Browsing by Author "Long, Justin M."

Now showing 1 - 6 of 6
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    Human primary mixed brain cultures: preparation, differentiation, characterization and application to neuroscience research
    (Springer Nature, 2014-09-16) Ray, Balmiki; Chopra, Nipun; Long, Justin M.; Lahiri, Debomoy K.; Psychiatry, School of Medicine
    Background: Culturing primary cortical neurons is an essential neuroscience technique. However, most cultures are derived from rodent brains and standard protocols for human brain cultures are sparse. Herein, we describe preparation, maintenance and major characteristics of a primary human mixed brain culture, including neurons, obtained from legally aborted fetal brain tissue. This approach employs standard materials and techniques used in the preparation of rodent neuron cultures, with critical modifications. Results: This culture has distinct differences from rodent cultures. Specifically, a significant numbers of cells in the human culture are derived from progenitor cells, and the yield and survival of the cells grossly depend on the presence of bFGF. In the presence of bFGF, this culture can be maintained for an extended period. Abundant productions of amyloid-β, tau and proteins make this a powerful model for Alzheimer's research. The culture also produces glia and different sub-types of neurons. Conclusion: We provide a well-characterized methodology for human mixed brain cultures useful to test therapeutic agents under various conditions, and to carry forward mechanistic and translational studies for several brain disorders.
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    Lessons from a BACE1 inhibitor trial: off-site but not off base
    (Elsevier, 2014-10) Lahiri, Debomoy K.; Maloney, Bryan; Long, Justin M.; Greig, Nigel H.; Department of Medical & Molecular Genetics, IU School of Medicine
    Alzheimer's disease (AD) is characterized by formation of neuritic plaque primarily composed of a small filamentous protein called amyloid-β peptide (Aβ). The rate-limiting step in the production of Aβ is the processing of Aβ precursor protein (APP) by β-site APP-cleaving enzyme (BACE1). Hence, BACE1 activity plausibly plays a rate-limiting role in the generation of potentially toxic Aβ within brain and the development of AD, thereby making it an interesting drug target. A phase II trial of the promising LY2886721 inhibitor of BACE1 was suspended in June 2013 by Eli Lilly and Co., due to possible liver toxicity. This outcome was apparently a surprise to the study's team, particularly since BACE1 knockout mice and mice treated with the drug did not show such liver toxicity. Lilly proposed that the problem was not due to LY2886721 anti-BACE1 activity. We offer an alternative hypothesis, whereby anti-BACE1 activity may induce apparent hepatotoxicity through inhibiting BACE1's processing of β-galactoside α-2,6-sialyltransferase I (STGal6 I). In knockout mice, paralogues, such as BACE2 or cathepsin D, could partially compensate. Furthermore, the short duration of animal studies and short lifespan of study animals could mask effects that would require several decades to accumulate in humans. Inhibition of hepatic BACE1 activity in middle-aged humans would produce effects not detectable in mice. We present a testable model to explain the off-target effects of LY2886721 and highlight more broadly that so-called off-target drug effects might actually represent off-site effects that are not necessarily off-target. Consideration of this concept in forthcoming drug design, screening, and testing programs may prevent such failures in the future.
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    MicroRNA-339-5p down-regulates protein expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) in human primary brain cultures and is reduced in brain tissue specimens of Alzheimer disease subjects
    (ASBMB, 2014-02-21) Long, Justin M.; Ray, Balmiki; Lahiri, Debomoy; Department of Medical & Molecular Genetics, IU School of Medicine
    Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β (Aβ) peptide as neuritic plaques in the brain. The short Aβ peptide is derived from the large transmembrane Aβ precursor protein (APP). The rate-limiting step in the production of Aβ from APP is mediated by the β-site APP-cleaving enzyme 1 (BACE1). Dysregulation of BACE1 levels leading to excess Aβ deposition is implicated in sporadic AD. Thus, elucidating the full complement of regulatory pathways that control BACE1 expression is key to identifying novel drug targets central to the Aβ-generating process. MicroRNAs (miRNAs) are expected to participate in this molecular network. Here, we identified a known miRNA, miR-339-5p, as a key contributor to this regulatory network. Two distinct miR-339-5p target sites were predicted in the BACE1 3'-UTR by in silico analyses. Co-transfection of miR-339-5p with a BACE1 3'-UTR reporter construct resulted in significant reduction in reporter expression. Mutation of both target sites eliminated this effect. Delivery of the miR-339-5p mimic also significantly inhibited expression of BACE1 protein in human glioblastoma cells and human primary brain cultures. Delivery of target protectors designed against the miR-339-5p BACE1 3'-UTR target sites in primary human brain cultures significantly elevated BACE1 expression. Finally, miR-339-5p levels were found to be significantly reduced in brain specimens isolated from AD patients as compared with age-matched controls. Therefore, miR-339-5p regulates BACE1 expression in human brain cells and is most likely dysregulated in at least a subset of AD patients making this miRNA a novel drug target.
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    Novel regulation of neuronal genes implicated in Alzheimer disease by microRNA
    (2013-12-11) Long, Justin M.; Zhou, Feng C.; Lahiri, Debomoy K.; Farlow, Martin R.; Nass, Richard M.; Du, Yansheng
    Alzheimer disease (AD) results, in part, from the excess accumulation of the amyloid-β peptide (Aβ) as neuritic plaques in the brain. The short Aβ peptide is derived from a large transmembrane precursor protein, APP. Two different proteolytic enzymes, BACE1 and the gamma-secretase complex, are responsible for cleaving Aβ peptide from APP through an intricate processing pathway. Dysregulation of APP and BACE1 levels leading to excess Aβ deposition has been implicated in various forms of AD. Thus, a major goal in this dissertation was to discover novel regulatory pathways that control APP and BACE1 expression as a means to identify novel drug targets central to the Aβ-generating process. MicroRNAs (miRNA) are short, non-coding RNAs that act as post-transcriptional regulators of gene expression through specific interactions with target mRNAs. Global analyses predict that over sixty percent of human transcripts contain evolutionarily conserved miRNA target sites. Therefore, the specific hypothesis tested was that miRNA are relevant regulators of APP and BACE1 expression. In this work, several specific miRNA were identified that regulate APP protein expression (miR-101, miR-153 and miR-346) or BACE1 expression (miR-339-5p). These miRNAs mediated their post-transcriptional effects via interactions with specific target sites in the APP and BACE1 transcripts. Importantly, these miRNA also altered secretion of Aβ peptides in primary human fetal brain cultures. Surprisingly, miR-346 stimulated APP expression via target sites in the APP 5’-UTR. The mechanism of this effect appears to involve other RNA-binding proteins that bind to the APP 5’-UTR. Expression analyses demonstrated that these miRNAs are expressed to varying degrees in the human brain. Notably, miR-101, miR-153 and miR-339-5p are dysregulated in the AD brain at various stages of the disease. The work in this dissertation supports the hypothesis that miRNAs are important regulators of APP and BACE1 expression and are capable of altering Aβ homeostasis. Therefore, these miRNA may possibly serve as novel therapeutic targets for AD.
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    Novel upregulation of amyloid-β precursor protein (APP) by microRNA-346 via targeting of APP mRNA 5′-untranslated region: Implications in Alzheimer’s disease
    (Nature, 2019) Long, Justin M.; Maloney, Bryan; Rogers, Jack T.; Lahiri, Debomoy K.; Psychiatry, School of Medicine
    In addition to the devastating symptoms of dementia, Alzheimer’s disease (AD) is characterized by accumulation of the processing products of the amyloid-β (Aβ) peptide precursor protein (APP). APP’s non-pathogenic functions include regulating intracellular iron (Fe) homeostasis. MicroRNAs are small (~ 20 nucleotides) RNA species that instill specificity to the RNA-induced silencing complex (RISC). In most cases, RISC inhibits mRNA translation through the 3′-untranslated region (UTR) sequence. By contrast, we report a novel activity of miR-346: specifically, that it targets the APP mRNA 5′-UTR to upregulate APP translation and Aβ production. This upregulation is reduced but not eliminated by knockdown of argonaute 2. The target site for miR-346 overlaps with active sites for an iron-responsive element (IRE) and an interleukin-1 (IL-1) acute box element. IREs interact with iron response protein1 (IRP1), an iron-dependent translational repressor. In primary human brain cultures, miR-346 activity required chelation of Fe. In addition, miR-346 levels are altered in late-Braak stage AD. Thus, miR-346 plays a role in upregulation of APP in the CNS and participates in maintaining APP regulation of Fe, which is disrupted in late stages of AD. Further work will be necessary to integrate other metals, and IL-1 into the Fe-miR-346 activity network. We, thus, propose a “FeAR” (Fe, APP, RNA) nexus in the APP 5′-UTR that includes an overlapping miR-346-binding site and the APP IRE. When a “healthy FeAR” exists, activities of miR-346 and IRP/Fe interact to maintain APP homeostasis. Disruption of an element that targets the FeAR nexus would lead to pathogenic disruption of APP translation and protein production.
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    Regulation of Proteins Implicated in Alzheimer’s Disease by MicroRNAs
    (Office of the Vice Chancellor for Research, 2013-04-05) Chopra, Nipun; Long, Justin M.; Ray, Balmiki; Obukhov, Alexander G.; Lahiri, Debomoy K.
    Alzheimer’s Disease (AD) is a neurodegenerative disorder characterized by the deposition of Amyloid-Beta (Aβ) peptide in the brain. This toxic peptide is generated by the sequential cleavage of Amyloid Precursor Protein (APP) by Beta-site APP-cleaving enzyme-1 (BACE-1) and γ-secretase. The disorder is also characterized by the perturbation of calcium homeostasis in neurons. MicroRNAs are short, single-stranded RNAs that are able to influence protein expression by targeting the 3’ Untranslated region (UTR) or 5’ UTR of mRNAs. Previous work in our laboratory has shown that miR-101, miR-153 and miR-346 can regulate APP whereas miR-339-5p can lower BACE1 expression. Here, we aim to reduce APP, BACE1 and Aβ levels, in vitro, by the addition of microRNAs that target the 3’ UTR of APP and BACE1. We show that in a human astrocytoma-glioblastoma (U373) cell line, the expression of BACE1 protein is significantly reduced compared to the mock condition upon transfecting miR-298, miR-328 and miR-144. miR-298 also reduces Aβ levels in these cells. Similarly, in HeLa cells, we show that miR-520c, miR-20b and miR-144 produce a reduction in APP expression compared to both mock and a negative control microRNA mimic. Additionally, we observed that knocking down APP using siRNA, but not knocking down BACE1, lowers basal intracellular calcium levels as well as changes the kinetics of Potassium Chloride (KCl)-induced intracellular calcium influx in a human fetal brain (HFB) culture, when compared to control. miR-346 increases basal calcium levels, but does not affect KCl-induced calcium transients in our HFB culture. Taken together, these results show that miRNAs can influence both the protein expression as well as calcium homeostasis in different human cell culture models. By reducing levels of proteins implicated in AD pathology and by reversing calcium dysregulation, our results will benefit AD research and generate possibilities for novel therapeutics.