Modeling the Competition between Misfolded Aβ Conformers That Produce Distinct Types of Amyloid Pathology in Alzheimer’s Disease

The amyloid pathology characteristic of Alzheimer’s disease (AD) can be broadly classified as either fibrillary amyloid or diffuse amyloid. Fibrillary amyloid is found in cored-neuritic deposits, fibrillar deposits, and vascular deposits, and binds strongly to the amyloid revealing dyes Thioflavin-S or Congo Red. Diffuse amyloid can appear as wispy dispersed deposits or compact tufted deposits dispersed in neuropil, and binds amyloid dyes weakly if at all. In AD brains, both types of pathology are detected. Homogenates from AD brains, or the brains of transgenic mice modeling AD-amyloidosis, have been used to seed pathology in vulnerable host transgenic models. These studies suggest that pathologies may arise from distinct conformers or strains of misfolded Aβ, similar to propagating prions. Using Aβ strains sourced from four different AD-amyloidosis models, we injected pathological seeds into the brains of newborn mice from three different transgenic hosts with distinctive Aβ pathologies. Two of the seeding sources were from mice that primarily develop cored-neuritic Aβ deposits (cored strain) while the other two seeding sources were from mice that develop diffuse Aβ deposits (diffuse strain). These seeds were injected into host APP mice in which the resident strain was either diffuse or cored-neuritic pathology. Seeding-homogenates were injected into the brains of newborn mice to initiate propagation as early as possible. Depending upon the level of transgene expression in the host, we show that the injected strains of misfolded Aβ from the seeding homogenate were able to outcompete the resident strain of the APP host model. In serial passaging experiments, it appeared that the diffuse strain was more easily propagated than the cored strain. Collectively, our studies align with the idea that different types of Aβ pathology in AD brains arise from different populations of Aβ conformers that compete to populate the brain

Multiple Factors Influence the Incubation Period of ALS Prion-like Transmission in SOD1 Transgenic Mice

Mutations in superoxide dismutase 1 (SOD1) that are associated with amyotrophic lateral sclerosis (ALS) cause its misfolding and aggregation. Prior studies have demonstrated that the misfolded conformation of ALS-SOD1 can template with naïve SOD1 “host proteins” to propagate, spread, and induce paralysis in SOD1 transgenic mice. These observations have advanced the argument that SOD1 is a host protein for an ALS conformer that is prion-like and experimentally transmissible. Here, we investigated the propagation of different isolates of G93A-SOD1 ALS conformers using a paradigm involving transmission to mice expressing human G85R-SOD1 fused to yellow fluorescent protein (G85R-SOD1:YFP). In these studies, we also utilized a newly developed line of mice in which the G85R-SOD1:YFP construct was flanked by loxp sites, allowing its temporal and spatial regulation. We used methods in which the G93A ALS conformers were injected into the sciatic nerve or hindlimb muscle of adult transgenic mice. We observed that the incubation period to paralysis varied significantly depending upon the source of inoculum containing misfolded G93A SOD1. Serial passage and selection produced stable isolates of G93A ALS conformers that exhibited a defined minimum incubation period of ~2.5 months when injected into the sciatic nerve of young adult mice. As expected, neuronal excision of the transgene in loxpG85R-SOD1:YFP mice blocked induction of paralysis by transmission of G93A ALS conformers. Our findings indicate that G93A ALS conformers capable of inducing disease require neuronal expression of a receptive host SOD1 protein for propagation, with a defined incubation period to paralysis

Differential induction of mutant SOD1 misfolding and aggregation by tau and α-synuclein pathology

Abstract Background Prior studies in C. elegans demonstrated that the expression of aggregation-prone polyglutamine proteins in muscle wall cells compromised the folding of co-expressed temperature-sensitive proteins, prompting interest in whether the accumulation of a misfolded protein in pathologic features of human neurodegenerative disease burdens cellular proteostatic machinery in a manner that impairs the folding of other cellular proteins. Methods Mice expressing high levels of mutant forms of tau and α-synuclein (αSyn), which develop inclusion pathologies of the mutant protein in brain and spinal cord, were crossed to mice expressing low levels of mutant superoxide dismutase 1 fused to yellow fluorescent protein (G85R-SOD1:YFP) for aging and neuropathological evaluation. Results Mice expressing low levels of G85R-SOD1:YFP, alone, lived normal lifespans and were free of evidence of inclusion pathology, setting the stage to use this protein as a reporter of proteostatic function. We observed robust induction of G85R-SOD1:YFP inclusion pathology in the neuropil of spinal cord and brainstem of bigenic mice that co-express high levels of mutant tau in the spinal axis and develop robust spinal tau pathology (JNPL3 mice). In contrast, in crosses of the G85R-SOD1:YFP mice with mice that model spinal α-synucleinopathy (the M83 model of αSyn pathology), we observed no G85R-SOD1:YFP inclusion formation. Similarly, in crosses of the G85R-SOD1:YFP mice to mice that model cortical tau pathology (rTg4510 mice), we did not observe induction of G85R-SOD1:YFP inclusions. Conclusion Despite robust burdens of neurodegenerative pathology in M83 and rTg4510 mice, the introduction of the G85R-SOD1:YFP protein was induced to aggregate only in the context of spinal tau pathology present in the JNPL3 model. These findings suggest unexpected specificity, mediated by both the primary protein pathology and cellular context, in the induced “secondary aggregation” of a mutant form of SOD1 that could be viewed as a reporter of proteostatic function

Distinctive features of the D101N and D101G variants of superoxide dismutase 1; two mutations that produce rapidly progressing motor neuron disease.

Mutations in superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis induce misfolding and aggregation of the protein with the inherent propensity of mutant SOD1 to aggregate generally correlating, with a few exceptions, to the duration of illness in patients with the same mutation. One notable exception was the D101N variant, which has been described as wild-type-like. The D101N mutation is associated with rapidly progressing motor neuron degeneration but shows a low propensity to aggregate. By assaying the kinetics of aggregation in a well-characterized cultured cell model, we show that the D101N mutant is slower to initiate aggregation than the D101G mutant. In this cell system of protein over-expression, both mutants were equally less able to acquire Zn than WT SOD1. In addition, both of these mutants were equivalently less able to fold into the trypsin-resistant conformation that characterizes WT SOD1. A second major difference between the two mutants was that the D101N variant more efficiently formed a normal intramolecular disulfide bond. Overall, our findings demonstrate that the D101N and D101G variants exhibit clearly distinctive features, including a different rate of aggregation, and yet both are associated with rapidly progressing disease. We sought to better characterize the biochemical features of two SOD1 mutants associated with rapidly progressing disease, the D101G and wild-type like D101N mutants. We observed using our cell model that that although similarities were observed when comparing the ability to bind metals and resist trypsin digestion, these mutants differed in their ability to initiate aggregation and to form the normal intramolecular disulfide bond. We conclude that these mutants exhibit distinct properties despite producing similar disease phenotypes in patients

Additional file 8: of Differential induction of mutant SOD1 misfolding and aggregation by tau and α-synuclein pathology

Figure S8. Primary pathology burden in the M83 transgenic mouse spinal cord relative to M83/G85R-SOD1:YFP mice. M83 only mice (a) compared to M83/G85R-SOD1:YFP mice (b) after injection with αSyn fibrils to induce αSyn pathology. Sections were stained with the 81A antibody (pSer129 αSyn). Lumbar spinal cord sections are shown. Scale bar; 900 μm. (TIF 2919 kb

Additional file 2: of Differential induction of mutant SOD1 misfolding and aggregation by tau and Îą-synuclein pathology

Figure S2. Low power views of G85R-SOD1:YFP pathology in the spinal cord of bigenic JNPL3-G85R-SOD1:YFP mice (a). The box marks the position of the image shown in Fig. 1d of the main text. Low power view of fluorescence in mice expressing G85R-SOD1:YFP alone (c). Images shows midsagittal brain section (b). Nuclei were stained with DAPI (blue). The left and right arrows are drawn to magnified regions that are shown in the top left and top right of (b), respectively. Images shown are representative of 8 JNPL3-G85R-SOD1:YFP mice and 3 G85R-SOD1:YFP mice. (TIF 8095 kb

Additional file 10: of Differential induction of mutant SOD1 misfolding and aggregation by tau and α-synuclein pathology

Figure S10. Solubility of αSyn and SOD1 in M83/G85R-SOD1:YFP mice. No changes in soluble versus NP40-insoluble αSyn were observed between M83/G85R-SOD1:YFP versus G85R-SOD1:YFP mice (a and b). For these immunoblots we used a sequential fractionation protocol that produced a PBS-soluble fraction and an NP40-insoluble fractions (see Methods). Here we controlled sample concentration by resuspending the NP40-insoluble fraction in a volume equivalent to the initial PBS soluble fraction. Equivalent amounts of each fraction were analyzed by SDS-PAGE (30 μL per sample). We used antibodies to GAPDH as a loading control in soluble fractions on the same blot (a). Soluble G85R-SOD1:YFP and endogenous mouse SOD1 was detected in both animal groups (c), and insoluble G85R-SOD1:YFP was not detected in either group (d). αSyn was detected using the 94-3A10 antibody (provided by the laboratory of Benoit Giasson [79]), while mouse/human SOD1 was detected using an in-house generated antibody. n = 3 per genotype. (TIF 284 kb

Soluble brain homogenates from diverse human and mouse sources preferentially seed diffuse Aβ plaque pathology when injected into newborn mouse hosts

Background: Seeding of pathology related to Alzheimer’s disease (AD) and Lewy body disease (LBD) by tissue homogenates or purified protein aggregates in various model systems has revealed prion-like properties of these disorders. Typically, these homogenates are injected into adult mice stereotaxically. Injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could direct the evolution of amyloid-β (Aβ) pathology co-mixed with either tau or α-synuclein (αSyn) pathology in susceptible mouse models. Methods: Homogenates of human pre-frontal cortex were injected into the lateral ventricles of newborn (P0) mice expressing a mutant humanized amyloid precursor protein (APP), human P301L tau, human wild type αSyn, or combinations thereof. The homogenates were prepared from AD and AD/LBD cases displaying variable degrees of Aβ pathology and co-existing tau and αSyn deposits. Behavioral assessments of APP transgenic mice injected with AD brain lysates were conducted. For comparison, homogenates of aged APP transgenic mice that preferentially exhibit diffuse or cored deposits were similarly injected into the brains of newborn APP mice. Results: We observed that lysates from the brains with AD (Aβ+, tau+), AD/LBD (Aβ+, tau+, αSyn+), or Pathological Aging (Aβ+, tau-, αSyn-) efficiently seeded diffuse Aβ deposits. Moderate seeding of cerebral amyloid angiopathy (CAA) was also observed. No animal of any genotype developed discernable tau or αSyn pathology. Performance in fear-conditioning cognitive tasks was not significantly altered in APP transgenic animals injected with AD brain lysates compared to nontransgenic controls. Homogenates prepared from aged APP transgenic mice with diffuse Aβ deposits induced similar deposits in APP host mice; whereas homogenates from APP mice with cored deposits induced similar cored deposits, albeit at a lower level. Conclusions: These findings are consistent with the idea that diffuse Aβ pathology, which is a common feature of human AD, AD/LBD, and PA brains, may arise from a distinct strain of misfolded Aβ that is highly transmissible to newborn transgenic APP mice. Seeding of tau or αSyn comorbidities was inefficient in the models we used, indicating that additional methodological refinement will be needed to efficiently seed AD or AD/LBD mixed pathologies by injecting newborn mice

Additional file 11: of Differential induction of mutant SOD1 misfolding and aggregation by tau and Îą-synuclein pathology

Table S1. RNAseq expression data for SOD1 in mouse and human tauopathies. Transcriptomic data from studies of the rTg4510 and JNPL3 mouse models, and from studies of humans brain tissues from Alzheimer disease (AD) and progressive supranuclear palsy (PSP) cases available in https://www.synapse.org/#!Synapse:syn2580853/wiki/409840 . (TIF 694 kb

Relationship between mutant Cu/Zn superoxide dismutase 1 maturation and inclusion formation in cell models

A common property of Cu/Zn superoxide dismutase 1 (SOD1), harboring mutations associated with amyotrophic lateral sclerosis, is a high propensity to misfold and form abnormal aggregates. The aggregation of mutant SOD1 has been demonstrated in vitro, with purified proteins, in mouse models, in human tissues, and in cultured cell models. In vitro translation studies have determined that SOD1 with amyotrophic lateral sclerosis mutations is slower to mature, and thus perhaps vulnerable to off-pathway folding that could generate aggregates. The aggregation of mutant SOD1 in living cells can be monitored by tagging the protein with fluorescent fluorophores. In this study, we have taken advantage of the Dendra2 fluorophore technology in which excitation can be used to switch the output color from green to red, thereby clearly creating a time stamp that distinguishes pre-existing and newly made proteins. In cells that transiently over-express the Ala 4 to Val variant of SOD1-Dendra2, we observed that newly made mutant SOD1 was rapidly captured by pathologic intracellular inclusions. In cell models of mutant SOD1 aggregation over-expressing untagged A4V-SOD1, we observed that immature forms of the protein, lacking a Cu co-factor and a normal intramolecular disulfide, persist for extended periods. Our findings fit with a model in which immature forms of mutant A4V-SOD1, including newly made protein, are prone to misfolding and aggregation