Enhanced Chemotherapy for Glioblastoma Multiforme Mediated by Functionalized Graphene Quantum Dots

Glioblastoma is the most aggressive and lethal brain cancer. Current treatments involve surgical resection, radiotherapy and chemotherapy. However, the life expectancy of patients with this disease remains short and chemotherapy leads to severe adverse effects. Furthermore, the presence of the blood–brain barrier (BBB) makes it difficult for drugs to effectively reach the brain. A promising strategy lies in the use of graphene quantum dots (GQDs), which are light-responsive graphene nanoparticles that have shown the capability of crossing the BBB. Here we investigate the effect of GQDs on U87 human glioblastoma cells and primary cortical neurons. Non-functionalized GQDs (NF-GQDs) demonstrated high biocompatibility, while dimethylformamide-functionalized GQDs (DMF-GQDs) showed a toxic effect on both cell lines. The combination of GQDs and the chemotherapeutic agent doxorubicin (Dox) was tested. GQDs exerted a synergistic increase in the efficacy of chemotherapy treatment, specifically on U87 cells. The mechanism underlying this synergy was investigated, and it was found that GQDs can alter membrane permeability in a manner dependent on the surface chemistry, facilitating the uptake of Dox inside U87 cells, but not on cortical neurons. Therefore, experimental evidence indicates that GQDs could be used in a combined therapy against brain cancer, strongly increasing the efficacy of chemotherapy and, at the same time, reducing its dose requirement along with its side effects, thereby improving the life quality of patients

Graphene Quantum Dots’ Surface Chemistry Modulates the Sensitivity of Glioblastoma Cells to Chemotherapeutics

Recent evidence has shown that graphene quantum dots (GQDs) are capable of crossing the blood–brain barrier, the barrier that reduces cancer therapy efficacy. Here, we tested three alternative GQDs’ surface chemistries on two neural lineages (glioblastoma cells and mouse cortical neurons). We showed that surface chemistry modulates GQDs’ biocompatibility. When used in combination with the chemotherapeutic drug doxorubicin, GDQs exerted a synergistic effect on tumor cells, but not on neurons. This appears to be mediated by the modification of membrane permeability induced by the surface of GQDs. Our findings highlight that GQDs can be adopted as a suitable delivery and therapeutic strategy for the treatment of glioblastoma, by both directly destabilizing the cell membrane and indirectly increasing the efficacy of chemotherapeutic drugs

CNG channel-like conductance in differentiating hippocampal NSCs.

<p>Representative whole-cell patch-clamp recordings from NSCs grown for three days in differentiation medium and exhibiting morphological features of neurons. Application of 8-Br-cGMP (1 mM) elicited inward currents at holding potential of -80 mV (<b>A</b>) which were inhibited by the CNG channel blockers, 50 µM LCD (<b>B</b>) and 3 mM Cd²⁺ (<b>C</b>). (<b>D</b>) Voltage ramp protocol used to evoke leakage currents. (<b>E</b>) Typical current traces under control conditions and in the presence of 1 mM 8-Br-cGMP. 8-Br-cGMP-activated current (net current) was obtained by subtraction. (<b>F</b>) I–V plot of mean 8-Br-cGMP net currents (<i>n</i>=10).</p

Brain insulin resistance impairs hippocampal synaptic plasticity and memory via FoxO3a/zDHHC3-dependent enhancement of GluA1 palmitoylation

High-fat diet (HFD) and metabolic diseases cause detrimental effects on hippocampal synaptic plasticity, learning, and memory through molecular mechanisms still poorly understood. Here, we demonstrate that HFD increases palmitic acid deposition in the hippocampus and induces hippocampal insulin resistance leading to FoxO3a-mediated overexpression of the palmitoyltransferase zDHHC3. The excess of palmitic acid along with higher zDHHC3 levels causes hyper-palmitoylation of AMPA glutamate receptor subunit GluA1, hindering its activity-dependent trafficking to the plasma membrane. Accordingly, AMPAR current amplitudes and, more importantly, their potentiation underlying synaptic plasticity were inhibited, as well as hippocampal-dependent memory. Hippocampus-specific silencing of Zdhhc3 and, interestingly enough, intranasal injection of the palmitoyltransferase inhibitor, 2-bromopalmitate, counteract GluA1 hyper-palmitoylation and restore synaptic plasticity and memory in HFD mice. Our data reveal a key role of FoxO3a/Zdhhc3/GluA1 axis in the HFD-dependent impairment of cognitive function and identify a novel mechanism underlying the cross talk between metabolic and cognitive disorder

Blockade of CNG channels reduces neuronal differentiation of hippocampal NSCs but does not affect their proliferation.

<p>(<b>A</b>,<b>B</b>) Representative images of BrdU-positive cells (red) in NSC cultures grown in (<b>A</b>) normal proliferation medium and (<b>B</b>) in the presence of the CNG channel blocker, LCD (50 µM). (<b>C</b>) Bar graph showing the percentages of cells incorporating the proliferation marker BrdU in control and LCD-exposed NSC cultures. (<b>D</b>–<b>G</b>) Representative images of MAP2⁺ NSCs at D6 in control differentiative medium (<b>D</b>) and after 3 day-exposure (D1-D3) to 50 µM LCD (<b>E</b>), 1 µM KT5823 (<b>F</b>) and LCD plus KT5823 (<b>G</b>). Cell nuclei (DAPI⁺) are labeled in blue. Scale bars: 75 µm. (<b>H</b>) Bar graph showing the percentages of NSCs differentiating towards the neuronal phenotype (MAP2⁺) at D3, D6 and D10 in control conditions and in the culturing conditions described in the graph legend. Error bars show SEM values. Statistical significance was assessed by ANOVA (F_3,52=3.80, <i>P</i><0.05, at D3; F_3,74=7.45, <i>P</i><0.0005, at D6; F_3,84=11.6, <i>P</i><0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: *<i>P</i>< 0.05 and **<i>P</i><0.001 vs. control; n.s., not significant <i>P</i>-value.</p

Cultured hippocampal NSCs express CNG channels.

<p>(<b>A</b>–<b>D</b>) Time course of hippocampal NSC differentiation assessed by immunofluorescence. The vast majority of neurosphere-derived cells grown in the proliferation medium (D0) were positive for nestin (<b>A</b>); during differentiation this immunoreactivity diminished (<b>B</b>) and ultimately disappeared on D10 (<b>C</b>). Cell nuclei (DAPI⁺) are labeled in blue. The percentages of cells identified as neurons by MAP2 immunoreactivity significantly increased during the 10 days of differentiation (<b>D</b>). Undifferentiated NSCs, characterized by positivity for nestin and lack of MAP2 immunoreactivity, showed both CNGA1 (<b>E</b>,<b>F</b>) and CNGA2 staining (<b>I</b>,<b>J</b>). NSCs differentiating toward neuronal phenotype (MAP2⁺) exhibited CNGA1 (<b>G</b>,<b>H</b>) and CNGA2 (<b>K</b>,<b>L</b>) labeling. Scale bars: 50 µm.</p

The cGMP analogue, 8-Br-cGMP, promotes NSC neuronal differentiation through CNG channel activation.

<p>(<b>A</b>) Summary bar graph showing an increased number of MAP2⁺ cells compared to controls following treatment with 8-Br-cGMP (8-Br). This effect was abolished by CNG channel and PKG blockers at D3, D6 and D10. Statistical significance was assessed by ANOVA (F_3,76=8.25, <i>P</i><0.0001, at D3; F_3,85=38.5, <i>P</i><0.0001, at D6; F_3,87=32.8, <i>P</i><0.0001, at D10). The Dunnett’s post-hoc test was used for multiple comparisons: #<i>P</i><0.05 and # #<i>P</i><0.001 vs. control; *<i>P</i><0.05 and **<i>P</i><0.001 vs. 8-Br-cGMP-treated NSCs. (<b>B</b>–<b>D</b>) Representative images of MAP2⁺ NSCs at D6 in control differentiation medium (<b>B</b>) and after exposure to 1 mM 8-Br-cGMP (<b>C</b>) or 8-Br-cGMP plus 50 µM LCD (<b>D</b>). Scale bar: 75 µm.</p

Western immunoblots showing <b>CNG A</b> protein expression in hippocampal NSCs.

<div><p>(<b>A</b>) Western immunoblots of whole-cell NSC lysates confirmed both CNGA1 and CNGA2 expression in undifferentiated (D0) and differentiating NSCs at D1 and D3. Loading control: actin.</p> <p>(<b>B</b>,<b>C</b>) Control experiments showing the specificity of CNG A immunoblotting. Bands corresponding to CNGA1 and CNGA2 proteins were detected in control samples (hippocampal tissue and NSCs at D3 (B) whereas no signals were detected when the antibodies were pre-absorbed with 5-fold excess of the appropriate control peptide (C).</p></div

Activation of mGluR5 induces spike afterdepolarization and enhanced excitability in medium spiny neurons of the nucleus accumbens by modulating persistent Na+ currents

The involvement of metabotropic glutamate receptors type 5 (mGluR5) in drug-induced behaviours is well-established but limited information is available on their functional roles in addiction-relevant brain areas like the nucleus accumbens (NAc). This study demonstrates that pharmacological and synaptic activation of mGluR5 increases the spike discharge of medium spiny neurons (MSNs) in the NAc. This effect was associated with the appearance of a slow afterdepolarization (ADP) which, in voltage-clamp experiments, was recorded as a slowly inactivating inward current. Pharmacological studies showed that ADP was elicited by mGluR5 stimulation via G-protein-dependent activation of phospholipase C and elevation of intracellular Ca2+ levels. Both ADP and spike aftercurrents were significantly inhibited by the Na+ channel-blocker, tetrodotoxin (TTX). Moreover, the selective blockade of persistent Na+ currents (INaP), achieved by NAc slice pre-incubation with 20 nm TTX or 10 μm riluzole, significantly reduced the ADP amplitude, indicating that this type of Na+ current is responsible for the mGluR5-dependent ADP. mGluR5 activation also produced significant increases in INaP, and the pharmacological blockade of this current prevented the mGluR5-induced enhancement of spike discharge. Collectively, these data suggest that mGluR5 activation upregulates INaP in MSNs of the NAc, thereby inducing an ADP that results in enhanced MSN excitability. Activation of mGluR5 will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of drug-induced behaviours

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway

Summary: Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3β (GSK3β) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions—models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3β and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3β and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3β prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3β with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3β. A GSK3β-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3β regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity. : Scala et al. show how vulnerability to reward-related behaviors associates with maladaptive plasticity of medium spiny neurons through phosphorylation of the voltage-gated Na+ channel Nav1.6 by the enzyme GSK3β. Keywords: GSK3β, Nav1.6, enriched environment, isolated condition, persistent sodium current, neuronal firing, medium spiny neurons, reward pathway, plasticit