Flipping the switches: CD40 and CD45 modulation of microglial activation states in HIV associated dementia (HAD)

Microglial dysfunction is associated with the pathogenesis and progression of a number of neurodegenerative disorders including HIV associated dementia (HAD). HIV promotion of an M1 antigen presenting cell (APC) - like microglial phenotype, through the promotion of CD40 activity, may impair endogenous mechanisms important for amyloid- beta (Aβ) protein clearance. Further, a chronic pro-inflammatory cycle is established in this manner. CD45 is a protein tyrosine phosphatase receptor which negatively regulates CD40L-CD40-induced microglial M1 activation; an effect leading to the promotion of an M2 phenotype better suited to phagocytose and clear Aβ. Moreover, this CD45 mediated activation state appears to dampen harmful cytokine production. As such, this property of microglial CD45 as a regulatory "off switch" for a CD40-promoted M1, APC-type microglia activation phenotype may represent a critical therapeutic target for the prevention and treatment of neurodegeneration, as well as microglial dysfunction, found in patients with HAD

Cardiac Amyloidosis Presenting as Biventricular Systolic Heart Failure

A previously healthy octogenarian presented with new onset heart failure symptoms. Comprehensive multimodality imaging including complete echocardiography with longitudinal strain analysis, cardiac magnetic resonance imaging (cMRI), nuclear medicine pyrophosphate (99-mcTcPYP) scan along with biomarker, monoclonal protein analysis, and fat pad biopsy confirmed diagnosis of transthyretin cardiac amyloidosis

Efavirenz promotes β-secretase expression and increased Aβ1-40,42 via oxidative stress and reduced microglial phagocytosis: implications for HIV associated neurocognitive disorders (HAND).

Efavirenz (EFV) is among the most commonly used antiretroviral drugs globally, causes neurological symptoms that interfere with adherence and reduce tolerability, and may have central nervous system (CNS) effects that contribute in part to HIV associated neurocognitive disorders (HAND) in patients on combination antiretroviral therapy (cART). Thus we evaluated a commonly used EFV containing regimen: EFV/zidovudine (AZT)/lamivudine (3TC) in murine N2a cells transfected with the human "Swedish" mutant form of amyloid precursor protein (SweAPP N2a cells) to assess for promotion of amyloid-beta (Aβ) production. Treatment with EFV or the EFV containing regimen generated significantly increased soluble amyloid beta (Aβ), and promoted increased β-secretase-1 (BACE-1) expression while 3TC, AZT, or, vehicle control did not significantly alter these endpoints. Further, EFV or the EFV containing regimen promoted significantly more mitochondrial stress in SweAPP N2a cells as compared to 3TC, AZT, or vehicle control. We next tested the EFV containing regimen in Aβ - producing Tg2576 mice combined or singly using clinically relevant doses. EFV or the EFV containing regimen promoted significantly more BACE-1 expression and soluble Aβ generation while 3TC, AZT, or vehicle control did not. Finally, microglial Aβ phagocytosis was significantly reduced by EFV or the EFV containing regimen but not by AZT, 3TC, or vehicle control alone. These data suggest the majority of Aβ promoting effects of this cART regimen are dependent upon EFV as it promotes both increased production, and decreased clearance of Aβ peptide

EFV/3TC/AZT inhibits microglial phagocytosis of Aβ_1-42 peptide.

<p>(A) Primary microglia (1×10⁵ cells/well in 24-well tissue culture plates) were treated with aged FITC tagged Aβ_1-42 (50 nM) in complete medium for 60 min with antiretroviral medications (10uM) combined or singly as indicated, or PBS (control). As a control for nonspecifically incorporated Aβ, microglial cells were incubated at 4°C with the same treatment followed by DAPI staining. EFV or 3TC/AZT/EFV inhibited microglia-colocalization by fluorescence microscopy. Green indicates Aβ_1-42 positive; blue indicates microglia nuclei. Addition of heat inactivated HIV-1 Tat yielded similar results as vehicle control (data not shown) (B) Cell supernatants and lysates were analyzed for extracellular (top) and cell associated (bottom) FITC-Aβ using a fluorimeter. Data are represented as the relative fold of mean fluorescence change (mean ± SD), calculated as the mean fluorescence for each sample at 37°C divided by mean fluorescence at 4°C (<i>n</i> = 6 for each condition presented). One-way ANOVA followed by <i>post-hoc</i> comparison showed a significant difference between EFV (***<i>P<</i>0.001) or EFV/3TC/AZT <i>(**P<</i>0.05) but not 3TC or AZT compared to control.</p

cART treatment of SweAPP N2a cells promotes mitochondrial dysfunction.

<p>(<i>A) ATP levels are reduced in EFV or EFV/3TC/AZT treated SweAPP N2a neuron cells:</i> SweAPP N2a cells were grown with 10 µM of each medication or all three medications combined for 48 h. We found a significant decrease in ATP levels in cells treated with EFV or 3TC/AZT/EFV (***<i>P</i><0.001). <i>(B)MMP is reduced in EFV or EFV/3TC/</i>AZT <i>SweAPP N2a cells:</i> In accord with reduced ATP levels we found a similar reduction in MMP in the EFV or EFV/3TC/AZT treated groups <i>(***</i>P<<i>0.001</i>) <i>(C</i>–<i>F) ROS levels are increased in EFV or EFV/3TC/AZT treated SweAPP N2a cells:</i> EFV-treated primary neuron cells have significantly higher ROS contents (^**<i>P</i><0.001) after incubation for 60 min than untreated primary neuron. (C–E)The average relative fluorescence units of DCFDA in neurons from each treatment group as indicated by the mean ± standard deviations (D, F) The ROS content in the antiretroviral treatment is expressed as % RFU ± standard deviations for each group compared to untreated control primary neuron cells (100%). (<i>*P</i><<i>0.05, *** P</i><<i>0.001</i>).</p

EFV/3TC/AZT increases soluble Aβ levels in Tg2576 mice <i>via</i> BACE-1 activation <i>in vivo.</i>

<p>(<b>A</b>) Aβ_{40, 42} peptides were analyzed in brain homogenates from 8 month old Tg2576 mice by ELISA (n = 5 mice for each group). One-way ANOVA followed by <i>post hoc</i> comparison revealed significant differences between control (Tg2576mice treated with PBS) and EFV or EFV/3TC/AZT -treated Tg2576 mice (<i>P<0.001</i> and <i>0.05</i> respectively with n = 5 mice/group). (<b>B</b>) Western Blot of brain homogenates using anti-Aβ_1-17 antibody (6E10) shows total APP and a bands corresponding to soluble Aβ oligomer species. β-actin was an internal control. A <i>t-test</i> revealed significant differences in soluble Aβ species between EFV-treated compared to 3TC/AZT/EFV, 3TC or AZT treated Tg2576 mice (<i>P</i><0.01) (<b>C</b>) BACE-1 expression in brain homogenate of Tg2576 mice significantly was increased in EFV or EFV/3TC/AZT -treated Tg2576 mice (<i>P</i><0.001).</p

EFV or EFV/3TC/AZT treatment promotes Aβ generation in cultured neuronal cells <i>via</i> BACE-1 activation <i>in vitro.</i>

<p>Aβ species were analyzed in cell lysates from SweAPP N2a cells (<b>A</b>) by ELISA. Data are represented as the mean ± of a percentage of Aβ peptides secreted 24 h after 3TC, AZT, EFV, or 3TC/EFV/AZT administration, relative fold over control (PBS treated). Significant increases in Aβ were observed in EFV or EFV/3TC/AZT treated cells were observed compared to control (***<i>P</i><0.001 and **<i>P<0.05</i> respectively by ANOVA). (<b>B</b>) Western blot (6E10 antibody) of conditioned media shows increased oligomeric Aβ species vs. s-APP-α (control) in the EFV or EFV/3TC/AZT treated cells (***<i>P</i><0.001 and **<i>P<0.05</i> respectively). (<b>D</b>) BACE-1 expression in cultured media revealed significant differences between EFV or EFV/3TC/AZT treated cells compared to untreated control (***<i>P</i><0.001). β-actin is used for the internal loading control. Results are representative of three independent experiments.</p

Proposed mechanism of EFV neurotoxicity.

<p>Our present work suggests that EFV promotes an increase in Aβ <i>in vitro</i> and <i>in vivo</i> on both the production and clearance fronts <i>via</i> its inhibition of neurnoal MMP resulting in reduced ATP stores and thus a high ROS environment in the CNS. Previous studies indicate such high ROS microenvironments in the CNS promote BACE-1 APP processing and also inhibit microglial Aβ clearance functions. These events in turn all promote production of Aβ species. (*Note: Red arrows = inhibition, Green arrows = promotion).</p