Effect Sizes of Cognitive and Locomotive Behavior Tests in the 5XFAD-J Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid ß (Aß) plaques in the brain, leading to cognitive impairment and other clinical symptoms. The 5XFAD mouse model is commonly used in AD research because it expresses five human transgenes that result in the accumulation of Aß plaques and cognitive decline at a relatively early age. Behavioral experiments are frequently conducted using this model; however, the effect size has not yet been reported. In this study, we examined basic cognition and locomotion in 5XFAD mice with a C57BL6/J background (5XFAD-J) at 6 months of age, a period in which impairments of cognitive function and locomotion are commonly observed. We analyzed the effect sizes of cognitive and locomotive experiments in the 5XFAD mice compared with those in the wild-type mice. Our results suggest that for long-term memory analysis, the novel object recognition test (p = 0.013, effect size 1.24) required a sample size of at least 12 to obtain meaningful results. Moreover, analysis of general locomotion over total distance with the Laboratory Animal Behavior Observation, Registration and Analysis System (LABORAS) test during the dark phase (p = 0.007, effect size -1.37) needed a sample size of 10 for a statistical power (1-ß) of 0.8. In conclusion, we can conduct more ethical and scientifically rigorous animal experiments using 5XFAD mice based on the effect and sample sizes suggested in this study.

The Distribution of Prion Protein Allotypes Differs Between Sporadic and Iatrogenic Creutzfeldt-Jakob Disease Patients.

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent of the human prion diseases, which are fatal and transmissible neurodegenerative diseases caused by the infectious prion protein (PrP(Sc)). The origin of sCJD is unknown, although the initiating event is thought to be the stochastic misfolding of endogenous prion protein (PrP(C)) into infectious PrP(Sc). By contrast, human growth hormone-associated cases of iatrogenic CJD (iCJD) in the United Kingdom (UK) are associated with exposure to an exogenous source of PrP(Sc). In both forms of CJD, heterozygosity at residue 129 for methionine (M) or valine (V) in the prion protein gene may affect disease phenotype, onset and progression. However, the relative contribution of each PrP(C) allotype to PrP(Sc) in heterozygous cases of CJD is unknown. Using mass spectrometry, we determined that the relative abundance of PrP(Sc) with M or V at residue 129 in brain specimens from MV cases of sCJD was highly variable. This result is consistent with PrP(C) containing an M or V at residue 129 having a similar propensity to misfold into PrP(Sc) thus causing sCJD. By contrast, PrP(Sc) with V at residue 129 predominated in the majority of the UK human growth hormone associated iCJD cases, consistent with exposure to infectious PrP(Sc) containing V at residue 129. In both types of CJD, the PrP(Sc) allotype ratio had no correlation with CJD type, age at clinical onset, or disease duration. Therefore, factors other than PrP(Sc) allotype abundance must influence the clinical progression and phenotype of heterozygous cases of CJD.

Relative Abundance of apoE and Aß1-42 Associated with Abnormal Prion Protein Differs between Creutzfeldt-Jakob Disease Subtypes.

Aggregated and protease-resistant mammalian prion protein (PrPSc) is the primary protein component of infectious prions. Enriched PrPSc preparations are often used to study the mechanisms that underly prion disease. However, most enrichment procedures are relatively nonspecific and tend to yield significant amounts of non-PrPSc components including various proteins that could confound functional and structural studies. It is thus important to identify these proteins and assess their potential relevance to prion pathogenesis. Following proteinase K treatment and phosphotungstic acid precipitation of brain homogenate, we have used mass spectrometry to analyze the protein content of PrPSc isolated from prion-infected mice, multiple cases of sporadic Creutzfeldt-Jakob disease (sCJD), and human growth hormone associated cases of iatrogenic CJD (iCJD). Creatine kinase was the primary protein contaminant in all PrPSc samples, while many of the other proteins identified were also found in non-CJD controls, which suggests that they are not CJD specific. Interestingly, the Alzheimer's disease associated peptide amyloid ß 1-42 (Aß1-42) was identified in the majority of the sCJD cases as well as non-CJD age-matched controls, while apoliprotein E was found in greater abundance in the sCJD cases. By contrast, while some of the iCJD cases showed evidence of higher molecular weight Aß oligomers, monomeric Aß1-42 peptide was not detected by immunoblot, and only one case had significant levels of apolipoprotein E. Our data are consistent with the age-associated deposition of Aß1-42 in older sporadic CJD and non-CJD patients and suggest that both apolipoprotein E and Aß1-42 abundance can differ depending upon the type of CJD.

Uptake and degradation of protease-sensitive and -resistant forms of abnormal human prion protein aggregates by human astrocytes.

Sporadic Creutzfeldt-Jakob disease is the most common of the human prion diseases, a group of rare, transmissible, and fatal neurologic diseases associated with the accumulation of an abnormal form (PrP(Sc)) of the host prion protein. In sporadic Creutzfeldt-Jakob disease, disease-associated PrP(Sc) is present not only as an aggregated, protease-resistant form but also as an aggregated protease-sensitive form (sPrP(Sc)). Although evidence suggests that sPrP(Sc) may play a role in prion pathogenesis, little is known about how it interacts with cells during prion infection. Here, we show that protease-sensitive abnormal PrP aggregates derived from patients with sporadic Creutzfeldt-Jakob disease are taken up and degraded by immortalized human astrocytes similarly to abnormal PrP aggregates that are resistant to proteases. Our data suggest that relative proteinase K resistance does not significantly influence the astrocyte's ability to degrade PrP(Sc). Furthermore, the cell does not appear to distinguish between sPrP(Sc) and protease-resistant PrP(Sc), suggesting that sPrP(Sc) could contribute to prion infection.

A specific population of abnormal prion protein aggregates is preferentially taken up by cells and disaggregated in a strain-dependent manner.

Prion diseases are characterized by the conversion of the soluble protease-sensitive host-encoded prion protein (PrP(C)) into its aggregated, protease-resistant, and infectious isoform (PrP(Sc)). One of the earliest events occurring in cells following exposure to an exogenous source of prions is the cellular uptake of PrP(Sc). It is unclear how the biochemical properties of PrP(Sc) influence its uptake, although aggregate size is thought to be important. Here we show that for two different strains of mouse prions, one that infects cells (22L) and one that does not (87V), a fraction of PrP(Sc) associated with distinct sedimentation properties is preferentially taken up by the cells. However, while the fraction of PrP(Sc) and the kinetics of uptake were similar for both strains, PrP(Sc) derived from the 87V strain was disaggregated more rapidly than that derived from 22L. The increased rate of PrP(Sc) disaggregation did not correlate with either the conformational or aggregate stability of 87V PrP(Sc), both of which were greater than those of 22L PrP(Sc). Our data suggest that the kinetics of disaggregation of PrP(Sc) following cellular uptake is independent of PrP(Sc) stability but may be dependent upon some component of the PrP(Sc) aggregate other than PrP. Rapid disaggregation of 87V PrP(Sc) by the cell may contribute, at least in part, to the inability of 87V to infect cells in vitro.

Lack of prion infectivity in fixed heart tissue from patients with Creutzfeldt-Jakob disease or amyloid heart disease.

In most forms of prion disease, infectivity is present primarily in the central nervous system or immune system organs such as spleen and lymph node. However, a transgenic mouse model of prion disease has demonstrated that prion infectivity can also be present as amyloid deposits in heart tissue. Deposition of infectious prions as amyloid in human heart tissue would be a significant public health concern. Although abnormal disease-associated prion protein (PrP(Sc)) has not been detected in heart tissue from several amyloid heart disease patients, it has been observed in the heart tissue of a patient with sporadic Creutzfeldt-Jakob Disease (sCJD), the most common form of human prion disease. In order to determine whether prion infectivity can be found in heart tissue, we have inoculated formaldehyde fixed brain and heart tissue from two sCJD patients, as well as prion protein positive fixed heart tissue from two amyloid heart disease patients, into transgenic mice overexpressing the human prion protein. Although the sCJD brain samples led to clinical or subclinical prion infection and deposition of PrP(Sc) in the brain, none of the inoculated heart samples resulted in disease or the accumulation of PrP(Sc). Thus, our results suggest that prion infectivity is not likely present in cardiac tissue from sCJD or amyloid heart disease patients.

Genetic deletion of nitric oxide synthase 2 ameliorates Parkinson's disease pathology and neuroinflammation in a transgenic mouse model of synucleinopathy.

Studies of mouse models of Alzheimer's disease (AD) have demonstrated that nitric oxide synthase 2 (NOS2) is involved in AD pathology. However, the effects of NOS2 on the pathology of Parkinson's disease (PD) are not well studied. To address this gap, we examined the impact of NOS2 on disease-associated phenotypes in a mouse model of PD. Transgenic mice carrying the A53T mutation of α-synuclein (SynA53T) and newly generated double transgenic mice with deletion of NOS2 (SynA53T/NOS2-/-) were used. Compared with SynA53T mice, the loss of nos2 decreased α-synuclein phosphorylation at serine 129 and reduced α-synuclein-induced microglial and astrocyte activation in SynA53T/NOS-/- mice. Additionally, neuroinflammation-related gene clusters in the deep mesencephalic nucleus (DpMe) were altered in SynA53T/NOS-/- mice compared with SynA53T mice. Taken together, our results suggest that deletion of nos2 alleviates α-synuclein pathology and α-synuclein-associated neuroinflammatory responses in the brain.

Autoclave treatment fails to completely inactivate DLB alpha-synuclein seeding activity.

Synucleinopathies are characterized by the deposition of alpha-synuclein (α-syn) aggregates in brain tissue. Pathological α-syn aggregates propagate in a prion-like manner and display prion-like biochemical properties. Using RT-QuIC, we measured α-syn seeding activity from brains of Dementia with Lewy body (DLB) patients post autoclave. Here, we show that autoclaving at 121 °C removes one to two log10 of α-syn seeding activity but the remaining 50% seeding dose (SD50) is more than 107/mg tissue. DLB brain samples autoclaved at 132 °C still revealed an SD50 of approximately 106/mg tissue. Our data suggest that DLB α-syn seeds are incompletely inactivated by standard autoclave, thus highlighting the need for evaluating laboratory procedures that fully inactivate them.

In vivo and autopsy validation of alpha-synuclein seeding activity using RT-QuIC assay in the gastrointestinal tract of patients with Parkinson's disease.

OBJECTIVE: In the present study, real-time quaking-induced conversion (RT-QuIC) assay was used to evaluate pathologic alpha-synuclein (AS) seeding activity in formalin-fixed paraffin-embedded (FFPE) tissue from the gastrointestinal (GI) tract of Parkinson's disease (PD) patients. METHODS: This study was conducted in two parts: Part I. a preliminary autopsy study that included four autopsy-confirmed patients with synucleinopathy (2 PD, 1 dementia with Lewy bodies [DLB], and 1 multiple system atrophy [MSA]) and two normal autopsy controls. Frozen and FFPE tissues of the brain were obtained. Part II. a clinical case-control study that included 20 clinically diagnosed PD patients and matched controls. Surgically resected FFPE tissues from the upper and lower GI tracts were used. The RT-QuIC assay was performed to evaluate pathologic seed amplification using frozen or FFPE tissues. The presence or absence of AS aggregation was confirmed by conventional phosphorylated AS (pAS) immunohistochemistry (IHC). RESULTS: In Part I, RT-QuIC assay showed pathologic AS amplification in frozen and FFPE brain tissues of PD and DLB patients, and FFPE stomach tissue of PD patients but not in the MSA patient and controls. In Part II, pathologic seeding activity was found in 10% (2/20) of the stomach tissues of clinical PD patients but in none of the matched controls. IHC showed pAS-positive staining in 55% of patients (11/20) and 15% of controls (3/20). CONCLUSION: The present study results showed that the RT-QuIC assay using FFPE tissue of the GI tract was inadequate as a biomarker in PD.

Comparison of the level, distribution and form of disease-associated prion protein in variant and sporadic Creutzfeldt-Jakob diseased brain using conformation-dependent immunoassay and Western blot.

Disease-associated prion protein (PrP(Sc)) can be distinguished from the cellular isoform (PrP(C)) by conformation-dependent immunoassay (CDI). This technique exploits the presence of an epitope, accessible in PrP(C), but only unmasked by denaturation in PrP(Sc). In this study, we investigated PrP(Sc) in different brain regions in variant and sporadic Creutzfeldt-Jakob disease (CJD) by using CDI, and directly compared the results with those obtained using the more commonly employed protease digestion and Western blotting. In general, there was good agreement between the results, although there were certain discrepancies in relative abundance when the regional distribution in variant CJD cases was considered. The results largely confirmed the previously described targeting of different brain regions by variant and sporadic CJD. Additionally, the combination of protease digestion and CDI detection demonstrated, for the first time, the presence of PrP(Sc) in variant CJD brains that is susceptible to proteolysis under standard conditions.

Distinct stability states of disease-associated human prion protein identified by conformation-dependent immunoassay.

The phenotypic and strain-related properties of human prion diseases are, according to the prion hypothesis, proposed to reside in the physicochemical properties of the conformationally altered, disease-associated isoform of the prion protein (PrP(Sc)), which accumulates in the brains of patients suffering from Creutzfeldt-Jakob disease and related conditions, such as Gerstmann-Straussler-Scheinker disease. Molecular strain typing of human prion diseases has focused extensively on differences in the fragment size and glycosylation site occupancy of the protease-resistant prion protein (PrP(res)) in conjunction with the presence of mutations and polymorphisms in the prion protein gene (PRNP). Here we report the results of employing an alternative strategy that specifically addresses the conformational stability of PrP(Sc) and that has been used previously to characterize animal prion strains transmitted to rodents. The results show that there are at least two distinct conformation stability states in human prion diseases, neither of which appears to correlate fully with the PrP(res) type, as judged by fragment size or glycosylation, the PRNP codon 129 status, or the presence or absence of mutations in PRNP. These results suggest that conformational stability represents a further dimension to a complete description of potentially phenotype-related properties of PrP(Sc) in human prion diseases.

Preclinical Detection of Alpha-Synuclein Seeding Activity in the Colon of a Transgenic Mouse Model of Synucleinopathy by RT-QuIC.

In synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy body (DLB), pathological alpha-synuclein (α-syn) aggregates are found in the gastrointestinal (GI) tract as well as in the brain. In this study, using real-time quaking-induced conversion (RT-QuIC), we investigated the presence of α-syn seeding activity in the brain and colon tissue of G2-3 transgenic mice expressing human A53T α-syn. Here we show that pathological α-syn aggregates with seeding activity were present in the colon of G2-3 mice as early as 3 months old, which is in the presymptomatic stage prior to the observation of any neurological abnormalities. In contrast, α-syn seeding activity was not detectable in 3 month-old mouse brains and only identified at 6 months of age in one of three mice. In the symptomatic stage of 12 months of age, RT-QuIC seeding activity was consistently detectable in both the brain and colon of G2-3 mice. Our results indicate that the RT-QuIC assay can presymptomatically detect pathological α-syn aggregates in the colon of G2-3 mice several months prior to their detection in brain tissue.

Mental Disorders Linked to Crosstalk between The Gut Microbiome and The Brain.

Often called the second brain, the gut communicates extensively with the brain and vice versa. The conversation between these two organs affects a variety of physiological mechanisms that are associated with our mental health. Over the past decade, a growing body of evidence has suggested that the gut microbiome builds a unique ecosystem inside the gastrointestinal tract to maintain the homeostasis and that compositional changes in the gut microbiome are highly correlated with several mental disorders. There are ongoing efforts to treat or prevent mental disorders by regulating the gut microbiome using probiotics. These attempts are based on the seminal findings that probiotics can control the gut microbiome and affect mental conditions. However, some issues have yet to be conclusively addressed, especially the causality between the gut microbiome and mental disorders. In this review, we focus on the mechanisms by which the gut microbiome affects mental health and diseases. Furthermore, we discuss the potential use of probiotics as therapeutic agents for psychiatric disorders.

RT-QuIC-based detection of alpha-synuclein seeding activity in brains of dementia with Lewy Body patients and of a transgenic mouse model of synucleinopathy.

RT-QuIC is a shaking-based cyclic amplification technique originally developed in the prion field to detect minute amounts of scrapie prion protein (PrPSc). In this study, we applied the RT-QuIC assay to investigate a-synuclein (a-syn) seeding activity in brains of Dementia with Lewy Body (DLB) patients and in brains of G2-3 transgenic mice expressing human a-syn with A53T mutation. The results show that a-syn seeding activity varies between patients with detectable dilutions ranging from 10-3 to 10-8 dilutions of brain tissue and is stable under exposures to the cycles of freezing, thawing and sonication. A53T a-syn aggregates from G2-3 transgenic mice greatly favoured A53T recombinant human a-syn as substrates in comparison to wild-type a-syn, suggesting that conformations for wild-type a-syn to be able to adopt are not compatible with that of A53T aggregates from G2-3.

In vitro generation of tau aggregates conformationally distinct from parent tau seeds of Alzheimer's brain.

Normal monomeric tau can be converted into pathogenic aggregates and acquire protease resistance in a prion-like manner. This acquisition of partial protease-resistance in tau aggregates has to date only been partially investigated in various studies exploring the prion-like properties of tau. In this study, we induced the aggregation of tau repeat domain (RD) in cultured cells using detergent insoluble fractions of Alzheimer's brain tissue as seeds. The seeded aggregation of tau RD in cultured cells formed a ~7 kDa protease-resistant fragment in contrast to the ~12 kDa tau fragment characteristic of the AD seeds, suggesting that the in vitro generated tau aggregates were conformationally distinct from parent seeds.

Altered distribution, aggregation, and protease resistance of cellular prion protein following intracranial inoculation.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Experimental chronic wasting disease in wild type VM mice.

Chronic wasting disease (CWD) is a naturally occurring prion disease in North American deer (Odocoileus species), Rocky mountain elk (Cervus elaphus nelsoni) and moose (Alces alces). The disease was first confirmed in the Republic of Korea in 2001, and subsequent cases were diagnosed in 2004, 2005 and 2010. The experimental host range of CWD includes ferrets, several species of voles, white-footed mice, deer mice and Syrian golden hamsters. In addition, CWD was transmitted to the transgenic mouse over-expressing elk or deer prion protein efficiently, but not to wild type mouse. Here, we report the experimental transmission of elk CWD to conventional VM/Dk mice reaching 100% attack rate after second passage. The CWD-prion-affected wild type mice will be a useful model for future CWD studies.

Correlation of polydispersed prion protein and characteristic pathology in the thalamus in variant Creutzfeldt-Jakob disease: implication of small oligomeric species.

The vacuolation, neuronal loss and gliosis that characterize human prion disease pathology are accompanied by the accumulation of an aggregated, insoluble and protease-resistant form (termed PrP(Sc)) of the host-encoded normal cellular prion protein (PrP(C)). In variant Creutzfeldt-Jakob disease the frontal cortex and cerebellum exhibit intense vacuolation and the accumulation of PrP(Sc) in the form of amyloid plaques and plaque-like structures. In contrast the posterior thalamus is characterized by intense gliosis and neuronal loss, but PrP(Sc) plaques are rare and vacuolation is patchy. We have used sucrose density gradient centrifugation coupled with conformation dependent immunoassay to examine the biochemical properties of the PrP(Sc) that accumulates in these different brain regions. The results show a greater degree of PrP(Sc) polydisperal in thalamus compared with frontal cortex or cerebellum, including a subpopulation PrP(Sc) molecules in the thalamus that have sedimentation properties resembling those of PrP(C). Much effort has focused on identifying aspects of PrP(Sc) biochemistry that distinguish between different forms of human prion disease and contribute to differential diagnosis. Here we show that PrP(Sc) sedimentation properties, which can depend on aggregation state, correlate with, and may underlie the distinct neurodegenerative processes occurring in different regions of the variant Creutzfeldt-Jakob disease brain.