High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Disease-Associated Q159X Mutant Prion Protein Is Sufficient to Cause Fatal Degenerative Disease in Mice.

PRNP Q160X is one of the five dominantly inheritable nonsense mutations causing familial prion diseases. Till now, it remains unclear how this type of nonsense mutations causes familial prion diseases with unique clinical and pathological characteristics. Human prion protein (PrP) Q160X mutation is equivalent to Q159X in mouse PrP, which produces the mutant fragment PrP1-158. Through intracerebroventricular injection of recombinant adeno-associated virus in newborn mice, we successfully overexpressed mouse PrP1-158-FLAG in the central nervous system. Interestingly, high level PrP1-158-FLAG expression in the brain caused death in these mice with an average survival time of 60 ± 9.1 days. Toxicity correlated with levels of PrP1-158-FLAG but was independent of endogenous PrP. Histopathological analyses showed microgliosis and astrogliosis in mouse brains expressing PrP1-158-FLAG and most of PrP1-158-FLAG staining appeared intracellular. Biochemical characterization revealed that the majority of PrP1-158-FLAG were insoluble and a significant part of PrP1-158-FLAG appeared to contain an un-cleaved signal peptide that may contribute to its cytoplasmic localization. Importantly, an ~10-kDa proteinase K-resistant PrP fragment was detected, which was the same as those observed in patients suffering from this type of prion diseases. To our knowledge, this is the first animal study of familial prion disease caused by Q159X that recapitulates key features of human disease. It will be a valuable tool for investigating the pathogenic mechanisms underlying familial prion diseases caused by nonsense mutations.

Genetic and pharmacological reduction of CDK14 mitigates synucleinopathy.

Parkinson's disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of α-Synuclein (α-Syn) protein. Currently, no treatment can slow nor halt the progression of PD. Multiplications and mutations of the α-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress α-Syn replicate several features of PD. Decreasing total α-Syn levels, therefore, is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of α-Syn levels and identified CDK14, a kinase of largely unknown function as a regulator of α-Syn. To test the potential therapeutic effects of CDK14 reduction in PD, we ablated Cdk14 in the α-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical α-Syn pathology, indicated by reduced numbers of pS129 α-Syn-containing cells. In primary neurons, we found that Cdk14 depletion protects against the propagation of toxic α-Syn species. We further validated these findings on pS129 α-Syn levels in PD patient neurons. Finally, we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated α-Syn in human neurons, in PFF-challenged rat neurons and in the brains of α-Syn-humanized mice. In summary, we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy.

Major depressive disease research in BRICS: A bibliometric analysis of publications from 2003 to 2022.

Major depressive disorder (MDD) seriously threatens human health. BRICS, known as an acronym for "Brazil, Russia, India, China, and South Africa," were also actively carrying out researches on MDD. This study aimed to conduct a bibliometric study of research on MDD conducted by the BRICS. By searching in the Web of Science and using the software Vosviewer and Citespace as analysis tools, this study analyzed the cooperation network at the country, institution, author-specific levels, the research hotspots and trends from BRICS between 2003-2022. A total of 10,911 articles were finally included. Our findings showed that researches on MDD from BRICS rapidly increased during the past two decades. China and India have shown explosive growth, while South Africa has the largest average "Usage Count" and "Time Cited". The current cooperation partners of the BRICS were mainly high-income countries and other developing countries with similar cultures, languages, and geographical locations. Institutions in high-income countries served as the main bridges for BRICS cooperation, while at the author level, some core authors in the BRICS countries serve as centers. China showed a flexible model in domestic partnership, but institutions and authors in the other four countries have gathered to cooperate within the group. BRICS research on MDD mainly focused on cognitive science, brain science, epidemiology, and disease mechanisms. The keywords"gut microbiota", "network analysis," "machine learning" and "sleep quality" showed explosive growth and might become research hotspots in the near future. This bibliometric analysis provided a science knowledge graph and references for other researchers.

Barriers to and Facilitators of the Implementation of a Micronutrient Powder Program for Children: A Systematic Review Based on the Consolidated Framework for Implementation Research.

BACKGROUND: As one of the most cost-effective investments for improving child nutrition, micronutrient powder (MNP) has been widely used in many countries to underpin the Sustainable Development Goals, yet challenges remain regarding its implementation on a large scale. However, few studies have explored the factors that facilitate or impede the implementation process using implementation science theories and frameworks. To address this gap, we adopted the Consolidated Framework of Implementation Research (CFIR) and conducted a systematic review of studies on the implementation barriers to and facilitators of MNP interventions. METHOD: Five publication databases, including EMBASE, Medline, PubMed, Web of Science, and Scopus, were searched for studies on the influencing factors of MNP interventions. Based on the CFIR framework, the facilitators and barriers for the MNP program implementation reported in the included studies were extracted and synthesized by five domains: intervention characteristics, outer setting, inner setting, individual characteristics, and process. RESULTS: A total of 50 articles were eligible for synthesis. The majority of the studies were conducted in lower-middle-income countries (52%) through the free delivery model (78%). The inner setting construct was the most prominently reported factor influencing implementation, specifically including available resources (e.g., irregular or insufficient MNP supply), structural characteristics (e.g., public-driven community-based approach), and access to information and knowledge (e.g., lack of training for primary-level workers). The facilitators of the engagement of private sectors, external guidelines, and regular program monitoring were also highlighted. On the contrary, monotonous tastes and occasional side effects impede intervention implementation. Additionally, we found that the inner setting had an interrelation with other contributing factors in the MNP program implementation. CONCLUSION: Our results suggest that MNP program implementation was prominently influenced by the available resources, organizational structure, and knowledge of both providers and users. Mobilizing local MNP suppliers, engaging public-driven free models in conjunction with market-based channels, and strengthening the training for primary-level health workers could facilitate MNP interventions.

Hippocampal subfield vulnerability to α-synuclein pathology precedes neurodegeneration and 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.

The priority areas and possible pathways for health cooperation in BRICS countries.

As one of the largest alliances of middle-income countries, the BRICS, known as an acronym for five countries including "Brazil, Russia, India, China, and South Africa", represents half of the global population. The health cooperation among BRICS countries will benefit their populations and other middle- and low-income countries. This study aims to summarize the current status of health cooperation in BRICS countries and identify opportunities to strengthen BRICS participation in global health governance. A literature review was conducted to analyze the status, progress, and challenges of BRICS' health cooperation. Content analysis was used to review the 2011-2021 annual joint declarations of the BRICS Health Ministers Meetings. The priority health areas were identified through segmental frequency analysis. Our research suggested that communicable diseases, access to medicine, and universal health coverage appeared most frequently in the content of declarations, indicating the possible top health priorities among BRICS' health collaboration. These priority areas align with the primary health challenges of each country, including the threats of double burden of diseases, as well as the need for improving health systems and access to medicines. Respective external cooperation, inter-BRICS health cooperation, and unified external cooperation are the main forms of health cooperation among BRICS countries. However, challenges such as the lack of a unified image and precise position, lack of practical impact, and weak discourse power have impeded the impact of BRICS on health governance. This study suggests that the BRICS countries should recognize their positioning, improve their unified image, and establish cooperative entities; at the same time, they should increase their practical strength, promote non-governmental cooperation, and expand the cooperation space through the "BRICS Plus" mechanism with countries with similar interests to join.

Air pollution nanoparticle and alpha-synuclein fibrils synergistically decrease glutamate receptor A1, depending upon nPM batch activity.

Background: Epidemiological studies have variably linked air pollution to increased risk of Parkinson's disease (PD). However, there is little experimental evidence for this association. Alpha-synuclein (α-syn) propagation plays central roles in PD and glutamate receptor A1 (GluA1) is involved in memory and olfaction function. Methods: Each mouse was exposed to one of three different batches of nano-particulate matter (nPM) (300 µg/m3, 5 h/d, 3 d/week), collected at different dates, 2017-2019, in the same urban site. After these experiments, these nPM batches were found to vary in activity. C57BL/6 female mice (3 mo) were injected with pre-formed murine α-synuclein fibrils (PFFs) (0.4 µg), which act as seeds for α-syn aggregation. Two exposure paradigms were used: in Paradigm 1, PFFs were injected into olfactory bulb (OB) prior to 4-week nPM (Batch 5b) exposure and in Paradigm 2, PFFs were injected at 4th week during 10-week nPM exposure (Batches 7 and 9). α-syn pSer129, microglia Iba1, inflammatory cytokines, and Gria1 expression were measured by immunohistochemistry or qPCR assays. Results: As expected, α-syn pSer129 was detected in ipsilateral OB, anterior olfactory nucleus, amygdala and piriform cortex. One of the three batches of nPM caused a trend for elevated α-syn pSer129 in Paradigm 1, but two other batches showed no effect in Paradigm 2. However, the combination of nPM and PFF significantly decreased Gria1 mRNA in both the ipsi- and contra-lateral OB and frontal cortex for the most active two nPM batches. Neither nPM nor PFFs alone induced responses of microglia Iba1 and expression of Gria1 in the OB and cortex. Conclusion: Exposures to ambient nPM had weak effect on α-syn propagation in the brain in current experimental paradigms; however, nPM and α-syn synergistically downregulated the expression of Gria1 in both OB and cortex.

Hippocampal subfield vulnerability to α-synuclein pathology precedes neurodegeneration and 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. Highlights: Mice injected with α-synuclein fibrils develop hippocampal and cortical α- synuclein pathology with a dynamic regional burden at 1-, 3-, and 6-months post-injection.Silver-positive neuronal processes are an early and enduring degenerative feature of the fibril model, while extensive neurodegeneration of the hippocampal CA2/3 subfield is detected at 6-months post-injection.Mice exhibit progressive hippocampal-dependent spatial learning and memory deficits.Forebrain injection of α-synuclein fibrils may be used to model aspects of Lewy-related cognitive dysfunction.

Study on properties of sewage sludge cemented paste backfill and leaching mechanism of heavy metals.

In order to achieve sustainable development and control environmental pollution, this paper proposes sewage sludge (SS) as an auxiliary cementitious material, which is mixed with ordinary Portland cement (OPC), fly ash (FA), and gangue to produce sewage sludge cemented paste backfill (SS-CPB) material. The fluidity and mechanical properties of backfill materials with different SS contents and the heavy metal leaching mechanism of SS-CPB are investigated. The results reveal that (1) with the increase of SS content from 10 to 30%, the slump of fresh SS-CPB mortar decreased from 21.7 to 18.2 cm, the initial setting time decreased from 2.83 to 0.58 h, and the final setting time decreased from 4.92 to 0.83 h. (2) Compared with the control group, the 3-day unconfined compressive strength (UCS) of the SS-CPB mixed with 10% SS increased by 49.5%, and the UCS decreased slightly in the later stage, but it also met the actual needs of coal mines. (3) Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy were used to study the SS-CPB samples. It was found that the free Al3+ in SS promoted the formation of ettringite (AFt), provided part of the early UCS, and accelerated the setting time. (4) The leaching rule of heavy metal ions was analyzed in combination with leaching kinetics, and the change of heavy metal ion mass concentration in the rising stage was in line with the contraction core model controlled by diffusion.

China's innovation and research contribution to combating neglected diseases: a secondary analysis of China's public research data.

BACKGROUND: Many emerging and developing economies, such as China, have played the important roles in combating global neglected diseases (NDs). This study aims to explore China's public landscape of research projects and funding of NDs and to provide empirical evidence on promoting China's participation in addressing global health priorities that disproportionately affect developing countries. METHODS: We systematically sourced China's public funding information from the National Natural Science Foundation of China and provincial science and technology agency websites up to August 16, 2019. Following the G-FINDER R&D scope, we screened projects of NDs for analysis. National-funded projects were reviewed on an annual basis for exploring the trends and distribution of funding flows. Information on provincial-funded projects was compared with national projects by disease, research type, and geographical distribution. RESULTS: A total of 1266 projects were included for analysis and categorized by year, funding source, recipient, disease, research type, region, and province. China's national public funding for ND research reached a historical peak of USD 16.22 million in 2018. But the proportion of ND research to all public-funded projects was less than 0.5%, and over half of the ND projects were allocated to "the big three," i.e., tuberculosis, HIV/AIDS, and malaria. About 58% of national and provincial ND projects focus on basic research. Economically developed regions and municipalities play dominant roles in leading national ND research, such as Beijing, Shanghai, and Guangdong. Provincial ND projects are primarily driven by endemic regions. CONCLUSIONS: As a new emerging high-tech innovator, China has gradually increased public input to ND-related innovation and research. But there is still a large funding gap among NDs that requires China's increased support and participation. National development plans and cooperative health needs should be taken into account for China's participation in promoting global research and development (R&D) for combating NDs.

Prion protein amino acid sequence influences formation of authentic synthetic 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.

Access to DTP-Based Combination Vaccines in Asia-Pacific Countries between 2019 and 2022.

The Asia-Pacific countries are highly diverse in health and economic conditions that may impact vaccine access and uptake. Our study aimed to characterize patterns of health access to DTP-based combination vaccines in 10 countries from 2019 to 2022 using the IQVIA-MIDAS database. The availability, affordability, and accessibility were compared across countries by national health and economic performance indicators using Spearman's rank correlation coefficient. Our findings showed that the three aspects of access to DTP-based vaccines varied substantially in the Asia-Pacific region, with higher levels in countries with better health and economic performance. Affected by the COVID-19 pandemic, vaccine accessibility fluctuates significantly in lower-income countries, with DTP coverage rates falling by more than 14% in the Philippines and Indonesia between 2019 and 2021. For availability and affordability, Singapore and Malaysia from high-income groups were largely affected, which may be related to health expenditure as a percentage of gross domestic product (Coefficient = 0.39, p = 0.03). Our study indicates that greater attention needs to be paid to national health expenditure and routine immunization services to improve vaccine disparities and increase the robustness and resilience of the vaccine supply chain during public health emergencies.

Improving Innovation and Access to Combination Vaccines for Childhood Immunization in China.

BACKGROUND: combination vaccines can improve timely vaccination coverage and mitigate the social and economic burdens of both caregivers and health systems. Compared to other countries with high immunization performance, China remains behind the curve in promoting the inclusion of new combination vaccines into national vaccination schedules. The domestic research and development pipeline faces many technical obstacles, regulatory pressures, and competitive opposition. In addition to this, health disparities regarding combination vaccines exist in each dimension of access and their determinants, including availability, accessibility, acceptability, and quality. Our study aims to provide a cross-disciplinary analysis of China's combination vaccines (from innovation to access) and identify the main factors that affect the attitudes and behavior choices for combination vaccines. METHOD: systematic reviews and secondary data analysis will be conducted to map the landscape of combination vaccines in China and the determinants influencing their innovation and access. A cross-sectional survey will be performed in seven provinces of China based on geo-economic representativeness among caregivers with children that are between 2 and 24 months old and are registered in the national immunization system. Questionnaires will be used to examine the relationship between each dimension of access and their determinants. These questionnaires will cover the caregivers' knowledge, attitude, and willingness to pay for combination vaccines, as well as their perceptions about vaccination services. Semi-structured interviews with the suppliers (public and private) and healthcare providers will help identify research gaps and the key challenges they face when developing and introducing combination vaccines in China. DISCUSSION: using a combined approach, with cross-country and multi-disciplinary support from experts, our research is designed to fill the information gaps in China's combination vaccine industry across the innovation-access spectrum. It will lead to evidence-based recommendations which will foster greater access to innovation-enhancing combination vaccines for childhood immunization in China. Moreover, the multi-dimensional approach could also be adapted beyond combination vaccines to assess innovation and other public goods for health among disadvantaged groups in the future.

Recombinant Mammalian Prions: The "Correctly" Misfolded Prion Protein Conformers.

Generating a prion with exogenously produced recombinant prion protein is widely accepted as the ultimate proof of the prion hypothesis. Over the years, a plethora of misfolded recPrP conformers have been generated, but despite their seeding capability, many of them have failed to elicit a fatal neurodegenerative disorder in wild-type animals like a naturally occurring prion. The application of the protein misfolding cyclic amplification technique and the inclusion of non-protein cofactors in the reaction mixture have led to the generation of authentic recombinant prions that fully recapitulate the characteristics of native prions. Together, these studies reveal that recPrP can stably exist in a variety of misfolded conformations and when inoculated into wild-type animals, misfolded recPrP conformers cause a wide range of outcomes, from being completely innocuous to lethal. Since all these recPrP conformers possess seeding capabilities, these results clearly suggest that seeding activity alone is not equivalent to prion activity. Instead, authentic prions are those PrP conformers that are not only heritable (the ability to seed the conversion of normal PrP) but also pathogenic (the ability to cause fatal neurodegeneration). The knowledge gained from the studies of the recombinant prion is important for us to understand the pathogenesis of prion disease and the roles of misfolded proteins in other neurodegenerative disorders.

Drug research and development opportunities in low- and middle-income countries: accelerating traditional medicine through systematic utilization and comprehensive synergy.

BACKGROUND: Though the utilization of traditional medicine has been proposed for modern drug research and development (R&D), limited research has discussed its feasible paths. In this commentary, we summarized key factors for new drug R&D under limited resources by reviewing China's discovery of artemisinin, and raised suggestions to utilize traditional medicines in low- and middle-income countries (LMICs). MAIN TEXT: We suggested that systematic utilization of traditional medicine, outstanding synergy of research units at all levels and timely information-sharing mechanism should be achieved to establish a comprehensive and efficient R&D system, especially under low-resource settings. In the case of artemisinin discovery, Chinese scientists integrated documented traditional medicine experiences and modern approaches to develop drug candidates timely. Due to limited R&D resources, China adopted a collaborative way, motivating nearly all domestic research units at different levels, to develop antimalarial products. Moreover, the excellent synergy among all units through efficient information-sharing mechanisms greatly avoided work repetition and accelerated the R&D process. CONCLUSION: Traditional medicines inspires drug discoveries in LMICs, while a comprehensive and efficient R&D system could accelerate its R&D process and save investment. The discovery of artemisinin in China gave a reliable pattern to promote sustainable development of traditional medicines and a good example to realize R&D of traditional medicine under low-resource settings.

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer.

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.

Efficient interspecies transmission of synthetic prions.

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.

Streamlined alpha-synuclein RT-QuIC assay for various biospecimens in Parkinson's disease and dementia with Lewy bodies.

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.

An extended release GLP-1 analogue increases α-synuclein accumulation in a mouse model of prodromal Parkinson's disease.

The repurposing of drugs developed to treat type 2 diabetes for the treatment of Parkinson's disease (PD) was encouraged by the beneficial effect exerted by the glucagon-like peptide 1 (GLP-1) analogue exenatide in a phase 2 clinical trial. The effects of GLP-1 analogues have been investigated extensively using rodent toxin models of PD. However, many of the toxin-based models used lack robust α-synuclein (α-syn) pathology, akin to the Lewy bodies and neurites seen in PD. One prior study has reported a protective effect of a GLP-1 analogue on midbrain dopamine neurons following injection of α-syn preformed fibrils (PFF) into the striatum. Here, we used olfactory bulb injections of PFF as a model of prodromal PD and monitored the effect of a long-acting GLP-1 analogue on the propagation of α-syn pathology in the olfactory system. Thirteen weeks after PFF injection, mice treated with long-acting the GLP-1 analogue had a significant increase in pathological α-syn in brain regions connected to the olfactory bulb, accompanied by signs of microglia activation. Our results suggest that the nature of the neuronal insult and intrinsic properties of the targeted neuronal population markedly influence the effect of GLP-1 analogues.