Jagdeep K. Sandhu

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells

BackgroundIn addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules. These vesicles play important roles in intercellular communication by acting as carrier for essential cell-specific information to target cells. Endothelial cells in the brain form the blood–brain barrier, a specialized interface between the blood and the brain that tightly controls traffic of nutrients and macromolecules between two compartments and interacts closely with other cells forming the neurovascular unit. Therefore, brain endothelial cell extracellular microvesicles could potentially play important roles in ‘externalizing’ brain-specific biomarkers into the blood stream during pathological conditions, in transcytosis of blood-borne molecules into the brain, and in cell-cell communication within the neurovascular unit.MethodsTo study cell-specific molecular make-up and functions of brain endothelial cell exosomes, methods for isolation of extracellular microvesicles using mass spectrometry-compatible protocols and the characterization of their signature profiles using mass spectrometry -based proteomics were developed.ResultsA total of 1179 proteins were identified in the isolated extracellular microvesicles from brain endothelial cells. The microvesicles were validated by identification of almost 60 known markers, including Alix, TSG101 and the tetraspanin proteins CD81 and CD9. The surface proteins on isolated microvesicles could potentially interact with both primary astrocytes and cortical neurons, as cell-cell communication vesicles. Finally, brain endothelial cell extracellular microvesicles were shown to contain several receptors previously shown to carry macromolecules across the blood brain barrier, including transferrin receptor, insulin receptor, LRPs, LDL and TMEM30A.ConclusionsThe methods described here permit identification of the molecular signatures for brain endothelial cell-specific extracellular microvesicles under various biological conditions. In addition to being a potential source of useful biomarkers, these vesicles contain potentially novel receptors known for delivering molecules across the blood–brain barrier.

MALDI mass spectrometry imaging of gangliosides in mouse brain using ionic liquid matrix.

Mass spectrometry imaging has emerged as a powerful tool for the direct detection of biomolecules, mainly phospholipids, proteins and peptides, in tissue samples. To date, there is very little information available on the direct analysis of gangliosides in brain tissue. One major hurdle for imaging gangliosides in tissue using mass spectrometry is that sialic acid residues can be dissociated in ionization process. In this report, we investigated an ionic liquid matrix for mass spectrometry imaging of gangliosides. This ionic liquid matrix offered excellent sensitivity for detection gangliosides without significant loss of sialic acid residues. Thus, it can be used to study the abundance and anatomical localization of gangliosides in mouse brain using mass spectrometry imaging technique. Mass spectrometry image analyses of the mouse brain tissue sections demonstrated that the N-fatty acyl chains of gangliosides were differentially distributed in mouse hippocampal regions, whereby the gangliosides with N-C(18) acyl chain were enriched in CA1 region, while gangliosides with N-C(20) acyl chain were enriched in dentate gyrus. In addition, this observation is true for mono-, di- and tri-sialylated gangliosides. Although the linkage information was not determined, the mass spectrometry imaging technique was capable of spatial tissue mapping of ceramide structures in gangliosides.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal–glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24–48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 μg/ml of coenzyme Q10 (functions both as a ROS scavenger and co‐factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q10 in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters. The overexpression of GDNF, regardless of the promoter employed, had no obvious adverse effects on astroglia and the engineered cells stably produced and secreted GDNF for extended periods of time (> or =3 weeks). These astrocytes very effectively protected neurons against 6OHDA, in both mouse and human co-culture systems. The neuroprotective effects were mediated not only by GDNF, but also by the antioxidant GSH since its depletion reduced the level of GDNF protection. Furthermore, neurons and astrocytes expressed different components of GDNF signaling complex, suggesting that they might utilize separate pathways to mediate autocrine and paracrine effects of GDNF.

Characterization of astrocytes derived from human NTera‐2/D1 embryonal carcinoma cells

Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via high‐affinity glutamate transporters, glutamate transporter‐1 (GLT‐1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate‐catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera‐2 (NT2/D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4‐week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell‐cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5‐bromo‐2′deoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100β. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT‐1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT‐1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of 3H‐glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non‐toxic, suggesting that these cells were capable of converting it to non‐toxic metabolites. NT2/A and NT2‐derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.

Paraquat induces oxidative stress, neuronal loss in substantia nigra region and Parkinsonism in adult rats: Neuroprotection and amelioration of symptoms by water-soluble formulation of Coenzyme Q10

BackgroundParkinsons disease, for which currently there is no cure, develops as a result of progressive loss of dopamine neurons in the brain; thus, identification of any potential therapeutic intervention for disease management is of a great importance.ResultsHere we report that prophylactic application of water-soluble formulation of coenzyme Q10 could effectively offset the effects of environmental neurotoxin paraquat, believed to be a contributing factor in the development of familial PD. In this study we utilized a model of paraquat-induced dopaminergic neurodegeneration in adult rats that received three weekly intra-peritoneal injections of the herbicide paraquat. Histological and biochemical analyses of rat brains revealed increased levels of oxidative stress markers and a loss of approximately 65% of dopamine neurons in the substantia nigra region. The paraquat-exposed rats also displayed impaired balancing skills on a slowly rotating drum (rotorod) evidenced by their reduced spontaneity in gait performance. In contrast, paraquat exposed rats receiving a water-soluble formulation of coenzyme Q10 in their drinking water prior to and during the paraquat treatment neither developed neurodegeneration nor reduced rotorod performance and were indistinguishable from the control paraquat-untreated rats.ConclusionOur data confirmed that paraquat-induced neurotoxicity represents a convenient rat model of Parkinsonian neurodegeneration suitable for mechanistic and neuroprotective studies. This is the first preclinical evaluation of a water-soluble coenzyme Q10 formulation showing the evidence of prophylactic neuroprotection at clinically relevant doses.

Epigenetic Modifications of SOX2 Enhancers, SRR1 and SRR2, Correlate With In Vitro Neural Differentiation

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2‐D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell‐type‐specific SOX2 expression. This study showed that 1) unmethylated DNA and acetylated histones at both enhancers correlated with a high level of SOX2 expression in proliferating neural progenitors and 2) reversible modifications of the SRR1 element were observed during gene reexpression in astrocytes, whereas permanent epigenetic marks on the SRR2 enhancer were seen in neurons where the gene was silenced. Taken together, these results are clear illustrations of cell‐type‐specific epigenomes and suggest mechanisms by which they may be created and maintained.

Attenuation of free radical production and paracrystalline inclusions by creatine supplementation in a patient with a novel cytochrome b mutation.

Mitochondrial cytopathies are associated with increased free radical generation and paracrystalline inclusions. Paracrystalline inclusions were serendipitously found in a young male athlete with a very high respiratory exchange ratio during steady‐state exercise; he also had an unusually low aerobic capacity. Direct sequencing of the mitochondrial DNA (mtDNA) coding regions revealed a novel missense mutation (G15497A) resulting in a glycine→serine conversion at a highly conserved site in the cytochrome b gene in the subject, his mother, and sister. Cybrids, prepared by fusion of the subjects platelets with either U87MG ρ° or SH‐SY5Y ρ° cells, generated higher basal levels of reactive oxygen species (ROS), had a lower adenosine triphosphate (ATP) content, and were more sensitive to oxygen and glucose deprivation and peroxynitrite generation compared to control cybrids with wild‐type mtDNA. Cell survival was significantly enhanced with 50 mmol/L creatine monohydrate (CM) administration. The subject was also treated with CM (10 g/d) for a period of 5 weeks and a repeat muscle biopsy showed no paracrystalline inclusions. The results suggest that the development of exercise‐induced paracrystalline inclusions may be influenced by the G15497A mtDNA mutation, and that CM mitigates against the pathological consequences of this mutation. Muscle Nerve 29: 537–547, 2004

Effects of nitric oxide donors on cybrids harbouring the mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) A3243G mitochondrial DNA mutation

Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential. However, endogenous levels of reactive oxygen species were very similar in all cybrids regardless of whether they carried the mtDNA defects or not. Furthermore, the cybrids were insensitive to the nitrosative and/or nitrative stress produced by either DETA-NO or SIN-1 alone. Cytotoxicity, however, was observed in response to SNP treatment and a combination of SIN-1 and glucose-deprivation. The mutant cybrids were significantly more sensitive to these insults compared with the wt controls. Ultrastructural examination of dying cells revealed several characteristic features of autophagic cell death. We concluded that nitrosative and/or nitrative stress alone were insufficient to trigger cytotoxicity in these cells, but cell death was observed with a combination of metabolic and nitrative stress. The vulnerability of the cybrids to these types of injury correlated with the cellular energy status, which were compromised by the MELAS mutation.

Cerebral ischemia causes dysregulation of synaptic adhesion in mouse synaptosomes

Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.