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

Functional assays for the assessment of the pathogenicity of variants of GOSR2, an ER-to-Golgi SNARE involved in progressive myoclonus epilepsies.

Progressive myoclonus epilepsies (PMEs) are inherited disorders characterized by myoclonus, generalized tonic-clonic seizures, and ataxia. One of the genes that is associated with PME is the ER-to-Golgi Qb-SNARE GOSR2, which forms a SNARE complex with syntaxin-5, Bet1 and Sec22b. Most PME patients are homo-zygous for a p.Gly144Trp mutation and develop similar clinical presentations. Recently, a patient who was compound heterozygous for p.Gly144Trp and a previously unseen p.Lys164del mutation was identified. Because this patient presented with a milder disease phenotype, we hypothesized that the p.Lys164del mutation may be less severe compared to p.Gly144Trp. To characterize the effect of the p.Gly144Trp and p.Lys164del mutations, both of which are present in the SNARE motif of GOSR2, we examined the corresponding mutations in the yeast ortholog Bos1. Yeasts expressing the orthologous mutants in Bos1 showed impaired growth, suggesting a partial loss of function, which was more severe for the Bos1 p.Gly176Trp mutation. Using anisotropy and gel filtration, we report that Bos1 p.Gly176Trp and p.Arg196del are capable of complex formation, but with partly reduced activity. Molecular dynamics (MD) simulations showed that the hydrophobic core, which triggers SNARE complex formation, is compromised due to the glycine-to-tryptophan substitution in both GOSR2 and Bos1. In contrast, the deletion of residue p.Lys164 (or p.Arg196del in Bos1) interferes with the formation of hydrogen bonds between GOSR2 and syntaxin-5. Despite these perturbations, all SNARE complexes stayed intact during longer simulations. Thus, our data suggest that the milder course of disease in compound heterozygous PME is due to less severe impairment of the SNARE function

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

Raw sequencing data of GBA1 (all exons) and LRRK2 (Exon 31 and 41)

Sequencing of GBA1 (all exons) and LRRK2 (Exon 31 and 41) for all lines included in the manuscript: LRRK2 kinase activity regulates lysosomal glucocerebrosidase in Parkinson's disease pathogenesis. We do not observed unexpected point mutations in theses genes in non-mutant cell lines

Impact of Tenancy on Land Management

Leasing of agricultural land is gaining in importance in North America. The impact of leasing on soil management practices is examined in an area in the Canadan province of Ontario. Prevailing land contracts are insecure and the rental land market appears to be imperfect in the area. Under these conditions leasing leads to undesirable soil management practices and consequently to a lower state of conservation and to lower crop productivity over time. A difference in soil management and crop productivity has been observed between owner-operated and rented land