Imiquimod induces a Toll-like receptor 7-independent increase in intracellular calcium via IP3 receptor activation

Imiquimod is an itch-promoting, small, synthetic compound that is generally used to treat genital warts and basal cell carcinoma. The pruritogenic effect of imiquimod is considered to be due to TLR7 activation; however that idea has been challenged by our studies showing intact pruritogenic effects of imiquimod in TLR7 KO mice. Thus, the signaling pathways of imiquimod have not been completely elucidated. Here we investigated the novel effects of imiquimod on intracellular calcium ([Ca2+]i) signaling. We found that imiquimod induces [Ca2+]i increases in PC12 and F11 cells, and even in NIH-3T3 and HEK293T cells, which do not express TLR7. This [Ca 2+]i increase was due to Ca2+ release from the internal store without extracellular Ca2+ influx. Neither FCCP, a mitochondrial Ca2+ reuptake inhibitor, nor dantrolene, a ryanodine receptor inhibitor, affected the imiquimod-induced [Ca2+]i increase. However, 2APB, an IP3 receptor blocker, inhibited the imiquimod-induced [Ca2+]i increase. U73122, a PLCβ inhibitor, failed to block the imiquimod-induced [Ca2+]i increase. These data indicate that imiquimod triggers IP3 receptor-dependent Ca2+ signaling independently of TLR7. © 2014 Elsevier Inc. All rights reserved.

Highly selective microglial uptake of ceria-zirconia nanoparticles for enhanced analgesic treatment of neuropathic pain

Neuropathic pain is a chronic and pathological pain caused by injury or dysfunction in the nervous system. Pro-inflammatory microglial activation with aberrant reactive oxygen species (ROS) generation in the spinal cord plays a critical role in the development of neuropathic pain. However, the efficacy of current therapeutic methods for neuropathic pain is limited because only neurons or neural circuits involved in pain transmission are targeted. Here, an effective strategy to treat pain hypersensitivity using microglia-targeting ceria-zirconia nanoparticles (CZ NPs) is reported. The CZ NPs are coated with microglia-specific antibodies to promote their delivery to microglia, and thus to improve their therapeutic efficacy. The targeted delivery facilitates the elimination of both pro-inflammatory cytokines and ROS in microglia, enabling the rapid and effective inhibition of microglial activation. As a result, greatly ameliorated mechanical allodynia is achieved in a spinal nerve transection (SNT)-induced neuropathic pain mouse model, proving the potent analgesic effect of the microglia-targeting CZ NPs. Given the generality of the approach used in this study, the microglia-targeting CZ NPs are expected to be useful for the treatment of not only neuropathic pain but also other neurological diseases associated with the vicious activation of microglia.

Lysosomal Enzyme Glucocerebrosidase Protects against Aβ_1-42 Oligomer-Induced Neurotoxicity

<div><p>Glucocerebrosidase (GCase) functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher’s disease (GD), Parkinson’s disease (PD), and Dementia with Lewy Bodies (DLB). However, there is little information about the role of GCase in the pathogenesis of Alzheimer’s disease (AD). Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ_1–42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ_1–42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ_1–42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD.</p></div

GCase promotes Aβ_1–42 oligomer degradation.

<p>(A) Primary cultured mouse cortical neurons were infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i>, and 5 μM oligomeric Aβ_1–42 or PBS was added for 48 h at 10 days <i>in vitro</i>. 10 μg/ml of E-64d and 10 μg/ml of PepA were treated 4 h before the treatment of Aβ_1–42 oligomers. The monomeric and oligomeric Aβ expressions were measured with anti-Aβ antibody (4G8) 24 h after the treatment of oligomeric Aβ_1–42. (B) The quantification of Aβ levels is shown (Student’s t test, <i>P</i>* < 0.05, <i>P</i>*** < 0.001).</p

GCase protein levels and activity are reduced in sporadic AD brain tissues and Aβ_1–42 oligomer-treated primary neurons.

<p>(A and B) GCase expression was decreased in the hippocampal region of brains affected by Alzheimer’s disease (n = 6), as opposed to controls (n = 6). GCase expression levels were normalized to β-actin and quantified. (C) The GCase enzymatic activities were measured using lysosome-enriched fractions for the brains affected by Alzheimer’s disease and the controls (n = 6 per group). (D) Expression of <i>GBA1</i> gene was determined in AD hippocampal regions compared to the controls using real-time PCR. The β-actin mRNA was used as an internal reference control to normalize relative mRNA levels (n = 6 per group). (E) Oligomeric and monomeric Aβ_1–42 were identified by SDS-PAGE using anti-Aβ antibody (4G8). (F) Primary cortical neurons were incubated with 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. The cell lysates were subjected to immunoblot using GCase and β-actin antibodies. (G) The expression level of GCase was quantified. Data are expressed as the mean ± SEM (Student’s t test, *<i>P</i> < 0.05, **<i>P</i> < 0.01).</p

Overexpression of GCase increases intracellular pH levels reduced by Aβ_1–42 oligomers in primary neurons.

<p>(A) Primary mouse cortical neurons were incubated with pHrodo™ Red or pHrodo™ Green at 37°C for 30 minutes. The fluorescence intensities in the intracellular area (dotted outline) were measured using various pH ranges (4.5, 5.5, 6.5, and 7.5) of intracellular pH calibration buffers. The fluorescence units of pHrodo™ Red and pHrodo™ Green signals are represented as a standard curve. (B) Primary mouse cortical neurons infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i> were incubated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. The neurons were loaded with pHrodo™ Red and pHrodo™ Green and live cell imaging solution, and further incubated at 37°C for 30 minutes. (C and D) The fluorescence units were quantified and represented as a graph. (E) The fluorescence units of pHrodo™ Green were converted to pH and represented as a graph (two-way ANOVA, Bonferroni posttest, **<i>P</i> < 0.01, ***<i>P</i> < 0.001).</p

Overexpression of GCase restores impairment of lysosomal biogenesis induced by Aβ_1–42 oligomers in neurons.

<p>(A) Primary mouse cortical neurons were infected with lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus at 7 days <i>in vitro</i>. 72 h after the infection, the cells were further incubated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h. The plasma membranes were labeled with CellLight plasma membrane-CFP (PM, blue) and the lysosomes were labeled with LysoTracker Red DND-99 (Lysosome, red). (B) The relative area of lysosomes in plasma membrane (dotted outline) was measured (two-way ANOVA, Bonferroni posttest, <i>P</i>* < 0.05, <i>P</i>*** < 0.001). (C) The diameters of LysoTracker positive-puncta were quantified and represented as a scatter plot (two-way ANOVA, Bonferroni posttest, *<i>P</i> < 0.05, **<i>P</i> < 0.01) versus control (LV-Cont, PBS).</p

Ectopic expression of GCase restores lysosomal cathepsin D activity reduced by Aβ_1–42 oligomer treatment in primary neurons.

<p>(A) The localization of cathepsin D is represented using lysosomal marker (LAMP1) and DAPI. (B) The cathepsin D activity levels were measured in lysosome-enriched and cytoplasmic fractions of lenti-control (LV-Cont, n = 3) or lenti-GCase (LV-GC, n = 3) virus infected primary mouse cortical neurons treated with PBS or 1 μM oligomeric Aβ_1–42 for 24 h at 10 days <i>in vitro</i>. (C) Immunoblot analysis for heavy chain subunit of mature cathepsin D (CatD heavy chain) in cytoplasmic and lysosome-enriched fractions was performed using anti-cathepsin D antibody. The quality of cytosolic and lysosome-enriched fractions was confirmed with immunoblotting using anti-MEK1/2 and LAMP1 antibodies, respectively. The values are the mean ± SEM of three independent experiments (two-way ANOVA, Bonferroni posttest, *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001).</p

Graphene quantum dots prevent α-synucleinopathy in Parkinsons disease

Though emerging evidence indicates that the pathogenesis of Parkinson's disease is strongly correlated to the accumulation(1,2) and transmission(3,4) of alpha-synuclein (alpha-syn) aggregates in the midbrain, no anti-aggregation agents have been successful at treating the disease in the clinic. Here, we show that graphene quantum dots (GQDs) inhibit fibrillization of alpha-syn and interact directly with mature fibrils, triggering their disaggregation. Moreover, GQDs can rescue neuronal death and synaptic loss, reduce Lewy body and Lewy neurite formation, ameliorate mitochondrial dysfunctions, and prevent neuron-to-neuron transmission of alpha-syn pathology provoked by alpha-syn preformed fibrils(5,6). We observe, in vivo, that GQDs penetrate the blood-brain barrier and protect against dopamine neuron loss induced by alpha-syn preformed fibrils, Lewy body/Lewy neurite pathology and behavioural deficits