Expression of somatostatin and somatostatin receptor subtypes in Apolipoprotein D (ApoD) knockout mouse brain: An immunohistochemical analysis.

Apolipoprotein D (ApoD) is widely distributed in central and peripheral nervous system. ApoD expression has been shown to increase in several neurodegenerative and neuropsychiatric disorders, as well as during regeneration in the nervous system. Like ApoD, in the central nervous system somatostatin (SST) is widely present and functions as neurotransmitter and neuromodulator. The biological effects of SST are mediated via binding to five high-affinity G-protein coupled receptors termed SSTR1-5. Mice lacking ApoD exhibit reduced SST labeling in cortex and hippocampus and increased expression in striatum and amygdala without any noticeable changes in substantia nigra. Changes in SSTRs expressions have been described in several neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's diseases. In the present study, using SSTR1-5 receptor-specific antibodies, we mapped their distribution in wild type (wt) and ApoD knockout (ApoD(-/-)) mouse brain. SSTR1-5 expression was observed both as membrane and cytoplasmic protein and display regions and receptor specific differences between wt and ApoD(-/-) mice brains. In cortex and hippocampus, SSTR subtypes like immunoreactivity are decreased in ApoD(-/-) mice brain. Unlike cortex and hippocampus, in the striatum of ApoD(-/-) mice, projection neurons showed increased SSTR immunoreactivity, as compared to wt. Higher SSTR subtypes immunoreactivity is seen in substantia nigra pars compacta (SNpc) whereas lower in substantia nigra pars reticulata (SNpr) of ApoD(-/-) mice brains as compared to wt. Whereas, amygdala displayed SSTR subtypes changes in different nuclei of ApoD(-/-) mice in comparison to wt mice brain. Taken together, our results describe receptor and region specific changes in SST and SSTR subtypes expression in ApoD(-/-) mice brain, which may be linked to specific neurological disorders.

Apolipoprotein D modulates F2-isoprostane and 7-ketocholesterol formation and has a neuroprotective effect on organotypic hippocampal cultures after kainate-induced excitotoxic injury.

Apolipoprotein D (apoD), a member of the lipocalin family of transporter proteins binds a number of small lipophilic molecules including arachidonic acid and cholesterol. Recent studies showed a protective function of mammalian apoD as well as its insect and plant homologs against oxidative stress. In this study we investigated the effect of direct addition of exogenous human apoD protein purified from breast cystic fluid to rat hippocampal slice cultures after excitotoxic injury induced by the glutamate analog kainate. ApoD at a concentration of 10 microg/ml partially prevented loss of MAP2 immunostaining and LDH release from injured hippocampal neurons after kainate injury. ApoD also attenuated the increase in oxidative products of arachidonic acid and cholesterol, F(2)-isoprostanes and 7-ketocholesterol, respectively, after kainate treatment. In view of the molecular structure of apoD which consists of an eight stranded beta barrel that forms a binding pocket for a number of small hydrophobic molecules, we propose that apoD promotes its neuroprotective effects by binding to arachidonic acid and cholesterol thus preventing their oxidation to neurotoxic products such as 4-hydroxynonenal (4-HNE) and 7-ketocholesterol.

Immunohistochemical localization of dopamine receptor subtypes (D1R-D5R) in Alzheimer's disease brain.

Among the neurotransmitter abnormalities that have been investigated in Alzheimer's disease (AD), deficits in the cholinergic system have been the most intensively studied. Another key neurotransmitter system involved with emotion and cognition is the dopaminergic system. Here we have investigated alterations in all five dopamine receptor subtypes in AD brain. Using antipeptide rabbit antibodies for each of the five dopamine receptors (D1-D5) we mapped the distribution of these receptors in postmortem AD and age-matched control brains in the frontal cortex, utilizing biotin-avidin immunocytochemistry. All five DR subtypes were expressed as cell surface and cytoplasmic proteins. Receptor-specific changes in control and AD brain were identified as follows: D4R and D3R were the predominant receptor subtypes in age-matched controls followed by D2R and D1R; D5R is the least expressed receptor subtype. In AD brain, D2R and D5R are well expressed in comparison to D1R, D3R and D4R. Expression of D1R, D3R and D4R was severely reduced in AD cortex. D2R expression is moderately reduced in the frontal cortex of AD brain. D5R is the only receptor subtype whose expression is increased in AD frontal cortex. Furthermore, in AD, we found comparable expression of D3R in astrocytes, whereas D5R-like immunoreactivity is significantly increased in astrocytes, in comparison to normal frontal cortex, where it was predominantly neuronal. These results demonstrate subtype-specific changes in dopamine receptors in AD that may be important in disease pathophysiology and that may also serve as potential targets for therapeutic intervention in AD.

Lovastatin modulates increased cholesterol and oxysterol levels and has a neuroprotective effect on rat hippocampal neurons after kainate injury.

This study was carried out to elucidate the effect of a brain-permeable statin (lovastatin) on cholesterol and oxysterol levels of the hippocampus after neuronal injury induced by the excitotoxin, kainic acid. Increased immunolabeling to cholesterol and the oxysterol biosynthetic enzyme, cholesterol 24-hydroxylase, was observed in the rat hippocampus after kainate lesions. This was accompanied by increased levels of cholesterol, 24-hydroxycholesterol (product of cholesterol 24-hydroxylase enzymatic activity), and 7-ketocholesterol in homogenates of the degenerating hippocampus as detected by gas chromatography/mass spectrometry. Hippocampi from rats or organotypic slices that had been treated with kainate plus lovastatin showed significantly lower levels of cholesterol, 24-hydroxycholesterol, and 7-ketocholesterol compared with those that had been treated with kainate only. Lovastatin also modulated hippocampal neuronal loss after kainate treatment in vivo and in vitro. The level of 24-hydroxycholesterol detected in vivo after kainate treatment (>50 microM) was found to be neurotoxic in hippocampal slice cultures. These results suggest that brain-permeable statins such as lovastatin could have a neuroprotective effect by limiting the levels of oxysterol in brain areas undergoing neurodegeneration.

Expression of the carrier protein apolipoprotein D in the mouse inner ear.

The cochlear portion of the inner ear converts movements produced by sound waves into electrical impulses. Transcripts enriched in the cochlea are likely to have an important role in hearing. In this paper, we report that microarray analyses of the Soares NMIE inner ear library revealed cochlear enriched expression of apolipoprotein D (apoD), a glycoprotein and member of the lipocalin family that transport small hydrophobic ligands. The cochlear enriched expression of Apod was validated by quantitative real time PCR analysis. To investigate the function of apoD in the inner ear the transcript and protein were localised in the cochlea. Apod messenger RNA (mRNA) expression was localised to the spiral ligament and spiral limbus, particularly in the suprastrial and supralimbral regions. The apoD protein was detected in the spiral ligament, spiral limbus and also in the outer hair cells of the organ of Corti. Investigation of cell lines exhibiting characteristics of hair and supporting cells revealed no Apod mRNA expression in these cells. This suggests transport of the protein within the cochlea, followed by internalisation into outer hair cells. The spiral limbus and ligament contain subpopulations of fibrocytes that are intimately involved in regulation of ion balance in the cochlear fluids and type I, II and III fibrocytes of the spiral ligament were all shown to be positive for apoD protein. On the basis of these results it was hypothesised that apoD could be involved in maintaining cochlear fluid homeostasis. To determine whether the apoD gene product was important for normal auditory function the hearing ability of an apoD knockout mouse was tested. The mouse was found to have a hearing threshold that was not significantly different to the control strain.

Increase in cholesterol and cholesterol oxidation products, and role of cholesterol oxidation products in kainate-induced neuronal injury.

Little is known about changes in sterols, in particular cholesterol, and cholesterol oxidation products (COPs) in oxidative injury in neural tissues. We have therefore examined changes in cholesterol and COPs using a model of excitotoxic injury. Intracerebroventricular injections of kainate in rats resulted in an increase in immunoreactivity to cholesterol in the affected CA fields of the hippocampus. The increase was confirmed by increased filipin staining of cholesterol in adjacent sections from the same animals, and in hippocampal slice or neuronal cultures after kainate treatment. In neuronal cultures, addition of lovastatin, an inhibitor of cholesterol synthesis, attenuated the increased filipin staining after kainate treatment, indicating that the increase in cholesterol could involve increased cholesterol synthesis. Furthermore, gas chromatographic mass spectrometric (GC/MS) analysis of cholesterol and COPs in kainate-injected rat brain showed a marked increase in cholesterol and COPs including 7-ketocholesterol, 3 days after kainate treatment. The addition of some COPs, including 7-ketocholesterol and cholesterol epoxides to hippocampal slices resulted in neuronal injury as reflected by decreased staining of a neuronal marker in the affected CA fields. The ability of these COPs to produce neuronal injury was attenuated by glutathione, suggesting that oxidative mechanisms are involved in neuronal injury induced by these products. These results, together with GC/MS results that showed significant increase in 7-ketocholesterol at 3 days post-kainate injury suggest that 7-ketocholesterol may be a factor in aggravating oxidative damage to neurons, after the initial stages of kainate-induced neuronal injury.

Differential expression of D2-like dopamine receptors in the kidney of the spontaneously hypertensive rat.

OBJECTIVE: To compare the expression and cellular distribution of D(2)-like dopamine receptors in the kidney of the spontaneously hypertensive rat (SHR) and normotensive Wistar-Kyoto (WKY) rat. DESIGN: Renal D(2)-like receptor protein expression and distribution has not been studied in the SHR. Since changes in D(2)-like receptor expression and/or distribution may contribute to the dysregulation of renal dopamine and D(1A) receptor function, we examined the expression of the three subtypes of D(2)-like receptors (D(2), D(3) and D(4)) in SHR and WKY rat kidneys. METHODS: Western blot analysis and confocal immunocytochemistry with specific polyclonal antipeptide antibodies directed against the receptor subtypes, were used to assess protein expression. RESULTS: There were no differences in protein expression and cellular immunolocalization of the D(2) receptor subtypes between SHR and WKY rats. Expression of the 50 kDa D(3) receptor was reduced in the cortex of the SHR; no differences in D(3) receptor levels were seen in the inner medulla of SHR and WKY rats. The D(4) receptor polypeptides were overexpressed in the cortex of SHR, while in the inner medulla no difference in expression of the D(4) receptor proteins was observed between SHR and WKY rats. Immunocytochemistry also showed increased immunostaining of D(4) receptors in tubular structures in the cortex, but diminished staining in the SHR inner medulla. CONCLUSION: The observed differences in expression and distribution of D(3) and D(4) dopamine receptors between cortex and inner medulla of the kidneys of SHR and WKY rats may contribute to the aberrant state of dopaminergic-mediated natriuresis in SHR.

Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells.

Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo- and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity not only within the SSTR family but more generally in the HHR family.

Apolipoprotein D in the Niemann-Pick type C disease mouse brain: an ultrastructural immunocytochemical analysis.

Enhanced apoD gene expression and abnormally high levels of apoD protein accumulation in the brain have been previously documented as features of the neurodegenerative disorder, Niemann-Pick Type C disease (NP-C). In the present study we have used immunocytochemistry and light and electron microscopy to elucidate the cellular and subcellular distribution of apoD in the Balb/c NIH npc1(-/-) mouse brain. The normal mouse brain demonstrates low levels of apoD-expressing glia particularly in the cerebellar white matter. In contrast, abundant, strongly apoD-immunolabeled cells were observed in select grey matter nuclei, including the globus pallidus, thalamus, and substantia nigra, and in white matter tracts within the internal capsule and cerebellum of NP-C mouse brain. These brains regions have been previously shown to display the most significant neurodegenerative changes in the NP-C mouse. Ultrastructural analysis revealed dense apoD immunoreactivity on the nuclear envelopes of cells that have the morphological features of oligodendrocyte precursor-like cells and light staining on astrocytes. These results define the cellular and subcellular pattern of expression of apoD in NP-C mouse brain and suggest a possible role for this lipocalin in the pathophysiology of this disorder.