Transplantation of neural stem cells modulates apolipoprotein E expression in a rat model of stroke

The expression of apolipoprotein E (apoE) after ischemic brain damage has been associated with plasticity involved in promoting functional recovery. We therefore examined the expression and distribution of apoE in rats that received intraparenchymal grafts of the conditionally immortal stem cell line MHP36 either ipsilateral or contralateral to the lesion or intraventricular grafts 4 months after transplantation. ApoE immunoreactivity was highly expressed in the striatum, somatosensory cortex, and thalamus of the lesioned hemisphere in all rats subjected to middle cerebral artery occlusion. Only in rats with intraparenchymal grafts, apoE was significantly upregulated in the contralateral hemisphere, whereas levels and distribution in rats with intraventricular grafts resembled those of ischemic controls. In ischemic rats, apoE was seen in both astrocytes and neurons on the lesioned side, and in grafted rats, apoE was present in host and transplanted neurons and astrocytes. Previously we have shown that intraparenchymal grafts reduced sensorimotor asymmetry, whereas intraventricular grafts improved cognitive dysfunction, with transplanted cells being widely distributed in cortex, striatum, and corpus callosum on both sides of the brain in all grafted groups. Thus, stem cells grafted in the parenchyma are not only capable of limited expression of apoE in the host brain but also trigger a robust increase on the side contralateral to stroke damage where this does not normally occur. Findings that parenchymal, but not ventricular, grafts facilitated sensorimotor recovery suggests that apoE might contribute to plastic changes in relevant pathways, possibly on both sides of the brain. In contrast, no evidence was found for an association between apoE and recovery of cognitive function in rats with intraventricular grafts.     

Presence of apolipoprotein E immunoreactivity in degenerating neurones of mice is dependent on the severity of kainic acid-induced lesion

Apolipoprotein E (apoE) is a major apolipoprotein in the central nervous system (CNS) that may play a role in various CNS disorders. ApoE is primarily localised in astrocytes, but neuronal apoE mRNA expression has been demonstrated in normal and diseased human brain, as well as in ischaemic rat brain. To obtain further insight into the role of apoE in neuronal degeneration in the CNS and conditions of neuronal apoE localisation, we have investigated in mice the distribution of apoE following neuronal injury induced by kainic acid (n=35, 25 or 35 mg kainic acid/kg BW). Consecutive series of brain sections were immunostained for apoE and markers for astroglia (GFAP) and microglia/macrophage cells (CR3). Degenerating neurones were identified with a silver-degeneration staining technique. The intensity and cellular distribution of apoE-immunoreactivity (apoE-ir) was dependent on the severity of neuronal injury. Mice that developed mild neuronal degeneration, restricted to a subset of neurones in the hippocampus, showed increased apoE-ir in astrocytes concomitant with increased GFAP-ir and mild microgliosis. In these mice, no neuronal apoE-ir was detected. In contrast, mice developing severe neuronal injury in the hippocampus - frequently also showing degeneration in other brain regions including cortex, thalamus, striatum and amygdala - showed intense apoE-ir in degenerating neurones. Surrounding the lesion, apoE-ir was increased in neuropil recurrently whereas GFAP-ir astrocytes disappeared. Thus, in mice apoE accumulates in degenerating neurones in conditions of severe neuronal injury putatively in association with disruption of the glial network.     

Apolipoprotein D levels are elevated in prefrontal cortex of subjects with Alzheimer's disease: no relation to apolipoprotein E expression or genotype.

BACKGROUND: Apolipoprotein E (apoE) has been implicated in the pathology of AD ever since inheritance of the epsilon4 allele was shown to be an important risk factor for the development of AD. Apolipoprotein D (apoD) is elevated in association with several central nervous system disorders, including Alzheimer's disease (AD), and has been proposed to be an especially robust marker for brain regions specifically affected by particular neuropathologies. Progressive cognitive decline is the core clinical feature of AD and is associated with disturbances in the prefrontal cortex. METHODS: We measured apoD levels in prefrontal cortex samples obtained postmortem from 20 autopsy-confirmed AD subjects and 40 control subjects. RESULTS: Enzyme-linked immunosorbent assay analysis revealed a significant increase in apoD expression in AD subjects compared with control subjects (.218+/-.029 microg/mg protein vs.117+/-.011 microg/mg protein; p=0003). There was no significant difference in apoD expression between early-onset and late-onset Alzheimer's subjects. Apolipoprotein D expression levels were not correlated with apoE levels, nor were they correlated with inheritance of the APOE epsilon4 allele. CONCLUSIONS: These findings suggest that apoD may be related to the cognitive decline observed in AD patients and that apoD and apoE likely play different roles in the pathogenesis of AD.     

Association of plasma apolipoproteins D with RBC membrane arachidonic acid levels in schizophrenia

Apolipoprotein D (apoD) is a member of the lipocalin superfamily of transporter proteins that bind small hydrophobic molecules, including arachidonic acid (AA). The ability of apoD to bind AA implicates it in pathways associated with membrane phospholipid signal transduction and metabolism. Recent findings of an increased expression of apoD in the mouse brain after clozapine treatment suggested a role for apoD in the pharmacological action of clozapine. Moreover, clozapine has been shown to increase membrane AA levels in RBC phospholipids from schizophrenic patients. ApoD levels have also been shown to be elevated in the CNS of subjects with chronic schizophrenia, a disorder associated with AA dysfunction. In this study, we examined whether plasma apoD levels are related to red blood cell membrane AA contents in the first-episode neuroleptic-naive schizophrenic (FENNS) patients. Plasma apoD levels as measured by enzyme-linked immunosorbent assay (ELISA) were not significantly different (F = 0.51, df = 2,86, p = 0.60) among healthy controls (n = 36), FENNS patients (n = 33) and patients with other psychiatric disorders (n = 19). However, plasma apoD levels were significantly correlated with RBC-AA (p = 0.0022) and docosapentaenoic acid (p = 0.0008) in FENNS patients. There are several known mechanisms that can lead to the type of membrane fatty acid defects that have been identified in schizophrenia. Whether plasma apoD alone is a major determinant of reduced RBC membrane AA levels in FENNS patients remains to be determined, although these preliminary data appear not to support this premise. Taken together with other in vitro studies, however, the present data support the view that an increased expression of apoD such as induced by atypical neuroleptic drug, may facilitate incorporation of AA into membrane phospholipids by its selective binding to AA.     

Apolipoprotein D in the aging brain and in Alzheimer's dementia

Apolipoprotein D (apoD) levels were examined in the temporal cortex as well as an assessment of the location of apoD positive cells within the brain by immunohistochemical and biochemical methods in young control (YC), aged control (AC), and Alzheimer's demented (AD) probands. Scattered apoD positive astrocytes and oligodendrocytes were found throughout the white matter by immunohistochemistry. ApoD immunoreactivity was also observed in the cerebellar oligodendrocytes of the YC group. There was faint positive apoD staining in scattered cortical astrocytes and a few neurons in the same group. In contrast, some of the AC and all of the AD probands had intense and frequent apoD immunostained cortical astrocytes and pyramidal neurons. The cortical senile plaques and neurofibrillary tangles were apoD immunonegative. No quantitative differences were found between the cortical apoD levels in the AC and AD groups, determined by immunoblotting. ApoD detected in the brain tissue was different in molecular weight (29 kDal) from that seen in CSF or in the serum (32 kDal). Our results indicate apoD is present in the human brain, especially in glial cells, and has increased abundance in the elderly and AD subjects.     

Clozapine specifically alters the arachidonic acid pathway in mice lacking apolipoprotein D

Apolipoprotein D (apoD), a member of the lipocalin superfamily of lipid-binding proteins, exhibits abundant expression within the CNS of many species, including humans; however, its physiological role remains unclear. Treatment with atypical antipsychotic drugs, especially clozapine, results in elevation of apoD expression levels in rodent brain and in human plasma samples. In order to further explore the role of apoD in mechanisms of clozapine function, we have measured a panel of membrane fatty acids and membrane lipids in brain from drug-treated apoD knock-out mice. Mice received clozapine (10 mg/kg/day) in their drinking water for 28 days and forebrain samples were analyzed using high performance liquid chromatography and capillary gas chromatography. We identified significant differences in the levels of membrane fatty acids in response to clozapine treatment specifically in the brains of apoD knock-out mice, but not wild-type (wt) mice. The most striking observations were decreases in the levels of fatty acids related to metabolism of arachidonic acid (AA), which is a known binding partner for apoD. These include the precursor to arachidonic acid, linoleic acid (LA; 18:2n6c), arachidonic acid itself (20:4n6) and the elongation product of arachidonic acid, adrenic acid (22:4n6). We further report increases in LA, eicosadienoic acid and docosahexaenoic acid in apoD knock-out compared to wild-type mice. These findings implicate an important apoD/AA interaction, which may be necessary for clozapine function.     

Differential expression between pilocytic and anaplastic astrocytomas: identification of apolipoprotein D as a marker for low-grade, non-infiltrating primary CNS neoplasms

Fibrillary astrocytoma, the most common primary central nervous system neoplasm, is infiltrating, rapidly proliferating, and almost invariably fatal. This contrasts with the biologically distinct pilocytic astrocytoma, which is circumscribed, often cystic, slowly proliferating, and associated with a favorable long-term outcome. Diagnostic markers for distinguishing pilocytic astrocytomas from infiltrating anaplastic astrocytomas are currently not available. To identify genes that might either serve as markers or explain these distinct biologic behaviors, cDNA microarray analysis was used to compare the expression of 7,073 genes (nearly one quarter of the human genome) between these 2 types of astrocytoma. Messenger RNAs pooled from 3 pilocytic astrocytomas and from 4 infiltrating anaplastic astrocytomas were compared. Apolipoprotein D (apoD), which expressed 8.5-fold higher in pilocytic astrocytomas, showed the greatest level of differential expression and emerged as a potential marker for pilocytic tumors. By immunohistochemistry, 10 of 13 pilocytic astrocytomas stained positively for apoD, while none of 21 infiltrating astrocytomas showed similar staining. ApoD immunostaining was also seen in 9 of 14 of gangliogliomas, 4 of 5 subependymal giant cell astrocytomas (SEGAs), and a single pleomorphic xanthoastrocytomas (PXAs). By in situ hybridization, pilocytic astrocytomas, in contrast with infiltrating astrocytomas, showed widespread increased apoD expression. SAGE analysis using the NCBI database showed a higher level of expression of apoD RNA in pilocytic astrocytoma than in any of the other 94 neoplastic and non-neoplastic tissues in the database. ApoD is associated with decreased proliferation in some cell lines, and is the protein found in highest concentration in cyst fluid from benign cystic disease of the breast. ApoD might play a role in either decreased proliferation or cyst formation in pilocytic astrocytomas, gangliogliomas, SEGAs, and PXAs.     

Apolipoprotein C-I Expression in the Brain in Alzheimer's Disease

The H2 allele of apolipoprotein (apo) C-I is associated with Alzheimer's disease (AD). However, this association is potentially confounded by the linkage disequilibrium of H2 with the epsilon2 and epsilon4 alleles of apoE and of H1 with the epsilon3 allele. To establish plausibility for a direct role for apoC-I in AD, we compared apoC-I and apoE protein and mRNA levels in postmortem specimens of frontal cortex and hippocampus from AD patients with levels in nondemented controls. In H2-allelic individuals (usually also epsilon4 carriers), apoC-I mRNA levels were strikingly lower with AD (by 65%, P < 0.05), but apoC-I protein levels in AD were significantly higher (by 34%, P < 0.05). The opposite direction of the apoC-I mRNA and apoC-I protein level changes in AD in the epsilon4/H2 genotype may reflect decreased clearance of CNS lipoproteins associated with apoE4. In H1/H1 (usually epsilon3/epsilon3) individuals, both apoC-I protein and mRNA were lower in AD. ApoC-I protein levels in hippocampus were nearly twice those in frontal cortex. Immunohistochemistry of hippocampus revealed colocalization of apoC-I protein with the astrocytic marker GFAP. In addition, cultured human astrocytes expressed the mRNA for apoC-I. This study confirms apoC-I expression in the CNS and identifies astrocytes as the source of apoC-I. In addition, it has revealed differences in apoC-I expression based on site, genotype, and disease status that may reflect a role for apoC-I in the pathogenesis of AD.     

ApoD,a glia‐derived apolipoprotein,is required for peripheral nerve functional integrity and a timely response to injury

Glial cells are a key element to the process of axonal regeneration, either promoting or inhibiting axonal growth. The study of glial derived factors induced by injury is important to understand the processes that allow or preclude regeneration, and can explain why the PNS has a remarkable ability to regenerate, while the CNS does not. In this work we focus on Apolipoprotein D (ApoD), a Lipocalin expressed by glial cells in the PNS and CNS. ApoD expression is strongly induced upon PNS injury, but its role has not been elucidated. Here we show that ApoD is required for: (1) the maintenance of peripheral nerve function and tissue homeostasis with age, and (2) an adequate and timely response to injury. We study crushed sciatic nerves at two ages using ApoD knock‐out and transgenic mice over‐expressing human ApoD. The lack of ApoD decreases motor nerve conduction velocity and the thickness of myelin sheath in intact nerves. Following injury, we analyze the functional recovery, the cellular processes, and the protein and mRNA expression profiles of a group of injury‐induced genes. ApoD helps to recover locomotor function after injury, promoting myelin clearance, and regulating the extent of angiogenesis and the number of macrophages recruited to the injury site. Axon regeneration and remyelination are delayed without ApoD and stimulated by excess ApoD. The mRNA and protein expression profiles reveal that ApoD is functionally connected in an age‐dependent manner to specific molecular programs triggered by injury. © 2010 Wiley‐Liss, Inc.     

A role for lipoprotein lipase during synaptic remodeling in the adult mouse brain

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules found in lipoprotein particles. This particular lipase also has a role in the binding of lipoprotein particles to different cell-surface receptors. LPL has been identified in the brain but has no specific function yet. This study aimed at elucidating the role of LPL in the brain in response to injury. Mice were subjected to hippocampal deafferentation using the entorhinal cortex lesion and mRNA and protein expression were assessed over a time-course of degeneration/reinnervation. Hippocampal LPL levels peaked at 2 days post-lesion (DPL) both at the mRNA and protein levels. No change was observed for receptors of the LDL-receptor family or RAP at DPL 2 in the hippocampus but the glia-specific syndecan-4 was found to be significantly upregulated at DPL 2. These results suggest that LPL is involved in the recycling of cholesterol and lipids released from degenerating terminals after a lesion through a syndecan-4-dependent pathway.     

A deficit in astroglial organization causes the impaired reactive sprouting in human apolipoprotein E4 targeted replacement mice

The epsilon4 allele of apolipoprotein (apo)E associates with an increased risk of developing Alzheimer's disease (AD) as well as an earlier age of onset. However, the exact mechanisms by which apoE4 confers such susceptibility is currently unknown. We used a human apoE targeted replacement (hE-TR) mouse model to investigate the allele-specific response to entorhinal cortex lesion (ECL). We observed a marked impairment in reactive sprouting in hE4 mice compared to hE3 mice. ApoE expression was similar between genotypes at days post-lesion (DPL) 2 and 14. Thirty days post-lesion, hE4 mice had more reactive astrocytes as well as a defective outward migration pattern of the astrocytes in the dentate gyrus. The expression of the anti-inflammatory cytokine IL-1ra was delayed in hE4 mice compared to hE3 mice. ApoE and beta-amyloid (Abeta) 1-40 accumulated at 30 DPL in hE4 mice. These results suggest that the presence of apoE4 delays the astroglial repair process and indirectly compromises synaptic remodeling.     

Basic fibroblast growth factor mRNA increases in specific brain regions following convulsive seizures.

Basic fibroblast growth factor (bFGF) is a trophic factor synthesized in the central nervous system (CNS), where it is believed to play a role in neuronal maintenance and repair. Little is known about the regulation of this growth factor in the CNS. To determine whether the expression of the bFGF gene in the brain of adult animals changes in response to alterations of neuronal activity, we examined bFGF mRNA levels in several brain regions of rats experiencing focally-evoked convulsive seizures. Seizures were induced by microinjecting bicuculline unilaterally into an epileptogenic site within the deep prepiriform cortex, area tempestas (AT). By 5 h after initiation of brief limbic motor seizures from AT, there was a four fold increase in the levels of bFGF mRNA in the entorhinal cortex, hippocampus and olfactory bulb, but not in the caudate-putamen. The maximal expression of bFGF mRNA was reached by 10 h after seizure onset. In the same animals, the mRNA encoding nerve growth factor (NGF) was increased in entorhinal cortex and hippocampus, but not in the olfactory bulb. Our results demonstrate that neuronal activity can influence bFGF expression in an anatomically selective fashion and that acute changes in bFGF can occur in the uninjured mature brain. The increase in bFGF expression in response to excessive activation of specific neuronal circuitry may represent an adaptive response to protect against potential injury in those circuits.     

Impairment of the blood-nerve and blood-brain barriers in apolipoprotein e knockout mice

Apolipoprotein E (apoE) is well characterized as a plasma lipoprotein involved in lipid and cholesterol metabolism. Recent studies implicating apoE in Alzheimer's disease and successful recovery from neurological injury have stimulated much interest in the functions of apoE within the brain. To explore the functions of apoE within the nervous system, we examined apoE knockout (KO) mice. Previously, we showed that apoE KO mice have a delayed response to noxious thermal stimuli associated with a loss and abnormal morphology of unmyelinated fibers in the sciatic nerve. From these data, we hypothesized that apoE KO mice could have an impaired blood-nerve barrier (BNB). In this report, we demonstrate functionally impaired blood-nerve and blood-brain barriers (BBB) in apoE KO mice using immunofluorescent detection of serum protein leakage into nervous tissue as a diagnostic for decreased BNB and BBB integrity. Extensive extravasation of serum immunoglobulin G (IgG) is detected in the sciatic nerve, spinal cord, and cerebellum of apoE KO but not WT mice. In a subpopulation of apoE KO mice, IgG also extravasates into discrete cortical and subcortical locations, including hippocampus. Loss of BBB integrity was additionally confirmed by the ability of exogenously supplied Evans blue dye to penetrate the BBB and to colocalize with IgG immunoreactivity in CNS tissue. These observations support a role for apoE in maintaining the integrity of the BNB/BBB and suggest a novel relationship between apoE and neural injury.     

Astrocytes and Microglia Respond to Estrogen with Increased apoE mRNAin Vivoandin Vitro

This study examined the regulation of apolipoprotein E (apoE) by 17β-estradiol (E2) in brain glia, using rats with regular ovulatory cycles as anin vivomodel and cultured astrocytes and mixed glia asin vitromodels. Two brain regions were examined which had demonstrated transient synaptic remodeling during the estrous cycle. In the hippocampal CA1 region and the hypothalamic arcuate nucleus, apoE mRNA was elevated at proestrus when plasma E2 was high and synaptic density was increasing. Both astrocytes and microglia contributed to this increase in apoE mRNA.In vitro,E2 treatment had no effect on apoE mRNA levels in monotypic cultures of either astrocytes or microglia. In contrast, mixed glial cultures responded to E2 with increased apoE mRNA and protein, suggesting that heterotypic cellular interactions are important in the brain response to estrogens.In situhybridization in combination with cell-specific markers showed that E2 increased apoE mRNA levels in both astrocytes and microglia. These results, which are the first evidence of apoE mRNA localization to microgliain vivoand the control of apoE expression in brain cells by estrogens, are discussed in terms of the possible protective role of E2 in Alzheimer's disease and prior findings that emphasize the expression of apoE mRNA in astrocytes within the brain.     

The process of reinnervation in the dentate gyrus of adult rats: time course of increases in mRNA for glial fibrillary acidic protein

The present study evaluates the time course of increased expression of the mRNA for glial fibrillary acidic protein (GFAP) within the dentate gyrus and hippocampus after unilateral lesions of the entorhinal cortex. Levels of GFAP mRNA were evaluated by dot blot hybridization of mRNA isolated from the hippocampus and quantitative in situ hybridization. For dot blot hybridization, RNA was isolated from pooled hippocampi obtained from animals killed at 12 hr, 1, 2, 4, 6, 8, 10, 14, and 30 d postlesion. A separate set of animals killed at 2, 4, 6, 8, 10, 12, 14, and 32 d were prepared for in situ hybridization. The dot blot analyses of mRNA isolated from the hippocampus revealed that on the side ipsilateral to the lesion, the levels of GFAP mRNA increased rapidly, reaching a peak at 2 d postlesion. The increases were not evident by 12 hr postlesion, but by 24 hr, levels of GFAP mRNA were 5-fold higher than control, and by 48 hr, the levels were over 6-fold higher than control. The levels of GFAP mRNA decreased after 2 d postlesion. At 4 and 6 d postlesion the levels were about 2-fold higher than control. At later postlesion intervals, mRNA levels were comparable to the control. At 2 d postlesion, the levels of GFAP mRNA were also increased about 2-fold over control levels on the contralateral side. After 2 d, the levels of GFAP mRNA on the contralateral side were comparable to the control. In situ hybridization revealed a complex pattern of changes in the levels of GFAP. At 2 d postlesion, the levels of GFAP mRNA increased dramatically throughout the hippocampus bilaterally. The increases were most pronounced in the denervated portions of the neuropil; however, the levels of GFAP mRNA were also substantially elevated in laminae that do not receive direct projections from the entorhinal cortex. GFAP mRNA levels were also increased in other areas that receive projections from the entorhinal cortex, including the septum, lateral-dorsal thalamus, and entorhinal cortex contralateral to the lesion. In addition, GFAP mRNA levels were increased in regions bordering the ventricles throughout the brain, and over the surface of the tectum. After 2 d postlesion, the increases in the levels of GFAP mRNA were for the most part restricted to areas containing terminal degeneration. The generalized increases throughout the hippocampus were no longer apparent. Areas bordering the ventricles continued to exhibit higher labeling than in control animals, but this effect was not as prominent as at 2 d postlesion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization of basic fibroblast growth factor and its mRNA after CNS injury

Basic fibroblast growth factor (FGF) mRNA is increased 4 h after cortical brain injury. In situ hybridization reveals that the increased mRNA persists for at least 2 weeks and that, in areas adjacent and ipsilateral to the lesion, the expression of basic FGF mRNA is also modified. As an example, at three days distal from the lesion, mRNA can be detected in ependymal cells of the lateral ventricle and in selected cells of the hippocampus and cortex. Endothelial cells also synthesize basic FGF mRNA. The increase in basic FGF mRNA is paralleled by similar changes in the localization of the basic FGF protein. Both the intensity and number of cells which stain for basic FGF are increased when they are compared to staining in either the contralateral side or to comparable areas of unlesioned brains. The pattern of mRNA expression is similar from 4 hours to 14 days. Early in the response (4 h to 3 days) on the border of the lesion, the presence of basic FGF is most obvious within the MAC-1-immunopositive population (macrophages and/or microglia). From 7 days to 2 weeks, there has been extensive hypertrophy of the reactive astrocytes which stain intensely for anti-basic FGF(1-24). We conclude that there is increased basic FGF as a function of injury to the CNS. In view of the observation that it is an early and persistent response, the possibility that it plays multiple functions in the regenerative capacity of the CNS is discussed.