Golde, Todd E.

Abstract Background: Genetics play an important role in late-onset Alzheimer’s Disease (AD) etiology and dozens of genetic variants have been implicated in AD risk through large-scale GWAS meta-analyses. However, the precise mechanistic effects of most of these variants have yet to be determined. Deeply phenotyped cohort data can reveal physiological changes associated with genetic risk for AD across an age spectrum that may provide clues to the biology of the disease.Methods: We utilized over 2000 high-quality quantitative measurements obtained from blood of 2831 cognitively normal adult clients of a consumer-based scientific wellness company, each with CLIA-certified whole-genome sequencing data. Measurements included: clinical laboratory blood tests, targeted chip-based proteomics, and metabolomics. We performed a phenome-wide association study utilizing this diverse blood marker data and 25 known AD genetic variants, adjusting for sex, age, vendor (for clinical labs), and the first four genetic principal components; sex-SNP interactions were also assessed.Results: We observed statistically significant SNP-analyte associations for five genetic variants after correction for multiple testing (for SNPs in or near NYAP1, ABCA7, INPP5D, and APOE), with effects detectable from early adulthood. The ABCA7 SNP and the APOE2 and APOE4 encoding alleles were associated with lipid variability, as seen in previous studies; in addition, six novel proteins were associated with the e2 allele. The most statistically significant finding was between the NYAP1 variant and PILRA and PILRB protein levels, supporting previous functional genomic studies in the identification of a putative causal variant within the PILRA gene. Sex modified the effects of four genetic variants, with multiple interrelated immune-modulating effects associated with the PICALM variant. In post-hoc analysis, sex-stratified GWAS results from an independent AD case-control meta-analysis supported sex-specific disease effects of the PICALM variant, highlighting the importance of sex as a biological variable.Conclusions: Known AD genetic variation influenced lipid metabolism and immune response systems in a population of non-AD individuals, with associations observed from early adulthood onward. Further research is needed to determine whether and how these effects are implicated in early-stage biological pathways to AD. These analyses aim to complement ongoing work on the functional interpretation of AD-associated genetic variants.

Aggregation and accumulation of amyloid-β (Aβ) is a defining feature of Alzheimer’s disease pathology. To study microglial responses to Aβ, we applied exogenous Aβ peptide, in either oligomeric or fibrillar conformation, to primary mouse microglial cultures and evaluated system-level transcriptional changes and then compared these with transcriptomic changes in the brains of CRND8 APP mice. We find that primary microglial cultures have rapid and massive transcriptional change in response to Aβ. Transcriptomic responses to oligomeric or fibrillar Aβ in primary microglia, although partially overlapping, are distinct and are not recapitulated in vivo where Aβ progressively accumulates. Furthermore, although classic immune mediators show massive transcriptional changes in the primary microglial cultures, these changes are not observed in the mouse model. Together, these data extend previous studies which demonstrate that microglia responses ex vivo are poor proxies for in vivo responses. Finally, these data demonstrate the potential utility of using microglia as biosensors of different aggregate conformation, as the transcriptional responses to oligomeric and fibrillar Aβ can be distinguished.

AbstractIntron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome‐wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership‐Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon‐level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron‐retained mRNAs were identified using mass spectrometry. Further, we established AD‐specific intron expression Quantitative Trait Loci, and identified splicing‐related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.

AbstractBackgroundPathological aging (PA) patients are individuals that are cognitively normal prior to death but have abundant and widespread amyloid‐β (Aβ) deposits. These individuals typically have few cored amyloid deposits with variable levels of diffuse parenchymal deposits and/or vascular deposits. There is little or no inflammatory reaction, neuritic pathology or neurofibrillary tangles in the cortex. Previously, we have shown that total Aβ levels in PA brain lysates were similar to the levels in Alzheimer’s disease (AD) brain lysates, with overlap in the length of the Aβ peptides that were deposited and no major differences in SDS‐stable Aβ oligomeric assemblies. Despite these similarities, it is unclear whether PA represents a prodromal phase of AD or if the amyloid pathology of PA patients represents a less pathogenic variant. To explore this idea, we examined the seeding capacity of AD and PA brain homogenates to induce Aβ deposition and to secondarily induce tau pathology.MethodWe determined the seeding capacity of two AD, four PA and two control brain homogenates in transgenic mice expressing APPswe/ind with and without Tau‐P301L by intracerebral injection at neonatal day 0. At either 6, 9, 12 or 18 months, we assessed Aβ deposition and neurofibrillary pathology in all 4 genotypes by immunohistochemical and biochemical methods. Furthermore we examined the seeding activity of human AD brain lysates in brain slice cultures from APP/Tau transgenic mice.ResultIn our newborn mouse injections, we observed widespread Aβ seeding as evidenced by accelerated onset of diffuse parenchymal Aβ deposition that was largely proportional to the burden of parenchyma deposits in patient brains. In all cases, high levels of pial deposition were observed and Aβ42 was the predominant peptide deposited. Tau pathology was not induced in any animal, and with the absence of neuritic plaques there was no induction of tau positive neurites.ConclusionThis study provides evidence that the Aβ conformers associated with diffuse Aβ deposits in the brains of cognitively normal individuals possess seeding activities similar to those found in the brains of AD patients. The findings are consistent with the idea that pathological aging is on a continuum with AD.

AbstractBackgroundOur understanding of the biological changes in the brain associated with Alzheimer’s disease (AD) pathology and cognitive impairment remains incomplete. Unbiased discovery proteomic analysis of AD brain can help to increase our understanding of these changes.MethodWe analyzed dorsolateral prefrontal cortex, precuneus, and temporal cortex of control, asymptomatic AD, and AD brains from multiple different centers by label‐free quantitative mass spectrometry and weighted protein co‐expression analysis to obtain a consensus protein co‐expression network of AD brain. We also analyzed cerebrospinal fluid to investigate potential biomarkers related to this network.ResultThe network consisted of 13 protein co‐expression modules. Six of these modules correlated with amyloid‐β plaque burden, tau neurofibrillary tangle burden, cognitive function, and clinical functional status, and were altered in asymptomatic AD, AD, or in both disease states. These six modules reflected synaptic, mitochondrial, sugar metabolism, extracellular matrix, cytoskeletal, and RNA binding/splicing biological functions. The identified protein network modules were preserved in a community‐based cohort analyzed by a different quantitative mass spectrometry approach. They were also preserved in temporal lobe and precuneus brain regions. Some of the modules were influenced by aging, and showed changes in other neurodegenerative diseases such as frontotemporal dementia and corticobasal degeneration. The module most strongly associated with AD pathology and cognitive impairment was the sugar metabolism module. This module was enriched in AD genetic risk factors, and was also highly enriched in microglia and astrocyte protein markers associated with an anti‐inflammatory state, suggesting that the biological functions it represents may serve a protective role in AD. Proteins from the sugar metabolism module were increased in cerebrospinal fluid from asymptomatic AD and AD cases, highlighting their potential as biomarkers of the altered brain network.ConclusionIn this study of >2000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brain that may serve as therapeutic targets and fluid biomarkers for the disease.

Background: Plasma NfL (pNfL) levels are elevated in many neurological disorders. However, the utility of pNfL in a clinical setting has not been established. Objective: In a cohort of diverse older participants, we examined: 1) the association of pNfL to age, sex, Hispanic ethnicity, diagnosis, and structural and amyloid imaging biomarkers; and 2) its association to baseline and longitudinal cognitive and functional performance. Methods: 309 subjects were classified at baseline as cognitively normal (CN) or with cognitive impairment. Most subjects had structural MRI and amyloid PET scans. The most frequent etiological diagnosis was Alzheimer’s disease (AD), but other neurological and neuropsychiatric disorders were also represented. We assessed the relationship of pNfL to cognitive and functional status, primary etiology, imaging biomarkers, and to cognitive and functional decline. Results: pNfL increased with age, degree of hippocampal atrophy, and amyloid load, and was higher in females among CN subjects, but was not associated with Hispanic ethnicity. Compared to CN subjects, pNfL was elevated among those with AD or FTLD, but not those with neuropsychiatric or other disorders. Hippocampal atrophy, amyloid positivity and higher pNfL levels each added unique variance in predicting greater functional impairment on the CDR-SB at baseline. Higher baseline pNfL levels also predicted greater cognitive and functional decline after accounting for hippocampal atrophy and memory scores at baseline. Conclusion: pNfL may have a complementary and supportive role to brain imaging and cognitive testing in a memory disorder evaluation, although its diagnostic sensitivity and specificity as a stand-alone measure is modest. In the absence of expensive neuroimaging tests, pNfL could be used for differentiating neurodegenerative disease from neuropsychiatric disorders.

Abstract Background Self-assembly of the amyloid-β (Aβ) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer’s disease (AD). However it is clear that not all forms of Aβ are equally harmful, and that linking a specific aggregate to toxicity also depends on the assays and model systems used [1, 2]. Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration. Methods In this study, we examined familial mutations of Aβ that increase aggregation and oligomerization, E22G and DE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aβ fusion technology and rAAV2/1 based somatic brain transgenesis in mice to selectively express individual mutant Aβ species in vivo. In parallel we generated PhiC31-based transgenic Drosophila melanogaster expressing wild type (WT) and Aβ40 and Aβ42 mutants, fused to the Argos signal peptide to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different Aβ40 and Aβ42 species.Results When expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of Drosophila. In contrast, flies expressing a copy of Aβ40 (WT or mutants) in addition to Aβ42WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40. Conclusions These studies suggest that while some Aβ40 mutants form unique amyloid aggregates in mouse brains, they do not exacerbate Aβ42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

Abstract BackgroundSelf-assembly of the amyloid-β (Aβ) peptide into aggregates, from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer’s disease (AD). However it is clear that not all forms of Aβ are equally harmful, and that linking a specific aggregate to toxicity also depends on the assays and model systems used [1, 2]. Though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration.MethodsIn this study, we examined familial mutations of Aβ that increase aggregation and oligomerization, E22G and DE22, and induce cerebral amyloid angiopathy, E22Q and D23N. We also investigated synthetic mutations that stabilize dimerization, S26C, and a phospho-mimetic, S8E, and non-phospho-mimetic, S8A. To that end, we utilized BRI2-Aβ fusion technology and rAAV2/1 based somatic brain transgenesis in mice to selectively express individual mutant Aβ species in vivo . In parallel we generated PhiC31-based transgenic Drosophila melanogaster expressing wild type (WT) and Aβ40 and Aβ42 mutants, fused to the Argos signal peptide to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different Aβ40 and Aβ42 species.ResultsWhen expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of Drosophila . In contrast, flies expressing a copy of Aβ40 (WT or mutants) in addition to Aβ42WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40.ConclusionsThese studies suggest that some Aβ40 mutants form unique amyloid aggregates in mouse brains, despite protecting against Aβ42 toxicity in Drosophila , which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.

Abstract BackgroundSelf-assembly of the amyloid-β (Aβ) peptide into aggregates, ranging from small oligomers to amyloid fibrils, is fundamentally linked with Alzheimer’s disease (AD). However it is clear that not all forms of Aβ are equally harmful, and that linking a specific aggregate to toxicity also depends on the assays and model systems used [1, 2]. Indeed, though a central postulate of the amyloid cascade hypothesis, there remain many gaps in our understanding regarding the links between Aβ deposition and neurodegeneration.MethodsIn this study we utilized BRI2-Aβ fusion technology and rAAV2/1 based somatic brain transgenesis to examine Aβ aggregates that form from selective expression of individual mutant Aβ species in vivo. In parallel we generated PhiC31-based transgenic Drosophila melanogaster expressing WT and mutant Aβ40 and Aβ42, fused to the Argos signal peptide and placed under the GAL4-upstream activation sequence (UAS) expression system in order to assess the extent of Aβ42-induced toxicity as well as to interrogate the combined effect of different forms of Aβ40 and Aβ42 species.ResultsWhen expressed in the mouse brain for 6 months, Aβ42 E22G, Aβ42 E22Q/D23N, and Aβ42 WT formed amyloid aggregates consisting of some diffuse material as well as cored plaques, whereas other mutants formed predominantly diffuse amyloid deposits. Moreover, while Aβ40WT showed no distinctive phenotype, Aβ40 E22G and E22Q/D23N formed unique aggregates that accumulated in mouse brains. This is the first evidence that mutant Aβ40 overexpression leads to deposition under certain conditions. Interestingly, we found that mutant Aβ42 E22G, E22Q, and S26C, but not Aβ40, were toxic to the eye of the flies and exacerbated their behavior. In contrast, flies expressing a copy of Aβ40 (wild type [WT] or mutants) in addition to Aβ42 WT, showed improved phenotypes, suggesting possible protective qualities for Aβ40.ConclusionsThese studies suggest that some Aβ40 mutants form unique amyloid aggregates in mouse brains, despite being protective against Aβ42 toxicity in Drosophila, which highlights the significance of using different systems for a better understanding of AD pathogenicity and more accurate screening for new potential therapies.