Developmentally Regulated Expression of mRNA for Neurotrophin High-Affinity (trk) Receptors within Chick Trigeminal Sensory Neurons

To investigate the distribution of neurons within the developing trigeminal sensory system which express mRNA for each of the three known high-affinity neurotrophin receptors (trk, trkB and trkC), we have performed in situ hybridization histochemistry on serial sections through the trigeminal ganglion and trigeminal mesencephalic nucleus at various ages of development using specific antisense oligonucleotide probes. We show that trkC mRNA is first expressed in the chicken embryo at stage 13, in presumptive neurons prior to the formation of the ganglion, that trkB mRNA labelling is initially observed within peripheral neurons slightly later, at stage 19, and that trk mRNA expression is not detectable until around embryonic day 3.5 (stage 21/22). The neurons which exhibit mRNA labelling for each of the high-affinity receptors occupy discrete regions within the ganglion, indicating that the ganglion comprises distinct neuronal subpopulations, each of which has a different capacity to respond to the different neurotrophins. Neurons which express trk mRNA are confined to the proximal region of the ganglion, whereas those which express trkB mRNA and trkC mRNA are located in two distinct regions within the distal aspect and also within the trigeminal mesencephalic nucleus. From the estimation of the number of neurons which exhibit labelling between embryonic days 9 and 18, we determined that the expression of mRNA for the high-affinity receptors changes during embryonic development of the ganglion. This is consistent with the observed differences in the response to neurotrophins in vitro.     

Dystrophin antisense oligonucleotides decrease expression of nNOS in human neurons

Nitric oxide (NO) plays an important role in the pathogenesis of neurodegenerative disease. It has been shown that neuronal NO synthase (nNOS), the enzyme that constitutively produces NO in brain, is a component of the dystrophin-associated protein complex. The absence of dystrophin causes Duchenne muscular dystrophy. Thus, we attempted to study whether or not a decrease of dystrophin expression would induce a modification in nNOS expression in cultured human neurons. Human fetal neuronal cultures were treated with antisense oligonucleotides against different isoforms of dystrophin and the expression of nNOS tested by RT-PCR and immunocytochemistry. Results showed that nNOS mRNA was significantly decreased by about 35% in neurons treated with brain-specific dystrophin (brain Dp427) antisense, whereas iNOS expression was not affected. Accordingly, a decrease in immunostaining for nNOS was observed in antisense treated neurons compared to controls. Expression of neuronal markers, such as bFGF or synaptophysin, was not affected by the same antisense treatment. Astrocytes were not affected by treatment, as shown by utrophin expression, a dystrophin-like protein that was not modified in pure astrocytic cultures. Thus, we conclude that a decrease of dystrophin in human neurons is associated with a decrease of nNOS expression.     

Cellular localization of messenger RNA encoding amyloid-beta-protein in normal tissue and in Alzheimer disease

Amyloid precursor protein (APP) gene encodes the short peptide fragment amyloid-beta-protein present in senile plaque cores, cerebrovascular amyloid, and intracellular neurofibrillary tangles in Alzheimer disease (AD). Using in situ hybridization with biotin-labeled RNA probes, we found distinctive patterns of APP gene expression in different regions of postmortem human brain. Strong hybridization signal for APP messenger RNA (mRNA) was detected in specific classes of neurons, fascicular oligodendroglia, satellite glia, and presumptive microglia. Weaker signal was seen in other neuronal classes, fascicular astrocytes, and vascular endothelial cells, but no signal was seen in protoplasmic astrocytes. Human thymus also shows a restricted pattern of hybridization with high signal in reticular epithelial cells, and much lower signal in lymphocytes. In AD patients, neuronal hybridization for APP mRNA was specifically increased in hippocampus, but not cerebellar and visual cortex when compared to hybridization for neuron-specific enolase mRNA. Most neurons with neurofibrillary tangles had strong APP mRNA signal. These results suggest that APP gene expression is highly regulated in normal tissue, that many different cell classes in brain express the APP gene, and that neuronal expression may increase specifically in brain regions where widespread injury occurs in AD. Amyloid deposits in brains of AD patients might be explained by local production of precursor protein in endothelial cells, neurons or glia.     

Analysis of human endothelial cells and cortical neurons for susceptibility to HIV-1 infection and co-receptor expression

Neuronal cell death is believed to be the underlying cause of neurological diseases and AIDS dementia often seen in human immunodeficiency virus (HIV) infected patients. The means by which HIV invades the brain is still unknown and the mechanism of neuronal cell death remains to be elucidated. The aim of this study was to determine if direct infection of human brain endothelial cells and neurons play a role in viral invasion of the brain and neuronal cell death, respectively. To this effect, we evaluated human brain microvascular endothelial cells (HBMEC) and human cortical neurons (HCN) for the expression of HIV co-receptors and their susceptibility to HIV-1 infection. While both HBMEC and HCN failed to express any CXCR4 and CCR5 on their cell surface, as assessed by flow cytometry, RT - PCR revealed the presence of CXCR4 and CCR5 mRNA in HBMEC but not in HCN. Two dual tropic HIV-1 primary isolates failed to infect both cell types as determined by p24 antigen capture ELISA, RT - PCR and DNA PCR. These data support the hypothesis that no productive infection of HBMEC and HCN occurs in vitro and suggest that other cell types are the primary focus of HIV-1 infection in the brain.     

Putative cyclooxygenase-3 expression in rat brain cells.

Cyclooxygenase-3 (COX-3), a new acetaminophen-sensitive isoform of the COX family, has recently been cloned from canine tissues. Canine COX-3 apparently is identical to the full-length form of COX-1, with the exception that the COX-3 mRNA retains intron 1. Additionally, COX-3 mRNA expression is high in the brain. We investigated the expression of the putative rat COX-3 mRNA in primary cultures of neurons, astrocytes, endothelial cells, pericytes, and choroidal epithelial cells from the rat brain. Specific RT-PCR primers were designed to detect putative rat COX-3 mRNA, and the RT-PCR products were sequenced and compared to the known sequence of the rat COX-1 gene. Our results demonstrate that the mRNA of the putative COX-3 is expressed in all of the cell types except neurons. Cerebral endothelial cells showed the highest COX-3 expression. Whereas COX-2 expression increased several-fold after lipopolysaccharide (LPS) challenge, COX-1 and COX-3 expression did not change significantly, suggesting that cells constitutively express COX-3. In summary, we report, for the first time to our knowledge, that the putative COX-3 mRNA is detectable in rats and is differentially expressed in various cell types from rat brain, as well as that its expression is not stimulated by LPS.     

Elevated complement C5a receptor expression on neurons and glia in astrocyte-targeted interleukin-3 transgenic mice

Evidence from several central nervous system (CNS) inflammatory disease models suggests that intrathecal complement synthesis may contribute to early inflammatory events in the brain. In this study, we examined the expression of the receptor for C5a (C5aR), a potent inflammatory and chemotactic factor, in the brains of transgenic mice with constitutive astrocyte expression of interleukin-3 (IL-3), a hematopoietic and immunomodulatory cytokine. By in situ hybridization, we demonstrated that cells infiltrating the cerebellar meninges, the cerebellum, and demyelinating lesions in the cerebellum were strongly positive for C5aR mRNA. By immunohistochemistry, the infiltrating cells expressing the C5aR were identified as macrophages based on staining with antibodies to the complement receptor type 3 and F4/80, a mouse macrophage-specific marker. In addition, some of the cells in cerebellar lesions were positive for the astrocyte-specific marker, glial fibrillary acidic protein, suggesting that a subpopulation of astrocytes in these lesions express elevated levels of the C5aR. Increased C5aR expression was also observed in cortical neurons in the occipital cortex and in pyramidal neurons in the cornu ammonis and subiculum of the hippocampus, at both the protein and mRNA levels. These data suggest that IL-3 may play an immunomodulatory role in C5aR expression on several cell types in the brain and that increased C5aR expression correlates with the pathology seen in this model. The transgenic mice used in this study provide a useful tool for characterizing the mechanism of regulation of the C5aR expression and for examining the functions of this chemotactic receptor in CNS inflammation. GLIA 24:338–345, 1998. © 1998 Wiley-Liss, Inc.     

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (～10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.     

Involvement of the calcium-independent receptor for alpha-latrotoxin in brain ischemia

Cerebral ischemia is caused by a reduced blood supply to neurons, and vulnerability to neurodegeneration varies considerably among neuronal types. In hippocampus, neurons in the CA1 region are more susceptible to ischemia-induced neuronal death than neurons in the CA3 region, and in response to transient forebrain ischemia a family of calcium-dependent receptors for alpha-latrotoxin is differentially expressed in the two regions. Here, we report that an ischemic insult up-regulated a family of calcium-independent receptors for alpha-latrotoxin (CIRL) mRNAs in CA1 neurons and down-regulated their mRNAs in CA3 neurons. Furthermore, antisense oligonucleotides complementary to CIRL-1 mRNA or CIRL-3 mRNA suppressed neuronal death associated with hypoxia in hippocampal and cortical cell cultures. The observed region-specific CIRL mRNA expression in hippocampus and an in vitro rescue experiment by antisense oligonucleotides against CIRL mRNAs suggest a functional importance of CIRL in neurodegeneration.     

CNS interleukin-3 (IL-3) expression and neurological syndrome in antisense-IL-3 transgenic mice.

Interleukin-3 (IL-3) is an important mediator of physiological and pathophysiological processes affecting the central nervous system (CNS). It stimulates the proliferation and activation of microglia and can enhance differentiation of cholinergic and sensory neurons. To examine the role of IL-3 in the CNS, we utilized transgenic mice expressing a murine antisense IL-3 (AS-IL-3) RNA under the control of the T cell B19 promoter so that expression is limited to hematopoietic cells. The AS-IL-3 transgenic mice develop either a progressive neurologic dysfunction, which includes ataxia, bradykinesia, and paralysis, or a lymphoproliferative syndrome. Histopathology demonstrated accumulations of reactive astrocytes in the cerebellum, brain stem, and spinal cord, accompanied by activated microglia. Partial loss of cerebellar nuclei neurons as well as neurons in the cranial nerve nuclei and spinal cord motor neurons is seen. Despite depletion of IL-3 peripherally, expression of IL-3 mRNA and protein is turned on in the CNS of the transgenic mice. Astrocytes cultured from the AS-IL-3 mice contain IL-3 mRNA and may thus be responsible for the activation of the microglia. This model should provide important insights into the role of cytokines in neurological disorders.     

The Adhesion Molecule on Glia (AMOG) Is Widely Expressed by Astrocytes in Developing and Adult Mouse Brain

Adhesion molecule on glia (AMOG) is a 45 - 50 kD cell surface glycoprotein structurally similar to the Na, K-ATPase beta-subunit and associated with the catalytic subunit of this enzyme. Previous immunofluorescence results had suggested that AMOG is transiently expressed on Bergmann glia during mouse cerebellar development, and antibody-inhibition results have implicated it in the migration of granule neurons. We report that, while AMOG mRNA is detected in Bergmann glia during the migratory period, this astrocyte derivative continues to express AMOG mRNA at similar levels in adult mice suggesting a functional role for AMOG in adulthood. Evidence from RNA and protein blot analyses that AMOG is present before birth, increasing about ten fold in adult mouse brain and cerebellum is also provided. RNA blot analysis of astrocyte-enriched cell populations and in situ hybridization results show that astrocytes synthesize AMOG mRNA in all regions of the developing and adult brain. In the adult, AMOG mRNA is more abundant in grey than white matter and, among grey matter regions, highest in cerebellar cortex. These results indicate a relationship between density of neuronal elements and AMOG expression. It is further speculated that AMOG is part of a Na,K-ATPase complex expressed preferentially by astrocytes in mouse brain.     

Systemic administration of kainic acid in adult rat stimulates expression of the chemokine receptor CCR5 in the forebrain.

As chemokines and their receptors are primarily expressed by glial cells in brain parenchyma, a model of glial cell proliferation may be useful to study the regulation of their expression in the brain. The well-established kainic acid seizure model was used in this study, focusing on the expression of the CCR5 chemokine receptor. Adult Sprague-Dawley rats were injected intraperitoneally with kainic acid (12 mg/kg), and in situ hybridization of CCR5 mRNA was performed at 12 h, 1, 3, or 7 days, posttreatment. Autoradiographic films and wet photographic emulsions demonstrated the very low expression of CCR5 mRNA in normal brain parenchyma, as well as in the microvasculature and ventricular/choroid plexus systems. After kainic acid treatment, brain CCR5 mRNA expression increased progressively from 12 h to 7 days, especially in the olfactory system, amygdaloid complex, thalamus, hippocampal formation, septum, and neocortex. This increase paralleled that of activated microglial cells as shown, using the microglial marker, OX-42. Moreover, CCR5 mRNA ISH combined with neuron-specific enolase immunocytochemistry showed that, in addition to its glial expression, CCR5 mRNA is expressed in neurons in the normal brain and, to a lesser extent, after kainate treatment due to neuronal losses. Finally, CCR5 protein is detected by immunocytochemistry in neurodegenerative areas in numerous glial cells, as well as in neurons, as clearly shown in the hippocampal formation. In summary, the chemokine receptor CCR5 is expressed by neuronal and non-neuronal cell types in the normal brain and is upregulated in both cell types after an insult.     

Expression of basic fibroblast growth factor receptor messenger RNA in the periinfarcted brain tissue

The present study examined whether expression of basic fibroblast growth factor receptor (bFGFR) messenger ribonucleic acid (mRNA) was upregulated by focal ischemia. We have studied the in situ hybridization autoradiography for bFGFR mRNA in the rat model of middle cerebral artery (MCA) occlusion. Male Wistar rats were used for occlusion of the left MCA, and were sacrificed 1, 3, 7 and 14 days after MCA occlusion. In situ hybridization was performed on the brain sections of these animals and sham controls by using 35S-labeled antisense and sense (control) RNA probes for rat bFGFR. Expression of bFGFR mRNA was observed in the periinfarcted area of the rats within 1-14 days after MCA occlusion. Expression was evident in the whole hemisphere of the infarcted side, especially at 1 and 3 days after ischemia, but no expression was detected in the contralateral side. On microautoradiograms, the signals of bFGFR mRNA were detected in both neurons and non-neural cells located in the periinfarcted area. Upregulation of bFGFR mRNA detected in the periinfarcted brain tissue suggests that receptor-mediated action of bFGF may be related to preservation of neurons injured by ischemia.     

Gene expression in TUNEL-positive neurons in human immunodeficiency virus-infected brain

Human immunodeficiency virus (HIV) infection of the central nervous system (CNS) results in neuronal damage and apoptosis, and both in vitro models and pathological studies suggest that a variety of neurotoxins released by HIV-infected and -activated macrophages/microglia selectively damage susceptible subsets of neurons. Confirmation of in vitro findings of mechanisms of neurodegeneration and neuronal cell dysfunction in vivo has been approached through detailed pathological analysis of regional structural damage, immunohistochemical detection of selected antigens in damaged cells, and, more recently, analysis of gene expression in whole tissue blocks or pooled populations (hundreds/thousands) of microdissected cells. Recently developed techniques of gene expression analysis through antisense mRNA amplification (aRNA) at the single-cell level may offer the potential to study pathways of neuronal cell death and to determine patterns of coordinated gene expression that may more specifically identify susceptible neuronal subclasses in vivo. Utilizing this unique technique, the authors have demonstrated, for the first time, RNA amplification and gene expression profiling in individual deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL)-reactive neurons microdissected from fixed, archival human brain tissue. RNA amplification was successful in >80% of TUNEL-positive neurons, and quantitative aRNA/cDNA hybridization slot-blot analysis demonstrated similar levels of actin RNA but significant differences in caspase-2 RNA expression between TUNEL-reactive and -nonreactive neurons. Reliable quantitative comparisons were achieved with modest numbers of sampled neurons (approximately 10). These studies suggest that analysis of coordinated gene expression in individual damaged neurons in vivo can be reliably used to identify neuronal subclasses that express certain susceptibility- or survival-promoting genes that may be targeted for more specific neuroprotective strategies against HIV.     

Expression of PTPRO during mouse development suggests involvement in axonogenesis and differentiation of NT-3 and NGF-dependent neurons

Competition and cooperation between type II and type III receptor protein tyrosine phosphatases (RPTPs) regulate axon extension and pathfinding in Drosophila. The first step to investigate whether RPTPs influence axon growth in the more complex vertebrate nervous system is to identify which neurons express a particular RPTP. We studied the expression of mouse PTPRO, a type III RPTP with an extracellular region containing eight fibronectin type III domains, during embryogenesis and after birth. Mouse PTPRO mRNA is expressed exclusively in two cell types: neurons and kidney podocytes. Maximal expression in the brain was coincident with mid to late gestation and axonogenesis in the brain. We cloned two cDNAs, including a splice variant without sequence coding of 28 amino acids within the juxtamembrane domain that was found mostly in kidney. In situ hybridization detected mPTPRO mRNA in the cerebral cortex, olfactory bulb and nucleus, hippocampus, motor neurons, and the spinal cord midline. In addition, mPTPRO mRNA was found throughout dorsal root, cranial, and sympathetic ganglia and within kidney glomeruli. Mouse PTPRO mRNA was observed in neuron populations expressing TrkA, the high-affinity nerve growth factor receptor, or TrkC, the neurotrophin-3 receptor, and immunoreactive mPTPRO and TrkC colocalized in large dorsal root ganglia proprioceptive neurons. Our results suggest that mPTPRO is involved in the differentiation and axonogenesis of central and peripheral nervous system neurons, where it is in a position to modulate intracellular responses to neurotrophin-3 and/or nerve growth factor.     

Rat brain 5-HT1C receptors are encoded by a 5-6 kbase mRNA size class and are functionally expressed in injected Xenopus oocytes

Injection of rat brain RNA into Xenopus laevis oocytes induces synthesis of receptors that show an electrophysiological response to bath application of serotonin. While there are at least 4 pharmacologically distinct subtypes of 5-HT binding sites in the rat brain, we find that the pharmacological characteristics of the predominant electrophysiologically active receptor synthesized in Xenopus oocytes are most consistent with those of the 5-HT1C subtype. Additional electrophysiologically active 5-HT receptor types could not be detected. Injection of mRNA isolated from a number of rat brain regions shows that the choroid plexus is particularly enriched for 5-HT1C mRNA. Oocytes injected with RNA isolated from this region respond 16 or 8 times more strongly to serotonin than do oocytes injected with RNA isolated from cortex or substantia nigra, respectively. In addition, by fractionation of rat brain mRNA through agarose gels, we have identified a single RNA size class of about 5-6 kbase that encodes this serotonin receptor.     

Colocalization of estrogen receptor alpha and NMDA-2D mRNAs in amygdaloid and hypothalamic nuclei of the mouse brain

Interactions between gonadal steroid hormones and glutamatergic neurons participate in limbic and hypothalamic functions. Glutamate receptors are divided into metabotropic and ionotropic receptors. Among ionotropic receptors, N-methyl-D-aspartate (NMDA) is involved in a variety of neurophysiological processes. In turn, NMDA receptors are composed of subunits from two families: NR1 and NR2. Recently, molecular studies have shown that the expression of NMDA-2D receptor is regulated by estrogen. Although the expression patterns of NMDA-2D and ERalpha in the rodent brain appear to overlap, it remained to be determined whether or not these two receptors co-exist, in vivo, at the level of single neurons. To test the hypothesis that NMDA-2D and ERalpha messenger ribonucleic acid (mRNA) are co-expressed in the same neurons of the adult mouse brain, we used a dual-label in situ hybridization technique. Neuronal populations were identified with digoxigenin-tagged complementary RNA probes for NMDA-2D and 35S-labeled cRNA probes for ERalpha. Our results demonstrate that a majority of the ERalpha-positive neurons also express NMDA-2D mRNA. Quantitative examination of the cellular expression in the ventromedial and arcuate nuclei of the hypothalamus (VMH and Arc) showed that 52.5% and 61.5%, respectively, of the neurons endowed with ERalpha mRNA also contain NMDA-2D mRNA. In the amygdala, 51% of ERalpha-positive cells also contain NMDA-2D mRNA. These findings provide the first anatomical evidence that ER and NMDA-2D receptors can be found in the same hypothalamic and amygdaloid neurons. Co-expression of ERalpha and NMDA-2D receptors supports the hypothesis of the interactions between glutamate receptors and estrogens in brain regions where estrogens control female reproductive behaviors and neuroendocrine functions.