Neither alpha-synuclein fibril strain nor host murine genotype influences seeding efficacy.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive motor symptoms and alpha-synuclein (αsyn) aggregation in the nervous system. For unclear reasons, PD patients with certain GBA1 mutations (GBA-PD) have a more aggressive clinical progression. Two testable hypotheses that can potentially account for this phenomenon are that GBA1 mutations promote αsyn spread or drive the generation of highly pathogenic αsyn polymorphs (i.e., strains). We tested these hypotheses by treating homozygous GBA1 D409V knockin (KI) mice with human α-syn-preformed fibrils (PFFs) and treating wild-type mice (WT) with several αsyn-PFF polymorphs amplified from brain autopsy samples collected from patients with idiopathic PD and GBA-PD patients with either homozygous or heterozygous GBA1 mutations. Robust phosphorylated-αsyn (PSER129) positive pathology was observed at the injection site (i.e., the olfactory bulb granule cell layer) and throughout the brain six months following PFF injection. The PFF seeding efficiency and degree of spread were similar regardless of the mouse genotype or PFF polymorphs. We found that PFFs amplified from the human brain, regardless of patient genotype, were generally more effective seeders than wholly synthetic PFFs (i.e., non-amplified); however, PFF concentration differed between these two studies, which might also account for the observed differences. To investigate whether the molecular composition of pathology differed between different seeding conditions, we performed Biotinylation by Antibody Recognition on PSER129 (BAR-PSER129). We found that for BAR-PSER129, the endogenous PSER129 pool dominated identified interactions, and thus, very few potential interactions were explicitly identified for seeded pathology. However, we found Dynactin Subunit 2 (Dctn2) interaction was shared across all PFF conditions, and NCK Associated Protein 1 (Nckap1) and Adaptor Related Protein Complex 3 Subunit Beta 2 (Ap3b2) were unique to PFFs amplified from GBA-PD brains of heterozygous mutation carriers. In conclusion, both the genotype and αsyn strain had little effect on overall seeding efficacy and global PSER129-interactions.

Neither alpha-synuclein-preformed fibrils derived from patients with GBA1 mutations nor the host murine genotype significantly influence seeding efficacy in the mouse olfactory bulb.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive motor symptoms and alpha-synuclein (αsyn) aggregation in the nervous system. For unclear reasons, PD patients with certain GBA mutations (GBA-PD) have a more aggressive clinical progression. Two testable hypotheses that can potentially account for this phenomenon are that GBA1 mutations promote αsyn spread or drive the generation of highly pathogenic αsyn polymorphs (i.e., strains). We tested these hypotheses by treating homozygous GBA1 D409V knockin (KI) mice with human α-syn-preformed fibrils (PFFs) and treating wild-type mice (WT) with several αsyn-PFF polymorphs amplified from brain autopsy samples collected from patients with idiopathic PD and GBA-PD patients with either homozygous or heterozygous GBA1 mutations. Robust phosphorylated-αsyn (PSER129) positive pathology was observed at the injection site (i.e., the olfactory bulb granular layer) and throughout the brain six months following PFF injection. The PFF seeding efficiency and degree of spread were similar regardless of the mouse genotype or PFF polymorphs. We found that PFFs amplified from the human brain, regardless of patient genotype, were generally more effective seeders than wholly synthetic PFFs (i.e., non-amplified); however, PFF concentration differed between these two studies, and this might also account for the observed differences. To investigate whether the molecular composition of pathology differed between different seeding conditions, we permed Biotinylation by Antibody Recognition on PSER129 (BAR-PSER129). We found that for BAR-PSER129, the endogenous PSER129 pool dominated identified interactions, and thus, very few potential interactions were explicitly identified for seeded pathology. However, we found Dctn2 interaction was shared across all PFF conditions, and Nckap1 and Ap3b2 were unique to PFFs amplified from GBA-PD brains of heterozygous mutation carriers. In conclusion, both the genotype and αsyn strain had little effect on overall seeding efficacy and global PSER129-interactions.

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis.

Synucleinopathies are neurodegenerative diseases characterized by pathological inclusions called "Lewy pathology" (LP) that consist of aggregated alpha-synuclein predominantly phosphorylated at serine 129 (PSER129). Despite the importance for understanding disease, little is known about the endogenous function of PSER129 or why it accumulates in the diseased brain. Here we conducted several observational studies using a sensitive tyramide signal amplification (TSA) technique to determine PSER129 distribution and function in the non-diseased mammalian brain. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb (OB) and several brain regions across the neuroaxis (i.e., OB to brainstem). In contrast, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. We found evidence of PSER129 positive structures in OB mitral cells of non-diseased mice, rats, non-human primates, and healthy humans. Using TSA multiplex fluorescent labeling, we showed that PSER129 positive punctate structures occur within inactive (i.e., c-fos negative) T-box transcription factor 21 (TBX21) positive mitral cells and PSER129 within these cells was spatially associated with PK-resistant alpha-synuclein. Ubiquitin was found in PSER129 mitral cells but was not closely associated with PSER129. Biotinylation by antibody recognition (BAR) identified 125 PSER129-interacting proteins in the OB of healthy mice, which were significantly enriched for presynaptic vesicle trafficking/recycling, SNARE, fatty acid oxidation, oxidative phosphorylation, and RNA binding. TSA multiplex labeling confirmed the physical association of BAR-identified protein Ywhag with PSER129 in the OB and in other regions across the neuroaxis. We conclude that PSER129 accumulates in the mitral cells of the healthy OB as part of alpha-synuclein normal cellular functions. Incidental LP has been reported in the OB, and therefore we speculate that for synucleinopathies, either the disease processes begin locally in OB mitral cells or a systemic disease process is most apparent in the OB because of the natural tendency to accumulate PSER129.

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber.

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome's functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon "Musculibacterium intestinale", which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations.

In situ proximity labeling identifies Lewy pathology molecular interactions in the human brain.

The intracellular misfolding and accumulation of alpha-synuclein into structures collectively called Lewy pathology (LP) is a central phenomenon for the pathogenesis of synucleinopathies, including Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Understanding the molecular architecture of LP is crucial for understanding synucleinopathy disease origins and progression. Here we used a technique called biotinylation by antibody recognition (BAR) to label total (BAR-SYN1) and pathological alpha-synuclein (BAR-PSER129) in situ for subsequent mass spectrometry analysis. Results showed superior immunohistochemical detection of LP following the BAR-PSER129 protocol, particularly for fibers and punctate pathology within the striatum and cortex. Mass spectrometry analysis of BAR-PSER129-labeled LP identified 261 significantly enriched proteins in the synucleinopathy brain when compared to nonsynucleinopathy brains. In contrast, BAR-SYN1 did not differentiate between disease and nonsynucleinopathy brains. Pathway analysis of BAR-PSER129-enriched proteins revealed enrichment for 718 pathways; notably, the most significant KEGG pathway was PD, and Gene Ontology (GO) cellular compartments were the vesicle, extracellular vesicle, extracellular exosome, and extracellular organelle. Pathway clustering revealed several superpathways, including metabolism, mitochondria, lysosome, and intracellular vesicle transport. Validation of the BAR-PSER129-identified protein hemoglobin beta (HBB) by immunohistochemistry confirmed the interaction of HBB with PSER129 Lewy neurites and Lewy bodies. In summary, BAR can be used to enrich for LP from formalin-fixed human primary tissues, which allowed the determination of molecular signatures of LP. This technique has broad potential to help understand the phenomenon of LP in primary human tissue and animal models.

Environmental triggers of Parkinson's disease - Implications of the Braak and dual-hit hypotheses.

Idiopathic Parkinson's disease (PD) may take decades to develop, during which many risk or protective factors may come into play to initiate the pathogenesis or modify its progression to clinical PD. The lack of understanding of this prodromal phase of PD and the factors involved has been a major hurdle in the study of PD etiology and preventive strategies. Although still controversial, the Braak and dual-hit hypotheses that PD may start peripherally in the olfactory structures and/or the gut provides a theoretical platform to identify the triggers and modifiers of PD prodromal development and progression. This is particularly true for the search of environmental causes of PD as the olfactory structures and gut are the major human mucosal interfaces with the environment. In this review, we lay out our personal views about how the Braak and dual-hit hypotheses may help us search for the environmental triggers and modifiers for PD, summarize available experimental and epidemiological evidence, and discuss research gaps and strategies.

Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.

The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.

Gut Microbiota Dysbiosis Is Associated with Elevated Bile Acids in Parkinson's Disease.

The gut microbiome can impact brain health and is altered in Parkinson's disease (PD). The vermiform appendix is a lymphoid tissue in the cecum implicated in the storage and regulation of the gut microbiota. We sought to determine whether the appendix microbiome is altered in PD and to analyze the biological consequences of the microbial alterations. We investigated the changes in the functional microbiota in the appendix of PD patients relative to controls (n = 12 PD, 16 C) by metatranscriptomic analysis. We found microbial dysbiosis affecting lipid metabolism, including an upregulation of bacteria responsible for secondary bile acid synthesis. We then quantitatively measure changes in bile acid abundance in PD relative to the controls in the appendix (n = 15 PD, 12 C) and ileum (n = 20 PD, 20 C). Bile acid analysis in the PD appendix reveals an increase in hydrophobic and secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). Further proteomic and transcriptomic analysis in the appendix and ileum corroborated these findings, highlighting changes in the PD gut that are consistent with a disruption in bile acid control, including alterations in mediators of cholesterol homeostasis and lipid metabolism. Microbially derived toxic bile acids are heightened in PD, which suggests biliary abnormalities may play a role in PD pathogenesis.

Activity-Based Protein Profiling of Bile Salt Hydrolysis in the Human Gut Microbiome with Beta-Lactam or Acrylamide-Based Probes.

Microbial bile salt hydrolases (BSHs) found in the intestine catalyze the deconjugation of taurine- and glycine-linked bile salts produced in the liver. The resulting bile salts are biological detergents and are critical in aiding lipophilic nutrient digestion. Therefore, the activity of BSHs in the gut microbiome is directly linked to human metabolism and overall health. Bile salt metabolism has also been associated with disease phenotypes such as liver and colorectal cancer. In order to reshape the gut microbiome to optimize bile salt metabolism, tools to characterize and quantify these processes must exist to enable a much-improved understanding of how metabolism goes awry in the face of disease, and how it can be improved through an altered lifestyle and environment. Furthermore, it is necessary to attribute metabolic activity to specific members and BSHs within the microbiome. To this end, we have developed activity-based probes with two different reactive groups to target bile salt hydrolases. These probes bind similarly to the authentic bile salt substrates, and we demonstrate enzyme labeling of active bile salt hydrolases by using purified protein, cell lysates, and in human stool.

Detecting differential protein abundance by combining peptide level P-values.

The majority of methods for detecting differentially abundant proteins between samples in label-free LC-MS bottom-up proteomics experiments rely on statistically testing inferred protein abundances derived from peptide ionization intensities or averaging peptide level statistics. Here, we statistically test peptide ionization intensities directly and combine the resulting dependent P-values using the Empirical Brown's Method (EBM), avoiding error introduced through the estimation of protein abundances or summarizing test statistics. We show that on a spike-in proteomics dataset, a peptide level approach using EBM outperforms differential abundance detection using a protein level approach and several analysis workflows, including MSstats. Additionally, we demonstrate the effectiveness of this approach by detecting enriched proteins from an activity-based protein profiling dataset.

Epigenomic analysis of Parkinson's disease neurons identifies Tet2 loss as neuroprotective.

Parkinson's disease (PD) pathogenesis may involve the epigenetic control of enhancers that modify neuronal functions. Here, we comprehensively examine DNA methylation at enhancers, genome-wide, in neurons of patients with PD and of control individuals. We find a widespread increase in cytosine modifications at enhancers in PD neurons, which is partly explained by elevated hydroxymethylation levels. In particular, patients with PD exhibit an epigenetic and transcriptional upregulation of TET2, a master-regulator of cytosine modification status. TET2 depletion in a neuronal cell model results in cytosine modification changes that are reciprocal to those observed in PD neurons. Moreover, Tet2 inactivation in mice fully prevents nigral dopaminergic neuronal loss induced by previous inflammation. Tet2 loss also attenuates transcriptional immune responses to an inflammatory trigger. Thus, widespread epigenetic dysregulation of enhancers in PD neurons may, in part, be mediated by increased TET2 expression. Decreased Tet2 activity is neuroprotective, in vivo, and may be a new therapeutic target for PD.

Endogenous alpha-synuclein monomers, oligomers and resulting pathology: let's talk about the lipids in the room.

Alpha-synuclein is an intrinsically disordered, highly dynamic protein that pathogenically aggregates into inclusion structures called Lewy bodies, in several neurogenerative diseases termed synucleinopathies. Despite its importance for understanding disease, the oligomerization status of alpha-synuclein in healthy cells remains unclear. Alpha-synuclein may exist predominantly as either a monomer or a variety of oligomers of different molecular weights. There is solid evidence to support both theories. Detection of apparent endogenous oligomers are intimately dependent on vesicle and lipid interactions. Here we consider the possibility that apparent endogenous alpha-synuclein oligomers are in fact conformations of membrane-bound alpha-synuclein and not a bona fide stable soluble species. This perspective posits that the formation of any alpha-synuclein oligomers within the cell is likely toxic and interconversion between monomer and oligomer is tightly controlled. This differs from the hypothesis that there is a continuum of endogenous non-toxic oligomers and they convert, through unclear mechanisms, to toxic oligomers. The distinction is important, because it clarifies the biological origin of synucleinopathy. We suggest that a monomer-only, lipid-centric view of endogenous alpha-synuclein aggregation can explain how alpha-synuclein pathology is triggered, and that the interactions between alpha-synuclein and lipids can represent a target for therapeutic intervention. This discussion is well-timed due to recent studies that show lipids are a significant component of Lewy pathology.

The Appendix in Parkinson's Disease: From Vestigial Remnant to Vital Organ?

Parkinson's disease (PD) has long been considered a brain disease, but studies now point to the gastrointestinal (GI) tract as a potential starting point for PD. In particular, the human vermiform appendix has been implicated in PD. The appendix is a tissue rich in immune cells, serving as part of the gut-associated lymphoid tissue and as a storehouse for the gut microbiome. The functions of the appendix converge with recent evidence demonstrating that gut inflammation and shifts in the microbiome are linked to PD. Some epidemiological studies have linked removal of the appendix to lowered PD risk, though there is controversy among these associations. What is apparent is that there is an abundance of aggregated forms of α-synuclein in the appendix relevant to PD pathology. α-Synuclein pathology is thought to propagate from gut to brain via the vagus nerve, which innervates GI tract locations, including the appendix. Remarkably, α-synuclein aggregates in the appendix occur not only in PD patients, but are also present in healthy individuals. This has led to the proposal that in the appendix α-synuclein aggregates are not unique to PD. Moreover, the molecular events leading to PD and the mechanisms by which α-synuclein aggregates transfers from gut to brain may be identifiable in the human appendix. The influence of the appendix on GI inflammation, autoimmunity, microbiome storage, and the lymphatic system may be yet unexplored mechanisms by which the appendix contributes to PD. Overall, the appendix represents a promising tissue site to advance our understanding of PD pathobiology.

Spreading of alpha-synuclein - relevant or epiphenomenon?

The intracellular accumulation of misfolded alpha-synuclein pathology, termed Lewy pathology, throughout the brain is a phenomenon central to Parkinson's disease pathogenesis. In recent years it has become apparent that Lewy pathology can spread from neuron-to-neuron and between interconnected brain regions. Understanding the phenomenon of Lewy pathology propagation holds great promise in its explanatory power to determine the etiology of Parkinson's disease and related synucleinopathies. However, it remains to be seen if the spread of Lewy pathology is critical for driving this disease. Here we discuss the spreading of Lewy pathology while highlighting some important concepts and experimental observations. We conclude that further studies are required to determine if, and how, the spreading behavior of Lewy pathology is involved in Parkinson's disease. "This article is part of the Special Issue Synuclein".

Differential methylation of enhancer at IGF2 is associated with abnormal dopamine synthesis in major psychosis.

Impaired neuronal processes, including dopamine imbalance, are central to the pathogenesis of major psychosis, but the molecular origins are unclear. Here we perform a multi-omics study of neurons isolated from the prefrontal cortex in schizophrenia and bipolar disorder (n = 55 cases and 27 controls). DNA methylation, transcriptomic, and genetic-epigenetic interactions in major psychosis converged on pathways of neurodevelopment, synaptic activity, and immune functions. We observe prominent hypomethylation of an enhancer within the insulin-like growth factor 2 (IGF2) gene in major psychosis neurons. Chromatin conformation analysis revealed that this enhancer targets the nearby tyrosine hydroxylase (TH) gene responsible for dopamine synthesis. In patients, we find hypomethylation of the IGF2 enhancer is associated with increased TH protein levels. In mice, Igf2 enhancer deletion disrupts the levels of TH protein and striatal dopamine, and induces transcriptional and proteomic abnormalities affecting neuronal structure and signaling. Our data suggests that epigenetic activation of the enhancer at IGF2 may enhance dopamine synthesis associated with major psychosis.

Benzo[ a]pyrene Induction of Glutathione S-Transferases: An Activity-Based Protein Profiling Investigation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules. Conjugative enzymes, such as glutathione S-transferases or UDP-glucuronosyltransferases, are responsible for the detoxification and/or facilitate the elimination of these carcinogens. While responses to PAH exposures have been extensively studied for the bioactivating cytochrome P450 enzymes, much less is known regarding the response of glutathione S-transferases in mammalian systems. In this study, we investigated the expression and activity responses of murine hepatic glutathione S-transferases to benzo[ a]pyrene exposure using global proteomics and activity-based protein profiling for chemoproteomics, respectively. Using this approach, we identified several enzymes exhibiting increased activity including GSTA2, M1, M2, M4, M6, and P1. The activity of one GST enzyme, GSTA4, was found to be downregulated with increasing B[ a]P dose. Activity responses of several of these enzymes were identified as being expression-independent when comparing global and activity-based data sets, possibly alluding to as of yet unknown regulatory post-translational mechanisms.

The vermiform appendix impacts the risk of developing Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) involves the accumulation of aggregated α-synuclein, which has been suggested to begin in the gastrointestinal tract. Here, we determined the capacity of the appendix to modify PD risk and influence pathogenesis. In two independent epidemiological datasets, involving more than 1.6 million individuals and over 91 million person-years, we observed that removal of the appendix decades before PD onset was associated with a lower risk for PD, particularly for individuals living in rural areas, and delayed the age of PD onset. We also found that the healthy human appendix contained intraneuronal α-synuclein aggregates and an abundance of PD pathology-associated α-synuclein truncation products that are known to accumulate in Lewy bodies, the pathological hallmark of PD. Lysates of human appendix tissue induced the rapid cleavage and oligomerization of full-length recombinant α-synuclein. Together, we propose that the normal human appendix contains pathogenic forms of α-synuclein that affect the risk of developing PD.

A Cobalamin Activity-Based Probe Enables Microbial Cell Growth and Finds New Cobalamin-Protein Interactions across Domains.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B12). Our knowledge of the roles of vitamin B12 is limited, because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B12 supports the growth of B12-auxotrophic bacteria and archaea. We demonstrate that a B12 activity-based probe (B12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B12-ABP akin to native B12 Identification of the proteins that bind the B12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B12 protein targets. The B12-ABP also regulates the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B12 interactions in living cells and can be used to discover the role of B12 in diverse microbial systems.IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B12 in microbial growth and regulation by supporting the growth of B12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B12-protein interactions. The B12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B12 The application of the B12-ABP at biologically relevant concentrations facilitates a unique way to measure B12 microbial dynamics and identify new B12 protein targets in bacteria and archaea. We demonstrate that the B12-ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms to the salt-tolerant archaea Haloferax volcanii.

Vertebrate food products as a potential source of prion-like α-synuclein.

The aberrant aggregation of the protein α-synuclein is thought to be involved in Parkinson's disease (PD). However, the factors that lead to initiation and propagation of α-synuclein aggregation are not clearly understood. Recently, the hypothesis that α-synuclein aggregation spreads via a prion-like mechanism originating in the gut has gained much scientific attention. If α-synuclein spreads via a prion-like mechanism, then an important question becomes, what are the origins of this prion-like species? Here we review the possibility that α-synuclein aggregation could be seeded via the ingestion of a prion-like α-synuclein species contained within food products originating from vertebrates. To do this, we highlight current evidence for the gut-to-brain hypothesis of PD, and put this in context of available routes of α-synuclein prion infectivity via the gastrointestinal (GI) tract. We then discuss meat as a ready exogenous source of α-synuclein and how certain risk factors, including inflammation, may allow for dietary α-synuclein to pass from the GI lumen into the host to induce pathology. Lastly, we review epidemiological evidence that dietary factors may be involved in PD. Overall, research to date has yet to directly test the contribution of dietary α-synuclein to the mechanism of initiation and progression of the disease. However, numerous experimental findings, including the potent seeding and spreading behavior of α-synuclein fibrils, seem to support, at least in part, the feasibility of an infection with a prion α-synuclein particle via the GI tract. Further studies are required to determine whether dietary α-synuclein contributes to seeding pathology in the gut.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.