Alzheimer's disease: From immunotherapy to immunoprevention.

Recent Aß-immunotherapy trials have yielded the first clear evidence that removing aggregated Aß from the brains of symptomatic patients can slow the progression of Alzheimer's disease. The clinical benefit achieved in these trials has been modest, however, highlighting the need for both a deeper understanding of disease mechanisms and the importance of intervening early in the pathogenic cascade. An immunoprevention strategy for Alzheimer's disease is required that will integrate the findings from clinical trials with mechanistic insights from preclinical disease models to select promising antibodies, optimize the timing of intervention, identify early biomarkers, and mitigate potential side effects.

Medin co-aggregates with vascular amyloid-ß in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-ß precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-ß deposits, and that in mice, medin deficiency reduces vascular amyloid-ß deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-ß burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-ß to promote its aggregation, as medin forms heterologous fibrils with amyloid-ß, affects amyloid-ß fibril structure, and cross-seeds amyloid-ß aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-ß deposition in the blood vessels of the brain.

Proteopathic Strains and the Heterogeneity of Neurodegenerative Diseases.

Most age-related neurodegenerative diseases are associated with the misfolding and aberrant accumulation of specific proteins in the nervous system. The proteins self-assemble and spread by a prion-like process of corruptive molecular templating, whereby abnormally folded proteins induce the misfolding and aggregation of like proteins into characteristic lesions. Despite the apparent simplicity of this process at the molecular level, diseases such as Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and others display remarkable phenotypic heterogeneity, both clinically and pathologically. Evidence is growing that this variability is mediated, at least in part, by the acquisition of diverse molecular architectures by the misfolded proteins, variants referred to as proteopathic strains. The structural and functional diversity of the assemblies is influenced by genetic, epigenetic, and local contextual factors. Insights into proteopathic strains gleaned from the classical prion diseases can be profitably incorporated into research on other neurodegenerative diseases. Their potentially wide-ranging influence on disease phenotype also suggests that proteopathic strains should be considered in the design and interpretation of diagnostic and therapeutic approaches to these disorders.

Neurodegenerative diseases: expanding the prion concept.

The prion paradigm has emerged as a unifying molecular principle for the pathogenesis of many age-related neurodegenerative diseases. This paradigm holds that a fundamental cause of specific disorders is the misfolding and seeded aggregation of certain proteins. The concept arose from the discovery that devastating brain diseases called spongiform encephalopathies are transmissible to new hosts by agents consisting solely of a misfolded protein, now known as the prion protein. Accordingly, "prion" was defined as a "proteinaceous infectious particle." As the concept has expanded to include other diseases, many of which are not infectious by any conventional definition, the designation of prions as infectious agents has become problematic. We propose to define prions as "proteinaceous nucleating particles" to highlight the molecular action of the agents, lessen unwarranted apprehension about the transmissibility of noninfectious proteopathies, and promote the wider acceptance of this revolutionary paradigm by the biomedical community.

Polyglucosan body disease in an aged chimpanzee (Pan troglodytes).

A 57-year-old female chimpanzee presented with a brief history of increasing lethargy and rapidly progressive lower-limb weakness that culminated in loss of use. Postmortem examination revealed no significant gross lesions in the nervous system or other organ systems. Histological analysis revealed round, basophilic to amphophilic polyglucosan bodies (PGBs) in the white and gray matter of the cervical, thoracic, lumbar, and coccygeal regions of spinal cord. Only rare PGBs were observed in forebrain samples. The lesions in the spinal cord were polymorphic, and they were positively stained with hematoxylin, periodic acid Schiff, Alcian blue, toluidine blue, Bielschowsky silver, and Grocott-Gomori methenamine-silver methods, and they were negative for von Kossa and Congo Red stains. Immunohistochemical evaluation revealed reactivity with antibodies to ubiquitin, but they were negative for glial fibrillary acidic protein, neuron-specific enolase, neurofilaments, tau protein, and Aß protein. Electron microscopy revealed non-membrane-bound deposits composed of densely packed filaments within axons and in the extracellular space. Intra-axonal PGBs were associated with disruption of the axonal fine structure and disintegration of the surrounding myelin sheath. These findings are the first description of PGBs linked to neurological dysfunction in a chimpanzee. Clinicopathologically, the disorder resembled adult PGB disease in humans.

Amyloid polymorphisms constitute distinct clouds of conformational variants in different etiological subtypes of Alzheimer's disease.

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-ß peptide (Aß) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aß can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of ß-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aß nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aß plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aß-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aß among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aß conformation and clinical phenotype.

Aß seeding potency peaks in the early stages of cerebral ß-amyloidosis.

Little is known about the extent to which pathogenic factors drive the development of Alzheimer's disease (AD) at different stages of the long preclinical and clinical phases. Given that the aggregation of the ß-amyloid peptide (Aß) is an important factor in AD pathogenesis, we asked whether Aß seeds from brain extracts of mice at different stages of amyloid deposition differ in their biological activity. Specifically, we assessed the effect of age on Aß seeding activity in two mouse models of cerebral Aß amyloidosis (APPPS1 and APP23) with different ages of onset and rates of progression of Aß deposition. Brain extracts from these mice were serially diluted and inoculated into host mice. Strikingly, the seeding activity (seeding dose SD50) in extracts from donor mice of both models reached a plateau relatively early in the amyloidogenic process. When normalized to total brain Aß, the resulting specific seeding activity sharply peaked at the initial phase of Aß deposition, which in turn is characterized by a temporary several-fold increase in the Aß42/Aß40 ratio. At all stages, the specific seeding activity of the APPPS1 extract was higher compared to that of APP23 brain extract, consistent with a more important contribution of Aß42 than Aß40 to seed activity. Our findings indicate that the Aß seeding potency is greatest early in the pathogenic cascade and diminishes as Aß increasingly accumulates in brain. The present results provide experimental support for directing anti-Aß therapeutics to the earliest stage of the pathogenic cascade, preferably before the onset of amyloid deposition.

Self-propagation of pathogenic protein aggregates in neurodegenerative diseases.

For several decades scientists have speculated that the key to understanding age-related neurodegenerative disorders may be found in the unusual biology of the prion diseases. Recently, owing largely to the advent of new disease models, this hypothesis has gained experimental momentum. In a remarkable variety of diseases, specific proteins have been found to misfold and aggregate into seeds that structurally corrupt like proteins, causing them to aggregate and form pathogenic assemblies ranging from small oligomers to large masses of amyloid. Proteinaceous seeds can therefore serve as self-propagating agents for the instigation and progression of disease. Alzheimer's disease and other cerebral proteopathies seem to arise from the de novo misfolding and sustained corruption of endogenous proteins, whereas prion diseases can also be infectious in origin. However, the outcome in all cases is the functional compromise of the nervous system, because the aggregated proteins gain a toxic function and/or lose their normal function. As a unifying pathogenic principle, the prion paradigm suggests broadly relevant therapeutic directions for a large class of currently intractable diseases.

Generation of Clickable Pittsburgh Compound B for the Detection and Capture of ß-Amyloid in Alzheimer's Disease Brain.

The benzothiazole-aniline derivative Pittsburgh Compound B (PiB) is the prototypical amyloid affinity probe developed for the in vivo positron emission tomography (PET) detection of amyloid beta (Aß) deposits in Alzheimer's disease (AD). Specific high-affinity binding sites for PiB have been found to vary among AD cases with comparable Aß load, and they are virtually absent on human-sequence Aß deposits in animal models, none of which develop the full phenotype of AD. PiB thus could be an informative probe for studying the pathobiology of Aß, but little is known about the localization of PiB binding at the molecular or structural level. By functionalizing the 6-hydroxy position of PiB with a PEG3 spacer and a terminal alkyne (propargyl) moiety, we have developed a clickable PiB compound that was derivatized with commercially available azide-labeled fluorophores or affinity-tags using copper-catalyzed azide-alkyne cycloaddition reactions, commonly referred to as "click" chemistry. We have determined that both the clickable PiB derivative and its fluorescently labeled conjugate have low nanomolar binding affinities for synthetic Aß aggregates. Furthermore, the fluorescent-PiB conjugate can effectively bind Aß aggregates in human AD brain homogenates and tissue sections. By covalently coupling PiB to magnetic beads, Aß aggregates were also affinity-captured from AD brain extracts. Thus, the clickable PiB derivative described herein can be used to generate a wide variety of covalent conjugates, with applications including the fluorescence detection of Aß, the ultrastructural localization of PiB binding, and the affinity capture and structural characterization of Aß and other cofactors from AD brains.

A distinct subfraction of Aß is responsible for the high-affinity Pittsburgh compound B-binding site in Alzheimer's disease brain.

The positron emission tomography (PET) ligand (11) C-labeled Pittsburgh compound B (PIB) is used to image ß-amyloid (Aß) deposits in the brains of living subjects with the intent of detecting early stages of Alzheimer's disease (AD). However, deposits of human-sequence Aß in amyloid precursor protein transgenic mice and non-human primates bind very little PIB. The high stoichiometry of PIB:Aß binding in human AD suggests that the PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions. In this study, (3) H-PIB was employed to track purification of the PIB-binding site in > 90% yield from frontal cortical tissue of autopsy-diagnosed AD subjects. The purified PIB-binding site comprises a distinct, highly insoluble subfraction of the Aß in AD brain with low buoyant density because of the sodium dodecyl sulfate-resistant association with a limited subset of brain proteins and lipids with physical properties similar to lipid rafts and to a ganglioside:Aß complex in AD and Down syndrome brain. Both the protein and lipid components are required for PIB binding. Elucidation of human-specific biological components and pathways will be important in guiding improvement of the animal models for AD and in identifying new potential therapeutic avenues. A lipid-associated subpopulation of Aß accounts for the high-affinity binding of Pittsburgh compound B (PIB) in Alzheimer's disease brain. Mass spectrometry of the isolated PIB-binding site from frontal cortex identified Aß peptides and a set of plaque-associated proteins in AD but not age-matched normal brain. The PIB-binding site may represent a particularly pathogenic entity and/or report local pathologic conditions.

Aß seeds resist inactivation by formaldehyde.

Cerebral ß-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated ß-amyloid (Aß) into young, pre-depositing Aß precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aß itself. Here we report that the ß-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aß are maintained in fixed tissues. The resistance of Aß seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aß aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

Corruption and spread of pathogenic proteins in neurodegenerative diseases.

With advancing age, the brain becomes increasingly susceptible to neurodegenerative diseases, most of which are characterized by the misfolding and errant aggregation of certain proteins. The induction of aggregation involves a crystallization-like seeding mechanism by which a specific protein is structurally corrupted by its misfolded conformer. The latest research indicates that, once formed, proteopathic seeds can spread from one locale to another via cellular uptake, transport, and release. Impeding this process could represent a unified therapeutic strategy for slowing the progression of a wide range of currently intractable disorders.

Context dependence of protein misfolding and structural strains in neurodegenerative diseases.

Vast arrays of structural forms are accessible to simple amyloid peptides and environmental conditions can direct assembly into single phases. These insights are now being applied to the aggregation of the Aß peptide of Alzheimer's disease and the identification of causative phases. We extend use of the imaging agent Pittsburgh compound B to discriminate among Aß phases and begin to define conditions of relevance to the disease state. Also, we specifically highlight the development of methods for defining the structures of these more complex phases.

Soluble Aß seeds are potent inducers of cerebral ß-amyloid deposition.

Cerebral ß-amyloidosis and associated pathologies can be exogenously induced by the intracerebral injection of small amounts of pathogenic Aß-containing brain extract into young ß-amyloid precursor protein (APP) transgenic mice. The probable ß-amyloid-inducing factor in the brain extract has been identified as a species of aggregated Aß that is generated in its most effective conformation or composition in vivo. Here we report that Aß in the brain extract is more proteinase K (PK) resistant than is synthetic fibrillar Aß, and that this PK-resistant fraction of the brain extract retains the capacity to induce ß-amyloid deposition upon intracerebral injection in young, pre-depositing APP23 transgenic mice. After ultracentrifugation of the brain extract, <0.05% of the Aß remained in the supernatant fraction, and these soluble Aß species were largely PK sensitive. However, upon intracerebral injection, this soluble fraction accounted for up to 30% of the ß-amyloid induction observed with the unfractionated extract. Fragmentation of the Aß seeds by extended sonication increased the seeding capacity of the brain extract. In summary, these results suggest that multiple Aß assemblies, with various PK sensitivities, are capable of inducing ß-amyloid aggregation in vivo. The finding that small and soluble Aß seeds are potent inducers of cerebral ß-amyloidosis raises the possibility that such seeds may mediate the spread of ß-amyloidosis in the brain. If they can be identified in vivo, soluble Aß seeds in bodily fluids also could serve as early biomarkers for cerebral ß-amyloidogenesis and eventually Alzheimer's disease.

Exogenous seeding of cerebral ß-amyloid deposition in ßAPP-transgenic rats.

Deposition of the amyloid-ß (Aß) peptide in senile plaques and cerebral Aß angiopathy (CAA) can be stimulated in Aß-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aß seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aß itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aß seeding have employed animal models that, as they age, eventually will generate Aß lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aß within the course of its median lifespan (30 months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aß. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aß-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aß.

Pathogenic protein seeding in Alzheimer disease and other neurodegenerative disorders.

The misfolding and aggregation of specific proteins is a seminal occurrence in a remarkable variety of neurodegenerative disorders. In Alzheimer disease (the most prevalent cerebral proteopathy), the two principal aggregating proteins are ß-amyloid (Aß) and tau. The abnormal assemblies formed by conformational variants of these proteins range in size from small oligomers to the characteristic lesions that are visible by optical microscopy, such as senile plaques and neurofibrillary tangles. Pathologic similarities with prion disease suggest that the formation and spread of these proteinaceous lesions might involve a common molecular mechanism-corruptive protein templating. Experimentally, cerebral ß-amyloidosis can be exogenously induced by exposure to dilute brain extracts containing aggregated Aß seeds. The amyloid-inducing agent probably is Aß itself, in a conformation generated most effectively in the living brain. Once initiated, Aß lesions proliferate within and among brain regions. The induction process is governed by the structural and biochemical nature of the Aß seed, as well as the attributes of the host, reminiscent of pathogenically variant prion strains. The concept of prionlike induction and spreading of pathogenic proteins recently has been expanded to include aggregates of tau, α-synuclein, huntingtin, superoxide dismutase-1, and TDP-43, which characterize such human neurodegenerative disorders as frontotemporal lobar degeneration, Parkinson/Lewy body disease, Huntington disease, and amyotrophic lateral sclerosis. Our recent finding that the most effective Aß seeds are small and soluble intensifies the search in bodily fluids for misfolded protein seeds that are upstream in the proteopathic cascade, and thus could serve as predictive diagnostics and the targets of early, mechanism-based interventions. Establishing the clinical implications of corruptive protein templating will require further mechanistic and epidemiologic investigations. However, the theory that many chronic neurodegenerative diseases can originate and progress via the seeded corruption of misfolded proteins has the potential to unify experimental and translational approaches to these increasingly prevalent disorders.

The presence of Aß seeds, and not age per se, is critical to the initiation of Aß deposition in the brain.

The deposition of the ß-amyloid (Aß) peptide in senile plaques and cerebral Aß-amyloid angiopathy can be seeded in ß-amyloid precursor protein (APP)-transgenic mice by the intracerebral infusion of brain extracts containing aggregated Aß. Previous studies of seeded ß-amyloid induction have used relatively short incubation periods to dissociate seeded ß-amyloid induction from endogenous ß-amyloid deposition of the host, thus precluding the analysis of the impact of age and extended incubation periods on the instigation and spread of Aß lesions in brain. In the present study using R1.40 APP-transgenic mice (which do not develop endogenous Aß deposition up to 15 months of age) we show that: (1) seeding at 9 months of age does not induce more Aß deposition than seeding at 3 months of age, provided that the incubation period (6 months) is the same; and (2) very long-term (12 months) incubation after a focal application of the seed results in the emergence of Aß deposits throughout the forebrain. These findings indicate that the presence of Aß seeds, and not the age of the host per se, is critical to the initiation of Aß aggregation in the brain, and that Aß deposition, actuated in one brain area, eventually spreads throughout the brain.

Mitochondrial DNA polymorphisms specifically modify cerebral ß-amyloid proteostasis.

Several lines of evidence link mutations and deletions in mitochondrial DNA (mtDNA) and its maternal inheritance to neurodegenerative diseases in the elderly. Age-related mutations of mtDNA modulate the tricarboxylic cycle enzyme activity, mitochondrial oxidative phosphorylation capacity and oxidative stress response. To investigate the functional relevance of specific mtDNA polymorphisms of inbred mouse strains in the proteostasis regulation of the brain, we established novel mitochondrial congenic mouse lines of Alzheimer's disease (AD). We crossed females from inbred strains (FVB/N, AKR/J, NOD/LtJ) with C57BL/6 males for at least ten generations to gain specific mitochondrial conplastic strains with pure C57BL/6 nuclear backgrounds. We show that specific mtDNA polymorphisms originating from the inbred strains differentially influence mitochondrial energy metabolism, ATP production and ATP-driven microglial activity, resulting in alterations of cerebral ß-amyloid (Aß) accumulation. Our findings demonstrate that mtDNA-related increases in ATP levels and subsequently in microglial activity are directly linked to decreased Aß accumulation in vivo, implicating reduced mitochondrial function in microglia as a causative factor in the development of age-related cerebral proteopathies such as AD.