apoD mRNA Research Articles

Identifying mRNAs Residing in Myelinating Oligodendrocyte Processes as a Basis for Understanding Internode Autonomy.

In elaborating and maintaining myelin sheaths on multiple axons/segments, oligodendrocytes distribute translation of some proteins, including myelin basic protein (MBP), to sites of myelin sheath assembly, or MSAS. As mRNAs located at these sites are selectively trapped in myelin vesicles during tissue homogenization, we performed a screen to identify some of these mRNAs. To confirm locations, we used real-time quantitative polymerase chain reaction (RT-qPCR), to measure mRNA levels in myelin (M) and 'non-myelin' pellet (P) fractions, and found that five (LPAR1, TRP53INP2, TRAK2, TPPP, and SH3GL3) of thirteen mRNAs were highly enriched in myelin (M/P), suggesting residences in MSAS. Because expression by other cell-types will increase p-values, some MSAS mRNAs might be missed. To identify non-oligodendrocyte expression, we turned to several on-line resources. Although neurons express TRP53INP2, TRAK2 and TPPP mRNAs, these expressions did not invalidate recognitions as MSAS mRNAs. However, neuronal expression likely prevented recognition of KIF1A and MAPK8IP1 mRNAs as MSAS residents and ependymal cell expression likely prevented APOD mRNA assignment to MSAS. Complementary in situ hybridization (ISH) is recommended to confirm residences of mRNAs in MSAS. As both proteins and lipids are synthesized in MSAS, understanding myelination should not only include efforts to identify proteins synthesized in MSAS, but also the lipids.

LCAT, ApoD, and ApoA1 Expression and Review of Cholesterol Deposition in the Cornea.

Lecithin:cholesterol acyltransferase (LCAT) is an enzyme secreted by the liver and circulates with high-density lipoprotein (HDL) in the blood. The enzyme esterifies plasma cholesterol and increases the capacity of HDL to carry and potentially remove cholesterol from tissues. Cholesterol accumulates within the extracellular connective tissue matrix of the cornea stroma in individuals with genetic deficiency of LCAT. LCAT can be activated by apolipoproteins (Apo) including ApoD and ApoA1. ApoA1 also mediates cellular synthesis of HDL. This study examined the expression of LCAT by epithelial cells, keratocytes, and endothelial cells, the cell types that comprise from anterior to posterior the three layers of the cornea. LCAT and ApoD were immunolocalized to all three cell types within the cornea, while ApoA1 was immunolocalized to keratocytes and endothelium but not epithelium. In situ hybridization was used to detect LCAT, ApoD, and ApoA1 mRNA to learn what cell types within the cornea synthesize these proteins. No corneal cells showed mRNA for ApoA1. Keratocytes and endothelium both showed ApoD mRNA, but epithelium did not. Epithelium and endothelium both showed LCAT mRNA, but despite the presence of LCAT protein in keratocytes, keratocytes did not show LCAT mRNA. RNA sequencing analysis of serum-cultured dedifferentiated keratocytes (commonly referred to as corneal stromal fibroblasts) revealed the presence of both LCAT and ApoD (but not ApoA1) mRNA, which was accompanied by their respective proteins detected by immunolabeling of the cultured keratocytes and Western blot analysis of keratocyte lysates. The results indicate that keratocytes in vivo show both ApoA1 and LCAT proteins, but do not synthesize these proteins. Rather, keratocytes in vivo must take up ApoA1 and LCAT from the corneal interstitial tissue fluid.

Increased Expression of Apo-J and Omi/HtrA2 After Intracerebral Hemorrhage in Rats

To investigate the changes of Apo-J and Omi/HtrA2 protein expression in rats with intracerebral hemorrhage. 150 Sprague-Dawley adult rats were randomly divided into 3 groups: (1) normal control (NC) group, (2) sham group, and (3) intracerebral hemorrhage (ICH) group. The data were collected at 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick-end labeling staining. The distributions of the Apo-J and Omi/HtrA2 proteins were determined by immunohistochemical staining. The levels of Apo-J mRNA and Omi/HtrA2 mRNA expressions were examined by real-time polymerase chain reaction. Apoptosis in the ICH group was higher than in the sham and NC groups (P < 0.05). Both the Apo-J and Omi/HtrA2 expression levels were increased in the peripheral region of hemorrhage, with a peak at 3 days. The Apo-J mRNA level positively correlated with the HtrA2 mRNA level in the ICH group (r= 0.883, P < 0.001). The expressions of Apo-J and Omi/HtrA2 increased in parallel in the peripheral region of rat cerebral hemorrhage. Local high expression of Apo-J in the peripheral regions may play a neuroprotective role by inhibiting apoptosis via the Omi/HtrA2 pathway after hemorrhage.

Differential expression of apolipoprotein D in male reproductive system of rats by high-fat diet.

Apolipoprotein D, a 29-kDa secreted glycoprotein that belongs to the lipocalin superfamily, is widely expressed in various tissues and associated with lipid metabolism as a component of high-density lipoproteins. Although Apolipoprotein D binds to small hydrophobic ligands including cholesterol, little is known about effects of high-fat diet with cholesterol on expression of Apolipoprotein D in the male reproductive tract. Therefore, we investigated Apod expression in penises, prostate glands, and testes from rats fed a high-fat diet including a high amount of cholesterol. Our previous research indicated that a high-fat diet induces dyslipidemia leading to histological changes and dysfunction of male reproduction in rats. Consistent with these results, Apod mRNA expression was significantly (p<0.001) decreased in penises and prostate glands (p<0.01) and testes (p<0.01) from rats fed a high-fat diet as compared with normal diet. In addition, Apod mRNA and protein were detected predominantly in urethral epithelium and penile follicle from rats. Moreover, changes in expression of specific microRNAs (miR-229b-3p, miR-423-3p, and miR-490-3p) regulating Apod in the penises and prostate glands were negatively associated with Apod expression. Collectively, results of this study suggest that Apod is a novel regulatory gene in the male reproductive system, especially in penises of rats fed a high-cholesterol diet, and that expression of Apod is regulated at the posttranscriptional level by target microRNAs.

Neuronal Uptake and Neuroprotective Properties of Curcumin-Loaded Nanoparticles on SK-N-SH Cell Line: Role of Poly(lactide-co-glycolide) Polymeric Matrix Composition.

Curcumin, a neuroprotective agent with promising therapeutic approach has poor brain bioavailability. Herein, we demonstrate that curcumin-encapsulated poly(lactide-co-glycolide) (PLGA) 50:50 nanoparticles (NPs-Cur 50:50) are able to prevent the phosphorylation of Akt and Tau proteins in SK-N-SH cells induced by H2O2 and display higher anti-inflammatory and antioxidant activities than free curcumin. PLGA can display various physicochemical and degradation characteristics for controlled drug release applications according to the matrix used. We demonstrate that the release of curcumin entrapped into a PLGA 50:50 matrix (NPs-Cur 50:50) is faster than into PLGA 65:35. We have studied the effects of the PLGA matrix on the expression of some key antioxidant- and neuroprotective-related genes such as APOE, APOJ, TRX, GLRX, and REST. NPs-Cur induced the elevation of GLRX and TRX while decreasing APOJ mRNA levels and had no effect on APOE and REST expressions. In the presence of H2O2, both NPs-Cur matrices are more efficient than free curcumin to prevent the induction of these genes. Higher uptake was found with NPs-Cur 50:50 than NPs-Cur 65:35 or free curcumin. By using PLGA nanoparticles loaded with the fluorescent dye Lumogen Red, we demonstrated that PLGA nanoparticles are indeed taken up by neuronal cells. These data highlight the importance of polymer composition in the therapeutic properties of the nanodrug delivery systems. Our study demonstrated that NPs-Cur enhance the action of curcumin on several pathways implicated in the pathophysiology of Alzheimer's disease (AD). Overall, these results suggest that PLGA nanoparticles are a promising strategy for the brain delivery of drugs for the treatment of AD.

Cell-type specific expression and regulation of apolipoprotein D and E in human endometrium

ObjectiveTo assess the expression and regulation of antilipoprotein D (ApoD) and antilipoprotein E (ApoE) in human endometrium. Study designEndometrial biopsies from healthy, regularly cycling women were collected during the late proliferative and mid-secretory phase. mRNA gene expression of ApoD and ApoE was determined using real-time PCR in whole tissue, in isolated stromal (ESC), epithelial (EEC) and CD45+ leukocytes (EIC), as well as after hormonal stimulation of ESC and EEC in vitro. Protein expression was analyzed using immunohistochemistry. ResultsApoD and ApoE mRNA was expressed in all cell types examined. A rise in ApoD mRNA expression was seen in whole endometrium, ESC, and EEC in the secretory phase, as well as after hormonal stimulation of ESC and EEC in vitro. ApoE mRNA was significantly upregulated in whole endometrium of secretory phase biopsies, while its expression was not altered by progesterone in vitro. Immunohistochemistry of whole endometrial tissue localized ApoD mainly in ESC and EEC. While ApoE was localized slightly in ESC, it was particularly noted on the surface of secretory phase endothelial cells. ConclusionWe demonstrate for the first time the cell-type and cycle dependent expression of ApoD and ApoE within human endometrium, suggesting their role in endometrial modulation.

Expression of apolipoprotein D in autosomal dominant polycystic kidney disease and its influence on proliferation of cyst-lining epithelial cells

Objective To investigate the role of apolipoprotein D(ApoD) in the pathogenesis of autosomal dominant polycystic kidney disease(ADPKD).Methods The expression of ApoD protein in renal cystic tissues of ADPKD patients was examined by immunofluorescence and Western blotting analysis.Western blotting analysis and real-time PCR were used to detect the expression of ApoD protein and mRNA in the renal tissues of Han:SPRD rats.The cystic lining immortalized epithelial cells(WT9-12 cells) of ADPKD were stimulated with human recombinant ApoD protein and their proliferation was examined by MTT assay;flow cytometry was used to determine the cell apoptosis and cell cycle distribution.Results Immunohistochemisty and Western blotting analysis showed that ApoD expression was significantly lower in the renal tissues of ADPKD patients than that in healthy controls,and that in the Han:SPRD cy/+ rats was significantly lower than that in the Han:SPRD +/+ rats(P0.05,P0.01).ApoD mRNA expression in the kidneys of Han: SPRD cy/+ rats of 8,12,16,and 24 weeks old was significantly lower than that of Han: SPRD +/+ rats of same ages(P0.05).Recombinant ApoD protein at 100,200,and 400 ng/ml inhibited the proliferation of WT9-12 cells by 8.21%,7.59% and 8.07% after 48 h stimulation(P0.05),and by 8.62%,6.43% and 9.42% after 72 h stimulation,respectively(P0.05).Treatment with 400 ng/ml human recombinant ApoD protein for 72 h increased G1 phase WT9-12 cells by 8.26% and decreased S phase cells by 8.09%(P0.05),but it had no significant effect on cell apoptosis.Conclusion ApoD may inhibit WT9-12 cell proliferation by regulating cell cycle,and decreased ApoD expression may participate in the pathogenesis of ADPKD.

Characterization of nuclear factors modulating the apolipoprotein D promoter during growth arrest: Implication of PARP-1, APEX-1 and ERK1/2 catalytic activities

Human Apolipoprotein D (apoD) is upregulated under several stress conditions and pathological situations such as neurodegenerative diseases and cancers. We previously showed that apoD mRNA expression is induced in growth-arrested cells and demonstrated the specific binding of nuclear proteins to the region −514 to −475 of the promoter. Such region contains a pair of Serum Responsive Elements (SRE), an Ets-Binding Site (EBS) and a Glucocorticoid Responsive Element (GRE). In this study, we show that Parp-1, HnRNP-U, CBF-A, BUB-3, Kif4, APEX-1 and Ifi204 bind these regulatory elements of the apoD promoter. Specific binding of HnRNP-U and Parp-1 was confirmed by Electrophoretic Mobility Shift Assay (EMSA). In a biotin pull-down assay, Kif4 and BUB-3 bind preferentially the SRE1 and the EBS-GRE sites, respectively, while APEX-1 seems recruited indirectly to these elements. We found that the mRNA expression of some of these binding factors is upregulated in growth-arrested cells and that these proteins also transactivate the apoD promoter. In agreement with these results, mutants of APEX-1 and of Parp-1 defective for their DNA-binding and catalytic activities could not transactivate the promoter. The knockdown of Parp-1 and HnRNP-U and the use of specific inhibitors of MEK1/2 and of Parp-1 also inhibited the induction of apoD gene expression. Moreover, ERK1/2 was found activated in a biphasic manner post serum-starvation and the inhibition of Parp-1 causes a sustained activation of ERK2 but not ERK1 for up to 2 h. Altogether, these findings demonstrate the importance of Parp-1, APEX-1 and ERK1/2 catalytic activities in the growth arrest-induced apoD gene expression.

Apolipoprotein D is Elevated in Oligodendrocytes in the Peri-Infarct Region after Experimental Stroke: Influence of Enriched Environment

Injury to the brain (e.g., stroke) results in a disruption of neuronal connectivity and loss of fundamental sensori-motor functions. The subsequent recovery of certain functions involves structural rearrangements in areas adjacent to the infarct. This remodeling of the injured brain requires trafficking of macromolecular components including cholesterol and phospholipids, a transport carried out by apolipoproteins including apolipoprotein D (apoD). We investigated the changes in the levels of apoD mRNA and protein, and its cellular localization during a recovery period up to 30 days after experimental stroke in the rat brain. In the core of the brain infarct, apoD immunoreactivity but not mRNA increased in dying pyramidal neurons, indicative of cellular redistribution of lipids. During 2 to 7 days of recovery after stroke, the apoD levels increased in the peri-infarct and white matter areas in cells identified as mature oligodendrocytes. The apoD expressing cells were conspicuously located along the rim of the infarct, suggesting a role for apoD in tissue repair. Furthermore, housing animals in an enriched environment improved sensori-motor function and increased the apoD levels. Our data strongly suggest that apoD is involved in regenerative processes and scar formation in the peri-infarct area presumably by enhancing lipid trafficking.

High‐fat diets impede the lowering effect of cyclosporine a on rat brain lipids and interact with the expression of apolipoproteins E and J

Cyclosporine A (CsA), a common immunosuppressive agent, produces hyperlipidemia and apolipoprotein profile alterations in plasma as well as neurological and psychiatric complications. In rats, 10 mg CsA/kg/d treatments for 3 wk induce alterations of the electroencephalogram, and of the blood and brain lipids. Using this model, we evaluated whether triacylglycerol (TG)- and lecithin (PC)-enriched diets, reported to decrease epileptic episodes (TG) and to improve memory, could modify the effects of CsA treatment on brain lipids and possibly change apolipoprotein (apo) E and apoJ gene expression. To evaluate this hypothesis, three groups of rats were treated for 3 wk with CsA and received a low-fat, PC, or TG diet. Three other groups were fed the above-mentioned diets and were treated with the CsA solvent. As a control, one group was fed only the low-fat diet. The CsA-mediated decreases in brain cholesterol and PC contents, under a low-fat diet, were eliminated by the TG and PC diets. These high-fat diets induced a global increase in hippocampal transcriptional activity, as revealed by elevated polyadenylated RNA levels. The apoE and apoJ mRNA levels in the cortex and hippocampus of rats receiving the solvent were not statistically different between the TG- and PC-enriched diets but showed important variations compared with the low-fat diet solvent-treated group. A differential effect between the two high-fat diets was observed in the hippocampus, resulting in a significant increase of the apoE to apoJ ratio with the PC diet. The balance between apoE and apoJ is presumed to be important in encephalopathic mechanisms, by its involvement through low levels of brain cholesterol and PC, that might be associated with mental disorders. Our results therefore suggest that diet enrichment with polyunsaturated fat might be beneficial during CsA therapy. However, if the high levels in PC used here are more beneficial on CsA peripheral side effects than similar enrichment in TG, this does not seem to be the case in the brain. Thus, lower levels in PC should be tested.

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.

Apolipoprotein D and platelet-derived growth factor-BB synergism mediates vascular smooth muscle cell migration.

We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice.

Apolipoprotein D (apoD) expression is known to be elevated in select regions of rodent and human brain in association with different types of CNS pathology. To investigate a potential role for apoD in the neuropathology of Alzheimer's disease, we have measured apoD mRNA expression in transgenic mice expressing mutated human amyloid precursor protein under control of platelet-derived growth factor promoter (PDAPP mice). In situ hybridization analysis revealed increased apoD mRNA expression in brains of aged (26 months) PDAPP transgenic mice compared to aged littermate controls. These increases were most prominent in the hippocampal fimbria, corpus callosum and other white matter tracts. No substantial increases in expression were observed in white matter regions in young (6 months) PDAPP transgenic mice compared to young controls. Comparison between aged and young control mice revealed increased apoD expression in similar white matter regions of the aged animals. These findings suggest that, although increases in apoD expression are a normal feature of brain aging, super-increases may represent a glial cell compensatory response to beta-amyloid deposition in Alzheimer's disease.

Apolipoprotein J/clusterin is induced in vascular smooth muscle cells after vascular injury.

Understanding the precise molecular mechanisms underlying the phenomenon of restenosis after PTCA may help us to develop a new strategy for the treatment of restenosis after PTCA. The purpose of this study was to identify the genes involved in vascular restenosis. Applying a differential hybridization method to a model of the balloon-injured rabbit aorta, we identified 6 cDNA clones that were upregulated after injury. Northern blot showed that 5 genes, but not apolipoprotein J (apoJ)/clusterin, were constitutively expressed in noninjured aorta and upregulated after balloon injury. ApoJ mRNA was not detectable in noninjured aorta (control), began to be expressed at 6 hours after injury, showed a peak level at 24 hours (a 48-fold increase), gradually declined, and returned to the control level at 24 weeks. Western blot and immunohistochemistry demonstrated no expression of apoJ protein in noninjured aorta, an expression of apoJ at 2 days after balloon injury, and a peak level (a 55-fold increase) at 2 to 8 weeks. The expression of apoJ protein continued until 24 weeks after injury. In situ hybridization revealed that apoJ mRNA was expressed in smooth muscle cells (SMCs) of media at 2 days after injury and in SMCs of media and neointima at 2 weeks. To analyze the function of apoJ, stably transfected rabbit SMCs were created. The expression of apoJ stimulated proliferation and migration of SMCs. ApoJ is dramatically induced in media and neointima after vascular injury, suggesting that apoJ contributes to restenosis after angioplasty.

Apoptotic Cell Debris and Phosphatidylserine-Containing Lipid Vesicles Induce Apolipoprotein J (Clusterin) Gene Expression in Vital Fibroblasts

The molecular events in cells undergoing programmed cell death (apoptosis) are well studied; however, the response of the surviving neighbor cells to local cell death is largely uncharacterized. Apolipoprotein J (clusterin) is an 80-kDa glycoprotein that has been implied in cytoprotection of the vital cells, presumably by assisting in the clearance of apoptotic vesicles and membrane remnants. Its mRNA is specifically up-regulated in the vital cells of apoptotic tissues. The molecular mechanisms, however, leading to this response are not known. We here show that exposure of vital fibroblasts to apoptotic vesicles, disrupted vital cells, and trypsin-treated membrane remnants induces apoJ mRNA. Moreover, lipid vesicles consisting of phosphatidylserine (PtSer) and dimyristoylphosphatidylcholine (PC), but not liposomes with PC alone nor with dimyristoylphosphatidylethanolamine or phosphatidic acid, did elevate apoJ mRNA level. These results suggest that, apart from mediating the endocytic uptake of the apoptotic vesicles, PtSer also serves as a trigger to stimulate the expression of genes that might be involved in the cellular clearance process.

Effects of Age on Gene Expression during Estrogen-Induced Synaptic Sprouting in the Female Rat

Age and estrogen treatment influenced fiber outgrowth and compensatory neuronal sprouting after unilateral entorhinal cortex lesions (ECL) which model Alzheimer disease-like deafferentation in the dentate gyrus of the hippocampus. In young F344 rats (3 months old), ovariectomy (OVX) decreased reactive fiber outgrowth by 60%. Sprouting in middle-aged rats (18 months old) was reduced in intact females; no further reduction was caused by OVX. Several astrocyte mRNAs were measured in the dentate gyrus of young and middle-aged female rats in three different estrogen states (sham OVX, OVX, or OVX + estradiol) 1 week after ECL. Glial fibrillary acidic protein (GFAP) mRNA was twofold greater in middle-aged rats than young, although both ages showed threefold increases in response to ECL. In prior studies GFAP was found to be decreased by estradiol treatment 3–4 days after ECL; in this study GFAP mRNA had returned to sham OVX levels in young rats by 7 days post-ECL. Surprisingly, estradiol treatment increased GFAP mRNA levels by 25% above OVX in middle-aged rats. Apolipoprotein E (apoE) mRNA was decreased 20% by age in the dentate, although both age groups showed a 25% increase in apoE mRNA in response to ECL. Apolipoprotein J (apoJ) mRNA was increased 20% in the dentate gyrus of middle-aged rats, and both age groups responded to ECL with a 65% increase in apoJ mRNA. The estrogen state did not alter levels of either apolipoprotein mRNA in the deafferented dentate. The data suggest that the estrogen-induced decrease of GFAP in response to lesions does not persist at 7 days post-ECL during sprouting. Overall effects of age on the dentate gyrus include elevated GFAP mRNA and decreased apoE mRNA. The cortical wound site showed consistent enhancement of GFAP mRNA in both age groups by estradiol above sham OVX and greater responses in middle-aged rats.

Increased expression of apolipoprotein D following experimental traumatic brain injury.

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.

Modulation of apolipoprotein D and apolipoprotein E expression in rat hippocampus after entorhinal cortex lesion

Apolipoprotein (apo) D is a member of the lipocalin family of proteins. Although its physiological function is unknown, apoD is thought to transport one or more small hydrophobic ligands. A second apolipoprotein, apoE is known to play an important role in lipid transport, and apoE genetic polymorphism has been shown to be associated with susceptibility to Alzheimer's disease. Both apoD and apoE are expressed in the central nervous system (CNS) and both proteins accumulate at sites of peripheral nerve injury due to increased local synthesis. The two proteins may have overlapping or complementary functions within nervous tissue. In order to define the role of apoD within the CNS, we have studied the regional distribution of apoD and apoE mRNA and protein within the normal rat brain and the changes in apoD and apoE expression in the hippocampus of rats after entorhinal cortex lesion (EC lesion). Within the brains of normal rats, apoD expression in the hippocampus was as high as 180-fold that of the liver. ApoD mRNA levels in other areas of the rat brain ranged from 40 to 120 times the hepatic levels. The distribution of apoE gene expression within the brain was similar to that of apoD, but was much lower than hepatic apoE expression. When rats were subjected to EC lesion, the apoD message increased by 54% at 4 days post lesion (DPL) in the ipsilateral region of hippocampus while apoE mRNA levels (ipsilateral and contralateral) decreased by 43%. At 6 to 8 DPL apoD mRNA in the ipsilateral hippocampus remained elevated (42% above controls) whereas the apoE mRNA levels increased to about 15% above those of controls. At 14 and 31 DPL, both apoD and apoE expression was similar to controls. The increase in immunoreactive apoD in hippocampal extracts was more dramatic. At 1 DPL, immunoreactive apoD levels were already 16-fold higher than those in extracts of non-lesioned animals and, at 31 DPL, levels were still 8-fold higher than those of control animals. Finally, we have demonstrated that the levels of apoD in the brains of apoE-deficient mice are 50-fold those of wildtype control mice. ApoD clearly has an important function within the CNS in both normal and pathological situations.

Hippocampal alterations of apolipoprotein E and D mRNA levels in vivo and in vitro following kainate excitotoxicity.

Alteration in the expression of apolipoprotein E (ApoE) and apolipoprotein D (ApoD) genes was evaluated in rat, 7 days following status epilepticus (SE) induced by intra-amygdala injection of kainate (KA), and in organotypic hippocampal cultures, 2 days after a single 1 h exposure to KA. Global polyadenylated RNA (poly A+) steady state, assessing global regulation of mRNA transcription was first measured in cortices and hippocampi from each animal and in the organotypic cultures. No alteration due to KA treatment was observed and individual concentrations of ApoE and ApoD mRNA species were therefore measured and comparative analysis performed. In the cortices of KA-treated animals, ApoE and ApoD mRNA levels did not show statistically significant changes. In contrast, in hippocampi, 7 days after SE, ApoE and ApoD mRNA levels were significantly increased, respectively, by 123 and 138%. This in vivo effect was confirmed in vitro on organotypic cultures, where KA treatment increased ApoE and ApoD mRNA expressions, respectively, by 72 and 61%. These observations indicate that lipidic metabolism is modified in the lesioned structure and suggest an increased traffic of lipids and a need for more ApoE and D in the hippocampus during the period of recovery and restructuration that follows severe seizures.

ANTAGONISTIC EFFECTS OF PYRROLIDINE DITHIOCARBAMATE AND N-ACETYL-L-CYSTEINE ON SURFACTANT PROTEIN A AND B mRNAs

Pulmonary surfactant, a mixture of phospholipids and specific associated proteins, reduces surface tension at the air-liquid interface of the lung and protects the large epithelial surface of the lung from infectious organisms. Surfactant proteins, SP-A and SP-B, are required for normal surfactant function. In the current work, increased levels of oxidized glutathione (GSSG) are demonstrated at doses of pyrrolidine dithiocarbamate (PDTC) which decrease SP-A and SP-B mRNAs, suggesting that cellular oxidation reduces surfactant protein expression. Similarly, reduction of SP-A and SP-B mRNA levels following accumulation of GSSG induced by glutathione reductase inhibitor 1, 3-bis-(2-chloroethyl)-1 nitrosourea (BCNU), supports the hypothesis that surfactant protein synthesis is reduced in response to oxidation of pulmonary epithelial glutathione. Concurrent induction of apolipoprotein J(apoJ) mRNA by PDTC demonstrates the selectivity of pulmonary gene regulation by the dithiocarbamate. In contrast, the glutathione precursor N-acetyl-l-cysteine (NAC) prevented PDTC-dependent increase in GSSG/GSH ratio, inhibition of SP-A and -B mRNAs, and induction of apoJ. Insufficiency of SP-A and -B, which occurs in inflammatory lung diseases, may result from the exposure of the pulmonary epithelium to oxidant stress and may be reversed by the antioxidant NAC.