α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies.SIGNIFICANCE STATEMENT Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.

Prion protein (PrP) is well studied for its pathogenic role in prion disease, but its potential contribution to other pathological processes is less understood. PrP is expressed in a variety of cancers and at least in pancreatic and breast cancers, its expression appears to be associated with poor prognosis. To understand the role of PrP in breast cancer cells, we knocked down PrP expression in MDA-MB-435 breast cancer cells with small interfering RNA and subjected these cells to a series of analyses. We found that PrP knockdown in these cells does not affect cell proliferation or colony formation, but significantly influences the cellular response to cytotoxic stimuli. Compared to control cells, PrP knockdown cells exhibited an increased susceptibility to serum deprivation induced apoptosis, no change to staurosporine- or paclitaxel-induced cell deaths, and a reduced susceptibility to chemotherapy drug doxorubicin-induced cell death. To understand the mechanism of unexpected role of PrP in exacerbating doxorubicin-induced cytotoxicity, we analyzed cell death related Bcl-2 family proteins. We found that PrP knockdown alters the expression of several Bcl-2 family proteins, correlating with increased resistance to doxorubicin-induced cytotoxicity. Moreover, the enhanced doxorubicin resistance is independent of DNA damage related p53 pathway, but at least partially through the ERK1/2 pathway. Together, our study revealed that silencing PrP in MDA-MB-435 breast cancer cells results in very different responses to various cytotoxic stimuli and ERK1/2 signaling pathway is involved in PrP silencing caused resistance to doxorubicin.

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

Synthetic prions, generated de novo from minimal, non-infectious components, cause bona fide prion disease in animals. Transmission of synthetic prions to hosts expressing syngeneic PrPC results in extended, variable incubation periods and incomplete attack rates. In contrast, murine synthetic prions (MSP) generated via PMCA with minimal cofactors readily infected mice and hamsters and rapidly adapted to both species. To investigate if hamster synthetic prions (HSP) generated under the same conditions as the MSP are also highly infectious, we inoculated hamsters with HSP generated with either hamster wild type or mutant (ΔG54, ΔG54/M139I, M139I/I205M) recombinant PrP. None of the inoculated hamsters developed clinical signs of prion disease, however, brain homogenate from HSPWT- and HSPΔG54-infected hamsters contained PrPSc, indicating subclinical infection. Serial passage in hamsters resulted in clinical disease at second passage accompanied by changes in incubation period and PrPSc conformational stability between second and third passage. These data suggest the HSP, in contrast to the MSP, are not comprised of PrPSc, and instead generate authentic PrPSc via deformed templating. Differences in infectivity between the MSP and HSP suggest that, under similar generation conditions, the amino acid sequence of PrP influences generation of authentic PrPSc.

Prions are comprised solely of PrPSc, the misfolded self-propagating conformation of the cellular protein, PrPC. Synthetic prions are generated in vitro from minimal components and cause bona fide prion disease in animals. It is unknown, however, if synthetic prions can cross the species barrier following interspecies transmission. To investigate this, we inoculated Syrian hamsters with murine synthetic prions. We found that all the animals inoculated with murine synthetic prions developed prion disease characterized by a striking uniformity of clinical onset and signs of disease. Serial intraspecies transmission resulted in a rapid adaptation to hamsters. During the adaptation process, PrPSc electrophoretic migration, glycoform ratios, conformational stability and biological activity as measured by protein misfolding cyclic amplification remained constant. Interestingly, the strain that emerged shares a strikingly similar transmission history, incubation period, clinical course of disease, pathology and biochemical and biological features of PrPSc with 139H, a hamster adapted form of the murine strain 139A. Combined, these data suggest that murine synthetic prions are comprised of bona fide PrPSc with 139A-like strain properties that efficiently crosses the species barrier and rapidly adapts to hamsters resulting in the emergence of a single strain. The efficiency and specificity of interspecies transmission of murine synthetic prions to hamsters, with relevance to brain derived prions, could be a useful model for identification of structure function relationships between PrPSc and PrPC from different species.

Definitive diagnosis of Parkinson's disease (PD) and dementia with Lewy bodies (DLB) relies on postmortem finding of disease-associated alpha-synuclein (αSynD) as misfolded protein aggregates in the central nervous system (CNS). The recent development of the real-time quaking induced conversion (RT-QuIC) assay for ultrasensitive detection of αSynD aggregates has revitalized the diagnostic values of clinically accessible biospecimens, including cerebrospinal fluid (CSF) and peripheral tissues. However, the current αSyn RT-QuIC assay platforms vary widely and are thus challenging to implement and standardize the measurements of αSynD across a wide range of biospecimens and in different laboratories. We have streamlined αSyn RT-QuIC assay based on a second generation assay platform that was assembled entirely with commercial reagents. The streamlined RT-QuIC method consisted of a simplified protocol requiring minimal hands-on time, and allowing for a uniform analysis of αSynD in different types of biospecimens from PD and DLB. Ultrasensitive and specific RT-QuIC detection of αSynD aggregates was achieved in million-fold diluted brain homogenates and in nanoliters of CSF from PD and DLB cases but not from controls. Comparative analysis revealed higher seeding activity of αSynD in DLB than PD in both brain homogenates and CSF. Our assay was further validated with CSF samples of 214 neuropathologically confirmed cases from tissue repositories (88 PD, 58 DLB, and 68 controls), yielding a sensitivity of 98% and a specificity of 100%. Finally, a single RT-QuIC assay protocol was employed uniformly to detect seeding activity of αSynD in PD samples across different types of tissues including the brain, skin, salivary gland, and colon. We anticipate that our streamlined protocol will enable interested laboratories to easily and rapidly implement the αSyn RT-QuIC assay for various clinical specimens from PD and DLB. The utilization of commercial products for all assay components will improve the robustness and standardization of the RT-QuIC assay for diagnostic applications across different sites. Due to ultralow sample consumption, the ultrasensitive RT-QuIC assay will facilitate efficient use and sharing of scarce resources of biospecimens. Our streamlined RT-QuIC assay is suitable to track the distribution of αSynD in CNS and peripheral tissues of affected patients. The ongoing evaluation of RT-QuIC assay of αSynD as a potential biomarker for PD and DLB in clinically accessible biospecimens has broad implications for understanding disease pathogenesis, improving early and differential diagnosis, and monitoring therapeutic efficacies in clinical trials.

Cognitive dysfunction is a salient feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The onset of dementia reflects the spread of Lewy pathology throughout forebrain structures. The mere presence of Lewy pathology, however, provides limited indication of cognitive status. Thus, it remains unclear whether Lewy pathology is the de facto substrate driving cognitive dysfunction in PD and DLB. Through application of α-synuclein fibrils in vivo, we sought to examine the influence of pathologic inclusions on cognition. Following stereotactic injection of α-synuclein fibrils within the mouse forebrain, we measured the burden of α-synuclein pathology at 1-, 3-, and 6-months post-injection within subregions of the hippocampus and cortex. Under this paradigm, the hippocampal CA2/3 subfield was especially susceptible to α-synuclein pathology. Strikingly, we observed a drastic reduction of pathology in the CA2/3 subfield across time-points, consistent with the consolidation of α-synuclein pathology into dense somatic inclusions followed by neurodegeneration. Silver-positive degenerating neurites were observed prior to neuronal loss, suggesting that this might be an early feature of fibril-induced neurotoxicity and a precursor to neurodegeneration. Critically, mice injected with α-synuclein fibrils developed progressive deficits in spatial learning and memory. These findings support that the formation of α-synuclein inclusions in the mouse forebrain precipitate neurodegenerative changes that recapitulate features of Lewy-related cognitive dysfunction.

Cognitive dysfunction is a salient feature of Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). The onset of dementia reflects the spread of Lewy pathology throughout forebrain structures. The mere presence of Lewy pathology, however, provides limited indication of cognitive status. Thus, it remains unclear whether Lewy pathology is the de facto substrate driving cognitive dysfunction in PD and DLB. Through application of α-synuclein fibrils in vivo , we sought to examine the influence of pathologic inclusions on cognition. Following stereotactic injection of α-synuclein fibrils within the mouse forebrain, we measured the burden of α-synuclein pathology at 1-, 3-, and 6-months post-injection within subregions of the hippocampus and cortex. Under this paradigm, the hippocampal CA2/3 subfield was especially susceptible to α- synuclein pathology. Strikingly, we observed a drastic reduction of pathology in the CA2/3 subfield across time-points, consistent with the consolidation of α-synuclein pathology into dense somatic inclusions followed by neurodegeneration. Silver-positive degenerating neurites were observed prior to neuronal loss, suggesting that this might be an early feature of fibril-induced neurotoxicity and a precursor to neurodegeneration. Critically, mice injected with α-synuclein fibrils developed progressive deficits in spatial learning and memory. These findings support that the formation of α-synuclein inclusions in the mouse forebrain precipitate neurodegenerative changes that recapitulate features of Lewy-related cognitive dysfunction.

Misfolded alpha-synuclein (αSyn) is a major constituent of Lewy bodies and Lewy neurites, which are pathological hallmarks of Parkinson's disease (PD). The contribution of αSyn to PD is well established, but the detailed mechanism remains obscure. Using a model in which αSyn aggregation in primary neurons was seeded by exogenously added, preformed αSyn amyloid fibrils (PFF), we found that a majority of pathogenic αSyn (indicated by serine 129 phosphorylated αSyn, ps-αSyn) was membrane-bound and associated with mitochondria. In contrast, only a minuscule amount of physiological αSyn was mitochondrial bound. In vitro, αSyn PFF displayed a stronger binding to purified mitochondria than did αSyn monomer, revealing a preferential mitochondria binding by aggregated αSyn. This selective mitochondrial ps-αSyn accumulation was confirmed in other neuronal and animal αSyn aggregation models that do not require exogenously added PFF and, more importantly, in postmortem brain tissues of patients suffering from PD and other neurodegenerative diseases with αSyn aggregation (α-synucleinopathies). We also showed that the mitochondrial ps-αSyn accumulation was accompanied by defects in cellular respiration in primary neurons, suggesting a link to mitochondrial dysfunction. Together, our results show that, contrary to physiological αSyn, pathogenic αSyn aggregates preferentially bind to mitochondria, indicating mitochondrial dysfunction as the common downstream mechanism for α-synucleinopathies. Our findings suggest a plausible model explaining the formation and the peculiar morphology of Lewy body and reveal that disrupting the interaction between ps-αSyn and the mitochondria is a therapeutic target for α-synucleinopathies.

Parkinson's disease (PD) is the second most common late-age onset neurodegenerative disease. Except for the symptomatic alleviating treatment, no disease modifying therapy is currently available. In this study, we investigated the potential neuroprotective role of hydroxytyrosol (HT), a major phenolic compound present in olive oil, against dopaminergic cell death. We found that HT effectively protected dopaminergic SH-SY5Y cells against dopamine (DA) and 6-hydroxydopamine (6-OHDA) induced cell death, but had no apparent effect on 1-methyl-4-phenylpyridinium (MPP(+))-induced cytotoxicity. Furthermore, we have shown that HT efficiently induced the expression of phase II detoxifying enzymes, including NAD(P)H quinone oxidoreductase 1 (NQO1). Using an NQO1 inhibitor, we revealed that increased NQO1 expression contributed to the protective effect of HT against dopaminergic cell death. Together, our findings suggest that HT has a protective effect against DA- and 6-OHDA-induced dopaminergic cell death, supporting the beneficial effect of olive oil in preventing DA-metabolism related dopaminergic neuron dysfunction.

The selective vulnerability of dopaminergic neurons in the substantia nigra pars compacta in Parkinson's disease, a late age onset neurodegenerative disorder, indicates the involvement of dopamine metabolism in the pathogenesis. Dopamine oxidation produces dopamine o-quinone, which covalently modifies cysteinyl proteins forming quinoprotein adduct. Although quinoprotein formation correlates with increased dopaminergic neurotoxicity, the in vivo protein targets for quinone modification remain unclear. Using two-dimensional gel electrophoresis and nitroblue tetrazolium/glycinate redox-cycling staining, we compared quinoprotein adducts in the substantia nigra of 2- and 15-month old rats and for the first time identified the in vivo protein targets with increased quinone modification in aged substantia nigra. Interestingly, several key enzymes in energy metabolism and mitochondrial function were selectively modified by quinone during aging. In vitro analyses confirmed that two of identified enzymes, l-lactate dehydrogenase (LDH) and malate dehydrogenase (MDH), were readily conjugated by dopamine o-quinone, resulting in a significant reduction in enzyme activity. Since the proteomic approach to detect quinoprotein adducts represents a single analysis comparing pools of substantia nigra from young or old rats, these findings need to be verified in the future. Nonetheless, our results reveal that the enzymatic activity of LDH and MDH can be compromised by quinone modification, suggesting a role of energy metabolism impairment in the selective vulnerability of aged substantia nigra dopaminergic neurons in Parkinson's disease.

Prions, characterized by self-propagating protease-resistant prion protein (PrP) conformations, are agents causing prion disease. Recent studies generated several such self-propagating protease-resistant recombinant PrP (rPrP-res) conformers. While some cause prion disease, others fail to induce any pathology. Here we showed that although distinctly different, the pathogenic and non-pathogenic rPrP-res conformers were similarly recognized by a group of conformational antibodies against prions and shared a similar guanidine hydrochloride denaturation profile, suggesting a similar overall architecture. Interestingly, two independently generated non-pathogenic rPrP-res were almost identical, indicating that the particular rPrP-res resulted from cofactor-guided PrP misfolding, rather than stochastic PrP aggregation. Consistent with the notion that cofactors influence rPrP-res conformation, the propagation of all rPrP-res formed with phosphatidylglycerol/RNA was cofactor-dependent, which is different from rPrP-res generated with a single cofactor, phosphatidylethanolamine. Unexpectedly, despite the dramatic difference in disease-causing capability, RT-QuIC assays detected large increases in seeding activity in both pathogenic and non-pathogenic rPrP-res inoculated mice, indicating that the non-pathogenic rPrP-res is not completely inert in vivo. Together, our study supported a role of cofactors in guiding PrP misfolding, indicated that relatively small structural features determine rPrP-res' pathogenicity, and revealed that the in vivo seeding ability of rPrP-res does not necessarily result in pathogenicity.

Alpha synuclein (α-syn) is central to the pathogenesis of a group of neurodegenerative disorders known as synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Aggregation of α-syn is the pathologic hallmark of these disorders and is intimately associated with the pathogenic changes. The prion-like hypothesis postulates that the aggregated α-syn provides a template to seed the aggregation of normal α-syn and spread the pathology. Thus far, it remains unclear whether aggregated α-syn can be a useful biomarker for diagnosis and/or tracking disease progression, which is mainly due to the lack of a suitable biochemical assay. The protein misfolding cyclic amplification (PMCA) technique is known for its enormous amplification power to detect the seeding activity of protein aggregates such as prions. In this study, we adapted PMCA for detecting the seeding activity of α-syn. By extensively optimizing the PMCA parameters, we developed a protocol that is able to sensitively and quantitatively detect the seeding activity of as little as 100 attomoles (10-16 mol) of α-syn aggregate. Using our protocol, we detected α-syn seeding activity from a histologically positive, formaldehyde-fixed MSA sample, but not with the histologically negative, formaldehyde-fixed control sample. Our results confirmed that the α-syn in MSA patient's brain does contain seeding activity, which remains active even after fixation. Moreover, we also established that PMCA with sonication is a sensitive and quantitative method for detecting α-syn seeding activity, which can be further adapted to more accessible patients' samples to evaluate α-syn aggregates as a biomarker for synucleinopathies.

α-Synuclein (α-syn) is the predominant protein in Lewy-body inclusions, which are pathological hallmarks of α-synucleinopathies, such as Parkinson's disease (PD) and multiple system atrophy (MSA). Other hallmarks include activation of microglia, elevation of pro-inflammatory cytokines, as well as the activation of T and B cells. These immune changes point towards a dysregulation of both the innate and the adaptive immune system. T cells have been shown to recognize epitopes derived from α-syn and altered populations of T cells have been found in PD and MSA patients, providing evidence that these cells can be key to the pathogenesis of the disease.ObjectiveTo study the role of the adaptive immune system with respect to α-syn pathology.

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.

Prion disease is a group of transmissible neurodegenerative disorders affecting humans and animals. The prion hypothesis postulates that PrPSc, the pathogenic conformer of host-encoded prion protein (PrP), is the unconventional proteinaceous infectious agent called prion. Supporting this hypothesis, highly infectious prion has been generated in vitro with recombinant PrP plus defined non-protein cofactors and the synthetically generated prion (recPrPSc) is capable of causing prion disease in wild-type mice through intracerebral (i.c.) or intraperitoneal (i.p.) inoculation. Given that many of the naturally occurring prion diseases are acquired through oral route, demonstrating the capability of recPrPSc to cause prion disease via oral transmission is important, but has never been proven. Here we showed for the first time that oral ingestion of recPrPSc is sufficient to cause prion disease in wild-type mice, which was supported by the development of fatal neurodegeneration in exposed mice, biochemical and histopathological analyses of diseased brains, and second round transmission. Our results demonstrate the oral transmissibility of recPrPSc and provide the missing evidence to support that the in vitro generated recPrPSc recapitulates all the important properties of naturally occurring prions.

Conversion of normal prion protein (PrPC) to the pathogenic PrPSc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrPC-to-PrPSc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrPSc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrPSc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrPSc mutants were significantly different from each other and from that of WT recPrPSc. Together, our results support that the NPR greatly influences PrPC-to-PrPSc conversion, but it is not essential for the generation of PrPSc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

Parkinson's disease (PD) is a synucleinopathy that has multiple neuropathological characteristics, with nigrostriatal dopamine system degeneration being a core feature. Current models of PD pathology typically fail to recapitulate several attributes of the pathogenic process and neuropathology. We aimed to define the effects of combining a mouse model exhibiting multiple PD-like changes with intrastriatal injections of α-synuclein (α-syn) pre-formed fibril (PFFs) aggregates. We employed the heterozygous Engrailed 1 (En1+/-) mouse that features several pathophysiological hallmarks of clinical PD.

For people with Parkinson's disease (PD), considered the most common neurodegenerative disease behind Alzheimer's disease, accurate diagnosis is dependent on many factors; however, misdiagnosis is extremely common in the prodromal phases of the disease, when treatment is thought to be most effective. Currently, there are no robust biomarkers that aid in the early diagnosis of PD. Following previously reported work by our group, we accurately measured the concentrations of 18 bile acids in the serum of a prodromal mouse model of PD. We identified three bile acids at significantly different concentrations (p < 0.05) when mice representing a prodromal PD model were compared with controls. These include ω-murichoclic acid (MCAo), tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA). All were down-regulated in prodromal PD mice with TUDCA and UDCA at significantly lower levels (17-fold and 14-fold decrease, respectively). Using the concentration of three bile acids combined with logistic regression, we can discriminate between prodromal PD mice from control mice with high accuracy (AUC (95% CI) = 0.906 (0.777⁻1.000)) following cross validation. Our study highlights the need to investigate bile acids as potential biomarkers that predict PD and possibly reflect the progression of manifest PD.

Hybrid crops produce higher yields than their inbred parents due to heterosis. For high purity of hybrid seeds, it is critical to eliminate self-pollination. Manual or mechanical removal of male parts (such as detasseling in maize) is labor-intensive, fuel and time-consuming, and can cause physical damage to female plants, resulting in significant seed yield reductions. Many male-sterility systems either require a maintainer for male-sterile line propagation or are often affected by environmental factors. Roundup® Hybridization System (RHS) utilizes glyphosate to induce male sterility, which effectively eliminates the need for maintainer lines and removal of male parts for commercial hybrid seed production. The first-generation RHS (RHS1) is based on low expression of a glyphosate-insensitive 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in pollen. This report presents the second-generation RHS (RHS2) technology built on RNA interference (RNAi) combined with CP4 EPSPS. It utilizes maize endogenous male tissue-specific small interfering RNAs (mts-siRNAs) to trigger cleavage of the CP4 EPSPS mRNA specifically in tassels, resulting in glyphosate-sensitive male cells due to lack of the CP4 EPSPS protein. Male sterility is then induced by glyphosate application at the stages critical for pollen development, and the male-sterile plants are used as the female parent to produce hybrid seed. The endogenous mts-siRNAs are conserved across maize germplasms, and the inducible male sterility was replicated in representative germplasms through introgression of a CP4 EPSPS transgene containing the mts-siRNA target sequence. This technology combines the relative simplicity and convenience of a systemic herbicide spray methodology with targeted protein expression to create an inducible male sterility system for industrial production of row crop hybrid seeds in an environmentally-independent manner.