Intact Brain Cells Research Articles

Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer’s disease cortex

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer’s disease (AD). New MethodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Monitoring gene expression in the brain with synthetic serum markers

Blood tests are among the most common clinical tools due to their low cost, simplicity, and ability to observe many markers at once. However, currently, blood tests can only monitor a fraction of physiological processes that happen to have a serum marker. Here, we will present our work on the development of synthetic serum markers that can track gene expression in intact brain cells with a simple blood test. The synthetic marker approach has several advantages. First, detection of existing markers may be challenging due to their low levels in blood. With a synthetic marker, one can design a reporter that is easy to detect allowing for superior sensitivity. Second, there are large numbers of genes in each cell, but available brain imaging techniques can at most represent only a few signals (e.g., a few colors of fluorescent proteins, or types of MRI contrast). Synthetic serum markers use biochemical detection and can be designed to be massively multiplexed similarly to how thousands of proteins can be detected in blood simultaneously with mass spectrometry. Third, synthetic serum markers can surveil large brain regions, unlike invasive locally implanted devices. Finally, these markers have the potential for inexpensive and simple readout. We show applications of this concept with markers that can cross through an intact blood–brain barrier to report on gene expression and markers whose release from the brain relies on sonobiopsy to provide spatial selectivity.

Protective and Therapeutic Capacities of Lactic Acid Bacteria Postmetabolites against Koi Herpesvirus Infection In Vitro.

The accumulation of data on beneficial biological effects of probiotics and their metabolic products favors their potential use in the prevention and treatment of various malaises. Nine postmetabolites from Lactic acid bacteria (LAB) of human or dairy origin and their antiviral activity were studied using the cytopathic effect inhibition test. The virucidal capacity, their influence on the adsorption stage of Koi herpes virus (KHV) and their preventive role against subsequent viral challenge on intact Common carp brain (CCB) cells were also determined by titration assay. Residual viral infectivity in postmetabolites-treated samples was compared to mock-treated controls and Δlgs were calculated. When administered during KHV replication, the microbial products isolated from Lactiplantibacillus plantarum showed remarkable activity with a selectivity index (SI) between 26.5 and 221.4, as those effects were dependent on the sample-virus incubation time. Postmetabolites from Lactobacillus gasseri and Lactiplantibacillus plantarum also demonstrated significant inhibition of KHV replication with SI of 24 and 16, respectively. The bioactive metabolites isolated from Limosilactobacillus fermentum had a minor effect on the viral replicative cycle. Compounds, produced during the fermentation by lactobacilli, grown on different nutritive media and collected at different time points, significantly inhibited extracellular KHV virions. All investigated postmetabolites remarkably blocked KHV attachment to the host cell (CCB), leading to a drop in viral titers by Δlg = 4.25-5.25, and exerted protective effects on CCB cells before they were subjected to viral infection. Our results open new horizons and promote LAB and their postbiotic products to be used in the prophylaxis and therapy of viral infections.

Immune Pathways in Etiology, Acute Phase, and Chronic Sequelae of Ischemic Stroke.

Inflammation and immune mechanisms are crucially involved in the pathophysiology of the development, acute damage cascades, and chronic course after ischemic stroke. Atherosclerosis is an inflammatory disease, and, in addition to classical risk factors, maladaptive immune mechanisms lead to an increased risk of stroke. Accordingly, individuals with signs of inflammation or corresponding biomarkers have an increased risk of stroke. Anti-inflammatory drugs, such as IL (interleukin)-1β blockers, methotrexate, or colchicine, represent attractive treatment strategies to prevent vascular events and stroke. Lately, the COVID-19 pandemic shows a clear association between SARS-CoV2 infections and increased risk of cerebrovascular events. Furthermore, mechanisms of both innate and adaptive immune systems influence cerebral damage cascades after ischemic stroke. Neutrophils, monocytes, and microglia, as well as T and B lymphocytes each play complex interdependent roles that synergize to remove dead tissue but also can cause bystander injury to intact brain cells and generate maladaptive chronic inflammation. Chronic systemic inflammation and comorbid infections may unfavorably influence both outcome after stroke and recurrence risk for further stroke. In addition, stroke triggers specific immune depression, which in turn can promote infections. Recent research is now increasingly addressing the question of the extent to which immune mechanisms may influence long-term outcome after stroke and, in particular, cause specific complications such as poststroke dementia or even poststroke depression.

Dissociation of Hexokinase II Binding to VDAC Increases State 3 Respiration and Reduces Membrane Potential Repolarization Time in Mitochondria Isolated From Brain and Heart

Hexokinase II (HKII) plays a critical role in ATP production by catalyzing the initial step in glucose metabolism and maintaining the gradient for glucose entry into the cell. HKII also closely interacts with the mitochondrial outer membrane protein, voltage dependent anion channel (VDAC), to permit the channeling of mitochondrial generated ATP, its substrate, for use in cellular metabolism. It has been reported that adding protease to mitochondrial isolation buffer may improve mitochondrial respiration. However, proteases could potentially degrade the HKII‐VDAC protein interaction at the interphase between mitochondrial outer membrane and extra‐matrix space. We hypothesize that protease interferes with the association of HKII to VDAC and this alters mitochondrial bioenergetics. To test this, we isolated mitochondria from brain and heart of guinea pigs to determine the consequences of HKII binding and unbinding to VDAC on mitochondrial respiration and membrane potential (ΔΨm). Mitochondria were isolated by differential centrifugation using the same isolation buffers except one with and one without the presence of protease. We examined the HKII binding to mitochondria, specifically to VDAC, between the two mitochondrial groups using Western blot (WB) and immunoprecipitation (IP). We measured the rate of O2 consumption using mitochondrial substrates before and after adding ADP and evaluated changes in ΔΨm using TMRM fluorescent dye. WB showed, in both heart and brain mitochondria, binding of HKII to mitochondria in the absence of protease was evident, but this was not detected in mitochondria isolated in the presence of protease. Moreover, IP experiments with cross‐linking revealed HKII‐VDAC association in the protease‐free isolation buffer, but not in the buffer containing protease. Mitochondrial respiration as determined by states 3 (ADP respiration) and 4 (ADP depleted respiration) respiration was significantly slower when HKII was bound to VDAC compared to when it was not bound to VDAC. ΔΨm repolarization following ADP‐induced depolarization (state 3 respiration) was substantially delayed when mitochondria were isolated in the absence, in contrast to the presence, of protease. Our results indicate that HKII binding to isolated mitochondria directly modulates oxidative phosphorylation, possibly by regulating the transfer of substrates and metabolites, including ADP/ATP, via VDAC. Overexpression of HKII has been implicated in cytoprotection; however, the underlying mechanisms is not well understood. The implications of the increased association or dissociation of HKII‐VDAC in cell physiology (survival) and/or pathophysiology (cell death) will be further explored in intact cardiac and brain cells.Support or Funding InformationThis work was supported in part by the Veterans Administration (BX‐002539‐01) and the National Institutes of Health (R01 HL131673‐01A1 and P01‐GM066730).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metal and complementary molecular bioimaging in Alzheimer's disease.

Alzheimer's disease (AD) is the leading cause of dementia in the elderly, affecting over 27 million people worldwide. AD represents a complex neurological disorder which is best understood as the consequence of a number of interconnected genetic and lifestyle variables, which culminate in multiple changes to brain structure and function. These can be observed on a gross anatomical level in brain atrophy, microscopically in extracellular amyloid plaque and neurofibrillary tangle formation, and at a functional level as alterations of metabolic activity. At a molecular level, metal dyshomeostasis is frequently observed in AD due to anomalous binding of metals such as Iron (Fe), Copper (Cu), and Zinc (Zn), or impaired regulation of redox-active metals which can induce the formation of cytotoxic reactive oxygen species and neuronal damage. Metal chelators have been administered therapeutically in transgenic mice models for AD and in clinical human AD studies, with positive outcomes. As a result, neuroimaging of metals in a variety of intact brain cells and tissues is emerging as an important tool for increasing our understanding of the role of metal dysregulation in AD. Several imaging techniques have been used to study the cerebral metallo-architecture in biological specimens to obtain spatially resolved data on chemical elements present in a sample. Hyperspectral techniques, such as particle-induced X-ray emission (PIXE), energy dispersive X-ray spectroscopy (EDS), X-ray fluorescence microscopy (XFM), synchrotron X-ray fluorescence (SXRF), secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled mass spectrometry (LA-ICPMS) can reveal relative intensities and even semi-quantitative concentrations of a large set of elements with differing spatial resolution and detection sensitivities. Other mass spectrometric and spectroscopy imaging techniques such as laser ablation electrospray ionization mass spectrometry (LA ESI-MS), MALDI imaging mass spectrometry (MALDI-IMS), and Fourier transform infrared spectroscopy (FTIR) can be used to correlate changes in elemental distribution with the underlying pathology in AD brain specimens. Taken together, these techniques provide new techniques to probe the pathobiology of AD and pave the way for identifying new therapeutic targets. The current review aims to discuss the advantages and challenges of using these emerging elemental and molecular imaging techniques, and highlight clinical achievements in AD research using bioimaging techniques.

7α-Hydroxylation of dehydroepiandrosterone does not interfere with the activation of glucocorticoids by 11β-hydroxysteroid dehydrogenase in EtC cerebellar neurons

The neuroprotective action of dehydroepiandrosterone (DHEA) in the absence of a known specific receptor has been attributed to its metabolism by different cell types in the brain to various steroids, with a preference to its 7-hydroxylated products. The EtC cerebellar granule cell line converts DHEA almost exclusively to 7α-hydroxy-DHEA (7α-OH-DHEA). It has been postulated that DHEA's 7-OH and 7-oxo metabolites can decrease glucocorticoid levels by an interactive mechanism involving 11β-hydroxysteroid dehydrogenase (11β-HSD). In order to study the relationship of 7-hydroxylation of DHEA and glucocorticoid metabolism in intact brain cells, we examined whether EtC cerebellar neurons, which are avid producers of 7α-OH-DHEA, could also metabolize glucocorticoids. We report that EtC neuronal cells exhibit 11β-HSD1 reductase activity, and are able to convert 11-dehydrocorticosterone into corticosterone, whereas they do not demonstrate 11β-HSD2 dehydrogenase activity. Consequently, EtC cells incubated with DHEA did not yield 7-oxo- or 7β-OH-DHEA. Our findings are supported by the reductive environment of EtC cells through expression of hexose-6-phosphate dehydrogenase (H6PDH), which fosters 11β-HSD1 reductase activity. To further explore the role of 7α-OH-DHEA in EtC neuronal cells, we examined the effect of preventing its formation using the CYP450 inhibitor ketoconazole. Treatment of the cells with this drug decreased the yield of 7α-OH-DHEA by about 75% without the formation of alternate DHEA metabolites, and had minimal effects on glucocorticoid conversion. Likewise, elevated levels of corticosterone, the product of 11β-HSD1, had no effect on the metabolic profile of DHEA. This study shows that in a single population of whole-cells, with a highly reductive environment, 7α-OH-DHEA is unable to block the reducing activity of 11β-HSD1, and that 7-hydroxylation of DHEA does not interfere with the activation of glucocorticoids. Our investigation on the metabolism of DHEA in EtC neuronal cells suggest that other alternate mechanisms must be at play to explain the in vivo anti-glucocorticoid properties of DHEA and its 7-OH-metabolites.

In Situ Mitochondrial Ca2+ Buffering Differences of Intact Neurons and Astrocytes from Cortex and Striatum

The striatum is particularly vulnerable to neurological disorders, such as Huntington disease. Previous studies, with nonsynaptic mitochondria isolated from cortical and striatal homogenates, suggest that striatal mitochondria are highly vulnerable to Ca(2+) loads, possibly influencing striatal vulnerability. However, whether and how neuronal and glial mitochondria from cortex and striatum differ in Ca(2+) vulnerability remains unknown. We test this hypothesis using a novel strategy allowing comparisons of mitochondrial Ca(2+) buffering capacity in cortical and striatal neuron-astrocyte co-cultures. We provide original evidence that mitochondria not only in neurons but also in astrocytes from striatal origin exhibit a decreased Ca(2+) buffering capacity when compared with cortical counterparts. The decreased mitochondrial Ca(2+) buffering capacity in striatal versus cortical astrocytes does not stem from variation in mitochondrial concentration or in the rate of intracellular Ca(2+) elevation, being mechanistically linked to an increased propensity to undergo cyclosporin A (CsA)-sensitive permeability transition. Indeed, 1 microm CsA selectively increased the mitochondrial Ca(2+) buffering capacity of striatal astrocytes, without modifying that of neurons or cortical astrocytes. Neither thapsigargin nor FK506 modified mitochondrial Ca(2+) buffering differences between cell types, excluding a predominant contribution of endoplasmic reticulum or calcineurin. These results provide additional insight into the mechanisms of striatal vulnerability, showing that the increased Ca(2+) vulnerability of striatal versus cortical mitochondria resides in both intact neurons and astrocytes, thus positioning the striatum at greater risk for disturbed neuron-astrocyte interactions. Also, the selective effect of CsA over striatal astrocytes suggests that in vivo neuronal sheltering with this compound may indirectly result from astrocytic protection.

Characterization of 1,4‐dideoxy‐1,4‐imino‐d‐arabinitol (DAB) as an inhibitor of brain glycogen shunt activity

The pharmacological properties of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB), a potent inhibitor of glycogen phosphorylase and synthase activity in liver preparations, were characterized in different brain tissue preparations as a prerequisite for using it as a tool to investigate brain glycogen metabolism. Its inhibitory effect on glycogen phosphorylase was studied in homogenates of brain tissue and astrocytes and IC50-values close to 400 nM were found. However, the concentration of DAB needed for inhibition of glycogen shunt activity, i.e. glucose metabolism via glycogen, in intact astrocytes was almost three orders of magnitude higher. Additionally, such complete inhibition required a pre-incubation period, a finding possibly reflecting a limited permeability of the astrocytic membrane. DAB did not affect the accumulation of 2-deoxyglucose-6-phosphate indicating that the transport of DAB is not mediated by the glucose transporter. DAB had no effect on enzymes involving glucose-6-phosphate, i.e. glucose-6-phosphate dehydrogenase, phosphoglucoisomerase and hexokinase. Furthermore, DAB was evaluated in a functional preparation of the isolated mouse optic nerve, in which its presence severely reduced the ability to sustain evoked compound action potentials in the absence of glucose, a condition in which glycogen serves as an important energy substrate. Based on the experimental findings, DAB can be used to evaluate glycogen shunt activity and its functional importance in intact brain tissue and cells at a concentration of 300-1000 muM and a pre-incubation period of 1 h.

Volumes-cibles anatomocliniques (GTV et CTV) des tumeurs gliales

Gliomas are the most frequent tumors of the central nervous system of the adult. These intraparenchymal tumors are infiltrative and the most important criterion for definition of GTV and CTV is the extent of infiltration. Delineation of GTV and CTV for untreated and resected glioma remains a controversial and difficult issue because of the discrepancy between real tumor invasion and that estimated by CT or MRI. Is particularly helpful a joint analysis of the four different methods as histopathological correlations with CT and MRI, use of new modality imaging, pattern of relapses after treatment and interobserver studies. The presence of isolated tumor cells in intact brain, oedema or adjacent structures requires the definition of two different options for CTV: i) a geometrical option with GTV defined as the tumor mass revealed by the contrast-enhanced zone on CT or MRI and a CTV with an expanded margin of 2 or 3 cm; ii) an anatomic option including the entire zone of oedema or isolated tumor cell infiltration extending at least as far as the limits of the hyperintense zone on T2-weighted MRI. Inclusion of adjacent structures (such as white matter, corpus callosum, subarachnoid spaces) in the CTV mainly depends on the site of the tumor and size of the volume is generally enlarged.

Molecular shuttle chelation: the use of ascorbate, desferrioxamine and Feralex-G in combination to remove nuclear bound aluminum.

1. Abundant data suggest that aluminum (Al(III)) exposure may be an environmental risk factor contributing to the development, progression and/or neuropathology of several human neurodegenerative disorders, including Alzheimer's disease (AD). 2. Nuclei appear to be one directed target for Al(III) binding, accumulation, and Al(III)-mediated dysfunction due in part to their high content of polyphosphorylated nucleic acids, nucleotides, and nucleoproteins. 3. The design of chelation therapies dealing with the removal of Al(III) from these genetic compartments therefore represents an attractive strategy to alleviate the development and/or progression of central nervous system dysfunction that may arise from excessive Al(III) exposure. 4. In this study we have investigated the potential application of 10 natural and synthetic Al(III) chelators, including ascorbate (AS), desferrioxamine (DF), and Feralex-G (FG), used either alone or in combination, to remove Al(III) preincubated with intact human brain cell nuclei. 5. Although nuclear bound Al(III) was found to be highly refractory to removal, the combination of AS+FG was found to be particularly effective in removing Al(III) from the nuclear matrix. 6. Our data suggest that chelators carrying cis-hydroxy ketone groups, such as FG, are particularly suited to the removal of Al(III) from complex biological systems. 7. We further suggest a mechanism whereby small chelating molecules may penetrate the nucleus, bind Al(III), diffuse to regions accessible by the larger DF or FG molecules and transfer their Al(III) to DF or FG. 8. The proposed mechanism, called molecular shuttle chelation may provide a useful pharmacotherapy in the potential treatment of Al(III) overload disease.

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response.

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 microl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.

Ketamine suppresses endotoxin-induced NF-kappaB expression.

Ketamine reduces endotoxin-induced production of proinflammatory cytokines, including tumour necrosis factor-alpha (TNF), in several types of inflammatory cells, including monocytes and macrophages. Transcription of the genes that encode production of these proinflammatory cytokines is regulated by nuclear factor-kappa B (NF-kappaB). Cytoplasmic B protein is activated by endotoxin (LPS) as well as by TNF, allowing B protein to migrate into the cell nucleus to activate gene transcription for these inflammatory mediators. Because NF-kappaB is likely involved in brain injury and inflammatory neurodegenerative disease, such as multiple sclerosis, we examined whether ketamine inhibits LPS-induced activation of NF-kappaB in human glioma cells in vitro and intact mouse brain cells in vivo. Endotoxin-induced NF-kappaB expression in both the human glioma cells in vitro and the intact mouse brain cells in vivo was determined by electrophoretic mobility shift assays (EMSA) of nuclear extracts and measurement of NF-kappaB expression by densitometry. Endotoxin was injected intracerebroventricularly in vivo and intact brain was harvested. Klenow fragment labeling was used to identify NF-kappaB protein for both the in vivo and vitro experiments. Endotoxin treatment increased NF-kappaB expression (P < 0.05) both in vivo and vitro compared with control (untreated) cells. Ketamine suppressed endotoxin-induced neuronal NF-kappaB activation in a dose-dependent manner (P < 0.05, except for the 10(-5) M concentration in vitro) both in vivo and vitro. Ketamine inhibits endotoxin-induced NF-kappaB expression in brain cells in vivo and vitro and it is suggested that this may have implications in the neuroprotective effects of ketamine reported by other investigators.

Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.

The physiological state of the cell is controlled by signal transduction mechanisms which regulate the balance between protein kinase and protein phosphatase activities. Here we report that a single protein can, depending on which particular amino-acid residue is phosphorylated, function either as a kinase or phosphatase inhibitor. DARPP-32 (dopamine and cyclic AMP-regulated phospho-protein, relative molecular mass 32,000) is converted into an inhibitor of protein phosphatase 1 when it is phosphorylated by protein kinase A (PKA) at threonine 34. We find that DARPP-32 is converted into an inhibitor of PKA when phosphorylated at threonine 75 by cyclin-dependent kinase 5 (Cdk5). Cdk5 phosphorylates DARPP-32 in vitro and in intact brain cells. Phospho-Thr 75 DARPP-32 inhibits PKA in vitro by a competitive mechanism. Decreasing phospho-Thr 75 DARPP-32 in striatal slices, either by a Cdk5-specific inhibitor or by using genetically altered mice, results in increased dopamine-induced phosphorylation of PKA substrates and augmented peak voltage-gated calcium currents. Thus DARPP-32 is a bifunctional signal transduction molecule which, by distinct mechanisms, controls a serine/threonine kinase and a serine/threonine phosphatase.

Inflammatory molecule release by β-amyloid-treated T98G astrocytoma cells: role of prostaglandins and modulation by paracetamol

Deposition of β-amyloid in the brain triggers an inflammatory response which accompanies the neuropathologic events of Alzheimer's disease and contributes to the destruction of brain tissue. The present study shows that β-amyloid can stimulate human astrocytoma cells (T98G) to secrete the proinflammatory factors interleukin-6 and prostaglandins. Furthermore, prostaglandins can stimulate T98G to secrete interleukin-6, which in turn triggers the formation of additional prostaglandins. Prostaglandins are, therefore, a key element in the induction and maintenance of a state of chronic inflammation in the brain which may exacerbate the fundamental pathology in Alzheimer patients. Paracetamol (0.01–1000 μM), an unusual analgesic/antipyretic drug which acts preferentially by reducing prostaglandin production within the central nervous system, and indomethacin (0.001–10 μM) caused a clear dose-dependent reduction of prostaglandin E 2 production by stimulated T98G cells whereas interleukin-6 release was not affected. These data provide further evidence of the involvement of non-steroidal anti-inflammatory drugs in the inflammatory processes that can be generated by glial cells in intact brain.

Aspects of human bioenergetics as studied in vivo by magnetic resonance spectroscopy

We outline the relevant capabilities of in vivo phosphorus MR spectroscopy by discussing some aspects of normal human biochemistry as studied by this technique. The transport of inorganic phosphate from cytosol into mitochondria in the human skeletal muscle was studied by exploiting a new experimental protocol. We found that Pi was transported into mitochondria in the absence of ATP biosynthesis and in the presence of a pH gradient. The control of CoQ on the efficiency of oxidative phosphorylation in the skeletal muscle and brain was studied by administering CoQ to patients with mitochondrial cytopathies due to known enzyme defects. Before CoQ we had detected a relevant reduction of mitochondrial functionality in the skeletal muscle as shown by the reduced rate of phosphocreatine recovery from exercise, and in the occipital lobes by reduced [phosphocreatine] and a high [ADP] and [Pi]. After CoQ all brain variables were remarkably improved. Treatment with CoQ also improved the rate of muscle phosphocreatine recovery from exercise. Our in vivo findings support the hypothesis that the concentration of CoQ rather than the rate of its lateral diffusion in the mitochondrial membrane controls the efficiency of oxidative phosphorylation. Other eperiments were undertaken to clarify the functional relationship between cytosolic free [Mg 2+] and cell bioenergetics in the intact human brain. In the same group of patients with mitochondrial cytopathies we found decreased ΔG of ATP hydrolysis and low cytosolic free [Mg 2+]. Treatment with CoQ resulted in improved brain bioenergetics and increased free [Mg 2+]. These findings strongly indicate that decreased free magnesium was secondary to defective mitochondrial respiration, and support the hypothesis that cytosolic free [Mg 2+] is regulated in the intact brain cell to equilibrate, at least in part, any changes in rapidly available free energy.

Free Radical Production After Exposure of Astrocytes and Astrocytic C6 Glioma Cells to Ethanol. Preliminary Results

Formation of the α-hydroxyethyl radical (CH3°CHOH) has already been extensively demonstrated after ethanol metabolism in the liver. Despite favourable conditions, this formation in the brain has remained speculative since there is no direct experimental evidence in intact brain cells. In this preliminary study, the formation of such a radical was observed after exposure of astrocytes and astrocytic C6 glioma cells to ethanol. These cells were studied because astrocyte integrity is essential for normal growth and functioning of neurons. The free radicals were detected by EPR spectroscopy using the spin trapping technique. Astrocytes appeared to be more sensitive than the C6 cells to free radical formation as the intensity of the signal was higher after exposure of the astrocytes and increased with time, a fact not observed after exposure of the C6 cells.

Alzheimer's beta-amyloid precursor protein is expressed on the surface of immediately ex vivo brain cells: a flow cytometric study.

Beta-amyloid precursor protein (beta APP) is ubiquitously expressed, but deposition of the beta APP proteolytic fragment A beta is virtually restricted to the brain, suggesting cell-specific processing of this molecule. Our laboratory has investigated expression of beta APP in mechanically dissociated, unfixed, immediately ex vivo cells from various mouse and rat organs by flow cytometry. Epitopes of predicted extracellular domains of beta APP recognized by the N-terminal 22C11 monoclonal antibody (mAb) and the juxtamembrane 4G8 mAb were not detectable on the surface of lymphoid cells, hepatocytes, or kidney cells. In contrast, surface 22C11 and 4G8 beta APP immunoreactivity was abundant on intact (propidium iodide-excluding) dissociated brain cells. The predicted C-terminal intracellular beta APP determinant recognized by the mAb Jonas was not detectable on the surface of intact brain cells, but was present in ethanol-permeabilized cells, consistent with a transmembrane configuration of beta APP in brain cells. Trypsinization of intact brain cells abolished cell surface immunoreactivity for 22C11, which was then reestablished by short-term culture. Augmentation of 22C11 and 4G8 surface immunoreactivity occurred when brain cells were cultured short-term in phenylarsine oxide, a general endocytosis inhibitor. By double staining protocols of brain cells with mAbs directed against beta APP ectodomain epitopes and the neuronal surface proteins Thy-1 or neural cell adhesion molecule (NCAM), we observed that all Thy-1+ and NCAM+ cells (approximately 50%) were immunoreactive for surface beta APP, but that some beta APP+ cells (approximately 20%) were negative for these neuronal markers. Our data suggest that neurons and a subpopulation of other brain cells, unlike peripheral cells, can support beta APP as a type 1 intrinsic membrane molecule with an intact ectodomain, and that beta APP surface abundance is regulated by an equilibrium between membranes vesicle insertion and endocytotic internalization. Transmembrane beta APP holoprotein may be a critical determinant of brain-predominant processing of beta APP to A beta, and may participate in a receptor/transducer function unique to brain cells.

Early changes in glucose metabolism in the cerebrum of senescence accelerated mouse: involvement of glucose transporter

The rate of 6-[ 14C] d-glucose oxidation in cerebral cells of SAMP8, a substrain of senescence accelerated mouse, was investigated in vitro. The production of 14CO 2 in dissociated intact brain cells prepared from the cerebrum of 4–8-week-old SAMP8 was higher than that of age-matched SAMR2 as a control mouse, while no difference between these two strains was observed in the production of 14CO 2 in the cerebral homogenates. These results indicated that the increased metabolism of glucose in SAMP8 might be associated with the glucose transport system across the cell membrane. Therefore, 2-deoxy- d-glucose (2-DG) uptake into the brain cells and cytochalasin B(CB) binding to cerebral crude membranes examined. Both the 2-DG uptake and the CB binding in SAMP8 were much greater than in SAMR2. Furthermore, the increased CB binding in SAMP8 was seen only in the cerebral cortex of 4- to 8-week-old mice, and neither in other regions of the cerebrum nor in other aged mice (2-week- and 40- to 48-week-old mice). These results suggest that the transient overproduction of the glucose transporter protein in the cerebral cortex is involved in the increased glucose metabolism in 4- to 8-week-old SAMP8.

Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro

In developing mammalian brain, many neurons migrate to their final position by moving in direct apposition to radially oriented glial cells. Glial-guided migration can be visualized in microcultures of mouse cerebellar cells by the combined use of cellular antigen markers and high resolution time-lapse video microscopy (Hatten et al., 1984; Edmondson and Hatten, 1987). Such studies have demonstrated the behavior of migrating cells and revealed a motile leading process on the migrating neuron that resembles an axonal growth cone and grows along extended glial fibers. To study the fine structural details of the migrating neuron and its neuron-glial apposition, we identified and monitored neurons in microcultures with video microscopy and examined the cytology and cellular contacts of the same cells with transmission electron microscopy. The cytology of the soma and leading process of migrating cells closely matches that described for granule cells in intact brain (Rakic, 1971). Newly observed structures include the presence of longitudinally oriented microtubules extending from a basal body in the soma into the leading process, and microfilament-rich filopodia arising from the soma and leading process. The most striking feature of actively migrating neurons is a specialized junction between the neuronal cell soma and apposing glial fibers. At this junction, here termed "interstitial density," the extracellular space is dilated to 20 nm and filamentous material in the intracellular cleft either spans the cleft or runs parallel to the cell membranes. Some interstitial fibrils are contiguous with, or are transmembranous extensions of, submembranous cytoskeletal elements that attach to microtubules. Interstitial junctions were not found between neurons that did not translocate in the observation period before fixation. Instead, stationary cells formed desmosomes (puncta and macula adhaerentia) at appositions with glial processes.