Modulating Amyloid Beta Clearance by Altering Cellular and Whole Brain Apolipoprotein E Metabolism

The aggregation and accumulation of the amyloid beta peptide in the brain is hypothesized to be an initial necessary event in the pathogenesis of Alzheimer\u27s disease (AD). Since the level of monomeric soluble amyloid beta as well as amyloid beta-binding molecules determine the onset and amount of amyloid beta aggregation, significant attention has been devoted to defining the molecular and systemic pathways that modulate amyloid beta synthesis and clearance in the brain. Extensive evidence exists that both the isoform and amount of apolipoprotein E (apoE), an amyloid beta-binding molecule, influence amyloid beta aggregation and clearance from the brain. Therefore, studying how the molecular mechanisms that modulate apoE levels in the brain affect amyloid beta clearance will enhance our insight into the disease process. The apoE receptor low-density lipoprotein receptor (LDLR) and ATP-binding cassette transporter A1 (ABCA1), a protein that regulates apoE lipidation, have previously been shown to modulate brain amyloid beta levels. In the work presented in this dissertation, we found that increasing LDLR levels enhanced the cellular uptake and degradation of amyloid beta by primary astrocytes, and increased amyloid beta transport to lysosomes. The effect of LDLR on amyloid beta uptake and clearance occurred independently of apoE, and potentially involved a direct interaction between amyloid beta and LDLR. To measure the clearance of apoE and amyloid beta in the mouse brain, we developed a technique that couples stable isotope-labeling kinetics (SILK) with mass spectrometry. We validated this technique by demonstrating that apoE clearance is enhanced in the brains of mice overexpressing LDLR. We also applied this technique to measure apoE clearance rates in the brains of human apoE targeted-replacement mice. Finally, we analyzed the effect of ABCA1 on apoE and amyloid beta clearance from the mouse brain. The fractional clearance rate of apoE was increased in amyloid precursor protein (APP) transgenic mice that either lacked or overexpressed ABCA1, while ABCA1 levels had no effect on amyloid beta clearance. Therefore, ABCA1 likely influences amyloid beta aggregation in vivo through a process other than modulating amyloid beta clearance. These data further our understanding of how proteins involved in apoE metabolism influence AD pathogenesis, and have important implications for future therapeutic strategies that target brain apoE levels and function

Characterizing Information Seeking Events in Health-Related Social Discourse

Social media sites have become a popular platform for individuals to seek and share health information. Despite the progress in natural language processing for social media mining, a gap remains in analyzing health-related texts on social discourse in the context of events. Event-driven analysis can offer insights into different facets of healthcare at an individual and collective level, including treatment options, misconceptions, knowledge gaps, etc. This paper presents a paradigm to characterize health-related information-seeking in social discourse through the lens of events. Events here are board categories defined with domain experts that capture the trajectory of the treatment/medication. To illustrate the value of this approach, we analyze Reddit posts regarding medications for Opioid Use Disorder (OUD), a critical global health concern. To the best of our knowledge, this is the first attempt to define event categories for characterizing information-seeking in OUD social discourse. Guided by domain experts, we develop TREAT-ISE, a novel multilabel treatment information-seeking event dataset to analyze online discourse on an event-based framework. This dataset contains Reddit posts on information-seeking events related to recovery from OUD, where each post is annotated based on the type of events. We also establish a strong performance benchmark (77.4% F1 score) for the task by employing several machine learning and deep learning classifiers. Finally, we thoroughly investigate the performance and errors of ChatGPT on this task, providing valuable insights into the LLM's capabilities and ongoing characterization efforts.Comment: Under review AAAI-2024. 10 pages, 6 tables, 2 figue

Measurement of apolipoprotein E and amyloid β clearance rates in the mouse brain using bolus stable isotope labeling

BACKGROUND: Abnormal proteostasis due to alterations in protein turnover has been postulated to play a central role in several neurodegenerative diseases. Therefore, the development of techniques to quantify protein turnover in the brain is critical for understanding the pathogenic mechanisms of these diseases. We have developed a bolus stable isotope-labeling kinetics (SILK) technique coupled with multiple reaction monitoring mass spectrometry to measure the clearance of proteins in the mouse brain. RESULTS: Cohorts of mice were pulse labeled with (13) C(6)-leucine and the brains were isolated after pre-determined time points. The extent of label incorporation was measured over time using mass spectrometry to measure the ratio of labeled to unlabeled apolipoprotein E (apoE) and amyloid β (Aβ). The fractional clearance rate (FCR) was then calculated by analyzing the time course of disappearance for the labeled protein species. To validate the technique, apoE clearance was measured in mice that overexpress the low-density lipoprotein receptor (LDLR). The FCR in these mice was 2.7-fold faster than wild-type mice. To demonstrate the potential of this technique for understanding the pathogenesis of neurodegenerative disease, we applied our SILK technique to determine the effect of ATP binding cassette A1 (ABCA1) on both apoE and Aβ clearance. ABCA1 had previously been shown to regulate both the amount of apoE in the brain, along with the extent of Aβ deposition, and represents a potential molecular target for lowering brain amyloid levels in Alzheimer's disease patients. The FCR of apoE was increased by 1.9- and 1.5-fold in mice that either lacked or overexpressed ABCA1, respectively. However, ABCA1 had no effect on the FCR of Aβ, suggesting that ABCA1 does not regulate Aβ metabolism in the brain. CONCLUSIONS: Our SILK strategy represents a straightforward, cost-effective, and efficient method to measure the clearance of proteins in the mouse brain. We expect that this technique will be applicable to the study of protein dynamics in the pathogenesis of several neurodegenerative diseases, and could aid in the evaluation of novel therapeutic agents

Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis

Apolipoprotein E ε4 (APOE ε4) is the strongest genetic risk factor for Alzheimer’s disease (AD). Evidence suggests that the effect of apoE isoforms on amyloid-β (Aβ) accumulation in the brain plays a critical role in AD pathogenesis. Like in humans, apoE4 expression in animal models that develop Aβ-amyloidosis results in greater Aβ and amyloid deposition than with apoE3 expression. However, whether decreasing levels of apoE3 or apoE4 would promote or attenuate Aβ-related pathology has not been directly addressed. To determine the effect of decreasing human apoE levels on Aβ accumulation in vivo, we generated human APOE isoform haploinsufficient mouse models by crossing APPPS1-21 mice with APOE isoform knock-in mice. By genetically manipulating APOE gene dosage, we demonstrate that decreasing human apoE levels, regardless of isoform status, results in significantly decreased amyloid plaque deposition and microglial activation. This differences in amyloid load between apoE3 and apoE4 expressing mice were not due to apoE4 protein being present at lower levels than apoE3. These data suggest that current therapeutic strategies to increase apoE levels without altering its lipidation state may actually worsen Aβ amyloidosis, while increasing apoE degradation or inhibiting its synthesis may be a more effective treatment approach

BCL11A deletions result in fetal hemoglobin persistence and neurodevelopmental alterations

A transition from fetal hemoglobin (HbF) to adult hemoglobin (HbA) normally occurs within a few months after birth. Increased production of HbF after this period of infancy ameliorates clinical symptoms of the major disorders of adult ß-hemoglobin: ß-thalassemia and sickle cell disease. The transcription factor BCL11A silences HbF and has been an attractive therapeutic target for increasing HbF levels; however, it is not clear to what extent BCL11A inhibits HbF production or mediates other developmental functions in humans. Here, we identified and characterized 3 patients with rare microdeletions of 2p15-p16.1 who presented with an autism spectrum disorder and developmental delay. Moreover, these patients all exhibited substantial persistence of HbF but otherwise retained apparently normal hematologic and immunologic function. Of the genes within 2p15-p16.1, only BCL11A was commonly deleted in all of the patients. Evaluation of gene expression data sets from developing and adult human brains revealed that BCL11A expression patterns are similar to other genes associated with neurodevelopmental disorders. Additionally, common SNPs within the second intron of BCL11A are strongly associated with schizophrenia. Together, the study of these rare patients and orthogonal genetic data demonstrates that BCL11A plays a central role in silencing HbF in humans and implicates BCL11A as an important factor for neurodevelopment

Anti-apoE immunotherapy inhibits amyloid accumulation in a transgenic mouse model of Aβ amyloidosis

The apolipoprotein E (APOE) ε4 allele is the strongest genetic risk factor for Alzheimer’s disease (AD). The influence of apoE on amyloid β (Aβ) accumulation may be the major mechanism by which apoE affects AD. ApoE interacts with Aβ and facilitates Aβ fibrillogenesis in vitro. In addition, apoE is one of the protein components in plaques. We hypothesized that certain anti-apoE antibodies, similar to certain anti-Aβ antibodies, may have antiamyloidogenic effects by binding to apoE in the plaques and activating microglia-mediated amyloid clearance. To test this hypothesis, we developed several monoclonal anti-apoE antibodies. Among them, we administered HJ6.3 antibody intraperitoneally to 4-mo-old male APPswe/PS1ΔE9 mice weekly for 14 wk. HJ6.3 dramatically decreased amyloid deposition by 60–80% and significantly reduced insoluble Aβ40 and Aβ42 levels. Short-term treatment with HJ6.3 resulted in strong changes in microglial responses around Aβ plaques. Collectively, these results suggest that anti-apoE immunization may represent a novel AD therapeutic strategy and that other proteins involved in Aβ binding and aggregation might also be a target for immunotherapy. Our data also have important broader implications for other amyloidosis. Immunotherapy to proteins tightly associated with misfolded proteins might open up a new treatment option for many protein misfolding diseases

Whole Genomes of Chandipura Virus Isolates and Comparative Analysis with Other Rhabdoviruses

The Chandipura virus (CHPV) belonging to the Vesiculovirus genus and Rhabdoviridae family, has recently been associated with a number of encephalitis epidemics, with high mortality in children, in different parts of India. No full length genome sequences of CHPV isolates were available in GenBank and little is known about the molecular markers for pathogenesis. In the present study, we provide the complete genomic sequences of four isolates from epidemics during 2003–2007. These sequences along with the deduced sequence of the prototype isolate of 1965 were analysed using phylogeny, motif search, homology modeling and epitope prediction methods. Comparison with other rhaboviruses was also done for functional extrapolations. All CHPV isolates clustered with the Isfahan virus and maintained several functional motifs of other rhabdoviruses. A notable difference with the prototype vesiculovirus, Vesicular Stomatitis Virus was in the L-domain flanking sequences of the M protein that are known to be crucial for interaction with host proteins. With respect to the prototype isolate, significant additional mutations were acquired in the 2003–2007 isolates. Several mutations in G mapped onto probable antigenic sites. A mutation in N mapped onto regions crucial for N-N interaction and a putative T-cell epitope. A mutation in the Casein kinase II phosphorylation site in P may attribute to increased rates of phosphorylation. Gene junction comparison revealed changes in the M-G junction of all the epidemic isolates that may have implications on read-through and gene transcription levels. The study can form the basis for further experimental verification and provide additional insights into the virulence determinants of the CHPV

In Vivo Human Apolipoprotein E Isoform Fractional Turnover Rates in the CNS

Apolipoprotein E (ApoE) is the strongest genetic risk factor for Alzheimer’s disease and has been implicated in the risk for other neurological disorders. The three common ApoE isoforms (ApoE2, E3, and E4) each differ by a single amino acid, with ApoE4 increasing and ApoE2 decreasing the risk of Alzheimer’s disease (AD). Both the isoform and amount of ApoE in the brain modulate AD pathology by altering the extent of amyloid beta (Aβ) peptide deposition. Therefore, quantifying ApoE isoform production and clearance rates may advance our understanding of the role of ApoE in health and disease. To measure the kinetics of ApoE in the central nervous system (CNS), we applied in vivo stable isotope labeling to quantify the fractional turnover rates of ApoE isoforms in 18 cognitively-normal adults and in ApoE3 and ApoE4 targeted-replacement mice. No isoform-specific differences in CNS ApoE3 and ApoE4 turnover rates were observed when measured in human CSF or mouse brain. However, CNS and peripheral ApoE isoform turnover rates differed substantially, which is consistent with previous reports and suggests that the pathways responsible for ApoE metabolism are different in the CNS and the periphery. We also demonstrate a slower turnover rate for CSF ApoE than that for amyloid beta, another molecule critically important in AD pathogenesis