Analysis of PD‐L1 expression and T cell infiltration in different molecular subgroups of diffuse midline gliomas

Diffuse midline gliomas (DMGs) are rare and devastating tumors with limited therapeutic options. Programmed death‐ligand 1 (PD‐L1) expression represents a potential predictive biomarker for immunotherapy. One hundred and twenty‐six DMGs (89 adult and 37 pediatric) were assessed for immune profile (PD‐L1, cluster of differentiation (CD3, CD8) and genetic markers (mutation in 27th amino acid of histone H3 (H3K27M), alpha thalassemia/mental retardation syndrome X‐linked (ATRX), isocitrate dehydrogenase 1 (IDH1), p53) by immunohistochemistry. Sanger sequencing was done for IDH1 and H3K27M. The thalamus was the commonest site. Four molecular subgroups of DMGs were identified. H3K27M mutation was more frequent in children (P = 0.0001). The difference in median overall survival (OS) was not significant in any of the four molecular subgroups (P > 0.05). PD‐L1 expression was significantly higher in H3K27M/IDH1 double‐negative adult glioblastomas (GBMs) (P = 0.002). Strong PD‐L1 expression was more frequent in grade IV tumors and thalamic location, although the difference was not significant (P = 0.14 and P = 0.19 respectively). Positive PD‐L1 expression was significantly associated with high tumor‐infiltrating lymphocytes count (P < 0.05). There was no significant difference in median OS in PD‐L1‐positive versus negative cases among four genetic subgroups (P > 0.05). On univariate analysis, there was no direct correlation of PD‐L1 with any genetic alteration, except H3K27M mutation (P = 0.01). CD3 infiltration was similar in both adults and pediatric ages (84.3% and 78.4%, respectively) while CD8 expression was significantly greater in adults compared to children (74.1% vs 37.8%, P = 0.0001). This is the first comprehensive analysis highlighting molecular and immune profiles of DMGs. Despite molecular and clinicopathological diversity, overall survival in DMGs remains dismal. Multicentric studies with larger numbers of cases should be undertaken for stratifying DMGs according to their age, immune and molecular profiles, to develop effective immunotherapies.     

Cell type-specific effects of p27<Superscript>KIP1</Superscript> loss on retinal development

Background

Cyclin-dependent kinase (CDK) inhibitors play an important role in regulating cell cycle progression, cell cycle exit and cell differentiation. p27^KIP1 (p27), one of the major CDK inhibitors in the retina, has been shown to control the timing of cell cycle exit of retinal progenitors. However, the precise role of this protein in retinal development remains largely unexplored. We thus analyzed p27-deficient mice to characterize the effects of p27 loss on proliferation, differentiation, and survival of retinal cells.

Methods

Expression of p27 in the developing and mature mouse retina was analyzed by immunohistochemistry using antibodies against p27 and cell type-specific markers. Cell proliferation and differentiation were examined in the wild-type and p27-deficient retinas by immunohistochemistry using various cell cycle and differentiation markers.

Results

All postmitotic retinal cell types expressed p27 in the mouse retinas. p27 loss caused extension of the period of proliferation in the developing retinas. This extra proliferation was mainly due to ectopic cell cycle reentry of differentiating cells including bipolar cells, Müller glial cells and cones, rather than persistent division of progenitors as previously suggested. Aberrant cell cycle activity of cones was followed by cone death resulting in a significant reduction in cone number in the mature p27-deficient retinas.

Conclusions

Although expressed in all retinal cell types, p27 is required to maintain the quiescence of specific cell types including bipolar cells, Müller glia, and cones while it is dispensable for preventing cell cycle reentry in other cell types.

     

Pediatric ganglioglioma with an H3 K27M mutation arising from the cervical spinal cord

The 2016 edition of the World Health Organization Classification of Tumors of the Central Nervous System introduced “diffuse midline glioma H3 K27M mutant” as a new diagnostic entity. These tumors predominately affect pediatric patients and arise from midline structures such as the brainstem, thalamus and spinal cord. Here, we report a rare patient with spinal ganglioglioma carrying an H3 K27M mutation. A 10‐year‐old boy presented with an intramedullary tumor in the cervical spinal cord. The lesion was partially removed and histologically diagnosed as ganglioglioma. After the remnant tumor grew within 3 months after surgery, the patient underwent radiotherapy. Genetic analyses revealed an H3F3A K27M mutation but no other genetic alterations such as IDH and BRAF mutations. This case may point to pathological heterogeneity in gliomas with H3 K27M mutations.     

DNA damage-associated oligodendrocyte degeneration precedes amyloid pathology and contributes to Alzheimer's disease and dementia

Introduction

In looking for novel non-amyloid-based etiologies for Alzheimer's disease, we explore the hypothesis that age-related myelin loss is an attractive explanation for age-associated cognitive decline and dementia.

Rhombomere-specific analysis reveals the repertoire of genetic cues expressed across the developing hindbrain

Background

Maintaining the correct balance of proliferation versus differentiation in retinal progenitor cells (RPCs) is essential for proper development of the retina. The cell cycle regulator cyclin D1 is expressed in RPCs, and mice with a targeted null allele at the cyclin D1 locus (Ccnd1 ^-/-) have microphthalmia and hypocellular retinas, the latter phenotype attributed to reduced RPC proliferation and increased photoreceptor cell death during the postnatal period. How cyclin D1 influences RPC behavior, especially during the embryonic period, is unclear.

Results

In this study, we show that embryonic RPCs lacking cyclin D1 progress through the cell cycle at a slower rate and exit the cell cycle at a faster rate. Consistent with enhanced cell cycle exit, the relative proportions of cell types born in the embryonic period, such as retinal ganglion cells and photoreceptor cells, are increased. Unexpectedly, cyclin D1 deficiency decreases the proportions of other early born retinal neurons, namely horizontal cells and specific amacrine cell types. We also found that the laminar positioning of horizontal cells and other cell types is altered in the absence of cyclin D1. Genetically replacing cyclin D1 with cyclin D2 is not efficient at correcting the phenotypes due to the cyclin D1 deficiency, which suggests the D-cyclins are not fully redundant. Replacement with cyclin E or inactivation of cyclin-dependent kinase inhibitor p27Kip1 restores the balance of RPCs and retinal cell types to more normal distributions, which suggests that regulation of the retinoblastoma pathway is an important function for cyclin D1 during embryonic retinal development.

Conclusion

Our findings show that cyclin D1 has important roles in RPC cell cycle regulation and retinal histogenesis. The reduction in the RPC population due to a longer cell cycle time and to an enhanced rate of cell cycle exit are likely to be the primary factors driving retinal hypocellularity and altered output of precursor populations in the embryonic Ccnd1 ^-/- retina.     

Stimulation of dopamine release by GABA in rat striatal slices

The effect of GABA on the release of dopamine (DA) from dopaminergic terminals was examined in striatal slices of the rat. The slices were continuously superfused withL-[³H]tyrosine and [³H]DA released was estimated in serial 2.5 min superfusate fractions. GABA from 10⁻⁵ M to 10⁻³ M stimulated the spontaneous release of [³H]DA. The intensity of the effect appeared to be concentration dependent. The GABA stimulatory effect on [³H]DA release persisted during the entire application of GABA for periods as long as 50 min. At a much higher concentration (10⁻¹ M) GABA inhibited the spontaneous release of [³H]DA.At a concentration (10⁻⁴ M) which stimulated [³H]DA release, GABA had no effect on [³H]DA uptake or [³H]tyrosine initial transport in striatal sucrose homogenates. Furthermore it did not affect DA synthesis as indicated by the estimation of the conversion index of tyrosine into DOPA (³H₂O formed fromL-[3,5-³H]tyrosine/tyrosine specific activity in tissues).The stimulatory effect of GABA (5 × 10⁻⁵ M) on the spontaneous release of [³H]DA was not detectable when slices were superfused in the absence of calcium or in the presence of tetrodotoxin (5 × 10⁻⁷ M). It was markedly reduced in the presence of picrotoxin (10⁻⁵ M), a GABA antagonist. Finally, like GABA, GABA agonists such as muscimol and compounds structurally related to GABA such as lioresaal and gammahydroxybutyrate stimulated [³H]DA spontaneous release. Gamma-hydroxylioresal, an inactive metabolite of lioresal, was without effect.These various results suggest that GABA stimulates specifically the release of DA endogenously synthesized in dopaminergic terminals. The blockade of the GABA stimulatory effect by tetrodotoxin (5 × 10⁻⁷ M) suggests that the GABA receptors mediating the action of GABA are not located presynaptically on dopaminergic terminals but on neurons or neuronal afferences within the striatum.     

Artemisinin exerts a protective effect in the MPTP mouse model of Parkinson's disease by inhibiting microglial activation via the TLR4/Myd88/NF-KB pathway

Aims

We performed cell and animal experiments to explore the therapeutic effect of artemisinin on Parkinson's disease (PD) and the TLR4/Myd88 signaling pathway.

Methods

C57 mice were randomly divided into the blank, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced and artemisinin-treated groups. Clinical symptoms, the number of dopaminergic (DAergic) neurons in the substantia nigra, and microglial cell activation were compared among the three groups. Subsequently, BV-2 cell activation and TLR4/Myd88 pathway component expression were compared among the blank, MPP⁺-treated, artemisinin-treated, and TLR4 activator-treated groups.

Results

Behavioral symptoms were improved, the number of DAergic neurons in the substantia nigra of the midbrain was increased, and microglial cell activation was decreased in artemisinin-treated MPTP-induced PD model mice compared with control-treated MPTP-induced PD model mice (p < 0.05). The cell experiments revealed that artemisinin treatment reduced MPP⁺-induced BV-2 cell activation and inhibited the TLR4/Myd88 signaling pathway. Moreover, the effect of artemisinin on the BV-2 cell model was inhibited by the TLR4 activator LPS (p < 0.05).

Conclusion

Artemisinin may reduce damage to DAergic neurons in a PD mouse model by decreasing microglial activation through the TLR4-mediated MyD88-dependent signaling pathway. However, this finding cannot explain the relationship between microglia and DAergic neurons.     

Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

Elevated Potassium Enhances Glutamate Vulnerability of Dopaminergic Neurons Developing in Mesencephalic Cell Cultures

This study examines the effects of high K⁺concentration on the growth and development of mesencephalic cells and their glutamate vulnerability. Mesencephalic cell cultures obtained from Wistar rat embryos on the 14th gestational day were maintained for 14 days in medium with either normal (4.2 mM) or elevated (24.2 mM) potassium concentration. There was no significant difference due to various K⁺concentration in cell growth and survival up to dayin vitro(DIV) 13–15. In order to test the glutamate (Glu) vulnerability, cultures were treated with 100 μMGlu for 15 min in salt solution on the DIV 3, 6, 8, and 13. Glu-induced neuronal damage was estimated 24 h later by measuring the neuron-specific enolase (NSE) content in the culture medium and by counting the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons. Glu had no damaging effect on the cells on DIV 3, but became pronounced beyond DIV 6. Elevated potassium concentration (24.2 mM) in the culture medium during development significantly increases neuronal vulnerability to Glu treatment, indicated by a higher increase of NSE content in the medium and by a more pronounced Glu-induced decrease of the number of TH-IR cells. The Glu-induced decrease of the number of TH-IR cells and of NSE-IR cells let us conclude that dopaminergic neurons are more vulnerable to glutamate than other neurons from mesencephalic culture.     

Neuroprotective effects of a lead compound from coral via modulation of the orphan nuclear receptor Nurr1

Aims

To screen coral-derived compounds with neuroprotective activity and clarify the potential mechanism of lead compounds.

Methods

The lead compounds with neuroprotective effects were screened by H₂O₂ and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP⁺)-induced cell damage models in SH-SY5Y cells. CCK8 and LDH assays were used to detect cell viability. The anti-apoptosis of lead compounds was evaluated by flow cytometry. JC-1 and MitoSox assays were performed to examine the changes in mitochondrial membrane potential and mitochondrial ROS level. Survival of primary cortical and dopaminergic midbrain neurons was measured by MAP2 and TH immunoreactivities. The Caenorhabditis elegans (C. elegans) model was established to determine the effect of lead compounds on dopaminergic neurons and behavior changes.

Results

Three compounds (No. 63, 68, and 74), derived from marine corals, could markedly alleviate the cell damage and notably reverse the loss of worm dopaminergic neurons. Further investigation indicated that compound 63 could promote the expression of Nurr1 and inhibit neuronal apoptosis signaling pathways.

Conclusion

Lead compounds from marine corals exerted significant neuroprotective effects， which indicated that coral might be a new and potential resource for screening and isolating novel natural compounds with neuroprotective effects. Furthermore, this study also provided a new strategy for the clinical treatment of neurodegenerative diseases such as Parkinson's disease.     

Dopamine D-2 Autoreceptors Regulating the Release of Dopamine from Cultured Rat Fetal Dopaminergic Neurons Do Not Desensitize upon Sustained Activation: Implications for the Combined Pharmaco- and Grafting Therapy in Parkinsonian Patients

Cultured rat fetal mesencephalic dopaminergic neurons exhibit specific high-affinity uptake for [³H]dopamine (DA) and express DA D-2 autoreceptors, functionally ally coupled to the inhibition of depolarization evoked [³H]DA release. In this study, we examined the effect of short- and long-term sustained agonist exposure on the responsiveness of these DA D-2 receptors, expressed on cultured fetal mesencephalic dopaminergic neurons. Therefore, cultures were incubated in the absence or presence of the selective D-2 receptor agonist LY 171555 (Quinpirole, 1 μM) before the DA D-2 receptor-mediated inhibition of depolarization-induced [³H]DA release was determined. Short-term preincubation of cultures for 4 h did not lead to any change in the K⁺-evoked [³H]DA release nor to a change in the efficacy of LY 171555 (1 μM) to inhibit this release. Similar results were obtained after long-term agonist exposure for 6 days. Only after exposure for 12 consecutive days was a small reduction of the LY 171555 mediated inhibitory effect on DA release observed, while the IC50 value was slightly shifted to the right as compared to control cultures. Additionally, in pretreated cultures, a statistically significant increase was observed in the depolarization-induced release of [³H]DA in the absence of drugs. In the same model, activation of muscarinic acetylcholine (M-ACh) receptors was shown to potentiate the depolarization-induced release of [³H]DA. Preincubation for 4 h with the muscarinic agonist carbachol (100 μM) induced a strong reduction in the M-ACh receptor-mediated effect on [³H]DA release, indicative of a rapid desensitization of M-ACh receptors. It is concluded that, while no functional desensitization of DA D-2 autoreceptors is apparent, the depolarization-induced release of DA from cultured fetal dopaminergic neurons is enhanced upon long-term sustained activation of DA D-2 receptors.     

Comprehensive behavioral phenotyping of calpastatin-knockout mice

Background

In previous work, we investigated dieldrin cytotoxicity and signaling cell death mechanisms in dopaminergic PC12 cells. Dieldrin has been reported to be one of the environmental factors correlated with Parkinson's disease and may selectively destroy dopaminergic neurons.

Methods

Here we further investigated dieldrin toxicity in a dopaminergic neuronal cell model of Parkinson's disease, namely N27 cells, using biochemical, immunochemical, and flow cytometric analyses.

Results

In this study, dieldrin-treated N27 cells underwent a rapid and significant increase in reactive oxygen species followed by cytochrome c release into cytosol. The cytosolic cytochrome c activated caspase-dependent apoptotic pathway and the increased caspase-3 activity was observed following a 3 hr dieldrin exposure in a dose-dependent manner. Furthermore, dieldrin caused the caspase-dependent proteolytic cleavage of protein kinase C delta (PKCδ) into 41 kDa catalytic and 38 kDa regulatory subunits in N27 cells as well as in brain slices. PKCδ plays a critical role in executing the apoptotic process in dieldrin-treated dopaminergic neuronal cells because pretreatment with the PKCδ inhibitor rottlerin, or transfection and over-expression of catalytically inactive PKCδ^K376R, significantly attenuates dieldrin-induced DNA fragmentation and chromatin condensation.

Conclusion

Together, we conclude that caspase-3-dependent proteolytic activation of PKCδ is a critical event in dieldrin-induced apoptotic cell death in dopaminergic neuronal cells.     

Muscarinic M1 and M3 receptors are present and increase intracellular calcium in adult rat anterior pituitary gland

Physiological and biochemical evidence indicates the existence of functional muscarinic cholinergic receptors in the anterior pituitary. The selectivity of these receptors has been characterised by studying the binding of [³H]quinuclidinyl benzilate ([³H]QNB) and [³H]diphenyl-acetoxy-N-methyl-piperidine ([³H]4-DAMP) in membrane preparation of male rat anterior pituitary at 25°C. Competition experiments with receptor selective muscarinic antagonists were used to characterise specific selective muscarinic receptor binding. Both [³H]QNB and [³H]4-DAMP bound to anterior pituitary membranes at low concentrations, binding was saturable and was potently displaced by 4-DAMP (M₁, M₃ subtypes selective antagonist) > atropine (general) > pirenzepine (M₁). Methoctramine (M₂) didn’t antagonise the [³H]QNB binding efficiently. Acetylcholine and carbachol increased the intracellular Ca²⁺ level in 62% and 65% of cultured rat anterior pituitary cells in a dose-dependent manner, and this effect was prevented by pirenzepine. Based on these results we suggest that both M₁ and M₃ muscarinic receptors are present and active in the majority of cells in the rat anterior pituitary gland, but their physiological role in the adult rat remains to be examined.     

Epigenetic modification after inhibition of IGF-1R signaling in human central nervous system atypical teratoid rhabdoid tumor (AT/RT)

Objective

This study investigated epigenetic modifications in human central nervous system atypical teratoid rhabdoid tumors (AT/RTs), in response to inhibition of insulin-like growth factor receptor 1 (IGF-1R).

Materials and methods

Tumor tissue was obtained from two pediatric patients, tissue was dissociated, and primary cultures were established. Cultured cells were treated with picropodophyllin (PPP; 0, 1, and 2 μM for 48 h), a selective IGF-1R inhibitor. Histone acetylation and methylation patterns (H3K9ac, H3K18ac, H3K4me3, H3K27me3) and levels of histone deacetylases (HDACs; HDAC1, HDAC3, and SirT1) and histone acetyl transferases (GCN5 and p300) were examined. H3K9ac and H3K18ac decreased in response to treatment with PPP. HDAC levels showed a biphasic response, increasing with 1 μM PPP, but then decreasing with 2 μM PPP.

Conclusion

Inhibition of IGF-1R modified epigenetic status in AT/RT. Determining the mechanisms behind these modifications will guide the development of novel therapeutic targets for this malignant embryonal cancer.     

APPswe/PS1ΔE9 mice exhibit low oxygen saturation and alterations of erythrocytes preceding the neuropathology and cognitive deficiency during Alzheimer's disease

Aim

The molecular mechanism underlying Alzheimer's disease (AD) pathologies remains unclear. The brain is extremely sensitive to oxygen deprivation, and brief interruptions in oxygen supply may lead to permanent brain damage. The objective here was to access the red blood cell (RBC) physiological alterations and the changes in blood oxygen saturation of an AD model as well as to explore the possible mechanism underlying these pathologies.

Methods

We used female APP^swe/PS1^ΔE9 mice as AD models. Data were collected at the age of 3, 6, and 9 months. In addition to examining classic features of AD, namely cognitive deficiency and Aβ depositions, 24 h blood oxygen saturation was monitored by Plus oximeters in real time. In addition, RBC physiological parameters were measured by blood cell counter using peripheral blood from the epicanthal veins. Furthermore, in the mechanism investigations, the expression of phosphorylated band 3 protein was examined by a series of Western blot analyses, and the levels of soluble Aβ40 and Aβ42 on the membrane of RBCs were determined by ELISA.

Results

Our results showed that the blood oxygen saturation in the AD mice was significantly reduced as early as at 3 months of age, preceding the neuropathological changes and cognitive impairments. Meanwhile, the expression of phosphorylated band 3 protein and levels of soluble Aβ40 and Aβ42 were all elevated in the erythrocytes of the AD mice.

Conclusion

APP^swe/PS1^ΔE9 mice exhibited decreased oxygen saturation together with reduced RBC counts and hemoglobin concentrations at the early stage, which may aid in the development of predictive markers for AD diagnosis. The increased expression of band 3 protein and elevated Aβ40 and Aβ42 levels may contribute to the deformation of RBCs and, in turn, cause the subsequent AD development.     

Myeloid cell tissue factor does not contribute to venous thrombogenesis in an electrolytic injury model

Introduction

Tissue factor (TF) is a potent initiator of the extrinsic coagulation cascade. The role and source of TF in venous thrombotic disease is not clearly defined. Our study objective was to identify the contribution of myeloid cell TF to venous thrombogenesis in mice.

Materials and methods

The mouse electrolytic inferior vena cava model was used to induce thrombosis. The following groups of mice were used (1) TF^flox/floxLysMCre⁺ mice that have reduced TF expression in myeloid cells, (2) TF^flox/floxLysMCre^- littermate controls, (3) Wild type mice given a monoclonal anti-mouse TF antibody (1H1) to inhibit TF activity, and (4) Wild type mice given rat IgG. Evaluations at baseline, day 2, and day 6 post thrombosis included thrombus weight, vein wall inflammatory cell migration, vein wall TF mRNA, and plasma D-dimer levels.

Results

Inhibition of TF significantly decreased thrombus weight 2 days post venous thrombosis. In contrast, TF^flox/floxLysMCre⁺ had no change in thrombus weight when compared to littermate controls. The absence of myeloid cell TF did not affect infiltration of neutrophils or monocytes into the vein wall. TF mRNA expression in the vein wall decreased at 2 days but then returned to baseline levels by 6 days post thrombosis. D-dimer levels peaked at 2 days post thrombosis in mice with or without myeloid cell TF.

Conclusions

TF is important in the formation of venous thrombi in the macrovasculature. However, TF expression by myeloid cells does not significantly contribute to venous thrombogenesis in this model.