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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nutrient deprivation induces α-synuclein aggregation through endoplasmic reticulum stress response and SREBP2 pathway.

Abnormal accumulation of filamentous α-synuclein (α-syn) in neurons, regarded as Lewy bodies (LBs), are a hallmark of Parkinson disease (PD). Although the exact mechanism(s) underlying LBs formation remains unknown, autophagy and ER stress response have emerged as two important pathways affecting α-syn aggregation. In present study we tested whether cells with the tetracycline-off inducible overexpression of α-syn and accumulating α-syn aggregates can benefit from autophagy activation elicited by nutrient deprivation (ND), since this approach was reported to effectively clear cellular polyglutamine aggregates. We found that nutrient deprivation of non-induced cells did not affect cell viability, but significantly activated autophagy reflected by increasing the level of autophagy marker LC3-II and autophagic flux and decrease of endogenous α-syn. Cells with induced α-syn expression alone displayed autophagy activation in an α-syn dose-dependent manner to reach a level comparable to that found in non-induced, nutrient deprived counterparts. Nutrient deprivation also activated autophagy further in α-syn induced cells, but the extent was decreased with increase of α-syn dose, indicating α-syn overexpression reduces the responsiveness of cells to nutrient deprivation. Moreover, the nutrient deprivation enhanced α-syn aggregations concomitant with significant increase of apoptosis as well as ER stress response, SREBP2 activation and cholesterolgenesis. Importantly, α-syn aggregate accumulation and other effects caused by nutrient deprivation were counteracted by knockdown of SREBP2, treatment with cholesterol lowering agent-lovastatin, or by GRP78 overexpression, which also caused decrease of SREBP2 activity. Similar results were obtained from studies of primary neurons with α-syn overexpression under nutrient deprivation. Together our findings suggested that down-regulation of SREBP2 activity might be a means to prevent α-syn aggregation in PD via reducing cholesterol levels.

Dopamine prevents lipid peroxidation-induced accumulation of toxic α-synuclein oligomers by preserving autophagy-lysosomal function.

The formation of Lewy bodies containing α-synuclein (α-syn), prominent loss of dopaminergic neurons and dopamine (DA) deficiency in substantia nigra and striatum are histopathological and biochemical hallmarks of Parkinson's disease (PD). Multiple lines of evidence have indicated that a critical pathogenic factor causing PD is enhanced production of reactive oxygen species (ROS), which reacts readily with polyunsaturated fatty acids to cause lipid peroxidation (LPO). LPO products have been shown to facilitate assembly of toxic α-syn oligomers in in vitro studies. Since DA is prone to autoxidation and cause ROS, it has been suggested that interactions among DA, LPO, and α-syn play an important role in neuronal loss in PD. However, the exact mechanism(s) remains unclear. We addressed this issue using a neuronal cell model which inducibly expresses human wild-type α-syn by the tetracycline off (Tet-Off) mechanism and stably expresses high levels of DA transporter. Under retinoic acid elicited neuronal differentiation, cells with or without overexpressing α-syn and with or without exposure to LPO inducer-arachidonic acid (AA), plus 0-500 µM of DA were assessed for the levels of LPO, α-syn accumulation, cell viability, and autophagy. AA exposure elicited similar LPO levels in cells with and without α-syn overexpression, but significantly enhanced the accumulation of α-syn oligomers and monomers only in cultures with Tet-Off induction and decreased cell survival in a LPO-dependent manner. Surprisingly, DA at low concentrations (<50 µM) protected cells from AA cytotoxicity and α-syn accumulation. Such effects were attributed to the ability of DA to preserve autophagic-lysosomal function compromised by the AA exposure. At high concentrations (>100 µM), DA exposure enhanced the toxic effects of AA. To our knowledge, this is the first report showing biphasic effects of DA on neuronal survival and α-syn accumulation.

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.

Characteristics of TBS-extractable hyperphosphorylated tau species: aggregation intermediates in rTg4510 mouse brain.

Conditional overexpression of four-repeat human tau containing the P301L missense mutation in the rTg4510 mouse model of tauopathy leads to progressive accumulation of neurofibrillary tangles and hyperphosphorylated, sarkosyl-insoluble tau species, which are biochemically comparable to abnormal tau characteristic of hereditary tauopathies termed FTDP-17. To fully understand the impact of tau species at different stages of self-assembly on neurodegeneration, we fractionated rTg4510 brain representing several stages of tauopathy to obtain TBS-extractable (S1), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble (P3) fractions. Under reducing condition, the S1 fraction was demonstrated by western blotting to contain both 50-60 kDa normally-sized and 64 kDa tau. Both are thermo-stable, but the 64 kDa tau showed a higher degree of phosphorylation. Under non-reducing condition, nearly all TBS-extractable 64 kDa tau were detected as ∼130 kDa species consistent with the size of dimer. Quantitative analysis showed ∼80 times more 64 kDa tau in S1 than P3 fraction. Immunoelectron microscopy revealed tau-positive granules/short filaments in S1 fraction. These structures displayed MC1 immunoreactivities indicative of conformational/pathological change of tau. MC1 immunoreactivity was detected by dot blotting in samples from 2.5 month-old mice, whereas Ab39 immunoreactivity indicative of late stages of tau assembly was detected only in P3 fraction. Quantitative analysis also demonstrated a significant inverse correlation between brain weight and 64 kDa tau, but the level of TBS-extractable 64 kDa tau reflects neurodegeneration better than that of sarkosyl-insoluble 64 kDa tau. Together, the findings suggest that TBS-extractable 64 kDa tau production is a potential target for therapeutic intervention of tauopathies.

Adenosine monophosphate-activated protein kinase overactivation leads to accumulation of α-synuclein oligomers and decrease of neurites.

Neuronal inclusions of α-synuclein (α-syn), termed Lewy bodies, are a hallmark of Parkinson disease (PD). Increased α-syn levels can occur in brains of aging human and neurotoxin-treated mice. Because previous studies have shown increased brain lactate levels in aging brains, in PD affected subjects when compared with age-matched controls, and in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP), we tested the effects of lactate exposure on α-syn in a cell-based study. We demonstrated that (1) lactate treatment led to α-syn accumulation and oligomerization in a time- and concentration-dependent manner; (2) such alterations were mediated via adenosine monophosphate-activated protein kinase (AMPK) and associated with increasing cytoplasmic phosphorylated AMPK levels; (3) AMPK activation facilitated α-syn accumulation and phosphorylation; (4) lactate treatment or overexpression of the active form of AMPK decreased α-syn turnover and neurite outgrowth; and (5) Lewy body-bearing neurons displayed abnormal cytoplasmic distribution of phosphorylated AMPK, which normally is located in nuclei. Together, our results suggest that chronic neuronal accumulation of α-syn induced by lactate-triggered AMPK activation in aging brains might be a novel mechanism underlying α-synucleinopathies in PD and related disorders.

Pitavastatin decreases tau levels via the inactivation of Rho/ROCK.

Epidemiological studies have shown that long-term treatment with statins decreases the risk of developing Alzheimer's disease. Statins have pleiotropic effects by lowering the concentration of isoprenoid intermediates. Although several studies have shown that statins may reduce amyloid beta protein levels, there have been few reports on the interaction between statins and tau. We report here that pitavastatin reduces total and phosphorylated tau levels in a cellular model of tauopathy, and in primary neuronal cultures. The decrease caused by pitavastatin is reversed by the addition of mevalonate, or geranylgeranyl pyrophosphate. The maturation of small G proteins, including RhoA was disrupted by pitavastatin, as was the activity of glycogen synthase kinase 3ß (GSK3ß), a major tau kinase. Toxin A, inhibitor of glycosylation of small G proteins, and Rho kinase (ROCK) inhibitor decreased phosphorylated tau levels. Rho kinase inhibitor also inactivated glycogen synthase kinase 3ß. Although the mechanisms responsible for the reduction in tau protein by pitavastatin require further examination, this report sheds light on possible therapeutic approaches to tauopathy.

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.

ER stress response plays an important role in aggregation of α-synuclein.

BACKGROUND: Accumulation of filamentous α-synuclein as Lewy bodies is a hallmark of Parkinson's disease. To identify the mechanisms involved in α-synuclein assembly and determine whether the assemblies are cytotoxic, we developed a cell model (3D5) that inducibly expresses wild-type human α-synuclein and forms inclusions that reproduce many morphological and biochemical characteristics of Lewy bodies. In the present study, we evaluated the effects of several histone deacetylase inhibitors on α-synuclein aggregation in 3D5 cells and primary neuronal cultures. These drugs have been demonstrated to protect cells transiently overexpressing α-synuclein from its toxicity. RESULTS: Contrary to transient transfectants, the drug treatment did not benefit 3D5 cells and primary cultures. The treated were less viable and contained more α-synuclein oligomers, active caspases 3 and 9, as well as ER stress markers than non-treated counterparts. The drug-treated, induced-3D5 cells, or primary cultures from transgenic mice overexpressing (<2 fold) α-synuclein, displayed more α-synuclein oligomers and ER stress markers than non-induced or non-transgenic counterparts. Similar effects were demonstrated in cultures treated with tunicamycin, an ER stressor. These effects were blocked by co-treatment with salubrinal, an ER stress inhibitor. In comparison, co-treatment with a pan caspase inhibitor protected cells from demise but did not reduce α-synuclein oligomer accumulation. CONCLUSIONS: Our results indicate that an increase of wild-type α-synuclein can elicit ER stress response and sensitize cells to further insults. Most importantly, an increase of ER stress response can promote the aggregation of wild type α-synuclein.

Phosphorylation regulates proteasomal-mediated degradation and solubility of TAR DNA binding protein-43 C-terminal fragments.

BACKGROUND: Inclusions of TAR DNA binding protein-43 (TDP-43) are the defining histopathological feature of several neurodegenerative diseases collectively referred to as TDP-43 proteinopathies. These diseases are characterized by the presence of cellular aggregates composed of abnormally phosphorylated, N-terminally truncated and ubiquitinated TDP-43 in the spinal cord and/or brain. Recent studies indicate that C-terminal fragments of TDP-43 are aggregation-prone and induce cytotoxicity. However, little is known regarding the pathways responsible for the degradation of these fragments and how their phosphorylation contributes to the pathogenesis of disease. RESULTS: Herein, we established a human neuroblastoma cell line (M17D3) that conditionally expresses an enhanced green fluorescent protein (GFP)-tagged caspase-cleaved C-terminal TDP-43 fragment (GFP-TDP220-414). We report that expression of this fragment within cells leads to a time-dependent formation of inclusions that are immunoreactive for both ubiquitin and phosphorylated TDP-43, thus recapitulating pathological hallmarks of TDP-43 proteinopathies. Phosphorylation of GFP-TDP220-414 renders it resistant to degradation and enhances its accumulation into insoluble aggregates. Nonetheless, GFP-TDP220-414 inclusions are reversible and can be cleared through the ubiquitin proteasome system. Moreover, both Hsp70 and Hsp90 bind to GFP-TDP220-414 and regulate its degradation. CONCLUSIONS: Our data indicates that inclusions formed from TDP-43 C-terminal fragments are reversible. Given that TDP-43 inclusions have been shown to confer toxicity, our findings have important therapeutic implications and suggest that modulating the phosphorylation state of TDP-43 C-terminal fragments may be a promising therapeutic strategy to clear TDP-43 inclusions.