Daniel Garcia-Ovejero

Glial expression of estrogen and androgen receptors after rat brain injury

Estrogens and androgens can protect neurons from death caused by injury to the central nervous system. Astrocytes and microglia are major players in events triggered by neural lesions. To determine whether glia are direct targets of estrogens or androgens after neural insults, steroid receptor expression in glial cells was assessed in two different lesion models. An excitotoxic injury to the hippocampus or a stab wound to the parietal cortex and hippocampus was performed in male rats, and the resultant expression of steroid receptors in glial cells was assessed using double‐label immunohistochemistry. Both lesions induced the expression of estrogen receptors (ERs) and androgen receptors (ARs) in glial cells. ERα was expressed in astrocytes immunoreactive (ERα‐ir) for glial fibrillary acidic protein or vimentin. AR immunoreactivity colocalized with microglial markers, such as Griffonia simplicifolia lectin‐1 or OX‐6. The time course of ER and AR expression in glia was studied in the stab wound model. ERα‐ir astrocytes and AR‐ir microglia were observed 3 days after lesion. The number of ERα‐ir and AR‐ir glial cells reached a maximum 7 days after lesion and returned to low levels by 28 days postinjury. The studies of ERβ expression in glia were inconclusive; different results were obtained with different antibodies. In sum, these results suggest that reactive astrocytes and reactive microglia are a direct target for estrogens and androgens, respectively. J. Comp. Neurol. 450:256–271, 2002.

Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury

Reactive microgliosis is a highly characteristic response to neural injury and disease, which may influence neurodegenerative processes and neural plasticity. We have investigated the origin and characteristics of reactive microglia in the acute phase of their activation in the dentate gyrus following transection of the entorhino‐dentate perforant path projection. To investigate the possible link between microglia and hematopoietic precursors, we analyzed the expression of the stem cell marker CD34 by lesion‐reactive microglia in conjunction with the proliferation marker bromodeoxyuridine (BrdU) and the use of radiation bone marrow (BM) chimeric mice. We found that CD34 is upregulated on early‐activated resident microglia, rather than by infiltrating bone marrow‐derived cells. The number of CD34+ microglia peaked at day 3 when 67% of the resident CD11b/Mac‐1+ microglia co‐expressed CD34, and all CD34+ cells co‐expressed Mac‐1, and decreased sharply toward day 5, unlike Mac‐1, which was maximally expressed at day 5. Approximately 80% of the CD34+ cells in the denervated dentate gyrus had incorporated BrdU into their nuclei at day 3. We also showed that CD34 is upregulated on early‐activated microglia in the facial motor nucleus following peripheral axotomy. The results suggest lesion‐reactive microglia to consist of functionally distinct subpopulations of cells; a major population of activated resident CD34+Mac‐1+ microglia with a high capacity for self‐renewal, and a subpopulation of CD34−Mac‐1+ microglia which has a mixed extrinsic and intrinsic origin and whose proliferative capacity is unknown.

Testosterone decreases reactive astroglia and reactive microglia after brain injury in male rats: role of its metabolites, oestradiol and dihydrotestosterone

Previous studies have shown that the neuroprotective hormone, testosterone, administered immediately after neural injury, reduces reactive astrogliosis. In this study we have assessed the effect of early and late therapy with testosterone or its metabolites, oestradiol and dihydrotestosterone, on reactive astroglia and reactive microglia after a stab wound brain injury in orchidectomized Wistar rats. Animals received daily s.c. injections of testosterone, oestradiol or dihydrotestosterone on days 0–2 or on days 5–7 after injury. The number of vimentin immunoreactive astrocytes and the volume fraction of major histocompatibility complex‐II (MHC‐II) immunoreactive microglia were estimated in the hippocampus in the lateral border of the wound. Both early and delayed administration of testosterone or oestradiol, but not dihydrotestosterone, resulted in a significant decrease in the number of vimentin‐immunoreactive astrocytes. The volume fraction of MHC‐II immunoreactive microglia was significantly decreased in the animals that received testosterone or oestradiol in both early and delayed treatments and in animals that received early dihydrotestosterone administration. Thus, both early and delayed administration of testosterone reduces reactive astroglia and reactive microglia and these effects may be at least in part mediated by oestradiol, while dihydrotestosterone may mediate part of the early effects of testosterone on reactive microglia. In conclusion, testosterone controls reactive gliosis and its metabolites, oestradiol and dihydrotestosterone, may be involved in this hormonal effect. The regulation of gliosis may be part of the neuroprotective mechanism of testosterone.

CB2 cannabinoid receptors promote mouse neural stem cell proliferation

Neurospheres are clonal cellular aggregates of neural stem/precursor cells that grow in culture as free‐floating clusters. Activation of CB1 cannabinoid receptors, which are expressed by these cells, promotes proliferation. In the present study we investigated the expression of CB2 cannabinoid receptors and the effect of exogenous cannabinoids on neural stem/precursor cell proliferation. Neurospheres containing nestin‐positive and sn‐1 diacylglycerol lipase α‐positive cells expressed both CB1 and CB2 receptors, which were maintained through several passages. Application of the non‐selective cannabinoid agonist (HU‐210, 0.5 µm) stimulated bromodeoxyuridine incorporation and neurosphere formation. This action involved both CB1 and CB2 receptors as neurosphere formation was stimulated by either selective CB1 [arachidonyl‐2’chloroethylamide/(all Z)‐N‐(2‐cycloethyl)‐5,8,11,14‐eicosatetraenamide (ACEA), 200 nm and 1 µm] or CB2 (JWH‐056, 0.5 µm) agonists. In addition, CB1 or CB2 antagonists (1 µm SR‐141716A and SR‐144528, respectively) blocked basal proliferation, suggesting that endogenous cannabinoids are implicated in neurosphere proliferation. In addition, cannabinoid agonist‐stimulated proliferation was reduced by the Akt translocation inhibitor BML‐257 (12.5 µm), suggesting a role for phosphoinositide‐3 kinase signalling. Together, our results suggest that cannabinoids stimulate proliferation of neural stem/precursor cells acting on both CB1 and CB2 cannabinoid receptors through a phosphoinositide‐3 kinase/Akt pathway.

The endocannabinoid system is modulated in response to spinal cord injury in rats

Endocannabinoids are lipid mediators with protective effects in many diseases of the nervous system. We have studied the modulation of the endocannabinoid system after a spinal cord contusion in rats. In early stages, lesion induced increases of anandamide and palmitoylethanolamide (PEA) levels, an upregulation of the synthesizing enzyme NAPE-phospholipase D and a downregulation of the degradative enzyme FAAH. In delayed stages, lesion induced increases in 2-arachidonoylglycerol and a strong upregulation of the synthesizing enzyme DAGL-alpha, that is expressed by neurons, astrocytes and immune infiltrates. The degradative enzyme MAGL was also moderately increased but only 7 days after the lesion. We have studied the cellular targets for the newly formed endocannabinoids using RT-PCR and immunohistochemistry against CB(1) and CB(2) receptors. We observed that CB(1) was constitutively expressed by neurons and oligodendrocytes and induced in reactive astrocytes. CB(2) receptor was strongly upregulated after lesion, and mostly expressed by immune infiltrates and astrocytes. The endocannabinoid system may represent an interesting target for new therapeutical approaches to spinal cord injury.

Classical androgen receptors in non-classical sites in the brain.

Androgen receptors are expressed in many different neuronal populations in the central nervous system where they often act as transcription factors in the cell nucleus. However, recent studies have detected androgen receptor immunoreactivity in neuronal and glial processes of the adult rat neocortex, hippocampal formation, and amygdala as well as in the telencephalon of eastern fence and green anole lizards. This review discusses previously published findings on extranuclear androgen receptors, as well as new experimental results that begin to establish a possible functional role for androgen receptors in axons within cortical regions. Electron microscopic studies have revealed that androgen receptor immunoreactive processes in the rat brain correspond to axons, dendrites and glial processes. New results show that lesions of the dorsal CA1 region by local administration of ibotenic acid reduce the density of androgen receptor immunoreactive axons in the cerebral cortex and the amygdala, suggesting that these axons may originate in the hippocampus. Androgen receptor immunoreactivity in axons is also decreased by the intracerebroventricular administration of colchicine, suggesting that androgen receptor protein is transported from the perikaryon to the axons by fast axonal transport. Androgen receptors in axons located in the cerebral cortex and amygdala and originating in the hippocampus may play an important role in the rapid behavioral effects of androgens.

CB2 cannabinoid receptors as an emerging target for demyelinating diseases : from neuroimmune interactions to cell replacement strategies

Amongst the various demyelinating diseases that affect the central nervous system, those induced by an inflammatory response stand out because of their epidemiological relevance. The best known inflammatory‐induced demyelinating disease is multiple sclerosis, but the immune response is a common pathogenic mechanism in many other less common pathologies (e.g., acute disseminated encephalomyelitis and acute necrotizing haemorrhagic encephalomyelitis). In all such cases, modulation of the immune response seems to be a logical therapeutic approach. Cannabinoids are well known immunomodulatory molecules that act through CB1 and CB2 receptors. While activation of CB1 receptors has a psychotropic effect, activation of CB2 receptors alone does not. Therefore, to bypass the ethical problems that could result from the treatment of inflammation with psychotropic molecules, considerable effort is being made to study the potential therapeutic value of activating CB2 receptors. In this review we examine the current knowledge and understanding of the utility of cannabinoids as therapeutic molecules for inflammatory‐mediated demyelinating pathologies. Moreover, we discuss how CB2 receptor activation is related to the modulation of immunopathogenic states.

Cannabinoids modulate Olig2 and polysialylated neural cell adhesion molecule expression in the subventricular zone of post-natal rats through cannabinoid receptor 1 and cannabinoid receptor 2.

The subventricular zone (SVZ) is a source of post‐natal glial precursors that can migrate to the overlying white matter, where they may differentiate into oligodendrocytes. We showed that, in the post‐natal SVZ ependymocytes, radial glia and astrocyte‐like cells express cannabinoid receptor 1 (CB1), whereas cannabinoid receptor 2 (CB2) is found in cells expressing the polysialylated neural cell adhesion molecule. To study CB1 and CB2 function, post‐natal rats were exposed to selective CB1 or CB2 agonists (arachidonyl‐2‐chloroethylamide and JWH‐056, respectively) for 15 days. Accordingly, we found that CB1 activation increases the number of Olig2‐positive cells in the dorsolateral SVZ, whereas CB2 activation increases polysialylated neural cell adhesion molecule expression in this region. As intense myelination occurs during the first weeks of post‐natal development, we examined how modulating these factors affected the expression of myelin basic protein. Pharmacological administration of agonists and antagonists of CB1 and CB2 showed that the activation of both receptors is needed to augment the expression of myelin basic protein in the subcortical white matter.

The constitutive production of the endocannabinoid 2-arachidonoylglycerol participates in oligodendrocyte differentiation

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid 2‐arachidonoylglycerol (2‐AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLβ), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and β abundantly, resulting in the stronger production of 2‐AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB2 receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC‐80267 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous 2‐AG. Likewise, 2‐AG synthesis disruption using specific siRNAs against DAGLα and DAGLβ significantly reduced myelin protein expression in vitro, whereas a pharmacological gain‐of‐function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC‐80267 all diminished basal ERK1/2 phosphorylation, effects that were partially reversed by the addition of 2‐AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of 2‐AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.

The endocannabinoid system modulates a transient TNF pathway that induces neural stem cell proliferation

Evidence is emerging that the tumour necrosis factor (TNF-alpha) is a potent signal that induces neural stem cell proliferation and migration. We show that NSC self-renewal is controlled by bi-directional cross-talk between the endocannabinoid system and the TNF signalling pathway. By blocking endogenous TNF-alpha activity, we demonstrate that the TNF system is critical for the proliferation of NSC. Furthermore, we show that pharmacological blockade of the CB1/CB2 cannabinoid receptors dramatically suppresses TNF-alpha-induced NSC proliferation. Interestingly, we found that CB1 or CB2 agonists induce NSC proliferation coupled to a significant increase in both TACE/ADAM 17 and TNF-alpha levels. Overall these data suggest a novel mode of action for the endocannabinoid system in NSC proliferation that is coupled to TNF signalling and that may be of therapeutic interest in the emerging field of brain repair.