Compartmentalized Neuronal Cultures

Darbinyan, Armine; Pozniak, Paul D.; Darbinian, Nune; White, Martyn K.; Khalili, Kamel

doi:10.1007/978-1-62703-640-5_13

Cited by 9 publications

(8 citation statements)

References 11 publications

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“…Our data suggest that either through transfection, or transduction, Tat causes mtDNA damage in neuronal cells. Previously, we confirmed that ultrapure Tat protein or short PTD-Tat (47-57 amino acids) was entering into neuronal cells or nucleus as early as after 40 minutes post-treatment [ 37 , 38 ]. Our and other research groups investigated features and characteristics of Tat protein to achieve an efficient transduction and to avoid degradation or cleavage prior its transportation into nucleus [ 39 – 43 ].…”

Section: Resultsmentioning

confidence: 77%

“…PCR conditions were as follows; activation 95°C 5 min, PCR 45 cycles: 95°C 10 sec, 60°C 20 sec, 72°C 30 sec, melting curve (95-65°C), cool to 40°C 30 sec. [ 37 ]. For relative quantification, the expression level of genes was normalized to the housekeeping gene β-actin.…”

Section: Methodsmentioning

confidence: 99%

“…Human primary cortical neurons were prepared in our laboratory by Dr. Darbinyan [4,[25][26][27][28]. Human fetal brains resulting from elective abortion were obtained from Advanced Bioscience Resources (ABR), Inc., Alameda, CA 94501 USA, under a protocol approved by Temple University's IRB.…”

Section: Cell Culturementioning

confidence: 99%

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HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

et al. 2020

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Introduction: Mitochondrial dysregulation is a key event in HIV-1 infection. Recent studies have suggested that age-related neurodegenerative disorders are associated with increased mitochondrial DNA (mtDNA) damage. As accelerated ageing was found in HIV-1 patients, we hypothesized that HIV-1 infection or HIV-1 proteins can lead to mtDNA damage. Unrepaired mtDNA impairs mitochondrial function, which can lead to oxidative stress and cell death. Investigations of mechanisms of mtDNA damage are limited by the lack of available human models.

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Section: Resultsmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

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HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

et al. 2020

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“…Fetal brain tissue (gestational age 16–18 weeks) was obtained from elective abortion procedures performed in full compliance with National Institutes of Health and Temple University ethical guidelines. Human Primary Fetal Neuronal Culture was prepared as described previously (Darbinyan et al, ,). The experiments were performed in triplicate with different fetal tissue from different donors for each experiment in single passage.…”

Section: Methodsmentioning

confidence: 99%

TNF‐α/TNFR2 Regulatory Axis Stimulates EphB2‐Mediated Neuroregeneration Via Activation of NF‐κB

Pozniak

Darbinyan

Khalili

2015

Journal Cellular Physiology

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HIV-1 infected individuals are at high risk of developing HIV-associated neurocognitive disorders (HAND) as HIV infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxic effects of HIV-1 are primarily a result of viral replication leading to the production of inflammatory chemokines and cytokines, including TNF-alpha. Given an important role of TNF-alpha in regulating synaptic plasticity, we investigated the effects of TNF-alpha on the development of neuronal processes after mechanical injury, and we showed that TNF-alpha treatment stimulates the regrowth of neuronal processes. To investigate transcriptional effects of TNF-alpha on synaptic plasticity, we analyzed both human neurosphere and isolated neuronal cultures for the regulation of genes central to synaptic alterations during learning and memory. TNF-alpha treatment upregulated Ephrin receptor B2 (EphB2), which is strongly involved in dendritic arborization and synaptic integrity. TNF-alpha strongly activates the NF-kappaB pathway, therefore, we propose that TNF-alpha-induced neurite regrowth occurs primarily through EphB2 signaling via stimulation of NF-kappaB. EphB2 promoter activity increased with TNF-alpha treatment and overexpression of NF-kappaB. Direct binding of NF-kappaB to the EphB2 promoter occurred in the ChIP assay, and site-directed mutagenesis identified binding sites involved in TNF-alpha-induced EphB2 activation. TNF-alpha induction of EphB2 was determined to occur specifically through TNF-alpha receptor 2 (TNFR2) activation in human primary fetal neurons. Our observations provide a new avenue for the investigation on the impact of TNF-alpha in the context of HIV-1 neuronal cell damage as well as providing a potential therapeutic target in TNFR2 activation of EphB2.

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“…A detailed protocol of using this type of device has been reported in a previous chapter [13]. Alternatively, a fabrication protocol can be followed if custom-designed patterns are preferred [14].…”

Section: Methodsmentioning

confidence: 99%

Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy

Zhang

Chowdary

Cui

2015

Methods in Molecular Biology

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Rab7 GTPase is known to regulate protein degradation and intracellular signaling via endocytic sorting and is also known to be involved in peripheral neurodegeneration. Mutations in the GTP-binding pocket of Rab7 cause Charcot–Marie–Tooth type 2B (CMT-2B) neuropathy. It has been suggested that the CMT-2B-associated Rab7 mutants may disrupt retrograde survival signaling by degrading the signaling endosomes carrying the nerve growth factor (NGF) and its TrkA receptor. Studying the cotrafficking of Rab7 and retrograde-TrkA endosomes in axons is therefore important to understand how Rab7 mutants affect the NGF signaling in neurons. However, tracking the axonal transport of Rab7 and TrkA with conventional microscopy and assigning the transport directionality in mass neuronal cultures pose some practical challenges. In this chapter, we describe the combination of a single-molecule imaging technique, pseudo-total internal reflection fluorescence (pTIRF) microscopy, with microfluidic neuron cultures that enables the simultaneous tracking of fluorescently labeled Rab7- and TrkA-containing endosomes in axons.

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Compartmentalized Neuronal Cultures

Cited by 9 publications

References 11 publications

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons

TNF‐α/TNFR2 Regulatory Axis Stimulates EphB2‐Mediated Neuroregeneration Via Activation of NF‐κB

Visualizing Directional Rab7 and TrkA Cotrafficking in Axons by pTIRF Microscopy

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