Cerebral Aß deposition in an Aß-precursor protein-transgenic rhesus monkey.

With the ultimate goal of developing a more representative animal model of Alzheimer's disease (AD), two female amyloid-ß-(Aß) precursor protein-transgenic (APPtg) rhesus monkeys were generated by lentiviral transduction of the APP gene into rhesus oocytes, followed by in vitro fertilization and embryo transfer. The APP-transgene included the AD-associated Swedish K670N/M671L and Indiana V717F mutations (APPSWE/IND) regulated by the human polyubiquitin-C promoter. Overexpression of APP was confirmed in lymphocytes and brain tissue. Upon sacrifice at 10 years of age, one of the monkeys had developed Aß plaques and cerebral Aß-amyloid angiopathy in the occipital, parietal, and caudal temporal neocortices. The induction of Aß deposition more than a decade prior to its usual emergence in the rhesus monkey supports the feasibility of creating a transgenic nonhuman primate model for mechanistic analyses and preclinical testing of treatments for Alzheimer's disease and cerebrovascular amyloidosis.

Correction to: Quantification of neurons in the hippocampal formation of chimpanzees: comparison to rhesus monkeys and humans.

The original version of this article contained a mistake in Figs. 3 and 4.

Quantification of neurons in the hippocampal formation of chimpanzees: comparison to rhesus monkeys and humans.

The hippocampal formation is important for higher brain functions such as spatial navigation and the consolidation of memory, and it contributes to abilities thought to be uniquely human, yet little is known about how the human hippocampal formation compares to that of our closest living relatives, the chimpanzees. To gain insight into the comparative organization of the hippocampal formation in catarrhine primates, we quantified neurons stereologically in its major subdivisions-the granular layer of the dentate gyrus, CA4, CA2-3, CA1, and the subiculum-in archival brain tissue from six chimpanzees ranging from 29 to 43 years of age. We also sought evidence of Aß deposition and hyperphosphorylated tau in the hippocampus and adjacent neocortex. A 42-year-old animal had moderate cerebral Aß-amyloid angiopathy and tauopathy, but Aß was absent and tauopathy was minimal in the others. Quantitatively, granule cells of the dentate gyrus were most numerous, followed by CA1, subiculum, CA4, and CA2-3. In the context of prior investigations of rhesus monkeys and humans, our findings indicate that, in the hippocampal formation as a whole, the proportions of neurons in CA1 and the subiculum progressively increase, and the proportion of dentate granule cells decreases, from rhesus monkeys to chimpanzees to humans. Because CA1 and the subiculum engender key hippocampal projection pathways to the neocortex, and because the neocortex varies in volume and anatomical organization among these species, these findings suggest that differences in the proportions of neurons in hippocampal subregions of catarrhine primates may be linked to neocortical evolution.

Comparative pathobiology of ß-amyloid and the unique susceptibility of humans to Alzheimer's disease.

The misfolding and accumulation of the protein fragment ß-amyloid (Aß) is an early and essential event in the pathogenesis of Alzheimer's disease (AD). Despite close biological similarities among primates, humans appear to be uniquely susceptible to the profound neurodegeneration and dementia that characterize AD, even though nonhuman primates deposit copious Aß in senile plaques and cerebral amyloid-ß angiopathy as they grow old. Because the amino acid sequence of Aß is identical in all primates studied to date, we asked whether differences in the properties of aggregated Aß might underlie the vulnerability of humans and the resistance of other primates to AD. In a comparison of aged squirrel monkeys (Saimiri sciureus) and humans with AD, immunochemical and mass spectrometric analyses indicate that the populations of Aß fragments are largely similar in the 2 species. In addition, Aß-rich brain extracts from the brains of aged squirrel monkeys and AD patients similarly seed the deposition of Aß in a transgenic mouse model. However, the epitope exposure of aggregated Aß differs in sodium dodecyl sulfate-stable oligomeric Aß from the 2 species. In addition, the high-affinity binding of (3)H Pittsburgh Compound B to Aß is significantly diminished in tissue extracts from squirrel monkeys compared with AD patients. These findings support the hypothesis that differences in the pathobiology of aggregated Aß among primates are linked to post-translational attributes of the misfolded protein, such as molecular conformation and/or the involvement of species-specific cofactors.

Wrapping it up in a person: Examining employment and earnings outcomes for Ph.D. recipients.

In evaluating research investments, it is important to establish whether the expertise gained by researchers in conducting their projects propagates into the broader economy. For eight universities, it was possible to combine data from the UMETRICS project, which provided administrative records on graduate students supported by funded research, with data from the U.S. Census Bureau. The analysis covers 2010-2012 earnings and placement outcomes of people receiving doctorates in 2009-2011. Almost 40% of supported doctorate recipients, both federally and nonfederally funded, entered industry and, when they did, they disproportionately got jobs at large and high-wage establishments in high-tech and professional service industries. Although Ph.D. recipients spread nationally, there was also geographic clustering in employment near the universities that trained and employed the researchers. We also show large differences across fields in placement outcomes.

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.

Nonhuman primate models of Alzheimer-like cerebral proteopathy.

Nonhuman primates are useful for the study of age-associated changes in the brain and behavior in a model that is biologically proximal to humans. The Aß and tau proteins, two key players in the pathogenesis of Alzheimer's disease (AD), are highly homologous among primates. With age, all nonhuman primates analyzed to date develop senile (Aß) plaques and cerebral ß-amyloid angiopathy. In contrast, significant tauopathy is unusual in simians, and only humans manifest the profound tauopathy, neuronal degeneration and cognitive impairment that characterize Alzheimer's disease. Primates thus are somewhat paradoxical models of AD-like pathology; on the one hand, they are excellent models of normal aging and naturally occurring Aß lesions, and they can be useful for testing diagnostic and therapeutic agents targeting aggregated forms of Aß. On the other hand, the resistance of monkeys and apes to tauopathy and AD-related neurodegeneration, in the presence of substantial cerebral Aß deposition, suggests that a comparative analysis of human and nonhuman primates could yield informative clues to the uniquely human predisposition to 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.

PIB binding in aged primate brain: enrichment of high-affinity sites in humans with Alzheimer's disease.

Aged nonhuman primates accumulate large amounts of human-sequence amyloid ß (Aß) in the brain, yet they do not manifest the full phenotype of Alzheimer's disease (AD). To assess the biophysical properties of Aß that might govern its pathogenic potential in humans and nonhuman primates, we incubated the benzothiazole imaging agent Pittsburgh Compound B (PIB) with cortical tissue homogenates from normal aged humans, humans with AD, and from aged squirrel monkeys, rhesus monkeys, and chimpanzees with cerebral Aß-amyloidosis. Relative to humans with AD, high-affinity PIB binding is markedly reduced in cortical extracts from aged nonhuman primates containing levels of insoluble Aß similar to those in AD. The high-affinity binding of PIB may be selective for a pathologic, human-specific conformation of multimeric Aß, and thus could be a useful experimental tool for clarifying the unique predisposition of humans to Alzheimer's disease.

SDS-PAGE/immunoblot detection of Abeta multimers in human cortical tissue homogenates using antigen-epitope retrieval.

The anomalous folding and polymerization of the beta-amyloid (Abeta) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis(1). Abeta is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into beta-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Abeta multimers are more toxic than fibrillar Abeta amyloid(2). The identification and quantification of low- and high-molecular weight multimeric Abeta species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies(3). Naturally occurring Abeta multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Abeta-specific antibodies. However, the separation and detection of multimeric Abeta requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Abeta peptide. Using this protocol, we consistently detect Abeta monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.

Deficient high-affinity binding of Pittsburgh compound B in a case of Alzheimer's disease.

Radiolabeled Pittsburgh compound B (PIB) is a benzothiazole imaging agent that usually binds with high affinity, specificity, and stoichiometry to cerebral beta-amyloid (Abeta) in patients with Alzheimer's disease. Among a cohort of ten AD subjects examined postmortem, we describe a case of idiopathic, end-stage Alzheimer's disease with heavy Abeta deposition yet substantially diminished high-affinity binding of (3)H-PIB to cortical homogenates and unfixed cryosections. Cortical tissue samples were analyzed by immunohistochemistry, electron microscopy, ELISA, immunoblotting, MALDI-TOF mass spectrometry, in vitro (3)H-PIB binding and (3)H-PIB autoradiography. The PIB-refractory subject met the histopathological criteria for AD. However, cortical tissue from this case contained more vascular beta-amyloidosis, higher levels of insoluble Abeta40 and Abeta42, and a higher ratio of Abeta40:Abeta42 than did tissue from the nine comparison AD cases. Furthermore, cerebral Abeta from the PIB-refractory subject displayed an unusual distribution of low- and high-molecular weight Abeta oligomers, as well as a distinct pattern of N- and C-terminally truncated Abeta peptides in both the soluble and insoluble cortical extracts. Genetically, the patient was apolipoprotein-E3/4 heterozygous, and exhibited no known AD-associated mutations in the genes for the beta-amyloid precursor protein, presenilin1 or presenilin2. Our findings suggest that PIB may differentially recognize polymorphic forms of multimeric Abeta in humans with Alzheimer's disease. In addition, while the prevalence of PIB-refractory cases in the general AD population remains to be determined, the paucity of high-affinity binding sites in this AD case cautions that minimal PIB retention in positron-emission tomography scans of demented patients may not always rule out the presence of Alzheimer-type Abeta pathology.

Tauopathy with paired helical filaments in an aged chimpanzee.

An enigmatic feature of age-related neurodegenerative diseases is that they seldom, if ever, are fully manifested in nonhuman species under natural conditions. The neurodegenerative tauopathies are typified by the intracellular aggregation of hyperphosphorylated microtubule-associated protein tau (MAPT) and the dysfunction and death of affected neurons. We document the first case of tauopathy with paired helical filaments in an aged chimpanzee (Pan troglodytes). Pathologic forms of tau in neuronal somata, neuropil threads, and plaque-like clusters of neurites were histologically identified throughout the neocortex and, to a lesser degree, in allocortical and subcortical structures. Ultrastructurally, the neurofibrillary tangles consisted of tau-immunoreactive paired helical filaments with a diameter and helical periodicity indistinguishable from those seen in Alzheimer's disease. A moderate degree of Abeta deposition was present in the cerebral vasculature and, less frequently, in senile plaques. Sequencing of the exons and flanking intronic regions in the genomic MAPT locus disclosed no mutations that are associated with the known human hereditary tauopathies, nor any polymorphisms of obvious functional significance. Although the lesion profile in this chimpanzee differed somewhat from that in Alzheimer's disease, the copresence of paired helical filaments and Abeta-amyloidosis indicates that the molecular mechanisms for the pathogenesis of the two canonical Alzheimer lesions--neurofibrillary tangles and senile plaques--are present in aged chimpanzees.

Diversity of Abeta deposits in the aged brain: a window on molecular heterogeneity?

The Abeta peptide forms a morphologically heterogeneous assortment of aggregates in the brains of patients with Alzheimer's disease. The reasons for the diversity of the histopathologically identified lesions (Abeta-plaques and cerebral beta-amyloid angiopathy) are uncertain. However, there is growing evidence for the existence of higher order structural heterogeneity in protein molecules with the same amino acid sequence and differential involvement in disease. Focused analysis of plaque morphotypes could yield novel insights into the organization and function of putative protein variants in the diseased brain. In addition to classical amyloid-selective dyes, new techniques are emerging to undertake such analyses, including selective, small molecule binding agents, specific antibodies, and conformationally sensitive optical probes. By illuminating the relationships between specific lesions and their molecular components, these agents can help to clarify the complex pathology of Alzheimer's disease.