Suppressor of Cytokine Signalling 2 (SOCS2) Regulates Numbers of Mature Newborn Adult Hippocampal Neurons and Their Dendritic Spine Maturation.

Overexpression of suppressor of cytokine signalling 2 (SOCS2) has been shown to promote hippocampal neurogenesis in vivo and promote neurite outgrowth of neurons in vitro. In the adult mouse brain, SOCS2 is most highly expressed in the hippocampal CA3 region and at lower levels in the dentate gyrus, an expression pattern that suggests a role in adult neurogenesis. Herein we examine generation of neuroblasts and their maturation into more mature neurons in SOCS2 null (SOCS2KO) mice. EdU was administered for 7 days to label proliferative neural precursor cells. The number of EdU-labelled doublecortin+ neuroblasts and NeuN+ mature neurons they generated was examined at day 8 and day 35, respectively. While no effect of SOCS2 deletion was observed in neuroblast generation, it reduced the numbers of EdU-labelled mature newborn neurons at 35 days. As SOCS2 regulates neurite outgrowth and dentate granule neurons project to the CA3 region, alterations in dendritic arborisation or spine formation may have correlated with the decreased numbers of EdU-labelled newborn neurons. SOCS2KO mice were crossed with Nes-CreERT2/mTmG mice, in which membrane eGFP is inducibly expressed in neural precursor cells and their progeny, and the dendrite and dendritic spine morphology of newborn neurons were examined at 35 days. SOCS2 deletion had no effect on total dendrite length, number of dendritic segments, number of branch points or total dendritic spine density but increased the number of mature "mushroom" spines. Our results suggest that endogenous SOCS2 regulates numbers of EdU-labelled mature newborn adult hippocampal neurons, possibly by mediating their survival and that this may be via a mechanism regulating dendritic spine maturation.

A novel role of suppressor of cytokine signaling-2 in the regulation of TrkA neurotrophin receptor biology.

Suppressor of cytokine signaling-2 (SOCS2) is a regulator of intracellular responses to growth factors and cytokines. Cultured dorsal root ganglia neurons from neonatal mice with increased or decreased SOCS2 expression were examined for altered responsiveness to nerve growth factor (NGF). In the presence of NGF, SOCS2 over-expression increased neurite length and complexity, whereas loss of SOCS2 reduced neurite outgrowth. Neither loss nor gain of SOCS2 expression altered the relative survival of these cells, suggesting that SOCS2 can discriminate between the differentiation and survival responses to NGF. Interaction studies in 293T cells revealed that SOCS2 immunoprecipitates with TrkA and a juxtamembrane motif of TrkA was required for this interaction. SOCS2 also immunoprecipitated with endogenous TrkA in PC12 Tet-On cells. Over-expression of SOCS2 in PC12 Tet-On cells increased total and surface TrkA expression. In contrast, dorsal root ganglion neurons which over-expressed SOCS2 did not exhibit significant changes in total levels but an increase in surface TrkA was noted. SOCS2-induced neurite outgrowth in PC12 Tet-On cells correlated with increased and prolonged activation of pAKT and pErk1/2 and required an intact SOCS2 SH2 domain and SOCS box domain. This study highlights a novel role for SOCS2 in the regulation of TrkA signaling and biology.

t-PA-specific modulation of a human blood-brain barrier model involves plasmin-mediated activation of the Rho kinase pathway in astrocytes.

Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.

Nanodiamonds with silicon vacancy defects for nontoxic photostable fluorescent labeling of neural precursor cells.

Nanodiamonds (NDs) containing silicon vacancy (SiV) defects were evaluated as a potential biomarker for the labeling and fluorescent imaging of neural precursor cells (NPCs). SiV-containing NDs were synthesized using chemical vapor deposition and silicon ion implantation. Spectrally, SiV-containing NDs exhibited extremely stable fluorescence and narrow bandwidth emission with an excellent signal to noise ratio exceeding that of NDs containing nitrogen-vacancy centers. NPCs labeled with NDs exhibited normal cell viability and proliferative properties consistent with biocompatibility. We conclude that SiV-containing NDs are a promising biomedical research tool for cellular labeling and optical imaging in stem cell research.

Transcriptional regulation and specification of neural stem cells.

With the discovery two decades ago that the adult brain contains neural stem cells (NSCs) capable of producing new neurons, a great deal of research has been undertaken to manipulate these cells to repair the damaged nervous system. Much progress has been made in understanding what regulates adult neural stem cell specification, proliferation and differentiation but much remains to be determined. Lessons can be learned from understanding how embryonic neural stem cells produce the exquisitely complicated organ that is the adult mammalian nervous system. This review will highlight the role of transcriptional regulation of mammalian neural stem cells during embryonic development and compare these to the adult neural stem cell/neural precursor cell (NPC) niches of the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Normal physiological NSC/NPC regulation will be explored, as well as their regulation and responses following neural injury and disease. Finally, transcriptional regulation of the endogenous NSC/NPCs will be compared and contrasted with embryonic stem/induced pluripotent stem (ES/iPS) cell-derived NSC/NPCs. Recapitulation of the embryonic sequence of transcriptional events in neural stem cell development into specific neuronal or glial lineages improves directed differentiation of ES/iPS cells and may be useful for activation and specification of endogenous adult neural stem cells for therapeutic purposes.

Bioinformatic prediction and confirmation of beta-adducin as a novel substrate of glycogen synthase kinase 3.

It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein ß-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), ß-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of ß-adducin by GSK3 promotes efficient neurite outgrowth in neurons.

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm.

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.

The Rho kinase pathway regulates mouse adult neural precursor cell migration.

Adult neural precursor cells (NPCs) in the subventricular zone (SVZ) normally migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB). Following neural injury, they also migrate to the site of damage. This study investigated the role of Rho-dependent kinase (ROCK) on the migration of NPCs in vitro and in vivo. In vitro, using neurospheres or SVZ explants, inhibition of ROCK using Y27632 promoted cell body elongation, process protrusion, and migration, while inhibiting NPC chain formation. It had no effect on proliferation, apoptosis, or differentiation. Both isoforms of ROCK were involved. Using siRNA, knockdown of both ROCK1 and ROCK2 was required to promote NPC migration and morphological changes; knockdown of ROCK2 alone was partially effective, with little/no effect of knockdown of ROCK1 alone. In vivo, infusion of Y27632 plus Bromodeoxyuridine (BrdU) into the lateral ventricle for 1 week reduced the number of BrdU-labeled NPCs in the OB compared with BrdU infusion alone. However, ROCK inhibition did not affect the tangential-to-radial switch of NPC migration, as labeled cells were present in all OB layers. The decrease in NPC number at the OB was not attributed to a decrease in NPCs at the SVZ. However, ROCK inhibition decreased the density of BrdU-labeled cells in the RMS and increased the distribution of these cells to ectopic brain regions, such as the accessory olfactory nucleus, where the majority differentiated into neurons. These findings suggest that ROCK signaling regulates NPC migration via regulation of cell-cell contact and chain migration.

Inflammatory regulators of redirected neural migration in the injured brain.

Brain injury following stroke or trauma induces the migration of neuroblasts derived from subventricular zone neural precursor cells (NPCs) towards the damaged tissue, where they then have the potential to contribute to repair. Enhancing the recruitment of new cells thus presents an enticing prospect for the development of new therapeutic approaches to treat brain injury; to this end, an understanding of the factors regulating this process is required. During the neuroinflammatory response to ischemic and traumatic brain injuries, a plethora of pro- and anti-inflammatory cytokines, chemokines and growth factors are released in the damaged tissue, and recent work indicates that a variety of these are able to influence injury-induced migration. In this review, we will discuss the contribution of specific chemokines and growth factors towards stimulating NPC migration in the injured brain.

LIF receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival.

Multiple sclerosis (MS) is a disabling inflammatory demyelinating disease of the central nervous system (CNS) that primarily affects young adults. Available therapies can inhibit the inflammatory component of MS but do not suppress progressive clinical disability. An alternative approach would be to inhibit mechanisms that drive the neuropathology of MS, which often includes the death of oligodendrocytes, the cells responsible for myelinating the CNS. Identification of molecular mechanisms that mediate the stress response of oligodendrocytes to optimize their survival would serve this need. This study shows that the neurotrophic cytokine leukemia inhibitory factor (LIF) directly prevents oligodendrocyte death in animal models of MS. We also demonstrate that this therapeutic effect complements endogenous LIF receptor signaling, which already serves to limit oligodendrocyte loss during immune attack. Our results provide a novel approach for the treatment of MS.

Eph receptor tyrosine kinases regulate astrocyte cytoskeletal rearrangement and focal adhesion formation.

EphA4 null mice have impaired astrocytic gliosis following spinal cord injury. This may be because of altered cytoskeletal regulation and is examined herein using cultured astrocytes from wildtype and EphA4 null mice. Under basal conditions EphA4 null astrocytes appeared relatively normal but following stimuli resulting in cytoskeletal rearrangement, EphA4 null cells responded more slowly. When F-actin stress fibers were collapsed using the Rho kinase inhibitor HA1077, fewer EphA4 null cells showed stress fiber collapse in response to HA1077 and recovered stress fibers more slowly following HA1077 removal. EphA4 null astrocytes were less adherent and had smaller focal adhesions, while activation of Eph receptors with ephrin-A5-Fc increased the numbers of focal adhesions in both wildtype and knockout astrocytes following serum starvation. Using scratch wound assays, EphA4 null astrocytes invading the scratch showed impaired glial fibrillary acidic protein expression, particularly in proliferative cells. Astrocytes did not express Ephexin, a major Eph-interacting Rho guanine exchange factor, but they expressed Vav proteins, with lower levels of phospho-Vav in EphA4 null compared to wildtype astrocytes. This may contribute to the slower cytoskeletal responses generally observed in the EphA4 null astrocytes. Eph receptor signaling therefore regulates astrocyte reactivity through modulation of cytoskeletal responses.

LPA receptor expression in the central nervous system in health and following injury.

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA(1-3)) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA(1) was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA(2) showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA(3) showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA(1-3) are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.

Species differences in reactivity of mouse and rat astrocytes in vitro.

Reactive astrogliosis constitutes a major obstacle to neuronal regeneration and is characterized by rearrangement and upregulation of expression of cytoskeletal proteins, increased proliferation and hypertrophy. Many approaches have been attempted to mimic astrogliosis by inducing reactive astrocytes in vitro. Such research is usually performed using astrocytes derived from Mus musculus or Rattus norvegicus, and results compared between species on the assumption that these cells behave equivalently. Therefore, we compared reactivity between mouse and rat astrocytes in scratch wound assays to gain further insight into how comparable these cell culture models are. Proliferation and migration, as well as expression of the cytoskeletal proteins glial fibrillary acidic protein (GFAP) and vimentin, were compared by immunocytochemistry and immunoblot. Further, we investigated migration of proliferating cells by 5-ethynyl-2'-deoxyuridine staining. Substantial differences in GFAP expression and proliferation between astrocytes of the two species were found: rat astrocytes showed different cytoskeletal morphology, expressed significantly more GFAP and vimentin of different molecular size and were more proliferative than comparable mouse astrocytes. Our results suggest that rat and mouse astrocytes may respond differently to various reactivity-triggering stimuli, which needs to be considered when general conclusions are drawn regarding effects of factors regulating astrocyte reactivity.

Cytokine-induced SOCS expression is inhibited by cAMP analogue: impact on regeneration in injured retina.

Injured adult retinal ganglion cells (RGCs) regrow axons into peripheral nerve (PN) grafted onto cut optic nerve. Survival and regeneration of RGCs is increased by intraocular injections of ciliary neurotrophic factor (CNTF) and axonal regeneration is further enhanced by co-injection of a cyclic AMP analogue (CPT-cAMP). Based on these data, and because cytokine signaling is negatively regulated by suppressor of cytokine signaling (SOCS) proteins, we set out to determine whether CNTF injections increase retinal SOCS expression and whether any changes are attenuated by co-injection with CPT-cAMP. Using quantitative PCR we found increased SOCS1, SOCS2 and SOCS3 mRNA levels at various times after a single CNTF injection. Expression remained high for many days. SOCS protein levels were also increased. In situ hybridization revealed that RGCs express SOCS3 mRNA, and SOCS expression in cultured RGCs was increased by CNTF. Co-injection of CPT-cAMP reduced CNTF induced expression of SOCS1 and SOCS3 mRNA and decreased SOCS3 protein expression. CNTF injection also transiently increased retinal leukemia inhibitory factor (LIF) expression, an effect that was also moderated by CPT-cAMP. We propose that, along with known reparative effects of elevated cAMP on neurons, reducing SOCS upregulation may be an additional way in which cyclic nucleotides augment cytokine-induced regenerative responses in the injured CNS.

Fibroblast growth factor-9 inhibits astrocyte differentiation of adult mouse neural progenitor cells.

Fibroblast growth factor-9 (FGF9) is expressed in the CNS and is reported to be a mitogen for glial cells, to promote neuronal survival, and to retard oligodendrocyte differentiation. Here we examined the effects of FGF9 on the differentiation, survival, and proliferation of adult neural progenitor cells derived from the adult mouse subventricular zone. FGF9 by itself induced neurosphere proliferation, but its effects were modest compared with those of epidermal growth factor and FGF2. When neurospheres were dissociated and plated for differentiation, FGF9 increased total cell number over time in a dose-dependent manner. Ki67 immunostaining and bromodeoxyuridine incorporation indicated that this was at least partially due to the continued presence of proliferative nestin-positive neural progenitor cells and betaIII tubulin-positive neuronal precursors. FGF9 also promoted cell survival as indicated by a decreased number of TUNEL-positive cells over time. Assessment of differentiation showed that FGF9 increased neuron generation that reflected the increase in total cell number; however, the percentage of progenitor cells differentiating into neurons was slightly decreased. FGF9 had a modest effect on oligodendrocyte generation, although it appeared to slow the maturation of oligodenrocytes at higher concentrations. The most marked effect on differentiation was an almost total lack of glial fibrillary acidic protein (GFAP)-positive astrocytes up to 7 days following FGF9 addition, indicating that astrocyte differentiation was strongly inhibited. Total inhibition required prolonged treatment, although a 1-hr pulse was sufficient for partial inhibition, and bone morphogenic protein-4 could partially overcome the FGF9 inhibition of astrocyte differentiation. FGF9 therefore has multiple effects on adult neural precursor cell function, enhancing neuronal precursor proliferation and specifically inhibiting GFAP expression.

Lysophosphatidic acid inhibits neuronal differentiation of neural stem/progenitor cells derived from human embryonic stem cells.

Lysophospholipids are signaling molecules that play broad and major roles within the nervous system during both early development and neural injury. We used neural differentiation of human embryonic stem cells (hESC) as an in vitro model to examine the specific effects of lysophosphatidic acid (LPA) at various stages of neural development, from neural induction to mature neurons and glia. We report that LPA inhibits neurosphere formation and the differentiation of neural stem cells (NSC) toward neurons, without modifying NSC proliferation, apoptosis, or astrocytic differentiation. LPA acts through the activation of the Rho/ROCK and the phosphatidylinositol 3-kinase/Akt pathways to inhibit neuronal differentiation. This study is the first demonstration of a role for LPA signaling in neuronal differentiation of hESC. As LPA concentrations increase during inflammation, the inhibition of neuronal differentiation by LPA might contribute to the low level of neurogenesis observed following neurotrauma.

c-Myb is required for neural progenitor cell proliferation and maintenance of the neural stem cell niche in adult brain.

Ongoing production of neurons in adult brain is restricted to specialized neurogenic niches. Deregulated expression of genes controlling homeostasis of neural progenitor cell division and/or their microenvironment underpins a spectrum of brain pathologies. Using conditional gene deletion, we show that the proto-oncogene c-myb regulates neural progenitor cell proliferation and maintains ependymal cell integrity in mice. These two cellular compartments constitute the neurogenic niche in the adult brain. Brains devoid of c-Myb showed enlarged ventricular spaces, ependymal cell abnormalities, and reduced neurogenesis. Neural progenitor cells lacking c-Myb showed a reduced intrinsic proliferative capacity and reduction of Sox-2 and Pax-6 expression. These data point to an important role for c-Myb in the neurogenic niche of the adult brain.

Erythropoietin promotes axonal growth in a model of neuronal polarization.

Erythropoietin (EPO) enhances neurogenesis, neuroprotection and regeneration. Here, we examined the effects of EPO on axonal and dendritic growth in a model of neuronal polarization. EPO did not effect survival or the polarized morphology of hippocampal neurons but its effect on neurite outgrowth depended upon the stage of polarization. When added to neurons in the process of establishing polarity (0-2 days in vitro (DIV)), it enhanced axonal and dendritic growth, while EPO added to early polarized cultures at 3-4 DIV promoted the growth of axons but not dendrites. EPO stimulated the phosphorylation of Akt at serine-473 and co-incubation of the Akt/PI-3 kinase pathway inhibitor LY294002 with EPO abolished its effects on Akt phosphorylation and axonal growth. However, while Leukemia Inhibitory Factor (LIF) similarly stimulated phosphorylation of Akt, it had no effect on axonal or dendritic growth, indicating that AKT phosphorylation is necessary but not sufficient for neurite outgrowth in hippocampal neurons.

Tropomyosin-Related Kinase B (TrkB) Regulates Neurite Outgrowth via a Novel Interaction with Suppressor of Cytokine Signalling 2 (SOCS2).

Brain-derived neurotrophic factor (BDNF) is highly expressed in the hippocampus, where it can initiate signalling pathways leading to neurite outgrowth, neuron survival, spine maturation and increased synapse strength. Although suppressor of cytokine signalling 2 (SOCS2) is primarily known to negatively regulate cytokine signalling, it is also highly expressed in the hippocampus and exerts neuron-specific functions in the brain, effecting the length and architecture of neurons. However, little is known about the role of SOCS2 in the hippocampus. In this study, we hypothesised that SOCS2 may have a regulatory role in BDNF-dependent neurite growth and hippocampal neuronal function. Here our data demonstrate that SOCS2 interacts with the kinase domain of the BDNF receptor TrkB. Germline overexpression of SOCS2 results in a BDNF-dependent increase in hippocampal neurite outgrowth, whereas deletion of SOCS2 results in shorter neurite outgrowth. Expression of SOCS2 also results in increased ubiquitination of the juxtamembrane region of TrkB, and alters the trafficking of TrkB into recycling endosomes. Collectively, our data suggest a novel role for SOCS2 in interacting with and regulating the trafficking of TrkB, leading to increased neurite outgrowth in hippocampus neurons.

Growth hormone signaling and hippocampal neurogenesis: insights from genetic models.

Adult hippocampal neurogenesis (AHN) is modulated by a variety of factors through effects on the proliferation-differentiation-survival regulatory axis. We have employed growth hormone receptor knockout (GH-R-/-) and suppressor of cytokine signaling-2 transgenic (SOCS-2 Tg) mice as models of altered GH-signaling to assess their affects on basal and exercised-induced hippocampal neurogenesis. Assessment of proliferation 24-h after 7-days of bromodeoxyuridine (BrdU) labeling with or without voluntary running showed that the density of BrdU(+) cells in the subgranular zone remained unchanged between genotypes in control housing, while running induced significant increases in BrdU-labeled cells in WT, GH-R-/-, and SOCS-2 Tg mice. The proportion of BrdU/doublecortin and BrdU/S100beta cells did not vary between genotype or running conditions at this time-point. Assessment of cell survival 28-days after BrdU labeling showed that SOCS-2 Tg animals had significantly higher BrdU(+) cell densities in the granule cell layer compared to WT and GH-R-/- animals in control housing and after voluntary running. There were no differences in cell survival between WT and GH-R-/- mice with or without running. Mature phenotype analysis showed similar proportions of BrdU/NeuN and BrdU/S100beta in all groups. While SOCS-2 Tg mice had similar social interaction behaviors and sensorimotor gating, they appeared to be less anxious with heightened basal locomotor activity and showed enhanced performance in the Morris watermaze test. Overall, our data indicated that mice over-expressing SOCS-2 showed increased survival of neurons generated during AHN, which correlated with improved performance in a hippocampal-dependent cognitive task. Furthermore, voluntary running increased AHN in WT, SOCS-2 Tg, and serum-IGF-1-deficient GH-R-/- mice.