Directly targeting ASC by lonidamine alleviates inflammasome-driven diseases.

BACKGROUND: Dysregulated activation of the inflammasome is involved in various human diseases including acute cerebral ischemia, multiple sclerosis and sepsis. Though many inflammasome inhibitors targeting NOD-like receptor protein 3 (NLRP3) have been designed and developed, none of the inhibitors are clinically available. Growing evidence suggests that targeting apoptosis-associated speck-like protein containing a CARD (ASC), the oligomerization of which is the key event for the assembly of inflammasome, may be another promising therapeutic strategy. Lonidamine (LND), a small-molecule inhibitor of glycolysis used as an antineoplastic drug, has been evidenced to have anti-inflammation effects. However, its anti-inflammatory mechanism is still largely unknown. METHODS: Middle cerebral artery occlusion (MCAO), experimental autoimmune encephalomyelitis (EAE) and LPS-induced sepsis mice models were constructed to investigate the therapeutic and anti-inflammasome effects of LND. The inhibition of inflammasome activation and ASC oligomerization by LND was evaluated using western blot (WB), immunofluorescence (IF), quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) in murine bone marrow-derived macrophages (BMDMs). Direct binding of LND with ASC was assessed using molecular mock docking, surface plasmon resonance (SPR), and drug affinity responsive target stability (DARTS). RESULTS: Here, we find that LND strongly attenuates the inflammatory injury in experimental models of inflammasome-associated diseases including autoimmune disease-multiple sclerosis (MS), ischemic stroke and sepsis. Moreover, LND blocks diverse types of inflammasome activation independent of its known targets including hexokinase 2 (HK2). We further reveal that LND directly binds to the inflammasome ligand ASC and inhibits its oligomerization. CONCLUSIONS: Taken together, our results identify LND as a broad-spectrum inflammasome inhibitor by directly targeting ASC, providing a novel candidate drug for the treatment of inflammasome-driven diseases in clinic.

TRIOL Inhibits Rapid Intracellular Acidification and Cerebral Ischemic Injury: The Role of Glutamate in Neuronal Metabolic Reprogramming.

As one of the key injury incidents, tissue acidosis in the brain occurs very quickly within several minutes upon the onset of ischemic stroke. Glutamate, an excitatory amino acid inducing neuronal excitotoxicity, has been reported to trigger the decrease in neuronal intracellular pH (pHi) via modulating proton-related membrane transporters. However, there remains a lack of clarity on the possible role of glutamate in neuronal acidosis via regulating metabolism. Here, we show that 200 µM glutamate treatment quickly promotes glycolysis and inhibits mitochondrial oxidative phosphorylation of primary cultured neurons within 15 min, leading to significant cytosolic lactate accumulation, which contributes to the rapid intracellular acidification and neuronal injury. The reprogramming of neuronal metabolism by glutamate is dependent on adenosine monophosphate-activated protein kinase (AMPK) signaling since the inhibition of AMPK activation by its selective inhibitor compound C significantly reverses these deleterious events in vitro. Moreover, 5α-androst-3ß,5α,6ß-TRIOL (TRIOL), a neuroprotectant we previously reported, can also remarkably reverse intracellular acidification and alleviate neuronal injury through the inhibition of AMPK signaling. Furthermore, TRIOL remarkably reduced the infarct volume and attenuated neurologic impairment in acute ischemic stroke models of middle cerebral artery occlusion in vivo. In summary, we reveal a novel role of glutamate in rapid intracellular acidification injury resulting from glutamate-induced lactate accumulation through AMPK-mediated neuronal reprogramming. Moreover, inhibition of the quick drop in neuronal pHi by TRIOL significantly reduces the cerebral damages, suggesting that it is a promising drug candidate for ischemic stroke.

Author Correction: Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Following the publication of our paper (Zhang et al., 2020), it has come to our attention that we erroneously listed two funding sources unrelated to this study in the "ACKNOWLEDGEMENTS" section. Hereby, we wish to update the "ACKNOWLEDGEMENTS" section as a correction.

SMARCB1 Promotes Ubiquitination and Degradation of NR4A3 via Direct Interaction Driven by ROS in Vascular Endothelial Cell Injury.

Nuclear receptor subfamily 4 group A member 3 (NR4A3) protects the vascular endothelial cell (VEC) against hypoxia stress, whose expression is primarily reported to be governed at a transcriptional level. However, the regulation of NR4A3 in the protein level is largely unknown. Here, we report that NR4A3 protein abundance is decreased immensely in VEC injury induced by reoxygenation after oxygen-glucose deprivation (OGD-R), which is significantly blocked by the administration of the antioxidative steroid TRIOL. Moreover, the notable improvement of NR4A3 and the alleviation of pulmonary endothelial barrier hyperpermeability induced by acute hypobaric hypoxia in cynomolgus monkeys are also observed after TRIOL administration. The overproduction of reactive oxygen species (ROS) decreases NR4A3 protein abundance in VEC under OGD-R condition, which is reversed by TRIOL and N-acetylcysteine (NAC). TRIOL dose-dependently increases the NR4A3 protein level by inhibiting ubiquitination and ubiquitin proteasome system- (UPS-) mediated degradation rather than promoting its transcription. Using yeast two-hybrid screening, we further identify the interaction between NR4A3 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SMARCB1), and the DNA-binding domain of NR4A3 is required for this interaction. Knockdown of SMARCB1 reduces ubiquitination and degradation of NR4A3, suggesting the proubiquitylation effect of this interaction which is enhanced by ROS in VEC injury induced by OGD-R. In summary, our study here for the first time reveals a posttranslational regulation in SMARCB1-mediated NR4A3 protein degradation which is driven by ROS, providing further understanding of the impaired regulation of NR4A3-mediated prosurvival pathways under pathological condition in VEC.

Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys.

Hypobaric hypoxia (HH) exposure can cause serious brain injury as well as life-threatening cerebral edema in severe cases. Previous studies on the mechanisms of HH-induced brain injury have been conducted primarily using non-primate animal models that are genetically distant to humans, thus hindering the development of disease treatment. Here, we report that cynomolgus monkeys ( Macacafascicularis) exposed to acute HH developed human-like HH syndrome involving severe brain injury and abnormal behavior. Transcriptome profiling of white blood cells and brain tissue from monkeys exposed to increasing altitude revealed the central role of the HIF-1 and other novel signaling pathways, such as the vitamin D receptor (VDR) signaling pathway, in co-regulating HH-induced inflammation processes. We also observed profound transcriptomic alterations in brains after exposure to acute HH, including the activation of angiogenesis and impairment of aerobic respiration and protein folding processes, which likely underlie the pathological effects of HH-induced brain injury. Administration of progesterone (PROG) and steroid neuroprotectant 5α-androst-3ß,5,6ß-triol (TRIOL) significantly attenuated brain injuries and rescued the transcriptomic changes induced by acute HH. Functional investigation of the affected genes suggested that these two neuroprotectants protect the brain by targeting different pathways, with PROG enhancing erythropoiesis and TRIOL suppressing glutamate-induced excitotoxicity. Thus, this study advances our understanding of the pathology induced by acute HH and provides potential compounds for the development of neuroprotectant drugs for therapeutic treatment.

Marine-Steroid Derivative 5α-Androst-3ß, 5α, 6ß-triol Protects Retinal Ganglion Cells from Ischemia⁻Reperfusion Injury by Activating Nrf2 Pathway.

High intraocular pressure (IOP)-induced retinal ischemia leads to acute glaucoma, which is one of the leading causes of irreversible visual-field loss, characterized by loss of retinal ganglion cells (RGCs) and axonal injury in optic nerves (ONs). Oxidative stress and the inflammatory response play an important role in the ischemic injury of retinal and optic nerves. We focus on 5α-androst-3ß, 5α, 6ß-triol (TRIOL), a synthetic neuroactive derivative of natural marine steroids 24-methylene-cholest-3ß, 5α, 6ß, 19-tetrol and cholestane-3ß, 5α, 6ß-triol, which are two neuroactive polyhydroxysterols isolated from the soft coral Nephthea brassica and the gorgonian Menella kanisa, respectively. We previously demonstrated that TRIOL was a neuroprotective steroid with anti-inflammatory and antioxidative activities. However, the potential role of TRIOL on acute glaucoma and its underlying mechanisms remains unclear. Here, we report TRIOL as a promising neuroprotectant that can protect RGCs and their axons/dendrites from ischemic-reperfusion (I/R) injury in an acute intraocular hypertension (AIH) model. Intravitreal injection of TRIOL significantly alleviated the loss of RGCs and the damage of axons and dendrites in rats and mice with acute glaucoma. As NF-E2-related factor 2 (Nrf2) is one of the most critical regulators in oxidative and inflammatory injury, we further evaluated the effect of TRIOL on Nrf2 knockout mice, and the neuroprotective role of TRIOL on retinal ischemia was not observed in Nrf2 knockout mice, indicating that activation of Nrf2 is responsible for the neuroprotection of TRIOL. Further experiments demonstrated that TRIOL can activate and upregulate Nrf2, along with its downstream hemeoxygenase-1 (HO-1), by negative regulation of Kelch-like ECH (Enoyl-CoA Hydratase) associated Protein-1 (Keap1). In conclusion, our study shed new light on the neuroprotective therapy of retinal ischemia and proposed a promising marine drug candidate, TRIOL, for the therapeutics of acute glaucoma.

A Novel Synthetic Steroid of 2ß,3α,5α-Trihydroxy-androst-6-one Alleviates the Loss of Rat Retinal Ganglion Cells Caused by Acute Intraocular Hypertension via Inhibiting the Inflammatory Activation of Microglia.

Neuroinflammation has been well recognized as a key pathological event in acute glaucoma. The medical therapy of acute glaucoma mainly focuses on lowering intraocular pressure (IOP), while there are still scarce anti-inflammatory agents in the clinical treatment of acute glaucoma. Here we reported that ß,3α,5α-trihydroxy-androst-6-one (sterone), a novel synthetic polyhydric steroid, blocked neuroinflammation mediated by microglia/macrophages and alleviated the loss of retinal ganglion cells (RGCs) caused by acute intraocular hypertension (AIH). The results showed that sterone significantly inhibited the morphological changes, the up-regulation of inflammatory biomarker ionized calcium-binding adapter molecule 1 (Iba-1), and the mRNA increase of proinflammatory tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) in BV2 microglia and RAW264.7 macrophages. Moreover, immunofluorescence and western blotting analysis revealed that sterone markedly abrogated the nuclear translocation and phosphorylation of nuclear factor-κB (NF-κB) p65 subunit. Furthermore, sterone significantly suppressed the inflammatory microglial activation and RGCs' reduction caused by retinal ischemia/reperfusion (I/R) injury in a rat AIH model. These results suggest sterone may be a potential candidate in the treatment of acute glaucoma caused by microglial activation-mediated neuroinflammatory injury.

Cholestane-3ß, 5α, 6ß-triol suppresses neuronal hyperexcitability via binding to voltage-gated sodium channels.

Neuronal hyperexcitability is identified as a critical pathological basis of epileptic seizures. Cholestane-3ß, 5α, 6ß-triol (Triol) is a major metabolic oxysterol of cholesterol. Although its neuroprotective effect on ischemia-induced neuronal injury and negative modulation of voltage-gated sodium (Nav) channels were well established, the physical binding site of triol to sodium channels and its effects on neuronal hyperexcitability have not yet been explored. In this study, we utilized molecular docking and molecular dynamics simulation to investigate the interaction between triol and Nav Channels. Our results demonstrated that triol binds to the indole ring of Trp122 of the Nav Channel in silico with a high biological affinity. We further found that triol negatively modulates the action potentials bursts of hippocampal neurons by cell-attached patch recording. Moreover, triol significantly inhibits low Mg2+-induced hyperexcitability in vitro. In addition, triol attenuates pentylenetetrazole (PTZ)-induced convulsive-form behavioral deficits in vivo. Together, our results suggest that triol suppresses neuronal hyperexcitability via binding to Nav channel, indicating that triol might be an attractive lead compound for the treatment of neuronal hyperexcitability-related neurological disorders, especially epileptic seizures.

Hexokinase 2-dependent hyperglycolysis driving microglial activation contributes to ischemic brain injury.

Hyperglycolysis, observed within the penumbra zone during brain ischemia, was shown to be detrimental for tissue survival because of lactate accumulation and reactive oxygen species overproduction in clinical and experimental settings. Recently, mounting evidence suggests that glycolytic reprogramming and induced metabolic enzymes can fuel the activation of peripheral immune cells. However, the possible roles and details regarding hyperglycolysis in neuroinflammation during ischemia are relatively poorly understood. Here, we investigated whether overactivated glycolysis could activate microglia and identified the crucial regulators of neuroinflammatory responses in vitro and in vivo. Using BV 2 and primary microglial cultures, we found hyperglycolysis and induction of the key glycolytic enzyme hexokinase 2 (HK2) were essential for microglia-mediated neuroinflammation under hypoxia. Mechanistically, HK2 up-regulation led to accumulated acetyl-coenzyme A, which accounted for the subsequent histone acetylation and transcriptional activation of interleukin (IL)-1ß. The inhibition and selective knockdown of HK2 in vivo significantly protected against ischemic brain injury by suppressing microglial activation and IL-1ß production in male Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (MCAo) surgery. We provide novel insights for HK2 specifically serving as a neuroinflammatory determinant, thus explaining the neurotoxic effect of hyperglycolysis and indicating the possibility of selectively targeting HK2 as a therapeutic strategy in acute ischemic stroke.

Lidocaine suppresses glioma cell proliferation by inhibiting TRPM7 channels.

BACKGROUND: Malignant glioma is the most common brain cancer with devastating prognosis. Recurrence of malignant glioma following surgery is very common with few preventive and therapeutic options. Novel targets and therapeutic agents are constantly sought for better outcome. Our previous study established that inhibition of transient receptor potential melastatin 7 (TRPM7) channels resulted in significant decrease of human glioma cell growth and proliferation. As local anesthetic lidocaine has been shown to inhibit TRPM7 currents, we hypothesize that lidocaine may suppress glioma cell proliferation through TRPM7 channel inhibition. METHODS: TRPM7 currents were recorded in rat C6 glioma cells using the whole cell patch clamp technique. Cell growth and proliferation were assessed under microscopic examination and biochemical assays. RESULTS: Lidocaine inhibits TRPM7-like currents in a dose-dependent and reversible manner. At 1 and 3 mM, it inhibits ~30% and ~50% of TRPM7 currents. At these concentrations, it is effective in inhibiting the proliferation of C6 cells. As expected, the TRPM7 inhibitors gadolinium and 2-Aminoethoxydiphenyl borate have similar effects on TRPM7 currents and proliferation of C6 cells. Similar to its effect on C6 cells, lidocaine inhibits the proliferation of A172 cells, a human glioblastoma cell line. CONCLUSIONS: Lidocaine significantly inhibits the proliferation of glioma cells. The effect of lidocaine is mediated, at least in part, by inhibiting TRPM7 channels.

Neuroprotectant androst-3ß, 5α, 6ß-triol suppresses TNF-α-induced endothelial adhesion molecules expression and neutrophil adhesion to endothelial cells by attenuation of CYLD-NF-κB pathway.

Neuroinflammation is one of key pathologic element in neurological diseases including stroke, traumatic brain injury, Alzheimer' s Disease, Parkinson's Disease, and multiple sclerosis as well. Up-regulation of endothelial adhesion molecules, which facilitate leukocyte adhesion to the endothelium, is the vital process of endothelial cells mediated neuroinflammation. Androst-3ß, 5α, 6ß-triol (Triol) is a synthetic steroid which has been reported to have neuroprotective effects in hypoxia/re-oxygenation-induced neuronal injury model. In the present study, we firstly investigated whether Triol inhibited the TNF-α-induced inflammatory response in rat brain microvascular endothelial cells (RBMECs). Our data showed that Triol decreased TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) and the adhesion of neutrophil to RBMECs. We also found that Triol inhibited TNF-α-induced degradation of IκBα and phosphorylation of NF-κBp65 that are required for NF-κB activation. Furthermore, Triol significantly reversed TNF-α-induced down-expression of CYLD, which is a deubiquitinase that negatively regulates activation of NF-κB. These results suggest that Triol displays an anti-inflammatory effect on TNF-α-induced RBMECs via downregulating of CYLD-NF-κB signaling pathways and might have a potential benefit in therapeutic neuroinflammation related diseases.

Comparison of bile salt/phosphatidylcholine mixed micelles in solubilization to sterols and stability.

Androst-3ß,5α,6ß-triol (Triol) is a promising neuroprotective agent, but its poor solubility restricts its development into parenteral preparations. In this study, Triol is significantly solubilized by bile salt/phosphatidylcholine mixed micelles (BS/PC-MM). All BS/PC-MM systems are tested to remarkably improve the drug solubility with various stabilities after drug loading. Among them, the sodium glycocholate (SGC)/egg phosphatidylcholine (EPC) system with 2:1 ratio in weight and the total concentration of SGC and EPC of 100 mg/mL is proved to produce stable mixed micelles with high drug loading. It is found that the stability of drug-loaded mixed micelles is quite different, which might be related to the change in critical micelle concentration (CMC) after incorporating drugs. SGC/EPC and SGC/soya phosphatidylcholine (SPC) remain transparent under accelerated conditions and manifest a decreased CMC (dropping from 0.105 to 0.056 mg/mL and from 0.067 to 0.024 mg/mL, respectively). In contrast, swine bile acid-sodium salt (SBA-Na)/PC and sodium deoxycholate (SDC)/PC are accompanied by drug precipitation and reached the maximum CMC on the first and the third days, respectively. Interestingly, the variation of CMC under accelerated testing conditions highly matches the drug-precipitating event in the primary stability experiment. In brief, the bile salt/phosphatidylcholine system exists as a potential strategy of improving sterol drug solubility. CMC variation under accelerated testing conditions might be a simple and easy method to predict the stability of drug-loaded mixed micelles.

Comparison in toxicity and solubilizing capacity of hydroxypropyl-ß-cyclodextrin with different degree of substitution.

Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has been widely used as an effective solubilizing agent in pharmaceutical industry for many years. However, the effect of degree of substitution (D.S.) of HP-ß-CD on solubilizing capacity and toxicity has not been concerned. In this study, solubilizing capacity of HP-ß-CDs with three different D.S. (4.55, 6.16 and 7.76) for 16 drugs were measured and their toxicities were compared by a 7-day i.v. administration (q.d.) study in rats. Generally, HP-ß-CD with high D.S. (7.76) showed weaker solubilizing capacity for steroids and BCS class II drugs, but lower hemolytic activity, compared with that of HP-ß-CD with low (4.55) or medium (6.16) D.S. HP-ß-CD with low D.S. resulted in more changes in hematological and biochemical parameters, but the effects were reversible after a 7-day recovery. Moreover, HP-ß-CD with medium D.S. may have slightly greater nephrotoxicity than the other two HP-ß-CDs. HP-ß-CDs with different D.S. had similar urine excretion percentage after i.v. administration and none of them was found to affect glomerular filtration function of rats. The results suggest that HP-ß-CD with low D.S. would be a better choice considering both the solubilizing capacity and toxicity. However, comparison in toxicity of HP-ß-CDs with different D.S. should be carried out in human in view of its species-dependence property.

Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons.

Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia­associated neuronal death in non­human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK­801 and the calcium chelator ethylene glycol­bis (2­aminoethylether)­N, N, N', N'­tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal­associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α­fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N­Acetyl­L­cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity.

A synthetic steroid 5α-androst-3ß,5,6ß-triol blocks hypoxia/reoxygenation-induced neuronal injuries via protection of mitochondrial function.

Ischemic stroke is a leading cause of death worldwide, yet therapies are limited. During periods of ischemia following reperfusion in ischemic stroke, not only loss of energy supply, but a few other factors including mitochondrial dysfunction and oxidative stress also make vital contribution to neuronal injury. Here we synthesized a steroid compound 5α-androst-3ß,5,6ß-triol by 3 steps starting from dehydroepiandrosterone and examined its effect on mitochondrial function and oxidative stress in primary cultured cortical neurons exposed to hypoxia followed by reoxygenation. 5α-Androst-3ß,5,6ß-triol dose-dependently protected cortical neurons from hypoxia/reoxygenation exposure. Rates of reduction in neuronal viability, loss of mitochondrial membrane potential, disruption of ATP production and oxidative stress were ameliorated in 5α-androst-3ß,5,6ß-triol pretreated cultures. In summary, these results suggest that 5α-androst-3ß,5,6ß-triol is neuroprotective against hypoxia/reoxygenation induced neuronal injuries through mediation of mitochondrial function and oxidative stress.

The effects of anesthetics on tumor progression.

More and more cancer patients receive surgery and chronic pain control. Cell-mediated immunosuppression from surgical stress renders perioperative period a vulnerable period for tumor metastasis. Retrospective studies suggest that regional anesthesia reduces the risk of tumor metastasis and recurrence. This benefit may be due to the attenuation of immunosuppression by regional anesthesia. On the other hand, accumulating evidence points to a direct role of anesthetics in tumor progression. A variety of malignancies exhibit increased activity of voltage-gated sodium channels. Blockade of these channels by local anesthetics may help inhibit tumor progression. Opioids promote angiogenesis, cancer cell proliferation and metastasis. It will be interesting to examine the therapeutic potential of peripheral opioid antagonists against malignancy. Volatile anesthetics are organ-protective against hypoxia, however; this very protective mechanism may lead to tumor growth and poor prognosis. In this review, we examine the direct effects of anesthetics in tumor progression in hope that a thorough understanding will help to select the optimal anesthetic regimens for better outcomes in cancer patients.

Diazepam inhibits proliferation of human glioblastoma cells through triggering a G0/G1 cell cycle arrest.

BACKGROUND: Glioblastoma (GBM), the most common primary brain tumor, is the most aggressive malignancy in humans. Its rapid proliferation is a major obstacle to successful treatment. Patients with GBM often suffer from psychological disturbances associated with poor prognosis and physical discomfort. Diazepam is one of the most frequently used benzodiazepines (BZs) in cancer patients for its desirable psychotropic effects. The central effects of BZs are mediated by the activation of central BZ receptors. This study investigates whether diazepam has inhibitory effect on proliferation of GBM cell line T98G and explores its possible mechanism. METHODS: Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay. Cell cycle distribution was examined by flow cytometry. Western blot with specific protein antibodies was used to detect regulatory proteins involved in cell cycle regulation. RESULTS: Diazepam significantly decreased the proliferation of T98G cells in a dose-dependent and time-dependent manner. This effect was not reversed by the central BZ receptor antagonist flumazenil or the peripheral BZ receptor antagonist PK11195, indicating that it was not mediated by BZ receptors. Flow cytometry indicated that diazepam caused a cell accumulation in G0/G1 phase, thereby contributing to cell proliferation inhibition. Furthermore, our findings showed that lessened phosphorylation of Rb accounted for diazepam-induced G0/G1 phase arrest. CONCLUSIONS: Diazepam inhibits the proliferation of human GBM T98G cells by inducing G0/G1 phase arrest. Diazepam has potential to be a lead for new drugs in GBM therapy because of its antitumor activity.

Emergence of a seizure phenotype in aged apolipoprotein epsilon 4 targeted replacement mice.

The apolipoprotein ε4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD) and is associated with earlier age of onset. The incidence of spontaneous seizures has been reported to be increased in sporadic AD as well as in the early onset autosomal dominant forms of AD. We now report the emergence of a seizure phenotype in aged apolipoprotein E4 (apoE4) targeted replacement (TR) mice but not in age-matched apoE2 TR or apoE3 TR mice. Tonic-clonic seizures developed spontaneously after 5 months of age in apoE4 TR mice and are triggered by mild stress. Female mice had increased seizure penetrance compared to male mice, but had slightly reduced overall seizure severity. The majority of seizures were characterized by head and neck jerks, but 25% of aged apoE4 TR mice had more severe tonic-clonic seizures which occasionally progressed to tonic extension and death. Aged apoE4 TR mice progressed through pentylenetetrazol-induced seizure stages more rapidly than did apoE3 TR and apoE2 TR mice. Electroencephalographic (EEG) recordings revealed more frequent bursts of synchronous theta activity in the hippocampus of apoE4 TR mice than in apoE2 TR or apoE3 TR mice. Cortical EEG recordings also revealed sharp spikes and other abnormalities in apoE4 TR mice. Taken together, these findings demonstrate the emergence of an age-dependent seizure phenotype in old apoE4 TR mice in the absence of human amyloid-ß peptide (Aß) overexpression, suggesting increased central nervous system neural network excitability.

Human APOE isoform-dependent effects on brain beta-amyloid levels in PDAPP transgenic mice.

To investigate the role of human apolipoprotein E (apoE) on Abeta deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and Abeta peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain Abeta and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain Abeta deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of Abeta42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain Abeta burden, and the majority of apoE was associated with Abeta. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 < E3 << E2), resulting in early and dramatic apoE isoform-dependent effects on brain Abeta levels (E4 >> E3 > E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimer's disease.

Macrophage-mediated degradation of beta-amyloid via an apolipoprotein E isoform-dependent mechanism.

Recent studies suggest that bone marrow-derived macrophages can effectively reduce beta-amyloid (Abeta) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Abeta, we cultured murine macrophages on top of Abeta plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP(717V>F) mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Abeta in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Abeta degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Abeta than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Abeta was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Abeta-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Abeta degradation by macrophages. These apoE isoform-dependent effects of macrophages on Abeta degradation suggest a novel "peripheral" mechanism for Abeta clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimer's disease.