transmissible αS aggregates form. In a previous study, we demonstrated that αS aggregates rapidly form in neurons with impaired nuclear membrane integrity due to the interaction between nuclear proaggregant factor(s) and αS and that such aggregates may serve as a source for αS seeding. In the present study, we identify histones as a potential nuclear proaggregant factor for αS aggregation in both apoptotic neurons and brains with αS pathology. We further demonstrate that histone-induced aggregates contain a range of αS oligomers, including protofibrils and mature fibrils, and that these αS aggregates can seed additional aggregation. Importantly, we demonstrate transmissibility in mouse brains from stereotaxic injection. This study provides new clues to the mechanism underlying initial pathological aggregation of αS in PD and related disorders, and could lead to novel diagnostic and therapeutic approaches.

Pioglitazone prevents tau oligomerization.

Tau aggregation and amyloid ß protein (Aß) deposition are the main causes of Alzheimer's disease (AD). Peroxisome proliferator-activated receptor γ (PPARγ) activation modulates Aß production. To test whether the PPARγ agonist pioglitazone (PIO) is also effective in preventing tau aggregation in AD, we used a cellular model in which wild-type tau protein (4R0N) is overexpressed (M1C cells) (Hamano et al., 2012) as well as primary neuronal cultures. PIO reduced both phosphorylated and total tau levels, and inactivated glycogen synthase kinase 3ß, a major tau kinase, associated with activation of Akt. In addition, PIO decreased cleaved caspase3 and C-terminal truncated tau species by caspase, which is expected to decrease tau aggregation. A fractionation study showed that PIO reduced high molecular-weight (120 kDa), oligomeric tau species in Tris Insoluble, sarkosyl-soluble fractions. Tau decrease was reversed by adding GW9662, a PPARγ antagonist. Together, our current results support the idea that PPARγ agonists may be useful therapeutic agents for AD.

Proaggregant nuclear factor(s) trigger rapid formation of α-synuclein aggregates in apoptotic neurons.

Cell-to-cell transmission of α-synuclein (αS) aggregates has been proposed to be responsible for progressive αS pathology in Parkinson disease (PD) and related disorders, including dementia with Lewy bodies. In support of this concept, a growing body of in vitro and in vivo experimental evidence shows that exogenously introduced αS aggregates can spread into surrounding cells and trigger PD-like pathology. It remains to be determined what factor(s) lead to initiation of αS aggregation that is capable of seeding subsequent propagation. In this study we demonstrate that filamentous αS aggregates form in neurons in response to apoptosis induced by staurosporine or other toxins-6-hydroxy-dopamine and 1-methyl-4-phenylpyridinium (MPP+). Interaction between αS and proaggregant nuclear factor(s) is associated with disruption of nuclear envelope integrity. Knocking down a key nuclear envelop constituent protein, lamin B1, enhances αS aggregation. Moreover, in vitro and in vivo experimental models demonstrate that aggregates released upon cell breakdown can be taken up by surrounding cells. Accordingly, we suggest that at least some αS aggregation might be related to neuronal apoptosis or loss of nuclear membrane integrity, exposing cytosolic α-synuclein to proaggregant nuclear factors. These findings provide new clues to the pathogenesis of PD and related disorders that can lead to novel treatments of these disorders. Specifically, finding ways to limit the effects of apoptosis on αS aggregation, deposition, local uptake and subsequent propagation might significantly impact progression of disease.

Genetic modification of H2AX renders mesenchymal stromal cell-derived dopamine neurons more resistant to DNA damage and subsequent apoptosis.

BACKGROUND AIMS: Aberrant production of reactive oxygen species (ROS) and its impact on the integrity of genomic DNA have been considered one of the major risk factors for the loss of dopaminergic neurons in Parkinson's disease (PD). Stem cell transplantation as a strategy to replenish new functional neurons has great potential for PD treatment. However, limited survival of stem cells post-transplantation has always been an obstacle ascribed to the existence of neurotoxic environment in PD patients. METHODS: To improve the survival of transplanted stem cells for PD treatment, we explored a new strategy based on the function of the H2AX gene (H2A histone family, member X) in determination of DNA repair and cell apoptosis. We introduced a mutant form Y142F of H2AX into dopamine (DA) neuron-like cells differentiated from bone marrow-derived mesenchymal stromal cells (BMSCs). RESULTS: Expression of H2AX(Y142F) renders DA neuron-like cells more resistant to DNA damage and subsequent cell death induced by ultraviolet irradiation and 1-methyl-4-phenylpyridinium (MPP+) treatment. DISCUSSION: This is a meaningful attempt to improve the sustainability of BMSC-derived dopamine neurons under a brain neurotoxic environment. Further studies are needed to evaluate the implications of our findings in stem cell therapy for PD and related diseases.

Physiologically relevant factors influence tau phosphorylation by leucine-rich repeat kinase 2.

Hyperphosphorylation and aggregation of tau are observed in multiple neurodegenerative diseases termed tauopathies. Tau has also been implicated in the pathogenesis of Parkinson's disease (PD) and parkinsonisms. Some PD patients with mutations in the leucine-rich repeat kinase 2 (LRRK2) gene exhibit tau pathology. Mutations in LRRK2 are a major risk factor for PD, but LRRK2 protein function remains unclear. The most common mutation, G2019S, is located in the kinase domain of LRRK2 and enhances kinase activity in vitro. This suggests that the kinase activity of LRRK2 may underlie its cellular toxicity. Recently, in vitro studies have suggested a direct interaction between tubulin-bound tau and LRRK2 that results in tau phosphorylation at one identified site. Here we present data suggesting that microtubules (MTs) enhance LRRK2-mediated tau phosphorylation at three different epitopes. We also explore the effect of divalent cations as catalytic cofactors for G2019S LRRK2-mediated tau phosphorylation and show that manganese does not support kinase activity but inhibits the efficient ability of magnesium to catalyze LRRK2-mediated phosphorylation of tau. These results suggest that cofactors such as MTs and cations in the cellular milieu have an important impact on LRRK2-tau interactions and resultant tau phosphorylation.

Corticobasal degeneration with olivopontocerebellar atrophy and TDP-43 pathology: an unusual clinicopathologic variant of CBD.

Corticobasal degeneration (CBD) is a disorder affecting cognition and movement due to a progressive neurodegeneration associated with distinctive neuropathologic features, including abnormal phosphorylated tau protein in neurons and glia in cortex, basal ganglia, diencephalon, and brainstem, as well as ballooned neurons and astrocytic plaques. We identified three cases of CBD with olivopontocerebellar atrophy (CBD-OPCA) that did not have α-synuclein-positive glial cytoplasmic inclusions of multiple system atrophy (MSA). Two patients had clinical features suggestive of progressive supranuclear palsy (PSP), and the third case had cerebellar ataxia thought to be due to idiopathic OPCA. Neuropathologic features of CBD-OPCA are compared to typical CBD, as well as MSA and PSP. CBD-OPCA and MSA had marked neuronal loss in pontine nuclei, inferior olivary nucleus, and Purkinje cell layer. Neuronal loss and grumose degeneration in the cerebellar dentate nucleus were comparable in CBD-OPCA and PSP. Image analysis of tau pathology showed greater infratentorial tau burden, especially in pontine base, in CBD-OPCA compared with typical CBD. In addition, CBD-OPCA had TDP-43 immunoreactive neuronal and glial cytoplasmic inclusions and threads throughout the basal ganglia and in olivopontocerebellar system. CBD-OPCA met neuropathologic research diagnostic criteria for CBD and shared tau biochemical characteristics with typical CBD. These results suggest that CBD-OPCA is a distinct clinicopathologic variant of CBD with olivopontocerebellar TDP-43 pathology.

A proteomic study identifies different levels of light chain ferritin in corticobasal degeneration and progressive supranuclear palsy.

Clinical and pathological evidence supports the notion that corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) are distinct, but overlapping neurodegenerative tauopathies. Although both disorders are characterized by abnormal accumulation of 4-repeat tau, they display distinct proteolytic profiles of tau species and they have distinct astrocytic lesions, astrocytic plaques in CBD and tufted astrocytes in PSP. To investigate other differences between these two disorders at the molecular level, we compared the profiles of proteins from caudate nucleus of CBD and PSP by quantitative two-dimensional difference gel electrophoresis. Twenty-one protein spots differentially expressed in CBD and PSP were dissected for mass spectrometry (MS). One of the spots was identified by MS to contain light chain (LC) ferritin. Western blot analysis verified the presence of LC ferritin in this spot and showed that this protein was two-fold higher in caudate of CBD than that of PSP samples. These results were confirmed by LC ferritin immunohistochemistry. Co-labeling of caudate nucleus with tau and LC ferritin antibodies showed the presence of LC ferritin immunoreactivity in astrocytic plaques of CBD, but minimal labeling of tufted astrocytes in PSP. This difference did not reflect the extent of gliosis. Analysis of other brain regions in CBD and PSP showed no difference in LC ferritin levels. Together the data suggest that LC ferritin is a unique marker of astrocytic lesions in CBD, adding further support to the notion that CBD and PSP are distinct clinicopathologic entities.

Nanoparticles With Affinity for α-Synuclein Sequester α-Synuclein to Form Toxic Aggregates in Neurons With Endolysosomal Impairment.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. It is characterized pathologically by the aggregation of α-synuclein (αS) in the form of Lewy bodies and Lewy neurites. A major challenge in PD therapy is poor efficiency of drug delivery to the brain due to the blood-brain barrier (BBB). For this reason, nanomaterials, with significant advantages in drug delivery, have gained attention. On the other hand, recent studies have shown that nanoparticles can promote αS aggregation in salt solution. Therefore, we tested if nanoparticles could have the same effect in cell models. We found that nanoparticle can induce cells to form αS inclusions as shown in immunocytochemistry, and detergent-resistant αS aggregates as shown in biochemical analysis; and nanoparticles of smaller size can induce more αS inclusions. Moreover, the induction of αS inclusions is in part dependent on endolysosomal impairment and the affinity of αS to nanoparticles. More importantly, we found that the abnormally high level of endogenous lysosomotropic biomolecules (e.g., sphingosine), due to impairing the integrity of endolysosomes could be a determinant factor for the susceptibility of cells to nanoparticle-induced αS aggregation; and deletion of GBA1 gene to increase the level of intracellular sphingosine can render cultured cells more susceptible to the formation of αS inclusions in response to nanoparticle treatment. Ultrastructural examination of nanoparticle-treated cells revealed that the induced inclusions contained αS-immunopositive membranous structures, which were also observed in inclusions seeded by αS fibrils. These results suggest caution in the use of nanoparticles in PD therapy. Moreover, this study further supports the role of endolysosomal impairment in PD pathogenesis and suggests a possible mechanism underlying the formation of membrane-associated αS pathology.

Proteomic profiling of phosphoproteins and glycoproteins responsive to wild-type alpha-synuclein accumulation and aggregation.

A tetracycline inducible transfectant cell line (3D5) capable of producing soluble and sarkosyl-insoluble assemblies of wild-type human alpha-synuclein (alpha-Syn) upon differentiation with retinoic acid was used to study the impact of alpha-Syn accumulation on protein phosphorylation and glycosylation. Soluble proteins from 3D5 cells, with or without the induced alpha-Syn expression were analyzed by two-dimensional gel electrophoresis and staining of gels with dyes that bind to proteins (Sypro ruby), phosphoproteins (Pro-Q diamond) and glycoproteins (Pro-Q emerald). Phosphoproteins were further confirmed by binding to immobilized metal ion affinity column. alpha-Syn accumulation caused differential phosphorylation and glycosylation of 16 and 12, proteins, respectively, whose identity was revealed by mass spectrometry. These proteins, including HSP90, have diverse biological functions including protein folding, signal transduction, protein degradation and cytoskeletal regulation. Importantly, cells accumulating alpha-Syn assemblies with different abilities to bind thioflavin S displayed different changes in phosphorylation and glycosylation. Consistent with the cell-based studies, we demonstrated a reduced level of phosphorylated HSP90 alpha/beta in the substantia nigra of subjects with Parkinson's disease as compared to normal controls. Together, the results indicate that alpha-Syn accumulation causes complex cellular responses, which if persist may compromise cell viability.

Rho-kinase ROCK inhibitors reduce oligomeric tau protein.

Neurofibrillary tangles, one of the pathological hallmarks of Alzheimer's disease, consist of highly phosphorylated tau proteins. Tau protein binds to microtubules and is best known for its role in regulating microtubule dynamics. However, if tau protein is phosphorylated by activated major tau kinases, including glycogen synthase kinase 3ß or cyclin-dependent kinase 5, or inactivated tau phosphatase, including protein phosphatase 2A, its affinity for microtubules is reduced, and the free tau is believed to aggregate, thereby forming neurofibrillary tangles. We previously reported that pitavastatin decreases the total and phosphorylated tau protein using a cellular model of tauopathy. The reduction of tau was considered to be due to Rho-associated coiled-coil protein kinase (ROCK) inhibition by pitavastatin. ROCK plays important roles to organize the actin cytoskeleton, an expected therapeutic target of human disorders. Several ROCK inhibitors are clinically applied to prevent vasospasm postsubarachnoid hemorrhage (fasudil) and for the treatment of glaucoma (ripasudil). We have examined the effects of ROCK inhibitors (H1152, Y-27632, and fasudil [HA-1077]) on tau protein phosphorylation in detail. A human neuroblastoma cell line (M1C cells) that expresses wild-type tau protein (4R0N) by tetracycline-off (TetOff) induction, primary cultured mouse neurons, and a mouse model of tauopathy (rTG4510 line) were used. The levels of phosphorylated tau and caspase-cleaved tau were reduced by the ROCK inhibitors. Oligomeric tau levels were also reduced by ROCK inhibitors. After ROCK inhibitor treatment, glycogen synthase kinase 3ß, cyclin-dependent kinase 5, and caspase were inactivated, protein phosphatase 2A was activated, and the levels of IFN-γ were reduced. ROCK inhibitors activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Collectively, these results suggest that ROCK inhibitors represent a viable therapeutic route to reduce the pathogenic forms of tau protein in tauopathies, including Alzheimer's disease.

Using leucine zipper to facilitate alpha-synuclein assembly.

The accumulation of filamentous alpha-synuclein (alpha-S) is associated with Parkinson's disease. It remains controversial as to the mode (antiparallel or parallel) of alpha-S self-assembly and whether an exact alignment of the central hydrophobic region is essential. In the present study, we performed in vitro assembly using alpha-S with or without the attachment of artificial leucine zippers (Zips) capable of forming either parallel or antiparallel coiled coils and included a spacer in one derivative. Results showed that Zips accelerate filament assembly in both the parallel and antiparallel fashions, that a precise alignment of the central hydrophobic region is not essential, and that the antiparallel pairs displayed the highest thioflavin T signals. More importantly, cells expressing Zip-fused alpha-S, but not alpha-S alone, formed alpha-S immunopositive and thioflavin S-positive inclusions in 7 days. The results suggest that alpha-S can assemble in both parallel and antiparallel modes but have a higher tendency to assemble in the latter mode and that cells overexpressing Zip-fused alpha-S may be used to screen alpha-S assembly inhibitors due to enhanced ability to form inclusions.

Cathepsin D is the main lysosomal enzyme involved in the degradation of alpha-synuclein and generation of its carboxy-terminally truncated species.

Alpha-synuclein is likely to play a key role in the development of Parkinson's disease as well as other synucleinopathies. In animal models, overexpression of full-length or carboxy-terminally truncated alpha-synuclein has been shown to produce pathology. Although the proteosome and lysosome have been proposed to play a role in the degradation of alpha-synuclein, the enzyme(s) involved in alpha-synuclein clearance and generation of its carboxy-terminally truncated species have not been identified. In this study, the role of cathepsin D and calpain I in these processes was analyzed. In vitro experiments, using either recombinant or endogenous alpha-synuclein as substrates and purified cathepsin D or lysosomes, demonstrated that cathepsin D degraded alpha-synuclein very efficiently, and that limited proteolysis resulted in the generation of carboxy-terminally truncated species. Purified calpain I also cleaved alpha-synuclein, but carboxy-terminally truncated species were not the main cleavage products, and calpain I activity present in cellular lysates was not able to degrade the protein. Knockdown of cathepsin D in cells overexpressing wild-type alpha-synuclein increased total alpha-synuclein levels by 28% and lysosomal alpha-synuclein by 2-fold. In in vitro experiments, pepstatin A completely blocked the degradation of alpha-synuclein in purified lysosomes. Furthermore, lysosomes isolated from cathepsin D knockdown cells showed a marked reduction in alpha-synuclein degrading activity, indicating that cathepsin D is the main lysosomal enzyme involved in alpha-synuclein degradation. Our findings suggest that upregulation of cathepsin D could be an additional therapeutic strategy to lessen alpha-synuclein burden in synucleinopathies.

Aggregates assembled from overexpression of wild-type alpha-synuclein are not toxic to human neuronal cells.

Filamentous alpha-synuclein (alpha-syn) aggregates form Lewy bodies (LBs), the neuropathologic hallmarks of Parkinson disease and related alpha-synucleinopathies. To model Lewy body-associated neurodegeneration, we generated transfectant 3D5 of human neuronal-type in which expression of human wild-type alpha-syn is regulated by the tetracycline off (TetOff)-inducible mechanism. Retinoic acid-elicited differentiation promoted assembly of alpha-syn aggregates after TetOff induction in 3D5 cells. The aggregates accumulated 14 days after TetOff induction were primarily soluble and showed augmented thioflavin affinity with concomitant phosphorylation and nitration of alpha-syn. Extension of the induction led to the formation of sarkosyl-insoluble aggregates that appeared concurrently with thioflavin-positive inclusions. Immunoelectron microscopy revealed that the inclusions consist of dense bundles of 8- to 12-nm alpha-syn fibrils that congregate in the perikarya and resemble Lewy bodies. Most importantly, accumulation of soluble and insoluble aggregates after TetOff induction for 14 and 28 days was reversible and did not compromise the viability of the cells or their subsequent survival. Thus, this chemically defined culture paradigm provides a useful means to elucidate how oxidative injuries and other insults that are associated with aging promote alpha-syn to self-assemble or interact with other molecules leading to neuronal degeneration in alpha-synucleinopathies.

Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression.

The intracellular assembly of tau aggregates is a pathological hallmark shared by Alzheimer's disease and other neurodegenerative disorders known collectively as tauopathies. To model how tau fibrillogenesis evolves in tauopathies, we previously established transfectant M1C cultures from human neuroblastoma BE(2)-M17D cells that inducibly express human tau. In the present study, these cells were used to determine the role of the autophagic-lysosomal system in the degradation and aggregation of wild-type tau. Tau induction for 5 days led to the accumulation of tau with nominal assembly of tau aggregates within cells. When the lysosomotropic agent, chloroquine (CQ), was added following the termination of tau induction, tau clearance was delayed. Decreased tau truncation and increased levels of intact tau were observed. When present during tau induction, CQ led to tau accumulation and promoted the formation of sarkosyl-insoluble aggregates containing both truncated and full-length tau. CQ treatment significantly decreased the activities of cathepsins D, B and L, and the inhibition of cathepsins B and L mimicked the effect of CQ and increased tau levels in cells. Additionally, exposure of cells to the autophagy inhibitor, 3-methyladenine, led to tau accumulation and aggregation. These results suggest that the autophagic-lysosomal system plays a role in the clearance of tau, and that dysfunction of this system results in the formation of tau oligomers and insoluble aggregates.

Ethanol enhances tau accumulation in neuroblastoma cells that inducibly express tau.

Chronic alcohol consumption causes pathological changes in the brain and neuronal loss. Ethanol toxicity may partially result from the perturbation of microtubule-associated proteins, like tau. Tau dysfunction is well known for its involvement in certain neurodegenerative diseases, such as Alzheimer's disease. In the present study, the effect of ethanol on tau was examined using differentiated human neuroblastoma cells that inducibly express the 4R0N isoform of tau via a tetracycline-off expression system. During tau induction, ethanol exposure (1.25-5mg/ml) dose-dependently increased tau protein levels and reduced cell viability. The increase in cell death likely resulted from tau accumulation since increased levels of tau were sufficient to reduce cell viability and ethanol was toxic to cells expressing tau but not to non-induced controls. Tau accumulation did not result from greater tetracycline-off induction since ethanol increased neither tau mRNA expression nor the expression of the tetracycline-controlled transactivator. Additionally, ethanol increased endogenous tau protein levels in neuroblastoma cells lacking the tetracycline-off induction system for tau. Ethanol delayed tau clearance suggesting ethanol impedes its degradation. Though ethanol inhibited neither cathepsin B, cathepsin D, nor chymotrypsin-like activity, it did significantly reduce calpain I expression and activity. Calpain I knockdown by shRNA increased tau levels indicating that calpain participates in tau degradation in this model. Moreover, the activation of calpain, by the calcium ionophore A23187, partially reversed the accumulation of tau resulting from ethanol exposure. Impaired calpain-mediated degradation may thus contribute to the increased accumulation of tau caused by ethanol.

Impaired endo-lysosomal membrane integrity accelerates the seeding progression of α-synuclein aggregates.

In neurodegenerative diseases, seeding is a process initiated by the internalization of exogenous protein aggregates. Multiple pathways for internalization of aggregates have been proposed, including direct membrane penetration and endocytosis. To decipher the seeding mechanisms of alpha-synuclein (αS) aggregates in human cells, we visualized αS aggregation, endo-lysosome distribution, and endo-lysosome rupture in real-time. Our data suggest that exogenous αS can seed endogenous cytoplasmic αS by either directly penetrating the plasma membrane or via endocytosis-mediated endo-lysosome rupture, leading to formation of endo-lysosome-free or endo-lysosome-associated αS aggregates, respectively. Further, we demonstrate that αS aggregates isolated from postmortem human brains with diffuse Lewy body disease (DLBD) preferentially show endocytosis-mediated seeding associated with endo-lysosome rupture and have significantly reduced seeding activity compared to recombinant αS aggregates. Colocalization of αS pathology with galectin-3 (a marker of endo-lysosomal membrane rupture) in the basal forebrain of DLBD, but not in age-matched controls, suggests endo-lysosome rupture is involved in the formation of αS pathology in humans. Interestingly, cells with endo-lysosomal membrane permeabilization (LMP) are more vulnerable to the seeding effects of αS aggregates. This study suggests that endo-lysosomal impairment in neurons might play an important role in PD progression.

Recent advances in experimental modeling of the assembly of tau filaments.

Intracellular assembly of microtubule-associated protein tau into filamentous inclusions is central to Alzheimer's disease and related disorders collectively known as tauopathies. Although tau mutations, posttranslational modifications and degradations have been the focus of investigations, the mechanism of tau fibrillogenesis in vivo still remains elusive. Different strategies have been undertaken to generate animal and cellular models for tauopathies. Some are used to study the molecular events leading to the assembly and accumulation of tau filaments, and others to identify potential therapeutic agents that are capable of impeding tauopathy. This review highlights the latest developments in new models and how their utility improves our understanding of the sequence of events leading to human tauopathy.