Takako Matsushima

Mouse brains deficient in neuronal PDGF receptor-beta develop normally but are vulnerable to injury.

Platelet‐derived growth factors (PDGFs) and PDGF receptors (PDGFRs) are widely expressed in the mammalian CNS, though their functional significance remains unclear. The corresponding null‐knockout mutations are lethal. Here, we developed novel mutant mice in which the gene encoding the β subunit of PDGFR (PDGFR‐β) was genetically deleted in CNS neurons to elucidate the role of PDGFR‐β, particularly in the post‐natal stage. Our mutant mice reached adulthood without apparent anatomical defects. In the mutant brain, immunohistochemical analyses showed that PDGFR‐β detected in neurons and in the cells in the subventricular zone of the lateral ventricle in wild‐type mice was depleted, but PDGFR‐β detected in blood vessels remained unaffected. The cerebral damage after cryogenic injury was severely exacerbated in the mutants compared with controls. Furthermore, TdT‐mediated dUTP‐biotin nick end labeling (TUNEL)‐positive neuronal cell death and lesion formation in the cerebral hemisphere were extensively exacerbated in our mutant mice after direct injection of NMDA without altered NMDA receptor expression. Our results clearly demonstrate that PDGFR‐β expressed in neurons protects them from cryogenic injury and NMDA‐induced excitotoxicity.

PDGFR-β as a positive regulator of tissue repair in a mouse model of focal cerebral ischemia

Although platelet-derived growth factors (PDGFs) and receptors (PDGFRs) are abundantly expressed in the central nervous system, their functions largely remain elusive. We investigated the role of PDGFR-β in tissue responses and functional recovery after photothrombolic middle cerebral artery occlusion (MCAO). In the normal adult mouse brain, PDGFR-β was mainly localized in neurons and in pericyte/vascular smooth muscle cells (PC/vSMCs). From 3 to 28 days after MCAO, postnatally induced systemic PDGFR-β knockout mice (Esr-KO) exhibited the delayed recovery of body weight and behavior, and larger infarction volume than controls. In Esr-KO, PC/vSMC coverage was decreased and vascular leakage of infused fluorescent-labeled albumin was extensive within the ischemic lesion, but not in the uninjured cerebral cortex. Angiogenesis levels were comparable between Esr-KO and controls. In another PDGFR-β conditional KO mouse (Nestin-KO), PDGFR-β was deleted in neurons and astrocytes from embryonic day 10.5, but was preserved in PC/vSMCs. After MCAO, vascular leakage and infarction volume in Nestin-KO were worse than controls, but partly improved compared with Esr-KO. Astroglial scar formation in both Esr-KO and Nestin-KO was similarly reduced compared with controls after MCAO. These data suggested that PDGFR-β signaling is crucial for neuroprotection, endogenous tissue repair, and functional recovery after stroke by targeting neurons, PC/vSMCs, and astrocytes.

Activation of MAP kinases, Akt and PDGF receptors in injured peripheral nerves

A number of receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol‐3‐kinase (PI3K)/Akt and mitogen‐activated protein (MAP) kinase signaling pathways have been critically involved in peripheral nerve regeneration. Here, we examined the activation of PI3K/Akt and MAP kinase pathways, and platelet‐derived growth factor receptors (PDGFRs) in the distal segments of crushed rat sciatic nerve from 3 to 28 days after injury. In Western blot analyses, the phosphorylated forms of extracellular signal‐regulated protein kinase (ERK) and c‐Jun NH2‐terminal kinases (JNKs) were highly augmented on days 3 and 7 and on days 7 and 14 after injury, respectively. Phosphorylated Akt and p38 consistently increased from 3 to 28 days after injury. Phosphorylated PDGFR‐α and ‐β were also increased from 3 to 14 days. In the immunohistological analyses, phosphorylated ERK and PDGFR‐α were co‐localized in many activated Schwann cells and regrowing axons 3 days after injury, while PDGFR‐β was localized in a few spindle‐shaped cells. The detected temporal profile of RTK signaling appears to be crucial for the regulation of Schwann cell proliferation and following redifferentiation. Furthermore, the immunohistological studies suggested a role of ERK and PDGFR‐α in axon regeneration as well.

Cognitive and socio-emotional deficits in platelet-derived growth factor receptor-β gene knockout mice.

Platelet-derived growth factor (PDGF) is a potent mitogen. Extensive in vivo studies of PDGF and its receptor (PDGFR) genes have reported that PDGF plays an important role in embryogenesis and development of the central nervous system (CNS). Furthermore, PDGF and the β subunit of the PDGF receptor (PDGFR-β) have been reported to be associated with schizophrenia and autism. However, no study has reported on the effects of PDGF deletion on mice behavior. Here we generated novel mutant mice (PDGFR-β KO) in which PDGFR-β was conditionally deleted in CNS neurons using the Cre/loxP system. Mice without the Cre transgene but with floxed PDGFR-β were used as controls. Both groups of mice reached adulthood without any apparent anatomical defects. These mice were further examined by conducting several behavioral tests for spatial memory, social interaction, conditioning, prepulse inhibition, and forced swimming. The test results indicated that the PDGFR-β KO mice show deficits in all of these areas. Furthermore, an immunohistochemical study of the PDGFR-β KO mice brain indicated that the number of parvalbumin (calcium-binding protein)-positive (i.e., putatively γ-aminobutyric acid-ergic) neurons was low in the amygdala, hippocampus, and medial prefrontal cortex. Neurophysiological studies indicated that sensory-evoked gamma oscillation was low in the PDGFR-β KO mice, consistent with the observed reduction in the number of parvalbumin-positive neurons. These results suggest that PDGFR-β plays an important role in cognitive and socioemotional functions, and that deficits in this receptor may partly underlie the cognitive and socioemotional deficits observed in schizophrenic and autistic patients.

Aberrant hippocampal spine morphology and impaired memory formation in neuronal platelet‐derived growth factor beta‐receptor lacking mice

The physiological role of platelet‐derived growth factor (PDGF) in the central nervous system (CNS) synaptic function remains uncharacterized. Here we identify physiological roles of PDGF receptor‐β (PDGFR‐β) in the CNS by conditional knockout of the gene encoding it. In the hippocampus, PDGFR‐β colocalized immunohistochemically with both presynaptic synaptophysin and postsynaptic density‐95 (PSD‐95). In the hippocampal CA1 region, expression levels of postsynaptic proteins, including spinophilin, drebrin, and PSD‐95, were significantly decreased in PDGFR‐β knockout mice, although presynaptic synaptophysin levels remained comparable to controls. Interestingly, in hippocampal CA1 pyramidal neurons, dendritic spine density in PDGFR‐β knockout mice was significantly decreased compared with that seen in wild‐type mice, although spine length and number of dendritic branches remained unchanged. Consistent with these findings, impairment in hippocampal long‐term potentiation (LTP) and in hippocampus‐dependent memory formation were seen in PDGFR‐β knockout mice. These results suggest PDGFR‐β plays critical roles in spine morphology and memory formation in mouse brain.

Localization and hormonal control of serine dehydratase during metabolic acidosis differ markedly from those of phosphoenolpyruvate carboxykinase in rat kidney.

Serine dehydratase (SDH) is abundant in the rat liver but scarce in the kidney. When administrated with dexamethasone, the renal SDH activity was augmented 20-fold, whereas the hepatic SDH activity was affected little. In situ hybridization and immunohistochemistry revealed that SDH was localized to the proximal straight tubule of the nephron. To address the role of this hormone, rats were made acidotic by gavage of NH(4)Cl. Twenty-two hours later, the SDH activity was increased three-fold along with a six-fold increment in the phosphoenolpyruvate carboxykinase (PEPCK) activity, a rate-limiting enzyme of gluconeogenesis. PEPCK, which is localized to the proximal tubules under the normal condition, spreads throughout the entire cortex to the outer medullary rays by acidosis, whereas SDH does not change regardless of treatment with dexamethasone or NH(4)Cl. When NH(4)Cl was given to adrenalectomized rats, in contrast to the SDH activity no longer increasing, the PEPCK activity responded to acidosis to the same extent as in the intact rats. A simultaneous administration of dexamethasone and NH(4)Cl into the adrenalectomized rats fully restored the SDH activity, demonstrating that the rise in the SDH activity during acidosis is primarily controlled by glucocorticoids. The present findings clearly indicate that the localization of SDH and its hormonal regulation during acidosis are strikingly different from those of PEPCK.

Serine racemase deletion attenuates neurodegeneration and microvascular damage in diabetic retinopathy

Diabetic retinopathy (DR) is a leading cause of blindness. DR is recognized as a microvascular disease and inner retinal neurodegeneration. In the course of retinal neurodegeneration, N-methyl-D-aspartate receptor (NMDAR)-mediated excitotoxicity is involved. Full activation of NMDAR requires binding of agonist glutamate and coagonist glycine or D-serine. D-Serine is produced from L-serine by serine racemase (SRR) and contributes to retinal neurodegeneration in rodent models of DR. However, the involvement of SRR in both neurodegeneration and microvascular damage in DR remains unclear. Here, we established diabetic model of SRR knockout (SRR-KO) and control wild-type (WT) mice by streptozotocin injection. Six months after the onset of diabetes, the number of survived retinal ganglion cells was higher in SRR-KO mice than that of WT mice. The reduction of thickness of inner retinal layer (IRL) was attenuated in SRR-KO mice than that of WT mice. Moreover, the number of damaged acellular capillaries was lower in SRR-KO mice than that of WT mice. Our results suggest the suppression of SRR activity may have protective effects in DR.

PDGFR-β restores blood-brain barrier functions in a mouse model of focal cerebral ischemia

Although platelet-derived growth factor receptor beta (PDGFR-β) mediates the recruitment of vascular pericytes into ischemic lesion to restore the blood-brain barrier (BBB) dysfunction, its mechanisms still remain elusive. Compared with control PDGFR-βfloxed/floxed mice (Floxed), postnatally induced systemic PDGFR-β knockout mice (Esr-KO) not only showed severe brain edema, neurologic functional deficits, decreased expression of tight junction (TJ) proteins, abundant endothelial transcytosis, and deformed TJs in the BBB, but also showed reduced expression of transforming growth factor-β (TGF-β) protein after photothrombotic middle cerebral artery occlusion (MCAO). In endothelial-pericyte co-culture, an in vitro model of BBB, the increment in the barrier function of endothelial monolayer induced by pericyte co-culture was completely cancelled by silencing PDGFR-β gene expression in pericytes, and was additively improved by PDGFR-β and TGF-β receptor signals under hypoxia condition. Exogenous PDGF-BB increased the expression of p-Smad2/3, while anti-TGF-β1 antibody at least partially inhibited the phosphorylation of Smad2/3 after PDGF-BB treatment in vitro. Furthermore, pre-administration of TGF-β1 partially alleviated edema formation, neurologic dysfunction, and TJs reduction in Esr-KO mice after MCAO. Accordingly, PDGFR-β signalling, via TGF-β signalling, may be crucial for restoration of BBB integrity after cerebral ischemia and therefore represents a novel potential therapeutic target.

The Novel Pathogenesis of Retinopathy Mediated by Multiple RTK Signals is Uncovered in Newly Developed Mouse Model

Pericyte desorption from retinal blood vessels and subsequent vascular abnormalities are the pathogenesis of diabetic retinopathy (DR). Although the involvement of abnormal signals including platelet-derived growth factor receptor-β (PDGFRβ) and vascular endothelial growth factor-A (VEGF-A) have been hypothesized in DR, the mechanisms that underlie this processes are largely unknown. Here, novel retinopathy mouse model (N-PRβ-KO) was developed with conditional Pdgfrb gene deletion by Nestin promoter-driven Cre recombinase (Nestin-Cre) that consistently reproduced through early non-proliferative to late proliferative DR pathologies. Depletion of Nestin-Cre-sensitive PDGFRβ+NG2+αSMA− pericytes suppressed pericyte-coverages and induced severe vascular lesion and hemorrhage. Nestin-Cre-insensitive PDGFRβ+NG2+αSMA+ pericytes detached from the vascular wall, and subsequently changed into myofibroblasts in proliferative membrane to cause retinal traction. PDGFRα+ astrogliosis was seen in degenerated retina. Expressions of placental growth factor (PlGF), VEGF-A and PDGF-BB were significantly increased in the retina of N-PRβ-KO. PDGF-BB may contribute to the pericyte-fibroblast transition and glial scar formation. Since VEGFR1 signal blockade significantly ameliorated the vascular phenotype in N-PRβ-KO mice, the augmented VEGFR1 signal by PlGF and VEGF-A was indicated to mediate vascular lesions. In addition to PDGF-BB, PlGF and VEGF-A with their intracellular signals may be the relevant therapeutic targets to protect eyes from DR.

PDGFR-β is essential for recruitment, but not for survival of pericyte in adult mouse brain

It has been long postulated that social decision making consists of at least two different levels: rapid intuitive and slow reflective processes. However, behavioral and neuronal evidence of this hypothesis remains poorly identified. Here, we address this issue by conducting behavioral and fMRI experiments of the ultimatum game with cognitive load. Prior to the experiments, subjects were classified into prosocials (n = 39) and individualists (n = 20) by the definition of social value orientation (Van Lange et al., 1989; Haruno and Frith, 2010), which has been proposed as characterizing rapid and intuitive attitudes towards the division of resources. Subjects were asked to respond whether to accept or reject a sequence of divisions of 500 yen offered by proposers. The subjects were also required to memorize a fivedigit number prior to the presentation of the offer, and had to answer the next number to the one displayed on the screen after the response. To control the availability of subject’s cognitive resource for reflective thinking, we compared the loaded (random number) and unloaded (always “01234”) conditions. Behaviorally, prosocials rejected more often than individualists (p < 0.00001; three-way ANOVA). More importantly, prosocials showed more rejections in the loaded condition than in the unloaded condition (p < 0.05; paired t-test), while individualists accepted more often in the loaded condition. In parallel with this behavioral difference, activity in the nucleus accumbens correlated with proposer’s payoff minus subject’s payoff revealed a statistically significant difference between the two groups in the loaded condition (p < 0.05; FWE corrected), but not in the unloaded condition. These results indicate that “social value orientation” indeed represents subject’s rapid intuitive attitude and the nucleus accumbens plays a key role in making behavioral decisions under the influence of the social value orientation. Research fund: This study was supported by PRESTO/JST, KAKENHI (22300139), and SRPBS.