Expression and Transport of a-Synuclein at the Blood-Cerebrospinal Fluid Barrier and Effects of Manganese Exposure

The choroid plexus maintains the homeostasis of critical molecules in the brain by regulating their transport between the blood and cerebrospinal fluid (CSF). The current study was designed to investigate the potential role of the blood-CSF barrier (BCSFB) in α-synuclein (a-Syn) transport in the brain as affected by exposure to manganese (Mn), the toxic metal implicated in Parkinsonian disorders. Immunohistochemistry was used to identify intracellular a-Syn expression at the BCSFB. Quantitative real-time PCR was used to quantify the change in a-Syn mRNA expression following Mn treatments at the BCSFB in vitro. ELISA was used to quantify a-Syn levels following in vivo and in vitro treatments of Mn, copper (Cu), and/or external a-Syn. Thioflavin-T assay was used to investigate a-Syn aggregation after incubating with Mn and/or Cu in vitro. A two-chamber Transwell system was used to study a-Syn transport by BCSFB monolayer.Data revealed the expression of endogenous a-Syn in rat choroid plexus tissue and immortalized choroidal epithelial Z310 cells. The cultured primary choroidal epithelia from rats showed the ability to take up a-Syn from extracellular medium and transport a-Syn across the cellular monolayer from the donor to receiver chamber. Exposure of cells with Mn induced intracellular a-Syn accumulation without causing any significant changes in a-Syn mRNA expression. A significant increase in a-Syn aggregation in a cell-free system was observed with the presence of Mn. Moreover, Mn exposure resulted in a significant uptake of a-Syn by primary cells.These data indicate that the BCSFB expresses a-Syn endogenously and is capable of transporting a-Syn across the BCSFB monolayer; Mn exposure apparently increases a-Syn accumulation in the BCSFB by facilitating its uptake and intracellular aggregation

Direct Detection of α-Synuclein Dimerization Dynamics: Single-Molecule Fluorescence Analysis

AbstractThe aggregation of α-synuclein (α-Syn) is linked to Parkinson’s disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways

Two C-terminal Sequence Variations Determine Differential Neurotoxicity Between Human and Mouse α-synuclein

BACKGROUND: α-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson\u27s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology. METHODS: Primary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey\u27s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn\u27s multiple comparisons test or a two-tailed Mann-Whitney test. RESULTS: Mouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology. CONCLUSIONS: Mouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders

Alpha Synuclein Membrane-Induced Aggregation: Its Role in Parkinson\u27s Disease and a Potential Therapeutic Target

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that causes significant hardship for patients and caregivers for many years. The classic motor symptoms observed in PD are caused by the death of dopaminergic neurons in the substantia nigra region of the brain. The surviving dopaminergic neurons contain cytoplasmic inclusions call Lewy bodies, which predominantly contain large amyloid-like fibrils of the protein alpha-synuclein (aSyn). The production of these amyloid fibrils can be easily replicated in a test tube using purified protein. It is widely believed that the aggregation of aSyn is a key event in the development PD. Detailed insights into the process by which aSyn aggregates in the cell are critical for our understanding of the disease and for the development of new therapeutics to treat PD. Despite considerable efforts to develop inhibitors of aSyn fibrillization over the past 15 years, this approach has met with limited success. This has led to the hypothesis that the aggregation of aSyn, at least in early stages, occurs by a mechanism independent of aSyn fibril formation. In the cell, a considerable fraction of aSyn is bound to lipid membranes, which render it resistant to aggregation. We and others have hypothesized that disruption of the normal membrane interaction could promote aggregation of aSyn on the membrane surface. To examine this hypothesis, we characterized the membrane interaction and membrane-induced self-assembly of the familial aSyn mutants A30P and G51D and other aSyn variants. This study, one of the first in-depth analyses of the G51D mutant, revealed that A30P and G51D display an increased propensity for aggregation at the membrane surface and increased dopaminergic neurotoxicity compared to WT aSyn. In contrast, aSyn variants with a weak propensity for membrane-induced aggregation are non-toxic. Taken together, these results suggested a correlation between aSyn aggregation propensity at the membrane surface and the ability of aSyn to elicit dopaminergic neurotoxicity. By extension of this hypothesis, reducing membrane-induced aSyn aggregation should protect against aSyn-mediated neurodegeneration. To test this idea, we investigated whether an interacting protein could interfere with membrane-induced aSyn aggregation and alleviate aSyn neurotoxicity. The goal of this study was in part to strengthen our initial hypothesis that membrane-induced aggregation is involved in aSyn-mediated dopaminergic cell death, but in addition we aimed to determine whether membrane-bound aSyn could be a valid therapeutic target. The aSyn binding partner examined here was endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn and to be down-regulated in the brains of individuals with Alzheimer’s disease and Down’s syndrome. We found that ENSA interfered with membrane-induced aSyn aggregation, vesicle permeabilization, and alleviated aSyn neurotoxicity in a primary midbrain culture model of PD. Both the inhibitory and the neuroprotective activities of ENSA were lost when we introduced a point mutation previously shown to disrupt the binding of ENSA to membrane-associated aSyn, suggesting that ENSA interactions with aSyn at the membrane surface are critical for the ability of ENSA to interfere with aSyn aggregation, permeabilization, and neurotoxicity. The inhibitory effects of ENSA on membrane-induced aSyn aggregation, vesicle permeabilization, and aSyn neurotoxicity led us to hypothesize that a small molecule could potentially have similar protective activities. To identify candidates, we performed a phage display screen for heptapeptides that interact with different membrane-bound aSyn conformers. High-frequency interacting peptides were synthesized and examined for their effects on membrane-induced aggregation and vesicle permeabilization of G51D aSyn. Moreover, peptidomimetic derivatives of the peptides with enhanced metabolic stability were tested for the ability to protect against neurotoxicity elicited by G51D and A30P in primary midbrain cultures. We found several peptides and peptidomimetcs that interfered with membrane-induced aggregation of G51D and alleviated A30P- and G51D-mediated dopaminergic cell death. Together these results point to a key role for the aggregation of aSyn on the membrane surface in the neurotoxic effects elicited by this protein. This conclusion is strengthened by our observation that the inhibition of membrane-induced aSyn aggregation by ENSA was sufficient to alleviate aSyn neurotoxicity. Additionally, we showed that small-molecule peptides and peptidomimetics could be designed to reduce aSyn aggregation and aSyn-mediated dopaminergic cell death. Our findings strongly implicate membrane-induced aSyn aggregation in aSyn neurotoxicity and suggest that small-molecule inhibitors of aSyn self-assembly at membrane surfaces could be viable therapies to slow progression of the disease

Alpha Synuclein Membrane-Induced Aggregation: Its Role in Parkinson\u27s Disease and a Potential Therapeutic Target

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that causes significant hardship for patients and caregivers for many years. The classic motor symptoms observed in PD are caused by the death of dopaminergic neurons in the substantia nigra region of the brain. The surviving dopaminergic neurons contain cytoplasmic inclusions call Lewy bodies, which predominantly contain large amyloid-like fibrils of the protein alpha-synuclein (aSyn). The production of these amyloid fibrils can be easily replicated in a test tube using purified protein. It is widely believed that the aggregation of aSyn is a key event in the development PD. Detailed insights into the process by which aSyn aggregates in the cell are critical for our understanding of the disease and for the development of new therapeutics to treat PD. Despite considerable efforts to develop inhibitors of aSyn fibrillization over the past 15 years, this approach has met with limited success. This has led to the hypothesis that the aggregation of aSyn, at least in early stages, occurs by a mechanism independent of aSyn fibril formation. In the cell, a considerable fraction of aSyn is bound to lipid membranes, which render it resistant to aggregation. We and others have hypothesized that disruption of the normal membrane interaction could promote aggregation of aSyn on the membrane surface. To examine this hypothesis, we characterized the membrane interaction and membrane-induced self-assembly of the familial aSyn mutants A30P and G51D and other aSyn variants. This study, one of the first in-depth analyses of the G51D mutant, revealed that A30P and G51D display an increased propensity for aggregation at the membrane surface and increased dopaminergic neurotoxicity compared to WT aSyn. In contrast, aSyn variants with a weak propensity for membrane-induced aggregation are non-toxic. Taken together, these results suggested a correlation between aSyn aggregation propensity at the membrane surface and the ability of aSyn to elicit dopaminergic neurotoxicity. By extension of this hypothesis, reducing membrane-induced aSyn aggregation should protect against aSyn-mediated neurodegeneration. To test this idea, we investigated whether an interacting protein could interfere with membrane-induced aSyn aggregation and alleviate aSyn neurotoxicity. The goal of this study was in part to strengthen our initial hypothesis that membrane-induced aggregation is involved in aSyn-mediated dopaminergic cell death, but in addition we aimed to determine whether membrane-bound aSyn could be a valid therapeutic target. The aSyn binding partner examined here was endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn and to be down-regulated in the brains of individuals with Alzheimer’s disease and Down’s syndrome. We found that ENSA interfered with membrane-induced aSyn aggregation, vesicle permeabilization, and alleviated aSyn neurotoxicity in a primary midbrain culture model of PD. Both the inhibitory and the neuroprotective activities of ENSA were lost when we introduced a point mutation previously shown to disrupt the binding of ENSA to membrane-associated aSyn, suggesting that ENSA interactions with aSyn at the membrane surface are critical for the ability of ENSA to interfere with aSyn aggregation, permeabilization, and neurotoxicity. The inhibitory effects of ENSA on membrane-induced aSyn aggregation, vesicle permeabilization, and aSyn neurotoxicity led us to hypothesize that a small molecule could potentially have similar protective activities. To identify candidates, we performed a phage display screen for heptapeptides that interact with different membrane-bound aSyn conformers. High-frequency interacting peptides were synthesized and examined for their effects on membrane-induced aggregation and vesicle permeabilization of G51D aSyn. Moreover, peptidomimetic derivatives of the peptides with enhanced metabolic stability were tested for the ability to protect against neurotoxicity elicited by G51D and A30P in primary midbrain cultures. We found several peptides and peptidomimetcs that interfered with membrane-induced aggregation of G51D and alleviated A30P- and G51D-mediated dopaminergic cell death. Together these results point to a key role for the aggregation of aSyn on the membrane surface in the neurotoxic effects elicited by this protein. This conclusion is strengthened by our observation that the inhibition of membrane-induced aSyn aggregation by ENSA was sufficient to alleviate aSyn neurotoxicity. Additionally, we showed that small-molecule peptides and peptidomimetics could be designed to reduce aSyn aggregation and aSyn-mediated dopaminergic cell death. Our findings strongly implicate membrane-induced aSyn aggregation in aSyn neurotoxicity and suggest that small-molecule inhibitors of aSyn self-assembly at membrane surfaces could be viable therapies to slow progression of the disease

Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes.

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies

[Around the World, Or the Belgian Conquest]

Figure S1. ENSA does not have a pronounced effect on the binding of aSyn to phospholipid membranes. Figure S2. A29E aSyn fails to elicit membrane disruption. Figure S3. ENSA exhibits no membrane disruption activity on its own. Figure S4. Results of Western blot analysis showing equal expression levels of WT ENSA and S109E in primary midbrain cultures. Figure S5. WT ENSA and S109E do not elicit neurotoxicity when expressed alone or in combination with β-gal. Figure S6. Western blot image showing a trend towards reduced ENSA expression levels in the substantia nigra region of PD patients versus non-diseased individuals. Table S1. P values for vesicle permeabilization data in Fig. 3a and b. Table S2. P values for vesicle permeabilization data in Fig. 3c and d. Table S3. Summary of demographic information for donors of substantia nigra samples. (DOCX 421 kb

Lysosomal integral membrane protein-2 as a phospholipid receptor revealed by biophysical and cellular studies

Lysosomal integral membrane protein-2 (LIMP-2) is a glucocerebrosidase receptor, which is linked to kidney failure and other diseases. Here the authors show that LIMP-2 is also a phospholipid receptor and present the lipid-bound structure of the LIMP-2 luminal domain dimer and discuss its lipid trafficking mechanism