Expression of nuclear Methyl-CpG binding protein 2 (Mecp2) is dependent on neuronal stimulation and application of Insulin-like growth factor 1.

Methyl-CpG binding protein 2 (MECP2) is a chromosome-binding protein that regulates the development and maintenance of brain circuits. Altered function of the protein product of MECP2 plays an important role in the etiology of many neurodevelopmental disorders. Mutations involving a loss of function are implicated in the etiology of Rett syndrome, intellectual disability, psychosis and severe encephalopathy. Conversely, MECP2 duplications have been identified in autism and intellectual disability. MECP2 action is dependent on neuronal function, as the DNA binding is modulated by activity, and it is phosphorylated in response to stimulation. Although MECP2 is considered a major risk factor for neurodevelopmental disorders, and it is a mediator of activity-dependent mechanisms, the expression levels in response to neuronal activity have never been measured. We studied the expression of Mecp2 protein and RNA in mice neuronal cultures in response to different stimulation conditions and in the presence of insulin-like growth factor1 (IGF1): a growth factor involved in brain development and plasticity. The stimulation protocols were selected according to their ability to induce different forms of synaptic plasticity: rapid depolarization, feed-forward plasticity (LTP, LTD) and feedback forms of plasticity (TTX, KCl). We find a significant reduction of Mecp2 protein nuclear expression in neurons in response to stimuli that induce a potentiation of neuronal response, suggesting that Mecp2 protein expression is modulated by neuronal activation. Application of IGF1 to the cultures induces an increase in the expression of Mecp2 transcript and nuclear Mecp2 protein in neurons. These results show that Mecp2 is responsive to neuronal stimulation and IGF1, and different stimuli have different effects on Mecp2 expression; this differential response may have downstream effects on functional mechanisms regulating brain development and plasticity.

An inherited duplication at the gene p21 Protein-Activated Kinase 7 (PAK7) is a risk factor for psychosis.

Identifying rare, highly penetrant risk mutations may be an important step in dissecting the molecular etiology of schizophrenia. We conducted a gene-based analysis of large (>100 kb), rare copy-number variants (CNVs) in the Wellcome Trust Case Control Consortium 2 (WTCCC2) schizophrenia sample of 1564 cases and 1748 controls all from Ireland, and further extended the analysis to include an additional 5196 UK controls. We found association with duplications at chr20p12.2 (P = 0.007) and evidence of replication in large independent European schizophrenia (P = 0.052) and UK bipolar disorder case-control cohorts (P = 0.047). A combined analysis of Irish/UK subjects including additional psychosis cases (schizophrenia and bipolar disorder) identified 22 carriers in 11 707 cases and 10 carriers in 21 204 controls [meta-analysis Cochran-Mantel-Haenszel P-value = 2 × 10(-4); odds ratio (OR) = 11.3, 95% CI = 3.7, ∞]. Nineteen of the 22 cases and 8 of the 10 controls carried duplications starting at 9.68 Mb with similar breakpoints across samples. By haplotype analysis and sequencing, we identified a tandem ~149 kb duplication overlapping the gene p21 Protein-Activated Kinase 7 (PAK7, also called PAK5) which was in linkage disequilibrium with local haplotypes (P = 2.5 × 10(-21)), indicative of a single ancestral duplication event. We confirmed the breakpoints in 8/8 carriers tested and found co-segregation of the duplication with illness in two additional family members of one of the affected probands. We demonstrate that PAK7 is developmentally co-expressed with another known psychosis risk gene (DISC1) suggesting a potential molecular mechanism involving aberrant synapse development and plasticity.

Growth cartilage expression of growth hormone/insulin-like growth factor I axis in spontaneous and growth hormone induced catch-up growth.

INTRODUCTION: Catch-up growth following the cessation of a growth inhibiting cause occurs in humans and animals. Although its underlying regulatory mechanisms are not well understood, current hypothesis confer an increasing importance to local factors intrinsic to the long bones' growth plate (GP). AIM: The present study was designed to analyze the growth-hormone (GH)-insulin-like growth factor I (IGF-I) axis in the epiphyseal cartilage of young rats exhibiting catch-up growth as well as to evaluate the effect of GH treatment on this process. MATERIAL AND METHODS: Female Sprague-Dawley rats were randomly grouped: controls (group C), 50% diet restriction for 3 days+refeeding (group CR); 50% diet restriction for 3 days+refeeding & GH treatment (group CRGH). Analysis of GH receptor (GHR), IGF-I, IGF-I receptor (IGF-IR) and IGF binding protein 5 (IGFBP5) expressions by real-time PCR was performed in tibial growth plates extracted at the time of catch-up growth, identified by osseous front advance greater than that of C animals. RESULTS: In the absence of GH treatment, catch-up growth was associated with increased IGF-I and IGFBP5 mRNA levels, without changes in GHR or IGF-IR. GH treatment maintained the overexpression of IGF-I mRNA and induced an important increase in IGF-IR expression. CONCLUSIONS: Catch-up growth that happens after diet restriction might be related with a dual stimulating local effect of IGF-I in growth plate resulting from overexpression and increased bioavailability of IGF-I. GH treatment further enhanced expression of IGF-IR which likely resulted in a potentiation of local IGF-I actions. These findings point out to an important role of growth cartilage GH/IGF-I axis regulation in a rat model of catch-up growth.

Insulin-like growth factor 1 (IGF1) and its active peptide (1-3)IGF1 enhance the expression of synaptic markers in neuronal circuits through different cellular mechanisms.

Insulin-like growth factor-1 (IGF1) and its active peptide (1-3)IGF1 modulate brain growth and plasticity and are candidate molecules for treatment of brain disorders. IGF1 N-terminal portion is naturally cleaved to generate the tri-peptide (1-3)IGF1 (glycine-praline-glutamate). IGF1 and (1-3)IGF have been proposed as treatment for neuropathologies, yet their effect on nerve cells has not been directly compared. In this study we examine the effects of IGF1 and (1-3)IGF1 in primary cortical cultures and measure the expression levels of markers for intracellular pathways and synaptic function. We find that both treatments activate the IGF1 receptor and enhance the expression of synaptic markers, however, they activate different intracellular pathways. Furthermore, (1-3)IGF1 administration increases the expression of endogenous IGF1, suggesting a direct interaction between the two molecules. The results show that the two molecules increase the expression of synaptic proteins through activating different cellular mechanisms.

Rapamycin retards growth and causes marked alterations in the growth plate of young rats.

Rapamycin is a potent immunosuppressant with antitumoral properties widely used in the field of renal transplantation. To test the hypothesis that the antiproliferative and antiangiogenic activity of rapamycin interferes with the normal structure and function of growth plate and impairs longitudinal growth, 4-week-old male rats (n = 10/group) receiving 2 mg/kg per day of intraperitoneal rapamycin (RAPA) or vehicle (C) for 14 days were compared. Rapamycin markedly decreased bone longitudinal growth rate (94 +/- 3 vs. 182 +/- 3 microm/day), body weight gain (60.2 +/- 1.4 vs. 113.6 +/- 1.9 g), food intake (227.8 +/- 2.6 vs. 287.5 +/- 3.4 g), and food efficiency (0.26 +/- 0.00 vs. 0.40 +/- 0.01 g/g). Signs of altered cartilage formation such as reduced chondrocyte proliferation (bromodeoxiuridine-labeled cells 32.9 +/- 1.4 vs. 45.2 +/- 1.1%), disturbed maturation and hypertrophy (height of terminal chondrocytes 26 +/- 0 vs. 29 +/- 0 microm), and decreased cartilage resorption (18.7 +/- 0.5 vs. 31.0 +/- 0.8 tartrate-resistant phosphatase alkaline reactive cells per 100 terminal chondrocytes), together with morphological evidence of altered vascular invasion, were seen in the growth plate of RAPA animals. This study indicates that rapamycin can severely impair body growth in fast-growing rats and distort growth-plate structure and dynamics. These undesirable effects must be kept in mind when rapamycin is administered to children.

Administration of ghrelin to young uraemic rats increases food intake transiently, stimulates growth hormone secretion and does not improve longitudinal growth.

BACKGROUND: Ghrelin administration stimulates appetite and growth hormone (GH) secretion. Whether these effects are preserved in young individuals with chronic renal failure (CRF) and their potential benefit on growth is questioned. METHODS: Three experiments were performed in subtotally nephrectomized young rats (Nx). (i) Food intake was monitored in CRF rats receiving saline intraperitoneally or a ghrelin dose (30 nmol) shown to increase food intake over 2 and 24 h in rats with normal renal function. (ii) Plasma levels of GH were measured after a dose of intravenous ghrelin (3 nmol) was given to three groups of five rats each: Nx, sham-operated fed ad libitum (SAL) and sham-operated pair-fed with Nx (SPF). (iii) Growth of Nx rats treated with intraperitoneal ghrelin (3 nmol) for 7 days (Nx-Ghr) was compared with that of SAL and Nx groups receiving saline (n=8-10 per group). RESULTS: In CRF rats, the dose of 30 nmol of ghrelin increased food consumption for 2 h (1.3+/-0.2 g vs 0.5+/-0.2 g, P<0.05) but not 24-h food intake (12.5+/-0.6 g vs 12.2+/-0.5 g). Ghrelin (3 nmol) increased plasma levels of GH, which were maximal 10 min after injection, no differences being observed among groups (SAL: 666.2+/-104.6 ng/ml; Nx: 691.6+/-90.7 ng/ml; SPF: 577.8+/-125.4 ng/ml). Return to basal GH levels was delayed in Nx. Ghrelin did not improve body length and weight gains, longitudinal bone growth rate or food intake in the Nx-Ghr group. CONCLUSIONS: In young uraemic rats, ghrelin increases appetite but not 24-h food intake, stimulates GH secretion and does not improve growth.

Alterations of the growth plate in chronic renal failure.

Chronic renal failure modifies the morphology and dynamics of the growth plate (GP) of long bones. In young uremic rats, the height of cartilage columns of GP may vary markedly. The reasons for this variation are unknown, although the severity and duration of renal failure and the type of renal osteodystrophy have been shown to influence the height of GP cartilage. Expansion of GP cartilage is associated with that of the hypertrophic stratum. The interference of uremia with the process of chondrocyte differentiation is suggested by some morphological features. However, analysis by immunohistochemistry and/or in situ hybridization of markers of chondrocyte maturation in the GP of uremic rats has yielded conflicting results. Thus, there have been reported normal and reduced mRNA levels for collagen X, parathyroid hormone/parathyroid hormone-related peptide receptor, and matrix metalloproteinase 9, as well as normal mRNA and protein expression for vascular endothelial growth factor and chondromodulin I, peptides related to the control of angiogenesis. In addition, a decreased immunohistochemical signal for growth hormone receptor and low insulin-like growth factor I mRNA in the proliferative zone of uremic GP are supportive of reduced chondrocyte proliferation. Growth hormone treatment improves chondrocyte maturation and activates bone metabolism in the primary spongiosa.

Chondromodulin-I expression in the growth plate of young uremic rats.

BACKGROUND: Growth retardation of chronic renal failure is associated with alterations in the growth plate suggestive of a disturbed chondrocyte maturation process and abnormal vascular invasion at the chondro-osseous interphase. Chondromodulin I (ChM-I) is a potent cartilage-specific angiostatic factor. Its pattern of expression in the uremic rat growth plate is unknown. Persistence of ChM-I synthesis and/or imbalance between ChM-I and vascular endothelial growth factor (VEGF) expressions might play a role in the alterations of uremic growth plate. METHODS: Growth cartilage ChM-I expression was investigated by immunohistochemistry, in situ hybridization, and reverse transcription-polymerase chain reaction (RT-PCR) in growth-retarded young uremic rats (UREM), control rats, fed ad libitum (SAL) or pair-fed with the UREM group (SPF), and uremic rats treated with growth hormone (UREM-GH). VEGF expression was analyzed by immunohistochemistry. RESULTS: ChM-I and ChM-I mRNA were confined to the proliferative and early hypertrophic zones of growth cartilage. A similar number of chondrocytes per column was positive for ChM-I in the 4 groups. In accordance with the elongation of the hypertrophic stratum in uremia, the distance (X+/-SEM, microm) between the extracellular ChM-I signal and the metaphyseal end of growth cartilage was higher (P < 0.003) in UREM (236 +/- 40) and UREM-GH (297 +/- 17) than in SAL (92 +/- 7) and SPF (113 +/- 6). No differences in ChM-I expression were appreciated by RT-PCR. Similar VEGF positivity was observed in the hypertrophic chondrocytes of all groups. CONCLUSION: In experimental uremia, expansion of growth cartilage does not result from increased or persistent expression of ChM-I or from reduced VEGF expression at the cartilage-metaphyseal bone interphase. GH treatment does not modify ChM-I and VEGF expressions.

Growth plate height of uremic rats is influenced by severity and duration of renal failure.

To understand the changes induced by uremia in the epiphyseal growth plate, two studies were performed in young rats. In study 1, the morphological features of the tibial growth cartilage of stunted rats with different degrees of reduction of renal function were analyzed 2 weeks after nephrectomy and compared with control rats. There was a negative ( r=-0.549, P<0.05) correlation between serum urea nitrogen (SUN) concentrations and longitudinal growth rate. The heights (mean+/-SEM) of growth cartilage (564+/-32 vs. 366+/-9 microm) and its hypertrophic zone (321+/-25 vs. 157+/-6 microm) were greater ( P<0.05) in uremic than control rats and were highly and positively correlated ( r=0.604, P<0.03 and r=0.706, P<0.01) with SUN levels. In study 2, the time course of growth plate alterations was investigated in uremic rats sacrificed 1 (NX-1), 2 (NX-2), and 4 weeks (NX-4) after nephrectomy compared with their corresponding control animals (C-1, C-2, C-4). Growth cartilage and hypertrophic zone heights were greater in NX-2 (533+/-60 and 264+/-32 microm) than in C-2 (345+/-10 and 131+/-11 microm), with no significant differences in the other groups. This report shows that enlargement of the growth plate and its hypertrophic stratum is greatly, although not exclusively, influenced by the severity and duration of renal insufficiency.