Brenda D. Moore

IL-10 Alters Immunoproteostasis in APP Mice, Increasing Plaque Burden and Worsening Cognitive Behavior

Anti-inflammatory strategies are proposed to have beneficial effects in Alzheimers disease. To explore how anti-inflammatory cytokine signaling affects Aβ pathology, we investigated the effects of adeno-associated virus (AAV2/1)-mediated expression of Interleukin (IL)-10 in the brains of APP transgenic mouse models. IL-10 expression resulted in increased Aβ accumulation and impaired memory in APP mice. A focused transcriptome analysis revealed changes consistent with enhanced IL-10 signaling and increased ApoE expression in IL-10-expressing APP mice. ApoE protein was selectively increased in the plaque-associated insoluble cellular fraction, likely because of direct interaction with aggregated Aβ in the IL-10-expressing APP mice. Ex vivo studies also show that IL-10 and ApoE can individually impair glial Aβ phagocytosis. Our observations that IL-10 has an unexpected negative effect on Aβ proteostasis and cognition in APP mouse models demonstrate the complex interplay between innate immunity and proteostasis in neurodegenerative diseases, an interaction we call immunoproteostasis.

Overlapping profiles of Aβ peptides in the Alzheimer's disease and pathological aging brains

IntroductionA hallmark of Alzheimers disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains.MethodsAβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies.ResultsIn almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups.ConclusionsWe found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.

Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of α‐synuclein

The discoveries of mutations in SNCA were seminal findings that resulted in the knowledge that α‐synuclein (αS) is the major component of Parkinsons disease‐associated Lewy bodies. Since the pathologic roles of these protein inclusions and SNCA mutations are not completely established, we characterized the aggregation properties of the recently identified SNCA mutations, H50Q and G51D, to provide novel insights. The properties of recombinant H50Q, G51D, and wild‐type αS to polymerize and aggregate into amyloid were studied using (trans,trans)‐1‐bromo‐2,5‐bis‐(4‐hydroxy)styrylbenzene fluorometry, sedimentation analyses, electron microscopy, and atomic force microscopy. These studies showed that the H50Q mutation increases the rate of αS aggregation, whereas the G51D mutation has the opposite effect. However, H50Q and G51D αS could still be similarly induced to form intracellular aggregates from the exposure to exogenous amyloidogenic seeds under conditions that promote their cellular entry. Both mutant αS proteins, but especially G51D, promoted cellular toxicity under cellular stress conditions. These findings reveal that the novel pathogenic SNCA mutations, H50Q and G51D, have divergent effects on aggregation properties relative to the wild‐type protein, with G51D αS demonstrating reduced aggregation despite presenting with earlier disease onset, suggesting that these mutants promote different mechanisms of αS pathogenesis.

Lysine 624 of the Amyloid Precursor Protein (APP) Is a Critical Determinant of Amyloid β Peptide Length SUPPORT FOR A SEQUENTIAL MODEL OF γ-SECRETASE INTRAMEMBRANE PROTEOLYSIS AND REGULATION BY THE AMYLOID β PRECURSOR PROTEIN (APP) JUXTAMEMBRANE REGION

Background: γ-Secretase modulators (GSMs) bind APP near lysine 624. Results: Mutation of lysine 624 shifts cleavage toward smaller Aβ with no effect on ϵ cleavage. Conclusion: The amino acid at 624 in substrates affects the final γ-secretase cut. Significance: γ-Secretase cleavage likely begins at ϵ and proceeds up the transmembrane until Aβ is released, and GSMs may modulate this process through lysine 624. γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid β precursor protein (APP) to release the amyloid β peptide (Aβ). Aβ is the primary component of senile plaques in Alzheimers disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aβ and the APP intracellular domain (AICD), referred to as γ and ϵ, respectively. The heterogeneous nature of the γ cleavage that produces various Aβ peptides is highly relevant to AD, as increased production of Aβ 1–42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aβ) that plays a critical role in determining the final length of Aβ peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1–40 to 1–33 without significant changes to ϵ cleavage. These results further support a model where ϵ cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Reversible pathologic and cognitive phenotypes in an inducible model of Alzheimer-amyloidosis.

Transgenic mice that express mutant amyloid precursor protein (APPsi) using tet-Off vector systems provide an alternative model for assessing short- and long-term effects of Aβ-targeting therapies on phenotypes related to the deposition of Alzheimer-type amyloid. Here we use such a model, termed APPsi:tTA, to determine what phenotypes persist in mice with high amyloid burden after new production of APP/Aβ has been suppressed. We find that 12- to 13-month-old APPsi:tTA mice are impaired in cognitive tasks that assess short- and long-term memories. Acutely suppressing new APPsi/Aβ production produced highly significant improvements in performing short-term spatial memory tasks, which upon continued suppression translated to superior performance in more demanding tasks that assess long-term spatial memory and working memory. Deficits in episodic-like memory and cognitive flexibility, however, were more persistent. Arresting mutant APPsi production caused a rapid decline in the brain levels of soluble APP ectodomains, full-length APP, and APP C-terminal fragments. As expected, amyloid deposits persisted after new APP/Aβ production was inhibited, whereas, unexpectedly, we detected persistent pools of solubilizable, relatively mobile, Aβ42. Additionally, we observed persistent levels of Aβ-immunoreactive entities that were of a size consistent with SDS-resistant oligomeric assemblies. Thus, in this model with significant amyloid pathology, a rapid amelioration of cognitive deficits was observed despite persistent levels of oligomeric Aβ assemblies and low, but detectable solubilizable Aβ42 peptides. These findings implicate complex relationships between accumulating Aβ and activities of APP, soluble APP ectodomains, and/or APP C-terminal fragments in mediating cognitive deficits in this model of amyloidosis.

Steroids as γ-secretase modulators

Aggregation and accumulation of Aβ42 play an initiating role in Alzheimers disease (AD); thus, selective lowering of Aβ42 by γ‐secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aβ production, we screened 170 steroids at 10 μM for effects on Aβ42 secreted from human APP‐overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aβ42, whereas many nonacidic steroids actually raised Aβ42. Studies on the more potent compounds showed that Aβ42‐lowering steroids were bonafide GSMs and Aβ42‐raising steroids were inverse GSMs. The most potent steroid GSM identified was 5β‐cholanic acid (EC50=5.7 μM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4‐androsten‐3‐one‐17β‐carboxylic acid ethyl ester (EC50=6.25 μM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aβ production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.—Jung, J. I., Ladd, T. B., Kukar, T., Price, A. R., Moore, B. D., Koo, E. H., Golde, T. E., Felsenstein, K. M., Steroids as γ‐secretase modulators. FASEB J. 27, 3775–3785 (2013). www.fasebj.org

Cytosolic proteins lose solubility as amyloid deposits in a transgenic mouse model of Alzheimer-type amyloidosis

The extracellular accumulation of β-amyloid peptide is a key trigger in the pathogenesis of Alzheimers disease (AD). In humans, amyloid deposition precedes the appearance of intracellular inclusion pathology formed by cytosolic proteins such as Tau, α-synuclein and TDP-43. These secondary pathologies have not been observed in mice that model Alzheimer-type amyloidosis by expressing mutant amyloid precursor protein, with or without mutant presenilin 1. The lack of secondary pathology in these models has made it difficult to establish how amyloid deposition initiates the cascade of events that leads to secondary intracellular pathology that characterizes human AD. In transgenic mice that model Alzheimer-type amyloidosis, we sought to determine whether there is evidence of altered cytosolic protein folding by assessing whether amyloid deposition causes normally soluble proteins to misfold. Using a method that involved detergent extraction and sedimentation coupled with proteomic approaches, we identified numerous cytosolic proteins that show specific losses in solubility as amyloid accumulates. The proteins identified included glycolytic enzymes and members of the 14-3-3 chaperone family. A substantial accumulation of lysine 48-linked polyubiquitin was also detected. Overall, the data demonstrate that the accumulation of amyloid by some manner causes the loss of solubility intracellular cytosolic proteins.

Retention in endoplasmic reticulum 1 (RER1) modulates amyloid-β (Aβ) production by altering trafficking of γ-secretase and amyloid precursor protein (APP)

Background: Aβ production is influenced by intracellular trafficking of secretases and amyloid precursor protein (APP). Results: Retention in endoplasmic reticulum 1 (RER1) regulates the trafficking of γ-secretase and APP, thereby influences Aβ production. Conclusion: RER1, an ER retention/retrieval factor for γ-secretase and APP, modulates Aβ production. Significance: RER1 and its influence on γ-secretase and APP may be implicated for a safe strategy to target Aβ production. The presence of neuritic plaques containing aggregated amyloid-β (Aβ) peptides in the brain parenchyma is a pathological hallmark of Alzheimer disease (AD). Aβ is generated by sequential cleavage of the amyloid β precursor protein (APP) by β- and γ-secretase, respectively. As APP processing to Aβ requires transport through the secretory pathway, trafficking of the substrate and access to the secretases are key factors that can influence Aβ production (Thinakaran, G., and Koo, E. H. (2008) Amyloid precursor protein trafficking, processing, and function. J. Biol. Chem. 283, 29615–29619). Here, we report that retention in endoplasmic reticulum 1 (RER1) associates with γ-secretase in early secretory compartments and regulates the intracellular trafficking of γ-secretase. RER1 overexpression decreases both γ-secretase localization on the cell surface and Aβ secretion and conversely RER1 knockdown increases the level of cell surface γ-secretase and increases Aβ secretion. Furthermore, we find that increased RER1 levels decrease mature APP and increase immature APP, resulting in less surface accumulation of APP. These data show that RER1 influences the trafficking and localization of both γ-secretase and APP, thereby regulating the production and secretion of Aβ peptides.

Short Aβ peptides attenuate Aβ42 toxicity in vivo

Processing of amyloid-&bgr; (A&bgr;) precursor protein (APP) by &ggr;-secretase produces multiple species of A&bgr;: A&bgr;40, short A&bgr; peptides (A&bgr;37–39), and longer A&bgr; peptides (A&bgr;42–43). &ggr;-Secretase modulators, a class of Alzheimer’s disease therapeutics, reduce production of the pathogenic A&bgr;42 but increase the relative abundance of short A&bgr; peptides. To evaluate the pathological relevance of these peptides, we expressed A&bgr;36–40 and A&bgr;42–43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on A&bgr;42 toxicity. In contrast to A&bgr;42, the short A&bgr; peptides were not toxic and, when coexpressed with A&bgr;42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus–mediated expression of A&bgr;38 and A&bgr;40 in mice. When expressed in nontransgenic mice at levels sufficient to drive A&bgr;42 deposition, A&bgr;38 and A&bgr;40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower A&bgr;42 by raising the levels of short A&bgr; peptides could attenuate the toxic effects of A&bgr;42.

A novel panel of α-synuclein antibodies reveal distinctive staining profiles in synucleinopathies

Synucleinopathies are a spectrum of neurodegenerative diseases characterized by the intracellular deposition of the protein α-synuclein leading to multiple outcomes, including dementia and Parkinsonism. Recent findings support the notion that across the spectrum of synucleinopathies there exist diverse but specific biochemical modifications and/or structural conformations of α-synuclein, which would give rise to protein strain specific prion-like intercellular transmission, a proposed model that could explain synucleinopathies disease progression. Herein, we characterized a panel of antibodies with epitopes within both the C- and N- termini of α-synuclein. A comprehensive analysis of human pathological tissue and mouse models of synucleinopathy with these antibodies support the notion that α-synuclein exists in distinct modified forms and/or structural variants. Furthermore, these well-characterized and specific tools allow the investigation of biochemical changes associated with α-synuclein inclusion formation. We have identified several antibodies of interest with diverse staining and epitope properties that will prove useful in future investigations of strain specific disease progression and the development of targeted immunotherapeutic approaches to synucleinopathies.