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IMPORTANCE: Deposition of the pathological α-synuclein (αSynP) in the brain is the hallmark of synucleinopathies, including Parkinson disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA). Whether real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA) assays can sensitively detect skin biomarkers for PD and non-PD synucleinopathies remains unknown. OBJECTIVE: To develop sensitive and specific skin biomarkers for antemortem diagnosis of PD and other synucleinopathies. DESIGN, SETTING, AND PARTICIPANTS: This retrospective and prospective diagnostic study evaluated autopsy and biopsy skin samples from neuropathologically and clinically diagnosed patients with PD and controls without PD. Autopsy skin samples were obtained at 3 medical centers from August 2016 to September 2019, and biopsy samples were collected from 3 institutions from August 2018 to November 2019. Based on neuropathological and clinical diagnoses, 57 cadavers with synucleinopathies and 73 cadavers with nonsynucleinopathies as well as 20 living patients with PD and 21 living controls without PD were included. Specifically, cadavers and participants had PD, LBD, MSA, Alzheimer disease, progressive supranuclear palsy, or corticobasal degeneration or were nonneurodegenerative controls (NNCs). A total of 8 approached biopsy participants either refused to participate in or were excluded from this study due to uncertain clinical diagnosis. Data were analyzed from September 2019 to April 2020. MAIN OUTCOMES AND MEASURES: Skin αSynP seeding activity was analyzed by RT-QuIC and PMCA assays. RESULTS: A total of 160 autopsied skin specimens from 140 cadavers (85 male cadavers [60.7%]; mean [SD] age at death, 76.8 [10.1] years) and 41 antemortem skin biopsies (27 male participants [66%]; mean [SD] age at time of biopsy, 65.3 [9.2] years) were analyzed. RT-QuIC analysis of αSynP seeding activity in autopsy abdominal skin samples from 47 PD cadavers and 43 NNCs revealed 94% sensitivity (95% CI, 85-99) and 98% specificity (95% CI, 89-100). As groups, RT-QuIC also yielded 93% sensitivity (95% CI, 85-97) and 93% specificity (95% CI, 83-97) among 57 cadavers with synucleinopathies (PD, LBD, and MSA) and 73 cadavers without synucleinopathies (Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, and NNCs). PMCA showed 82% sensitivity (95% CI, 76-88) and 96% specificity (95% CI, 85-100) with autopsy abdominal skin samples from PD cadavers. From posterior cervical and leg skin biopsy tissues from patients with PD and controls without PD, the sensitivity and specificity were 95% (95% CI, 77-100) and 100% (95% CI, 84-100), respectively, for RT-QuIC and 80% (95% CI, 49-96) and 90% (95% CI, 60-100) for PMCA. CONCLUSIONS AND RELEVANCE: This study provides proof-of-concept that skin αSynP seeding activity may serve as a novel biomarker for antemortem diagnoses of PD and other synucleinopathies.

Missense mutations in Leucine-Rich Repeat Kinase 2(LRRK2) cause the majority of familial and some sporadic forms of Parkinson’s disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive—some with unwanted side effects and unclear clinical outcome—alternative types of LRRK2 inhibitors are lacking. Herein we identify 5′-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegansand D. melanogasterexpressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

Parkinson’s disease (PD) is the second most common neurodegenerative disease worldwide, caused by the degeneration of the dopaminergic neurons in the substantia nigra.Mutations in PARK7(DJ-1) result in early onset autosomal recessive PD, and oxidative modification of DJ-1 has been reported to regulate the protective activity of DJ-1 in vitro. Glutathionylation is a prevalent redox modification of proteins resulting from the disulfide adduction of the glutathione moiety to a reactive cysteine-SH, and glutathionylation of specific proteins has been implicated in regulation of cell viability. Glutaredoxin 1 (Grx1) is the principal deglutathionylating enzyme within cells, and it has been reported to mediate protection of dopaminergic neurons in Caenorhabditis elegans; however many of the functional downstream targets of Grx1 in vivoremain unknown. Previously, DJ-1 protein content was shown to decrease concomitantly with diminution of Grx1 protein content in cell culture of model neurons (SH-SY5Y and Neuro-2A lines). In the current study we aimed to investigate the regulation of DJ-1 by Grx1 in vivoand characterize its glutathionylation in vitro. Here, with Grx–/–mice we provide show that Grx1 regulates protein levels of DJ-1 in vivo. Furthermore, with model neuronal cells (SH-SY5Y) we observed decreased DJ-1 protein content in response to treatment with known glutathionylating agents, and with isolated DJ-1 we identified two distinct sites of glutathionylation. Finally, we found that overexpression of DJ-1 in the dopaminergic neurons partly compensates for the loss of the Grx1 homologue in a C. elegans in vivomodel of PD. Therefore, our results reveal a novel redox modification of DJ-1 and suggest a novel regulatory mechanism for DJ-1 content in vivo.

ABSTRACTOXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacterspp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low Kivalues for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant Kiimprovements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low Kis for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [tn] = 0) and tns ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.

Our previous studies have shown an essential role played by the octapeptide repeat region (OR) and the N‐terminal half of hydrophobic region (HR) in the anti‐apoptotic activity of prion protein (PrP). As PrP‐like protein Doppel (Dpl), which structurally resembles an N‐terminally truncated PrP, did not show any anti‐apoptotic activity, we examined apoptosis of HpL3–4 cells expressing Dpl fused to various lengths of the N‐terminal region of PrP to investigate whether the PrP/Dpl fusion proteins retain anti‐apoptotic function. HpL3–4 cells expressing Dpl fused to PrP(1–124) with the OR and N‐terminal half of HR of PrP showed anti‐apoptotic function, whereas Dpl fused to PrP(1–95) with OR did not rescue cells from apoptotic cell death induced by serum deprivation. These results indicate that the OR and N‐terminal half of HR of PrP retains anti‐apoptotic activity similar to full‐length PrP.

DNA aptamers were selected against recombinant human (rhu) cellular prion protein (PrPc) 23–231 by systematic evolution of llgands via a systematic evolution of ligands by exponential (SELEX) enrichment procedure using lateral flow chromatography. The SELEX procedure was performed with an aptamer library consisting of a randomized 40-nucleotide core flanked by 28-mer primer-binding sites that, theoretically, represented approximately 1024distinct nucleic acid species. Sixty nanograms of rhuPrPc23–231 immobilized in the center of a lateral flow device was used as the target molecule for SELEX. At the end of 6 iterations of SELEX, 13 distinct candidate aptamers were Identified, of which, 3 aptamers represented 32%, 8%, and 5% of the sequences respectively. Eight aptamers, including the three most frequently occurring candidates, were selected for further evaluation. Selected aptamers bound to rhuPrPc23–231 at 10–6M to 10–8M concentrations. Two of the eight aptamers bound at higher concentrations to rhuPrPc90–231. Theoretical thermodynamic modeling of selected aptamer sequences Identified several common motifs among the selected aptamers that could play a role in PrP binding. Binding affinity to rhuPrPc23–231 was both aptamer sequence and structure dependent. Further, selected aptamers bound to mammalian PrPs derived from brain of healthy sheep, calf, piglet, and deer, and to PrPcexpressed in mouse neuroblastoma cells. None of the aptamers bound to proteinase K-digested scrapie-infected mouse neuroblastoma cells or untreated PrP-null cells, which further confirmed the PrPcspecificity of the aptamers. In summary, we enriched and selected DNA aptamers that bind specifically to rhuPrPcand mammalian PrPcwith varying affinities and can be applied to biological samples for PrPcenrichment and as diagnostic tools in double ligand assay systems.

Creutzfeldt-Jakob disease (CJD), the most common human prion disease, includes sporadic (s) and familial (f) forms. Regardless of etiology, both forms are thought to share the pathogenic mechanism whereby the cellular prion protein (PrPC) converts into its pathogenic isoform (PrPSc). While PrPCconversion is thought to be random in sCJD, conversion in fCJD is facilitated by the congenital presence of mutated PrP. Differences in PrP genotype (PRNP) and in conversion circumstances lead to PrPScwith distinct characteristics that elicit different disease phenotypes. Here, we describe a case of fCJD with a substitution of histidine (H) for arginine (R) at codon 148 (R148H) and heterozygosity of the methionine/valine (M/V) polymorphic codon 129, with the 129M allele coupled with the mutation. The disease phenotype and all major characteristics of PrPScof fCJDR148Hwere virtually indistinguishable from those of sCJDMV2, which has features different from those of any other sCJD. Therefore, despite the differences in etiology, PRNP, and conversion process, the two forms of PrPSchad similar characteristics. Furthermore, comparison of fCJDR148Hwith a recently reported case carrying R148H and homozygosity at codon 129 suggests that codon 129 coupled with the mutation as well as that located on the normal allele can modify major phenotypic and PrPScfeatures of fCJDR148H.

Transmissible spongiform encephalopathies are a group of infectious diseases typically associated with the accumulation of a protease-resistant and ß-sheet-rich prion protein, PrPSc, in affected brains. PrPScis an altered isoform derived from the host-encoded glycoprotein, PrPc. The expression of PrPcis the highest in brain tissue, but it can also be detected at low levels in peripheral tissue. However, it is unclear whether a significant amount of PrPcis released into body fluid and excreted into urine. We have developed a simple, rapid method for the reliable detection of PrPcin urine from normal subjects by Western blotting. Our method can easily and reliably detect PrPcin apparently healthy individuals using less than 1 ml of urine in which the amount of urinary PrPcis estimated to be in the range of low micrograms/liter.

Creutzfeldt–Jakob disease (CJD), believed to be caused by a protease‐resistant isoform of prion protein (PrPSc), usually manifests itself as a clinically distinctive age‐related dementia because of its rapid progression, occasionally accompanied by cerebellar ataxia. Recently, a variant CJD (vCJD) has been described, which has prominent early psychiatric symptoms and an earlier age of death. Although cerebrospinal fluid (CSF) is part of the extracellular fluid of the central nervous system (CNS), the bulk of its proteins are derived from the plasma and there is increasing concern about possible transmission of prion disease by blood. As investigation of CSF has played a significant role in the diagnosis and management of several neurological diseases, it was decided to characterize PrP present in the CSF of CJD individuals. Significant variation was observed in the level of PrP in the CSF from both non‐CJD and CJD (including vCJD) patients, and the detected PrP forms are protease‐sensitive. Using a conformation‐dependent immunoassay, it was further demonstrated that the PrP detected in the CSF from CJD patients was broadly similar in conformation to that found in non‐CJD patients. Taken together, the results of this study fail to demonstrate any correlation between the presence of protease‐resistant PrP isoform (PrPSc) in the CSF and disease manifestation. Copyright © 2001 John Wiley & Sons, Ltd.

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp0/0) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp0/0mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp0/0mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.

Fatal familial insomnia (FFI) is linked to a mutation at codon 178 of the prion protein gene, coupled with the methionine codon at position 129, the site of a methionine/valine polymorphism. The D178N mutation coupled with the 129 valine codon is linked to a subtype of Creutzfeldt‐Jakob disease (CJD178) with a different phenotype. Two protease resistant fragments of the pathogenic PrP (PrPres), which differ in molecular mass, are associated with FFI and CJD178, respectively, suggesting that the two PrPreshave different conformations and hence they produce different disease phenotypes. FFI transmission experiments, which show that the endogenous PrPresrecovered in affected syngenic mice specifically replicates the molecular mass of the FFI PrPresinoculated and is associated with a phenotype distinct from that of the CJD178inoculated mice, support this idea. The second distinctive feature of the FFI PrPresis the underrepresentation of the unglycosylated PrPresform. Cell models indicate that the underrepresentation of this PrPresform results from the PrP dys‐metabolism caused by the D178N mutation and not from the preferential conversion of the glycosylated forms. Codon 129 on the normal allele further modifies the FFI phenotype determining patient subpopulations of 129 homozygotes and heterozygotes: disease duration is generally shorter, insomnia more severe and histopathology more restricted to the thalamus in the homozygotes than in the heterozygotes The allelic origin of PrPresfails to explain this finding since in both cases FFI PrPresis expressed only by the mutant allele. Despite remarkable advances, many issues remain unsolved precluding full understanding of the FFI pathogenesis.

The hallmark of prion diseases is the presence of an aberrant isoform of the prion protein (PrPres) that is insoluble in nondenaturing detergents and resistant to proteases. We investigated the allelic origin of PrPresin brains of subjects heterozygous for the D178N mutation linked to fatal familial insomnia (FFI) and a subtype of Creutzfeldt-Jakob disease (CJD178), as well as for insertional mutations associated with another CJD subtype. We found that in FFI and CJD178subjects, only mutant PrP was detergent-insoluble and protease-resistant. Therefore, PrPresderives exclusively from the mutant allele carrying the D178N mutation. In contrast, in the CJD subtype harboring insertional mutations, wild-type PrP was also detergent-insoluble and likely to be protease-resistant. Our findings indicate that the participation of the wild-type PrP in the formation of PrPresdepends on the type of mutations, providing an insight into the molecular mechanisms underlying the phenotypic heterogeneity in familial prion diseases.

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC), a glycoprotein that is anchored to the cell membrane by a glycosylphosphatidylinositol moiety, into an isoform that is protease-resistant (PrPres) and pathogenic. In inherited prion diseases, mutations in the prion protein (PrPM) engender the conversion of PrPMinto PrPres. We developed a cell model of Gerstmann-Sträussler-Scheinker disease, a neurodegenerative condition characterized by PrPM-containing amyloid deposits and neuronal loss, by expressing the Gerstmann-Sträussler-Scheinker haplotype Q217R-129V in human neuroblastoma cells. By comparison to PrPC, this genotype results in the following alterations of PrPM: 1) expression of an aberrant form lacking the glycosylphosphatidylinositol anchor, 2) increased aggregation and protease resistance, and 3) impaired transport to the cell surface. Most of these alterations are temperature-sensitive, indicating that they are due to misfolding of PrPM.

The cellular form of the prion protein (PrPc) is a glycoprotein anchored to the cell membrane by a glycosylphosphatidylinositol moiety. An aberrant form of PrPcthat is partially resistant to proteases, PrPres, is a hallmark of prion diseases, which in humans include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. We have characterized the major forms of PrP in normal and pathological human brains. A COOH-terminal fragment of PrPc, designated C1, is abundant in normal and CJD brains as well as in human neuroblastoma cells. Sequence analysis revealed that C1 contains alternative NH2termini starting at His-111 or Met-112. Like PrPc, C1 is glycosylated, anchored to the cell membrane, and is heat-stable. Consistent with the lack of the NH2-terminal region of PrPc, C1 is more acidic than PrPcand does not bind heparin. An additional fragment longer than C1, designated C2, is present in substantial amounts in CJD brains. Like PrPres, C2 is resistant to proteases and is detergent-insoluble. Our data indicate that C1 is a major product of normal PrPcmetabolism, generated by a cleavage that disrupts the neurotoxic and amyloidogenic region of PrP comprising residues 106-126. This region remains intact in C2, suggesting a role for C2 in prion diseases.

Gerstmann-Sträussler-Scheinker (GSS) is a hereditary prion disease typically associated with prion protein (PrP)-containing plaques. The protease-resistant, scrapie PrP (PrPSc) is represented by internal fragments, whereas the C-terminal fragments associated with the other prion diseases are generally underrepresented. Different histopathologic and PrPSc features associated with at least 13 PrP gene (PRNP) mutations have been described in GSS. We report the histopathology and PrP characteristics in a father and son carrying a mutation at PRNP codon 187 that substitutes histidine (H) with arginine (R) and is coupled with valine (V) at position 129 (H187R-129V). The PrP plaques were present in both cases but with different structure and topography and minimal spongiform degeneration. A distinctive, "curly" PrP immunostaining was prominent in one case. The protease-resistant PrPSc differed in amount in the 2 cases, possibly depending on whether plaques or the curly immunostain was present. Two protease-resistant PrP fragments of 14 kDa and 7 kDa with, in at least one case, N-terminus between residues 90-99 and 82-90, respectively, codistributed with the plaques, whereas only very small amounts of the PK-resistant PrP were present in the curly staining regions. PK-resistant PrP recovered from the plaque and curly staining regions appeared to be full length.