Multiple system atrophy-associated oligodendroglial protein p25α stimulates formation of novel α-synuclein strain with enhanced neurodegenerative potential.

Pathology consisting of intracellular aggregates of alpha-Synuclein (α-Syn) spread through the nervous system in a variety of neurodegenerative disorders including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. The discovery of structurally distinct α-Syn polymorphs, so-called strains, supports a hypothesis where strain-specific structures are templated into aggregates formed by native α-Syn. These distinct strains are hypothesised to dictate the spreading of pathology in the tissue and the cellular impact of the aggregates, thereby contributing to the variety of clinical phenotypes. Here, we present evidence of a novel α-Syn strain induced by the multiple system atrophy-associated oligodendroglial protein p25α. Using an array of biophysical, biochemical, cellular, and in vivo analyses, we demonstrate that compared to α-Syn alone, a substoichiometric concentration of p25α redirects α-Syn aggregation into a unique α-Syn/p25α strain with a different structure and enhanced in vivo prodegenerative properties. The α-Syn/p25α strain induced larger inclusions in human dopaminergic neurons. In vivo, intramuscular injection of preformed fibrils (PFF) of the α-Syn/p25α strain compared to α-Syn PFF resulted in a shortened life span and a distinct anatomical distribution of inclusion pathology in the brain of a human A53T transgenic (line M83) mouse. Investigation of α-Syn aggregates in brain stem extracts of end-stage mice demonstrated that the more aggressive phenotype of the α-Syn/p25α strain was associated with an increased load of α-Syn aggregates based on a Förster resonance energy transfer immunoassay and a reduced α-Syn aggregate seeding activity based on a protein misfolding cyclic amplification assay. When injected unilaterally into the striata of wild-type mice, the α-Syn/p25α strain resulted in a more-pronounced motoric phenotype than α-Syn PFF and exhibited a "tropism" for nigro-striatal neurons compared to α-Syn PFF. Overall, our data support a hypothesis whereby oligodendroglial p25α is responsible for generating a highly prodegenerative α-Syn strain in multiple system atrophy.

APP depletion alters selective pre- and post-synaptic proteins.

The normal role of Alzheimer's disease (AD)-linked amyloid precursor protein (APP) in the brain remains incompletely understood. Previous studies have reported that lack of APP has detrimental effects on spines and electrophysiological parameters. APP has been described to be important in synaptic pruning during development. The effect of APP knockout on mature synapses is complicated by this role in development. We previously reported on differential changes in synaptic proteins and receptors in APP mutant AD transgenic compared to wild-type neurons, which revealed selective decreases in levels of pre- and post-synaptic proteins, including of surface glutamate receptors. In the present study, we undertook a similar analysis of synaptic composition but now in APP knockout compared to wild-type mouse neurons. Here we demonstrate alterations in levels of selective pre- and post-synaptic proteins and receptors in APP knockout compared to wild-type mouse primary neurons in culture and brains of mice in youth and adulthood. Remarkably, we demonstrate selective increases in levels of synaptic proteins, such as GluA1, in neurons with APP knockout and with RNAi knockdown, which tended to be opposite to the reductions seen in AD transgenic APP mutant compared to wild-type neurons. These data reinforce that APP is important for the normal composition of synapses.

ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-ß accumulation.

Intracellular amyloid-ß (Aß) accumulation is a key feature of early Alzheimer's disease and precedes the appearance of Aß in extracellular plaques. Aß is generated through proteolytic processing of amyloid precursor protein (APP), but the intracellular site of Aß production is unclear. APP has been localized to multivesicular bodies (MVBs) where sorting of APP onto intraluminal vesicles (ILVs) could promote amyloidogenic processing, or reduce Aß production or accumulation by sorting APP and processing products to lysosomes for degradation. Here, we show that APP localizes to the ILVs of a subset of MVBs that also traffic EGF receptor (EGFR), and that it is delivered to lysosomes for degradation. Depletion of the endosomal sorting complexes required for transport (ESCRT) components, Hrs (also known as Hgs) or Tsg101, inhibited targeting of APP to ILVs and the subsequent delivery to lysosomes, and led to increased intracellular Aß accumulation. This was accompanied by dramatically decreased Aß secretion. Thus, the early ESCRT machinery has a dual role in limiting intracellular Aß accumulation through targeting of APP and processing products to the lysosome for degradation, and promoting Aß secretion.

The inside-out amyloid hypothesis and synapse pathology in Alzheimer's disease.

Cumulative evidence in brains and cultured neurons of Alzheimer's disease (AD) transgenic mouse models, as well as in human postmortem AD brains, highlights that age-related increases in ß-amyloid peptide (Aß), particularly in endosomes near synapses, are involved in early synapse dysfunction. Our immunoelectron microscopy and high-resolution immunofluorescence microscopy studies show that this early subcellular Aß accumulation leads to progressive Aß aggregation and pathology, particularly within dystrophic neurites and synapses. These studies confirm that neuritic/synaptic Aß accumulation is the nidus of plaque formation. Aß-dependent synapse pathology in AD models is modulated by synaptic activity and is plaque independent. The amyloid precursor protein (APP) is normally transported down neurites and appears to be preferentially processed to Aß at synapses. Synapses are sites of early Aß accumulation and aberrant tau phosphorylation in AD, which alter the synaptic composition at early stages of the disease. Elucidating the normal role of APP, and potentially of Aß, at synapses should provide important insights into the mechanism(s) of Aß-induced synapse dysfunction in AD and how to therapeutically mitigate these dysfunctions.

Heterogeneous Association of Alzheimer's Disease-Linked Amyloid-ß and Amyloid-ß Protein Precursor with Synapses.

Alzheimer's disease (AD) is increasingly viewed as a disease of synapses. Loss of synapses correlates better with cognitive decline than amyloid plaques and neurofibrillary tangles, the hallmark neuropathological lesions of AD. Soluble forms of amyloid-ß (Aß) have emerged as mediators of synapse dysfunction. Aß binds to, accumulates, and aggregates in synapses. However, the anatomical and neurotransmitter specificity of Aß and the amyloid-ß protein precursor (AßPP) in AD remain poorly understood. In addition, the relative roles of Aß and AßPP in the development of AD, at pre- versus post-synaptic compartments and axons versus dendrites, respectively, remain unclear. Here we use immunogold electron microscopy and confocal microscopy to provide evidence for heterogeneity in the localization of Aß/AßPP. We demonstrate that Aß binds to a subset of synapses in cultured neurons, with preferential binding to glutamatergic compared to GABAergic neurons. We also highlight the challenge of defining pre- versus post-synaptic localization of this binding by confocal microscopy. Further, endogenous Aß42 accumulates in both glutamatergic and GABAergic AßPP/PS1 transgenic primary neurons, but at varying levels. Moreover, upon knock-out of presenilin 1 or inhibition of γ-secretase AßPP C-terminal fragments accumulate both pre- and post-synaptically; however earlier pre-synaptically, consistent with a higher rate of AßPP processing in axons. A better understanding of the synaptic and anatomical selectivity of Aß/AßPP in AD can be important for the development of more effective new therapies for this major disease of aging.

Aß accumulation causes MVB enlargement and is modelled by dominant negative VPS4A.

BACKGROUND: Alzheimer's disease (AD)-linked ß-amyloid (Aß) accumulates in multivesicular bodies (MVBs) with the onset of AD pathogenesis. Alterations in endosomes are among the earliest changes associated with AD but the mechanism(s) that cause endosome enlargement and the effects of MVB dysfunction on Aß accumulation and tau pathology are incompletely understood. METHODS: MVB size and Aß fibrils in primary neurons were visualized by electron microscopy and confocal fluorescent microscopy. MVB-dysfunction, modelled by expression of dominant negative VPS4A (dnVPS4A), was analysed by biochemical methods and exosome isolation. RESULTS: Here we show that AD transgenic neurons have enlarged MVBs compared to wild type neurons. Uptake of exogenous Aß also leads to enlarged MVBs in wild type neurons and generates fibril-like structures in endocytic vesicles. With time fibrillar oligomers/fibrils can extend out of the endocytic vesicles and are eventually detectable extracellularly. Further, endosomal sorting complexes required for transport (ESCRT) components were found associated with amyloid plaques in AD transgenic mice. The phenotypes previously reported in AD transgenic neurons, with net increased intracellular levels and reduced secretion of Aß, were mimicked by blocking recycling of ESCRT-III by dnVPS4A. DnVPS4A further resembled AD pathology by increasing tau phosphorylation at serine 396 and increasing markers of autophagy. CONCLUSIONS: We demonstrate that Aß leads to MVB enlargement and that amyloid fibres can form within the endocytic pathway of neurons. These results are consistent with the scenario of the endosome-lysosome system representing the site of initiation of Aß aggregation. In turn, a dominant negative form of the CHMP2B-interacting protein VPS4A, which alters MVBs, leads to accumulation and aggregation of Aß as well as tau phosphorylation, mimicking the cellular changes in AD.

Direct High Affinity Interaction between Aß42 and GSK3α Stimulates Hyperphosphorylation of Tau. A New Molecular Link in Alzheimer's Disease?

Amyloid ß peptide (Aß42) assemblies are considered central to the development of Alzheimer's disease, but the mechanism of this toxicity remains unresolved. We screened protein microarrays with on-pathway oligomeric Aß42 to identify candidate proteins interacting with toxic Aß42 species. Samples prepared from Alexa546-Aß42 and Aß42 monomers at 1:5 molar ratio were incubated with the array during a time window of the amyloid fibril formation reaction during which the maximum number of transient oligomers exist in the reaction flux. A specific interaction was detected between Aß42 and glycogen synthase kinase 3α (GSK3α), a kinase previously implicated in the disease pathology. This interaction was validated with anti-GSK3α immunoprecipitation assays in neuronal cell lysates. Confocal microscopy studies further identified colocalization of Aß42 and GSK3α in neurites of mature primary mouse neurons. A high binding affinity (KD = 1 nM) was measured between Alexa488-Aß42 and GSK3α in solution using thermophoresis. An even lower apparent KD was estimated between GSK3α and dextran-immobilized Aß42 in surface plasmon resonance experiments. Parallel experiments with GSK3ß also identified colocalization and high affinity binding to this isoform. GSK3α-mediated hyperphosphorylation of the protein tau was found to be stimulated by Aß42 in in vitro phosphorylation assays and identified a functional relationship between the proteins. We uncover a direct and functional molecular link between Aß42 and GSK3α, which opens an important avenue toward understanding the mechanism of Aß42-mediated neuronal toxicity in Alzheimer's disease.

Critical role of intraneuronal Aß in Alzheimer's disease: technical challenges in studying intracellular Aß.

AIMS: Multiple lines of evidence have implicated ß-amyloid (Aß) in the pathogenesis of Alzheimer's disease (AD). However, the mechanism(s) whereby Aß is involved in the disease process remains unclear. The dominant hypothesis in AD has been that Aß initiates the disease via toxicity from secreted, extracellular Aß aggregates. More recently, an alternative hypothesis has emerged focusing on a pool of Aß that accumulates early on within AD vulnerable neurons of the brain. Although the topic of intraneuronal Aß has been of major interest in the field, technical difficulties in detecting intraneuronal Aß have also made this topic remarkably controversial. Here we review evidence pointing to the critical role of intraneuronal Aß in AD and provide insights both into challenges faced in detecting intracellular Aß and the prion-like properties of Aß. MAIN METHODS: Immunoprecipitation and Western blot are used for Aß detection. KEY FINDINGS: We highlight that a standard biochemical method can underestimate intraneuronal Aß and that extracellular Aß can up-regulate intracellular Aß. We also show that detergent can remove intraneuronal Aß. SIGNIFICANCE: There is a growing awareness that intraneuronal Aß is a key pathogenic pool of Aß involved in causing synapse dysfunction. Difficulties in detecting intraneuronal Aß are an insufficient reason for ignoring this critical pool of Aß.