Potent prion-like behaviors of pathogenic α-synuclein and evaluation of inactivation methods

The concept that abnormal protein aggregates show prion-like propagation between cells has been considered to explain the onset and progression of many neurodegenerative diseases. Indeed, both synthetic amyloid-like fibrils and pathogenic proteins extracted from patients’ brains induce self-templated amplification and cell-to-cell transmission in vitro and in vivo. However, it is unclear whether exposure to exogenous prion-like proteins can potentially cause these diseases in humans. Here, we investigated in detail the prion-like seeding activities of several kinds of pathogenic α-synuclein (α-syn), including synthetic fibrils and detergent-insoluble fractions extracted from brains of patients with α-synucleinopathies. Exposure to synthetic α-syn fibrils at concentrations above 100 pg/mL caused seeded aggregation of α-syn in SH-SY5Y cells, and seeded aggregation was also observed in C57BL/6 J mice after intracerebral inoculation of at least 0.1 μg/animal. α-Syn aggregates extracted from brains of multiple system atrophy (MSA) patients showed higher seeding activity than those extracted from patients with dementia with Lewy bodies (DLB), and their potency was similar to that of synthetic α-syn fibrils. We also examined the effects of various methods that have been reported to inactivate abnormal prion proteins (PrPSc), including autoclaving at various temperatures, exposure to sodium dodecyl sulfate (SDS), and combined treatments. The combination of autoclaving and 1% SDS substantially reduced the seeding activities of synthetic α-syn fibrils and α-syn aggregates extracted from MSA brains. However, single treatment with 1% SDS or generally used sterilization conditions proved insufficient to prevent accumulation of pathological α-syn. In conclusion, α-syn aggregates derived from MSA patients showed a potent prion-like seeding activity, which could be efficiently reduced by combined use of SDS and autoclaving

ALS clinically presenting with PMA

Amyotrophic lateral sclerosis (ALS) primarily affects upper and lower motor neurons. Phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) inclusion bodies are reportedly a pathological hallmark of sporadic ALS. Here, we present an atypical case of sporadic ALS that progressed very slowly, persisted for 19 years, and clinically appeared to only affect the lower motor neurons; however, upper motor neuron degeneration was detected on autopsy. Furthermore, no inclusion bodies positive for phosphorylated TDP-43, ubiquitin, fused in sarcoma, or SOD1 were detected in the CNS. We performed exome-sequencing data analysis but found no genetic disorders. This was therefore an unusual case of lower motor neuron-predominant ALS without TDP-43 pathology or known gene-disease associations. We also reviewed autopsied ALS cases that progressed slowly and had no phosphorylated TDP-43 or ubiquitin positive inclusions and present the clinicopathological features of such cases. Based on these results, there may be a sporadic ALS subgroup that progresses slowly and shows 76 no accumulation of phosphorylated TDP-43

Sulfated Dextrans Enhance In Vitro Amplification of Bovine Spongiform Encephalopathy PrPSc and Enable Ultrasensitive Detection of Bovine PrPSc

Prions, infectious agents associated with prion diseases such as Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep and goats, are primarily comprised of PrP(Sc), a protease-resistant misfolded isoform of the cellular prion protein PrP(C). Protein misfolding cyclic amplification (PMCA) is a highly sensitive technique used to detect minute amounts of scrapie PrP(Sc). However, the current PMCA technique has been unsuccessful in achieving good amplification in cattle. The detailed distribution of PrP(Sc) in BSE-affected cattle therefore remains unknown.We report here that PrP(Sc) derived from BSE-affected cattle can be amplified ultra-efficiently by PMCA in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrP(Sc) from the saliva, palatine tonsils, lymph nodes, ileocecal region, and muscular tissues of BSE-affected cattle. Individual differences in the distribution of PrP(Sc) in spleen and cerebrospinal fluid samples were observed in terminal-stage animals. However, the presence of PrP(Sc) in blood was not substantiated in the BSE-affected cattle examined.The distribution of PrP(Sc) is not restricted to the nervous system and can spread to peripheral tissues in the terminal disease stage. The finding that PrP(Sc) could be amplified in the saliva of an asymptomatic animal suggests a potential usefulness of this technique for BSE diagnosis. This highly sensitive method also has other practical applications, including safety evaluation or safety assurance of products and byproducts manufactured from bovine source materials

Hornerin deposits in neuronal intranuclear inclusion disease : direct identification of proteins with compositionally biased regions in inclusions

Neuronal intranuclear inclusion disease (NIID) is a neurodegenerative disorder, characterized by the presence of eosinophilic inclusions (NIIs) within nuclei of central and peripheral nervous system cells. This study aims to identify the components of NIIs, which have been difficult to analyze directly due to their insolubility. In order to establish a method to directly identify the components of NIIs, we first analyzed the huntingtin inclusion-rich fraction obtained from the brains of Huntington disease model mice. Although the sequence with expanded polyglutamine could not be identified by liquid-chromatography mass spectrometry, amino acid analysis revealed that glutamine of the huntingtin inclusion-rich fraction increased significantly. This is compatible with the calculated amino acid content of the transgene product. Therefore, we applied this method to analyze the NIIs of diseased human brains, which may have proteins with compositionally biased regions, and identified a serine-rich protein called hornerin. Since the analyzed NII-rich fraction was also serine-rich, we suggested hornerin as a major component of the NIIs. A specific distribution of hornerin in NIID was also investigated by Matrix-assisted laser desorption/ionization imaging mass spectrometry and immunofluorescence. Finally, we confirmed a variant of hornerin by whole-exome sequencing and DNA sequencing. This study suggests that hornerin may be related to the pathological process of this NIID, and the direct analysis of NIIs, especially by amino acid analysis using the NII-rich fractions, would contribute to a deeper understanding of the disease pathogenesis.Peer reviewe

A postmortem study suggests a revision of the dual-hit hypothesis of Parkinson's disease

The dual-hit hypothesis of Parkinson's disease (PD) originally postulated that a neurotropic pathogen leads to formation of alpha-synuclein pathology in the olfactory bulb (OB) and dorsal motor nucleus of the vagus (DMV) and then invades the brain from these two entry points. Little work has been conducted to validate an important underlying premise for the dual-hit hypothesis, namely that the initial Lewy pathology does arise simultaneously in the OB and the enteric nervous system (ENS) plexuses and DMV at the earliest disease stage. We conducted a focused re-analysis of two postmortem datasets, which included large numbers of mild Lewy body disease (LBD) cases. We found that cases with alpha-synuclein pathology restricted to the peripheral autonomic nervous system and/or lower brainstem (early body-first LBD cases) very rarely had any OB pathology, suggesting that Lewy pathology commonly arises in the ENS without concomitant involvement of the OB. In contrast, cases with mild amygdala-predominant Lewy pathology (early brain-first LBD cases) nearly always showed OB pathology. This is compatible with the first pathology being triggered in the OB or amygdala followed by secondary spreading to connected structures, but without early involvement of the ENS or lower brainstem. These observations support that the pathologic process starts in either the olfactory bulb or the ENS, but rarely in the olfactory bulb and gut simultaneously. More studies on neuropathological datasets are warranted to reproduce these findings. The agreement between the revised single-hit hypothesis and the recently proposed brain-first vs. body-first model of LBD is discussed.Peer reviewe

Two distinct prions in fatal familial insomnia and its sporadic form

Abstract Fatal familial insomnia is a genetic prion disease, which is associated with the aspartic acid to asparagine substitution at codon 178 of the prion protein gene. Although the hallmark pathological feature is thalamic and olivary degeneration, there is a patient with an atypical fatal familial insomnia without the hallmark feature. The cause of the pathological variability is unclear. We analysed a Japanese fatal familial insomnia kindred and compared one atypical clinicopathological fatal familial insomnia phenotype case and typical fatal familial insomnia phenotype cases with transmission studies using multiple lines of knock-in mice and with protein misfolding cyclic amplification. We also analysed the transmissibility and the amplification properties of sporadic fatal insomnia. Transmission studies revealed that the typical fatal familial insomnia with thalamic and olivary degeneration showed successful transmission only using knock-in mice expressing human–mouse chimeric prion protein gene. The atypical fatal familial insomnia with spongiform changes showed successful transmission only using knock-in mice expressing bank vole prion protein gene. Two sporadic fatal insomnia cases with thalamic and olivary degeneration showed the same transmissibility as the typical fatal familial insomnia phenotype. Interestingly, one sporadic fatal insomnia case with thalamic/olivary degeneration and spongiform changes showed transmissibility of both the typical and atypical fatal familial insomnia phenotypes. Protein misfolding cyclic amplification could amplify both typical fatal familial insomnia cases and sporadic fatal insomnia cases but not the atypical fatal familial insomnia phenotype or other sporadic Creutzfeldt–Jakob disease subtypes. In addition to clinical findings and neuropathological features, the transmission properties and the amplification properties were different between the typical and atypical fatal familial insomnia phenotypes. It is suggested that two distinct prions were associated with the diversity in the fatal familial insomnia phenotype, and these two prions could also be detected in sporadic fatal insomnia

Co-morbidity of progressive supranuclear palsy and amyotrophic lateral sclerosis : a clinical-pathological case report

Background: The coexistence of distinct neurodegenerative diseases in single cases has recently attracted greater attention. The phenotypic co-occurrence of progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) has been documented in several cases. That said, the clinicopathological comorbidity of these two diseases has not been demonstrated. Case presentation: A 77-year-old man presented with gait disturbance for 2 years, consistent with PSP with progressive gait freezing. At 79 years old, he developed muscle weakness compatible with ALS. The disease duration was 5 years after the onset of PSP and 5months after the onset of ALS. Neuropathological findings demonstrated the coexistence of PSP and ALS. Immunohistochemical examination confirmed 4-repeat tauopathy, including globose-type neurofibrillary tangles, tufted astrocytes, and oligodendroglial coiled bodies as well as TAR DNA-binding protein 43 kDa pathology in association with upper and lower motor neuron degeneration. Immunoblotting showed hyperphosphorylated full-length 4-repeat tau bands (64 and 68 kDa) and C-terminal fragments (33 kDa), supporting the diagnosis of PSP and excluding other parkinsonian disorders, such as corticobasal degeneration. Genetic studies showed no abnormalities in genes currently known to be related to ALS or PSP. Conclusions: Our case demonstrates the clinicopathological comorbidity of PSP and ALS in a sporadic patient. The possibility of multiple proteinopathies should be considered when distinct symptoms develop during the disease course

Accumulation of L-type Bovine Prions in Peripheral Nerve Tissues

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues

Multicentre multiobserver study of diffusion-weighted and fluid-attenuated inversion recovery MRI for the diagnosis of sporadic Creutzfeldt–Jakob disease: a reliability and agreement study

Objectives: To assess the utility of the display standardisation of diffusion-weighted MRI (DWI) and to compare the effectiveness of DWI and fluid-attenuated inversion recovery (FLAIR) MRI for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD). Design: A reliability and agreement study. Setting: Thirteen MRI observers comprising eight neurologists and five radiologists at two universities in Japan. Participants: Data of 1.5-Tesla DWI and FLAIR were obtained from 29 patients with sCJD and 13 controls. Outcome measures: Standardisation of DWI display was performed utilising b0 imaging. The observers participated in standardised DWI, variable DWI (the display adjustment was observer dependent) and FLAIR sessions. The observers independently assessed each MRI for CJD-related lesions, that is, hyperintensity in the cerebral cortex or striatum, using a continuous rating scale. Performance was evaluated by the area under the receiver operating characteristics curve (AUC). Results: The mean AUC values were 0.84 (95% CI 0.81 to 0.87) for standardised DWI, 0.85 (95% CI 0.82 to 0.88) for variable DWI and 0.68 (95% CI 0.63 to 0.72) for FLAIR, demonstrating the superiority of DWI (p<0.05). There was a trend for higher intraclass correlations of standardised DWI (0.74, 95% CI 0.66 to 0.83) and variable DWI (0.72, 95% CI 0.62 to 0.81) than that of FLAIR (0.63, 95% CI 0.53 to 0.74), although the differences were not statistically significant. Conclusions: Standardised DWI is as reliable as variable DWI, and the two DWI displays are superior to FLAIR for the diagnosis of sCJD. The authors propose that hyperintensity in the cerebral cortex or striatum on 1.5-Tesla DWI but not FLAIR can be a reliable diagnostic marker for sCJD

Reliability of DWI and FLAIR for diagnosis of sporadic CJD

Objectives: To assess the utility of the display standardisation of diffusion-weighted MRI (DWI) and to compare the effectiveness of DWI and fluid-attenuated inversion recovery (FLAIR) MRI for the diagnosis of sporadic Creutzfeldte–Jakob disease (sCJD). Design: A reliability and agreement study. Setting: Thirteen MRI observers comprising eight neurologists and five radiologists at two universities in Japan. Participants: Data of 1.5-Tesla DWI and FLAIR were obtained from 29 patients with sCJD and 13 controls. Outcome measures: Standardisation of DWI display was performed utilising b0 imaging. The observers participated in standardised DWI, variable DWI (the display adjustment was observer dependent) and FLAIR sessions. The observers independently assessed each MRI for CJD-related lesions, that is, hyperintensity in the cerebral cortex or striatum, using a continuous rating scale. Performance was evaluated by the area under the receiver operating characteristics curve (AUC). Results: The mean AUC values were 0.84 (95% CI 0.81 to 0.87) for standardised DWI, 0.85 (95% CI 0.82 to 0.88) for variable DWI and 0.68 (95% CI 0.63 to 0.72) for FLAIR, demonstrating the superiority of DWI (p<0.05). There was a trend for higher intraclass correlations of standardised DWI (0.74, 95% CI 0.66 to 0.83) and variable DWI (0.72, 95% CI 0.62 to 0.81) than that of FLAIR (0.63, 95% CI 0.53 to 0.74), although the differences were not statistically significant. Conclusions: Standardised DWI is as reliable as variable DWI, and the two DWI displays are superior to FLAIR for the diagnosis of sCJD. The authors propose that hyperintensity in the cerebral cortex or striatum on 1.5-Tesla DWI but not FLAIR can be a reliable diagnostic marker for sCJD