Endocytosis following dopamine D2 receptor activation is critical for neuronal activity and dendritic spine formation via Rabex-5/PDGFRß signaling in striatopallidal medium spiny neurons.

Aberrant dopamine D2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/platelet-derived growth factor receptor-ß (PDGFRß)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFRß are then transported to the Golgi apparatus, where those complexes trigger Gαi3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFRß is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.

Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Localization of PDGF-B protein in macrophages in all phases of atherogenesis.

Lesions of atherosclerosis occur in the innermost layer of the artery wall and consist primarily of proliferated smooth muscle cells surrounded by large amounts of connective tissue, numerous lipid-laden macrophages, and varying numbers of lymphocytes. Growth-regulatory molecules may be involved in intimal accumulation and proliferation of smooth muscle cells responsible for the occlusive lesions of atherosclerosis. Platelet-derived growth factor (PDGF) B-chain protein was found within macrophages in all stages of lesion development in both human and nonhuman primate atherosclerosis. Thus macrophages may play a critical role in the disease by providing PDGF, a potent chemotactic and growth-stimulatory molecule, to the intimal smooth muscle cells.

Inhibition of hypercholesterolemia-induced atherosclerosis in the nonhuman primate by probucol. I. Is the extent of atherosclerosis related to resistance of LDL to oxidation?

Lipoprotein oxidation is believed to play an important role in atherogenesis. To investigate whether inhibition of oxidation of low density lipoprotein (LDL) would alter atherogenesis in the nonhuman primate, we administered probucol, a potent antioxidant, to Macaca nemestrina fed a high-fat, high-cholesterol diet. Probucol was administered to half of the 16 monkeys 14 wk after starting the hypercholesterolemic diet, and was given daily until they were sacrificed after 11 mos. To evaluate the antioxidant effect of probucol, the resistance of isolated plasma LDL to in vitro oxidation was evaluated. Probucol significantly increased the resistance of LDL to oxidative modification, as shown by an increase in the lag time required for conjugated diene formation. Lesions in the probucol-treated animals appeared less mature, and increased accumulation of lipid was observed in smooth muscle cells. Comparison of all control and probucol-treated monkeys demonstrated that intimal lesion areas in the thoracic aortas of the probucol-treated monkeys were reduced by 43% (P < 0.0001), but no significant difference in lesion area was found in the abdominal aortas or in the iliac arteries. However, the lag phase of conjugated diene formation was not prolonged in 2 of the 8 probucol-treated animals. A plot of intimal lesion size versus lag phase of all 16 animals showed a trend that lesion size was inversely related to oxidation resistance for all anatomic sites. The strong inverse relationship between intimal lesion size and resistance of LDL to oxidation supports a role for lipoprotein oxidation in the development and progression of lesions of atherosclerosis. The possibility that some of the effect is due to other biological properties of probucol cannot be ruled out.

Normal developing rat brain expresses a platelet-derived growth factor B chain (c-sis) mRNA truncated at the 5' end.

The 5' untranslated sequence (5' UTS) of platelet-derived growth factor B (PDGF-B/c-sis) mRNA is highly preserved through evolution, and inhibits translation of downstream coding sequences. In this study, using Northern analysis we identified two PDGF-B/c-sis mRNAs (3.5 kb and 2.6 kb) expressed in normal developing rat brain. In contrast to the constitutive expression of 3.5 kb mRNA, the expression of 2.6 kb mRNA increased markedly in accordance with those stages of brain development at which we had previously demonstrated an increased immunoreactivity for PDGF-B/c-SIS in neurons (Sasahara et al., 1992). By PCR cloning and the RNase protection assay, we determined the complete sequence of rat PDGF-B/c-sis, and found that the 2.6 kb transcript was a form of the 3.5 kb message truncated at the 5' end, and that the predominant 2.6 kb mRNA commenced 15 nt upstream of the signal peptide. Accordingly, it is suggested that the truncation of 5' UTS contributes to the expression of PDGF-B/c-SIS protein in the CNS. Lack of translational inhibitory 5' UTS of PDGF-B/c-sis transcript and resultant efficient protein translation have been reported in only a few transformed cells and cultured umbilical vein endothelial cells. We have extended this knowledge to the developing rat brain, and suggest that a similar mechanism could operate widely in non-transformed tissue in vivo.

DNA/RNA-dependent ATPase activity is associated with ATBF1, a multiple homeodomain-zinc finger protein.

The AT motif-binding factor 1 (ATBF1)-A is a large transcription factor containing four homeodomains and 23 zinc finger motifs. It has a number of motifs involved in transcriptional regulation, and in addition, several motifs found in enzymes, such as ATPases and helicases. In this study, we examined whether ATPase activity is associated with the ATBF1-A molecule. A 263-amino acid segment of the ATBF1-A molecule, termed AHZ, which contains the ATPase A-motif, homeodomain IV and zinc finger 21, was expressed in Escherichia coli in the form of glutathione S-transferase fusion protein and analyzed for ATPase activity. We found that AHZ was able to hydrolyze ATP with K(m) 10.6 microM and K(cat) 0.055 min(-1) at 5 mM Mg(2+) and pH 7.75. AHZ retained bacterial DNA and removal of the DNA resulted in 70% decrease in ATPase activity. The addition of double- or single-stranded DNAs restored 70-75% ATPase activity and that of RNA restored 50-55% activity. Site-directed mutagenesis of the A-motif resulted in 34% reduction of ATPase activity with no significant loss of bound DNA. In contrast, mutation of homeodomain IV and zinc finger 21 resulted in 90 and 80% reduction of ATPase, respectively, with the loss of the ability to bind to DNA and RNA. These results show that ATBF1 has at least one enzyme activity in addition to regulation of DNA transcription. The ATPase activity associated with ATBF1-A is DNA/RNA-dependent and unique in that it requires both homeodomain and zinc finger motifs.

Induction of platelet-derived growth factor beta-receptor in focal ischemia of rat brain.

Our previous study on the ischemia-induced expression of platelet-derived growth factor (PDGF)-B chain in the rat brain prompted us to examine expression of PDGF beta-receptor in the ischemic brain. Focal ischemia was induced by permanent tandem occlusion of middle cerebral and common carotid arteries in spontaneously hypertensive rats. Northern analysis revealed that ischemia significantly increased expression of the receptor in the ischemic neocortex at 4 and 7 days (328 +/- 109%; 323 +/- 119%, respectively, over control: n = 4, p < 0.05 versus sham). Neurons in infarct transiently showed increased immunostaining for the receptor at 1 day, whereas neurons in periinfarct area showed sustained and increased immunoreactivity from 1 to 14 days post-ischemia. Reactive glial cells in the external capsule and in molecular layer of the neocortex adjacent to infarct possessed enhanced immunoreactivity from 1 to 21 days. Furthermore, marked immunoreactivity was observed on brain macrophages in infarct and on the abluminal side of capillaries surrounding infarct from 4 to 7 days. These results demonstrated that ischemic insult increases expression of the PDGF beta-receptor at both the mRNA and protein level in the brain, suggesting its important role in cellular cascade of the ischemic brain.

Ischemia induces the expression of the platelet-derived growth factor-B chain in neurons and brain macrophages in vivo.

To elucidate the role of the platelet-derived growth factor (PDGF)-B chain in the brain, we examined its expression in rat brains with focal ischemia. Focal ischemia was induced by permanent tandem occlusion of the middle cerebral and common carotid arteries in spontaneously hypertensive rats (SHRs). Northern analysis demonstrated that ischemia transiently increased mRNA expression of the PDGF-B chain, but not the PDGF-A chain, in the injured neocortex. The larger transcript (3.5 kb) of the B chain gradually increased to threefold by 16 h, whereas the smaller transcript (2.6 kb) of the B chain markedly increased sixfold by 4 h. Immunohistochemistry revealed enhanced immunoreactivity in the neurons in the infarct and in the periinfarct area from 16 h to days 4-7, with a peak at 24 h. Furthermore, the brain macrophages that accumulated in the infarct showed intense immunostaining in their perinuclear region from days 2 to 14, with a peak at days 5-6. The present study demonstrates that ischemia induces the expression of the PDGF-B chain, first in neurons and later in brain macrophages, and suggests an important role of the PDGF-B chain in the healing process of the injured brain.

A mutation in the microtubule-associated protein tau in pallido-nigro-luysian degeneration.

We detected a missense mutation in exon 10 of tau that causes a substitution at codon 279 (N279K) in a Japanese patient with a familial background of parkinsonism and dementia originally described as pallido-nigro-luysian degeneration. This mutation is the same as one seen in a Caucasian family with pallido-ponto-nigral degeneration. The similarities between these two families suggest a common genetic mechanism that may account for the peculiar distribution of neuroglial degeneration with tauopathy.

Expression of platelet-derived growth factor B-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries.

In order to obtain information about the developmental mechanisms of restenosis after angioplasty, we investigated an association between the expression of platelet-derived growth factors (PDGFs) and neointimal cell accumulation in rabbit femoral arteries subjected to balloon angioplasty. Northern analysis demonstrated that mRNA expression of PDGF B-chain (PDGF-B) increased markedly in the injured arteries, peaking at day 7 (sevenfold), and the transcripts remained augmented until day 21. Also transcripts of PDGF beta-receptor (PDGFR-beta) and alpha-receptor increased by 3- and 2.5-fold, respectively, but those of PDGF A-chain showed only a slight increase (1.5-fold). In situ hybridization and immunohistochemistry demonstrated the concordant expression of mRNA and protein for PDGF-B in the smooth muscle cells (SMCs) of injured vessels throughout the experiment. PDGF-B expression peaked in neointimal SMCs at day 7. In accordance with PDGF-B expression, cellular proliferation in neointima peaked at day 7, being followed by a dramatic increase of neointimal areas thereafter. Further, we demonstrated PDGFR-beta immunoreactivity in these neointimal cells with PDGF-B expression. Our data provide evidence that PDGF-B may stimulate vascular SMC proliferation and contribute to neointimal formation after angioplasty.

Effect of hypertension on lysosomal enzyme activities in aortic endothelial cells.

In order to obtain information about the changes in lysosomal enzyme activities in arterial endothelial cells under hypertensive conditions, a biochemical study was performed on 5 lysosomal enzymes, acid phosphatase, N-acetyl-beta-glucosaminidase (NAGase), cathepsin B, cathepsin D and beta-glucuronidase, in endothelial cells isolated by an enzymatic technique from the aorta of spontaneously and renal hypertensive rats, and normotensive control rats. The aortic endothelial cells in the old spontaneously and the renal hypertensive rats showed increased activities of enzymes examined in comparison with those in the age-matched control rats. Endothelial cells in young spontaneously hypertensive rats did not show any elevated enzyme activities compared with those in the controls, and the enzyme activities tended to increase with aging. From this, it is deduced that hypertension activates lysosomal enzyme activities in aortic endothelial cells. The differences in the activities of NAGase, cathepsin B and cathepsin D between hypertensive and control animals increased markedly with advancing age. These activated lysosomal enzymes seem to be involved in the developmental mechanism of arterial endothelial cell injury in hypertension and in further development of hypertensive vascular changes.

Semiquantitative histochemical investigation of lysosomal enzyme activities in the aortic endothelial cells of spontaneously hypertensive rats.

In order to obtain information about changes in lysosomal enzyme activities in the aortic endothelial cells under hypertensive conditions, semiquantitative histochemical investigations of acid phosphatase (Ac-Pase) and N-acetyl-beta-glucosaminidase (NAGase) activities in the aorta of spontaneously hypertensive rats (SHR) were performed on 'Häutchen' monolayer preparations. The aortic endothelial cells in SHR showed increased Ac-Pase and NAGase activities as compared with those in control normotensive rats, and the activities tended to increase with advancing age. The degenerating process of endothelial cells expressed by lysosomal enzyme activity seems to be accelerated by hypertension. The increased lysosomal enzymes may participate in the further development of hypertensive vascular changes.

Expression of platelet-derived growth factor B-chain and beta-receptor in hypoxic/ischemic encephalopathy of neonatal rats.

Expression of platelet-derived growth factor B-chain and of its specific receptor (beta-receptor) was investigated in immature brains with hypoxic/ischemic injury. After the left common carotid arteries of seven-day-old rats were ligated and pups were placed in a hypoxic chamber, the protein and messenger RNA of both B-chain and beta-receptor were assessed using immunocytochemistry and northern analysis, respectively. Transcripts for B-chain were localized by in situ hybridization. Faint but definite expression of B-chain and beta-receptor was seen in the brains of untreated neonatal controls. Three to 48 h after hypoxia B-chain protein was generally increased above control levels, but focally decreased expression was seen in infarcted areas. Enhanced induction of messenger RNA of B-chain was seen in the both sides of cerebral cortices and hippocampi at 3 h. Strongly increased positivity for B-chain protein and mRNA occurred in the neurons surrounding the infarct. In situ hybridization still showed this up-regulation seven days after hypoxia. Beta-receptor protein expression was enhanced in some neurons immediately surrounding the infarct at 3 h of hypoxia, and marked up-regulation was seen at 16 h. Beta-receptor messenger RNA remained at control levels. Immunocytochemistry showed strong immunoreactivity for the beta-receptor on the neurons surrounding the infarct at 72 h. These results indicate that a neonatal hypoxic/ischemic insult induces neuronal up-regulation of the platelet-derived growth factor B-chain as well as beta-receptor immediately after hypoxia. While this up-regulation is relatively transient in most neurons, sublethal damage to neurons immediately surrounding an infarct induces sustained up-regulation. Through autocrine and paracrine mechanisms, platelet-derived growth factor B-chain molecules may act as a neuroprotective factor in immature brain experiencing with hypoxic/ischemic injury.

Expression of platelet-derived growth factor B-chain in the mature rat brain and pituitary gland.

For better understanding of the role of platelet-derived growth factor (PDGF) B-chain in the brain, the expression of PDGF B-chain was studied in the mature rat brain at both protein and mRNA levels, by assay of PDGF B-chain-related mitogenic activity, Northern blot, in situ hybridization and immunohistochemistry. It was shown that (1) mature rat brain contained substantial PDGF B-chain-related mitogenic activity, (2) significant amounts of two sizes of transcripts (3.5 kb, widely, and 2.6 kb, weakly and in narrower areas) were expressed in the brain, and (3) the transcripts were localized in ubiquitous neurons by in situ hybridization, with the strongest signal in hippocampal pyramidal cells, which distribution almost corresponded with that of the immunoreactive products. The abundant neuronal localization of the transcript and protein, as well as the neuronal expression of the receptor reported elsewhere, suggests the role of the growth factor in neuronal cells as a neuronal regulatory and/or trophic agent acting by autocrine loop or by neuron to neuron interaction. However, there was an apparent discrepancy in part, in the distribution between transcripts and immunoreactivity; that is, transcripts were expressed intensely in the intermediate pituitary lobe with only a scattered immunoreactivity, and the opposite situation was observed in the accessory olfactory nerve and posterior pituitary lobe. This might suggest that PDGF B-chain is transported or secreted in these foci.

Increased 5'-nucleotidase activity induced by dibutyryl cyclic AMP treatment of cultured glial cells.

In an attempt to clarify the relationship between ecto-5'-nucleotidase (5'-N) activity and cell differentiation of glial cells, dibutyryl cyclic AMP (dBcAMP), which induces cell differentiation, was administrated to cultured rat glioma cells in logarithmic and confluent phases of cultivation. To evaluate the cellular differentiation, cell morphology and the number of glial fibrillary acidic protein (GFAP) positive cells were examined. Treatment with 1 mM dBcAMP decreased cell proliferation and induced cell differentiation in both the logarithmic and the confluent phases. The number of GFAP-positive cells increased with cellular aging and this tendency was enhanced by dBcAMP administration. Ecto-5'-N activity was higher in dBcAMP treated cells than in non-treated cells in both the logarithmic and the confluent phases. These findings suggest that ecto-5'-N activity of C6 glioma cells is increased by dBcAMP actions and is accompanied by cell differentiation.

Platelet-derived growth factor B-chain-like immunoreactivity in injured rat brain.

Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cells and glial cells. For a better understanding of the role of PDGF B-chain in the brain, the expression of PDGF B-chain was examined immunohistochemically in penetrating injury to the rat brain. Shrunken neurons were distributed with enhanced PDGF B-chain-related immunoreactivity (PBRI) in the vicinity of the lesion during a period from day 1 to day 4 post injury. Platelet-derived growth factor B-chain-related immunoreactivity was transiently observed also in the cytoplasm of the numerous brain macrophages in the lesion on day 3 and day 4. These distributions of PBRI in the lesion were closely related to the neovascularization and astrogliosis there. The close time and spatial correlation between the expression of PBRI and cellular responses to injury seen in this study suggests PDGF B-chain has an important role in the healing process of cerebral wound.

Heparin-binding epidermal growth factor-like growth factor mRNA expression in neonatal rat brain with hypoxic/ischemic injury.

The neuronal expression of mRNA of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was investigated in immature rat brains. Two rat models were used in this study. One was a hypoxic/ischemic (HI) brain injury model, and the other was an N-methyl-d-aspartate (NMDA) intracerebral injection model. The former model was made by permanent ligation of the left carotid artery and subsequent exposure to 2 h of hypoxia. After the HI insult, the HB-EGF mRNA was assessed by a Northern blot analysis. The levels of transcripts for HB-EGF in the cerebral cortex and the hippocampus of the ligated side were significantly higher than those of non-treated rats from 3 to 24 h after the insult. The spatial distribution of the mRNA of HB-EGF was also studied using in situ hybridization. Three to 24 h after the hypoxia, hybridization signals were intense in neuronal cytoplasm on the ligated side, but a focally decreased signal was seen in infarcted areas. Strongly increased mRNA expression was observed in the neurons surrounding the infarct. These results indicate that a neonatal HI insult induces a neuronal upregulation of HB-EGF immediately after hypoxia. In the latter model, the intracerebral NMDA injection also induced an immediate, strong upregulation of HB-EGF transcripts. Our results indicate that HB-EGF may act as a neuroprotective factor in the immature brain with HI injury by modulating the neurotoxic process which is mediated by overactivation of the NMDA receptor.

Mossy fiber sprouting in the dentate gyrus in a newly developed epileptic mutant, Ihara epileptic rat.

We examined the correlation between seizure activity and development of mossy fiber sprouting in the hippocampal formation using Timm staining in a newly developed Ihara epileptic rat (IER). The sprouting of mossy fibers were clearly shown in the inner molecular portion of the dentate gyrus and in the stratum oriens of CA3 pyramidal cell layer with repeated seizures. A positive correlation between the frequency of generalized tonic and clonic convulsions and the Timm staining score in molecular layer of dentate gyrus was revealed. Sprouting of mossy fiber in IER seems to be linked with seizure activities resulting from epileptic bursts, not to the genetic mutation.

Expression of heparin-binding epidermal growth factor-like growth factor in rat brain.

According to a recent report, messenger RNA coding for a member of the epidermal growth factor (EGF) family, heparin-binding EGF-like growth factor (HB-EGF), is expressed in the central nervous system (CNS). To obtain information about the role of HB-EGF in the brain, we carried out Northern analysis, in situ hybridization, and immunohistochemical studies evaluating the distribution and amounts of the growth factor using cDNA HB-EGF probes and an antibody raised against synthetic HB-EGF propeptide. Northern analysis revealed transcripts for HB-EGF in all regions of normal rat brain. Immunohistochemically, HB-EGF was demonstrated extensively in neurons at levels varying according to location. HB-EGF mRNA also was detected in neurons, suggesting that the growth factor is produced in these cells. HB-EGF mRNA and immunoreactivity were also demonstrated in interfascicular oligodendrocytes. These findings suggest that HB-EGF is a physiologic ligand for brain EGF receptors, and is likely to be important in neural function.

Olfactory ensheathing glia and platelet-derived growth factor B-chain reactivity in the transplanted rat olfactory bulb.

Using a monoclonal antibody against the B-chain of platelet-derived growth factor as a marker, we have examined the behavior of olfactory ensheathing glia in the normal and transplanted rat olfactory bulb. In the normal postnatal olfactory bulb, these glia are found to ensheath the bundles of incoming primary olfactory nerve fibers as well as those in the olfactory nerve layer. Olfactory marker protein antibody was used to identify the olfactory nerve proper. Within the transplant, the same glia: (1) ensheath bundles of both primary olfactory and non-primary olfactory axons, (2) ensheath axonal bundles deep within the donor tissue, and (3) eventually permit radiation of individual axons from bundles to surrounding neuropil. We believe that ensheathing glia (being rich in growth-related factors and extracellular matrix molecules) may be useful in providing trophic support and guidance for the reconstruction of developmentally or traumatically damaged neuronal pathways not directly related to the olfactory system. The evidence presented here indicates that ensheathing glia are capable of existing in deep brain areas and ensheathing other than primary olfactory axons. The special molecular characteristics of these glia along with the morphological findings presented here provide a foundation for further studies of these unique glia and their potential utility in the restoration of damaged neural pathways.