Spinophilin Expression Research Articles

Functional and structural recovery of the injured spinal cord in rats treated with gonadotropin-releasing hormone.

Several studies have shown that gonadotropin-releasing hormone (GnRH) have extra-pituitary roles, including neurotrophic effects. This study was to evaluate the effects of GnRH treatment on the spinal cord injury (SCI) of rats. Ovariectomized rats were divided into: sham SCI surgery (Sham), SCI treated with saline solution (SCI + SS), and SCI treated with GnRH (SCI + GnRH). The SCI was induced by compression. One day after the lesion, SCI + GnRH group was injected with GnRH (60 µg/kg/twice/day; i.m.) for 15 days and the other groups with saline solution. To kinematic gait analysis, length and velocity of the stride were measured. In spinal cord, axonal morphometry and spared white and gray matter were analyzed by histochemistry. Protein expression of spinophilin was evaluated by western blot. The results showed that, 5 weeks after the injury, the group of animals treated with GnRH, significantly increased the length and velocity of the stride compared to SCI + SS group and they were similar to Sham group. In spinal cord, GnRH treatment increased the number and caliber of nerve axons and in the case of white matter, spared tissue was significantly higher than those animals treated with saline solution. The expression of spinophilin in spinal cord of SCI + GnRH group was slightly increased with respect to those not treated. In conclusion, GnRH treatment improves recovery of gait and decreases histopathological damage in the injured spinal cord of rat. These findings suggest that GnRH acts as a neurotrophic factor and can be used as a potential therapeutic agent for treatment of SCI.

Spinophilin expression determines cellular growth, cancer stemness and 5-flourouracil resistance in colorectal cancer.

The putative tumor suppressor gene spinophilin has been involved in cancer progression in several types of cancer. In this study, we explored the prognostic value of spinophilin expression in 162 colon adenocarcinoma patients. In addition, we generated stably expressing spinophilin-directed shRNA CRC cell lines and studied the influence of spinophilin expression on cellular phenotypes and molecular interactions. We independently confirmed that low spinophilin expression levels are associated with poor prognosis in CRC patients (p = 0.038). A reduction of spinophilin levels in p53 wild-type HCT116 and p53-mutated Caco-2 cells led to increased cellular growth rates and anchorage-independent growth (p<0.05). At molecular level, reduced spinophilin levels increased the expression of the transcription factor E2F-1. In addition, we observed an increased formation of tumor spheres, increased number of CD133 positive cells and an increased resistance to 5-flourouracil (p<0.05). Finally, treatment with the de-methylating agent 5-aza-dC increased spinophilin expression in CRC cells (p<0.05), corroborated by a correlation of spinophilin expression and extent of methylated CpG sites in the gene promoter region (p<0.001). In conclusion, gain of aggressive biological properties of CRC cells including cellular growth, cancer stem cell features and 5-flourouracil resistance partly explains the role of spinophilin in CRC.

Loss of the putative tumor suppressor protein spinophilin is associated with poor prognosis in head and neck cancer

The putative tumor suppressor protein spinophilin has been recently involved in the pathogenesis of lung, liver, and other types of cancer. Previous studies also indicate that a loss of spinophilin in combination with functional impairment of p53 drives tumor progression. To date, no data exist about the role of spinophilin in head and neck squamous cell carcinoma (HNSCC). In the present study, we evaluated spinophilin and p53 expression by immunohistochemistry in 85 patients with nonmetastatic HNSCC. Kaplan-Meier curves and multivariate Cox proportional models were used to define the prognostic relevance of spinophilin for patients with HNSCC. Overall, immunoreactivity for spinophilin was reduced in 40 tumors (47%). Nine cases (10.5%) showed complete loss of spinophilin. Kaplan-Meier curve analysis demonstrated that reduced spinophilin expression is associated with poor overall survival (P = .022). Concomitant analysis of spinophilin and p53 further showed that patients with reduced spinophilin expression and nuclear p53 staining have a significantly decreased overall survival (hazard ratio, 1.96; 95% confidence interval, 1.06-3.61; P = .030). In conclusion, the combination of reduced spinophilin expression and nuclear p53 staining indicates a poor prognosis in HNSCC patients. Based on our results, spinophilin might play a previously unrecognized role in the pathogenesis of HNSCC.

Correlation of loss of spinophilin expression with increased proliferation and viability as a prognostic factor in patients with hepatocellular carcinoma.

e15064 Background: A loss of the putative tumorsuppressor protein spinophilin has been recently involved in the pathogenesis of lung and other carcinoma types. However, a detailed study of the clinical significance and functional cellular effects of spinophilin in hepatocellular carcinoma (HCC) has never been performed. Methods: In this study, we determined for the first time the expression pattern of spinophilin and some of it´s known interacting/down-stream genes (p53, p14ARF, pRB) and proliferation markers (MIB-1 and Cyclin D) in human hepatocellular carcinoma (HCC). The prognostic relevance of spinophilin expression was determined on a cohort of 104 HCC patients who underwent primary tumor resection, followed by functional gene silencing studies. Results: Reduced spinophilin expression was detected by three independent methods (Western blot, RT-PCR and IHC) in a substantial number of patients (70%, p&lt;0.05). Loss of spinophilin expression in HCC tissue was correlated with increased MIB-1 proliferation marker (p=0.01). In terms of clinical outcome, the disease-free survival (DFS) rate was 14.8% and 39.1% (p=0.010) and the cancer-specific survival (CSS) rate was 23.5% and 43.5% (p=0.006) for patients with low and high spinophilin expression, respectively. Multivariate Cox regression analysis identified low expression of spinophilin as an independent poor prognostic factor with respect to DFS (HR=1.8, CI=1.04-3.40, p=0.043) and CSS (HR=2.0, CI=1.1-3.8, p=0.025). Silencing of spinophilin expression in HepG2 cells was associated with increased cellular growth rate (p&lt;0.05). Conclusions: According to our data, the loss of this putative tumor suppressor protein is an independent negative prognosticator for the clinical outcome of HCC patients. Reduced levels of spinophilin expression are associated with higher proliferation rates of HCC cells.

Low expression of the putative tumour suppressor spinophilin is associated with higher proliferative activity and poor prognosis in patients with hepatocellular carcinoma

Background:Spinophilin, a multifunctional intracellular scaffold protein, is reduced in certain types of cancer and is regarded as a novel putative tumour suppressor protein. However, the role of spinophilin in hepatocellular carcinoma (HCC) has never been explored before.Methods:In this study, we determined for the first time the expression pattern of spinophilin in human HCC by immunohistochemistry and quantitative reverse transcriptase–PCR analysis. In addition, we performed immunohistochemical analysis of p53, p14ARF and the proliferation marker Ki-67. Kaplan–Meier curves and multivariate Cox proportional models were used to study the impact on clinical outcome. Small interfering RNA (siRNA) was used to silence spinophilin and to explore the effects of reduced spinophilin expression on cellular growth.Results:In our study, complete loss of spinophilin immunoreactivity was found in 44 of 104 HCCs (42.3%) and reduced levels were found in an additional 37 (35.6%) cases. After adjusting for other prognostic factors, multivariate Cox regression analysis identified low expression of spinophilin as an independent prognostic factor with respect to disease-free (hazard ratio (HR)=1.8; 95% confidence interval (CI)=1.04–3.40; P=0.043) and cancer-specific survival (HR=2.0; CI=1.1–3.8; P=0.025). Reduced spinophilin expression significantly correlated with higher Ki-67 index in HCC (P=0.014). Reducing spinophilin levels by siRNA induced a higher cellular growth rate and increased cyclin D2 expression in tumour cells (P<0.05).Conclusion:This is the first study of the expression pattern and distribution of spinophilin in HCC. According to our data, the loss of spinophilin is associated with higher proliferation and might be useful as a prognostic marker in patients with HCC.

Changes of cognitive function and spinophilin expression after partial hepatectomy in APP/PS1 transgenic mice

Objective To investigate the effects of partial hepatectomy on spatial learning and memory abilities and the expression of spinophilin, which was used as a marker to quantify changes in dendritic spine density in the hippocampus of APP/PS1 transgenic mice. Methods 3-month-old and 6-month-old male APP/PS1 transgenic mice and 3-month-old wild-type male littermates were divided into five groups based on their genetic background: 3-month-old control (group A, n=8), anesthesia (group B, n=32), surgery (group C, n=32), littermates control(group D, n=8), 6-month-old control (group E, n=8). According to the different time points at day 1, 3, 7, 14 after anesthesia or surgery, group B and C were divided randomly into 4 subgroups: B1, B3, B7, B14 and C1, C3, C7, C14. The spatial learning and memory abilities were detected by Morris water maze test in each group at the corresponding time points. Meanwhile, spinophilin expression was detected by Western blot and Immunocytochemistry in the hippocampus. Results Compared with the same time point subgroup B, the latency to find a hidden platform was longer and the frequency of passing through the platform was decreased in Group C at day 3, 7, 14 (P 0.05). The grey value and Western blot relative integrated optical density of spinophilin in group A( 1 609 453±92 801/0.471±0.015 ), approached the Group D( 1 329 398.4±77 783/0.434±0.008 )(P>0.05), were significantly increased compared with that( 801 678±10 505/0.053±0.003 ) in group E (P<0. 01). Compared with the Group A, the same time point subgroup B and subgroup C14, the grey value and Western blot relative integrated optical density of spinophilin were decreased in Group C at day 1, 3, 7(P<0. 05). Conclusion Partial hepatectomy can impair spatial learning and memory abilities of the APP/PS1 transgenic mice, which is closely related to the decrease expression of spinophilin in hippocampus. Key words: Partial hepatectomy; APP/PS1 transgenic mice; Cognitive function; Spinophilin

Estrogen regulation of spine density and excitatory synapses in rat prefrontal and somatosensory cerebral cortex

The steroid hormone, 17β-estradiol (E2) has been reported to enhance executive functions that are known to be mediated by the prefrontal cortex (PFC), although the underlying mechanisms remain unclear. To shed light on the potential mechanisms, we examined the effect of E2 in vivo upon spine density in the rat PFC and the somatosensory cortex (SSC), which has been implicated to be a transient storage site for information that can also contribute to working memory. The results revealed that E2 significantly enhanced the number of dendritic spines in both the SSC and PFC, as well as the expression of spinophilin. In vitro studies revealed further mechanistic insights by demonstrating that E2 enhanced AMPA GluR1 receptor expression and excitatory glutamatergic synapse formation in rat cortical neurons, without an effect upon inhibitory GABAergic synapse formation. Furthermore, E2 rapidly enhanced ERK and Akt activation in cortical neurons, and inhibitors of ERK and Akt activation significantly attenuated E2 induction of excitatory glutamatergic synapses. Administration of E2-BSA likewise significantly enhanced excitatory glutamatergic synapses in cortical neurons, and administration of an ER antagonist, ICI182,780 and a non-NMDA receptor antagonist (NBQX) significantly attenuated the effect of E2 upon enhancement of excitatory glutamatergic synapses, suggesting mediation by extranuclear estrogen receptors and involvement of non-NMDA receptor activation and signaling. As a whole, the studies demonstrate that E2 enhances spine density in both the PFC and SSC, and that E2 enhances excitatory glutamatergic synapse formation in cortical neurons via a rapid extranuclear ER-mediated signaling mechanism that involves up-regulation of GluR1 and mediation by Akt and ERK signaling pathways.

Dopamine facilitates dendritic spine formation by cultured striatal medium spiny neurons through both D1 and D2 dopamine receptors

Variations of dopamine (DA) levels induced by drugs of abuse or in the context of Parkinson's disease modulate the number of dendritic spines in medium spiny neurons (MSNs) of the striatum, showing that DA plays a major role in the structural plasticity of MSNs. However, little is presently known regarding early spine development in MSNs occurring before the arrival of cortical inputs and in particular about the role of DA and D1 (D1R) and D2 (D2R) DA receptors. A cell culture model reconstituting early cellular interactions between MSNs, intrinsic cholinergic interneurons and DA neurons was used to study the role of DA in spine formation. After 5 or 10 days in vitro, the presence of DA neurons increased the number of immature spine-like protrusions. In MSN monocultures, chronic activation of D1R or D2R also increased the number of spines and spinophilin expression in MSNs, suggesting a direct role for these receptors. In DA-MSN cocultures, chronic blockade of D1R or D2R reduced the number of dendritic spines. Interestingly, the combined activation or blockade of both D1R and D2R failed to elicit more extensive spine formation, suggesting that both receptors act through a mechanism that is not additive. Finally, we found increased ionotropic glutamate receptor responsiveness and miniature excitatory postsynaptic current (EPSC) frequency in DA-MSN co-cultures, in parallel with a higher number of spines containing PSD-95, suggesting that the newly formed spines present functional post-synaptic machinery preparing the MSNs to receive additional glutamatergic contacts. These results represent a first step in the understanding of how dopamine neurons promote the structural plasticity of MSNs during the development of basal ganglia circuits.

Selective interactions of spinophilin with the C-terminal domains of the δ- and μ-opioid receptors and G proteins differentially modulate opioid receptor signaling

Previous studies have shown that the intracellular domains of opioid receptors serve as platforms for the formation of a multi-component signaling complex consisting of various interacting partners (Leontiadis et al., 2009, Cell Signal. 21, 1218–1228; Georganta et al., 2010, Neuropharmacology, 59(3), 139–148). In the present study we demonstrate that spinophilin a dendritic-spine enriched scaffold protein associates with δ- and μ-opioid receptors (δ-ΟR, μ-OR) constitutively in HEK293 an interaction that is altered upon agonist administration and enhanced upon forskolin treatment for both μ-OR and δ-ΟR. Spinophilin association with the opioid receptors is mediated via the third intracellular loop and a conserved region of the C-terminal tails. The portion of spinophilin responsible for interaction with the δ-OR and μ-OR is narrowed to a region encompassing amino acids 151–444. Spinophilin, RGS4, Gα and Gβγ subunits of G proteins form a multi-protein complex using specific regions of spinophilin and a conserved amino acid stretch of the C-terminal tails of both δ-μ-ORs. Expression of spinophilin in HEK293 cells potentiated DPDPE-mediated adenylyl-cyclase inhibition of δ-OR leaving unaffected the levels of cAMP accumulation mediated by the μ-OR. Moreover, measurements of extracellular signal regulated kinase (ERK1,2) phosphorylation indicated that the presence of spinophilin attenuated agonist-driven ERK1,2 phosphorylation mediated upon activation of the δ-OR but not the μ-OR. Collectively, these findings suggest that spinophilin associates with both δ- and μ-ΟR and G protein subunits in HEK293 cells participating in a multimeric signaling complex that displays a differential regulatory role in opioid receptor signaling.

Wistar rats subjected to chronic restraint stress display increased hippocampal spine density paralleled by increased expression levels of synaptic scaffolding proteins

The aim of this study was to investigate whether the previously reported effect of chronic restraint stress (CRS) on hippocampal neuron morphology and spine density is paralleled by a similar change in the expression levels of synaptic scaffolding proteins. Adult male Wistar rats were subjected either to CRS (6 h/day) for 21 days or to control conditions. The resulting brains were divided and one hemisphere was impregnated with Golgi-Cox before coronal sectioning and autometallographic development. Neurons from CA1, CA3b, CA3c, and dentate gyrus (DG) area were reconstructed and subjected to Sholl analysis and spine density estimation. The contralateral hippocampus was used for quantitative real-time polymerase chain reaction and protein analysis of genes associated with spine density and morphology (the synaptic scaffolding proteins: Spinophilin, Homer1-3, and Shank1-3). In the CA3c area, CRS decreased the number of apical dendrites and their total length, whereas CA1 and DG spine density were significantly increased. Analysis of the contralateral hippocampal homogenate displayed an increased gene expression of Spinophilin, Homer1, Shank1, and Shank2 and increased protein expression of Spinophilin and Homer1 in the CRS animals. In conclusion, CRS influences hippocampal neuroplasticity by modulation of dendrite branching pattern and spine density paralleled by increased expression levels of synaptic scaffolding proteins.

Enhanced Hypotensive, Bradycardic, and Hypnotic Responses to α2-Adrenergic Agonists in Spinophilin-Null Mice Are Accompanied by Increased G Protein Coupling to the α2A-Adrenergic Receptor

We previously identified spinophilin as a regulator of alpha(2) adrenergic receptor (alpha(2)AR) trafficking and signaling in vitro and in vivo (Science 304:1940-1944, 2004). To assess the generalized role of spinophilin in regulating alpha(2)AR functions in vivo, the present study examined the impact of eliminating spinophilin on alpha(2)AR-evoked cardiovascular and hypnotic responses, previously demonstrated to be mediated by the alpha(2A)AR subtype, after systemic administration of the alpha(2)-agonists 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304) and clonidine in spinophilin-null mice. Mice lacking spinophilin expression display dramatically enhanced and prolonged hypotensive, bradycardic, and sedative-hypnotic responses to alpha(2)AR stimulation. Whereas these changes in sensitivity to alpha(2)AR agonists occur independent of any changes in alpha(2A)AR density or intrinsic affinity for agonist in the brains of spinophilin-null mice compared with wild-type control mice, the coupling of the alpha(2A)AR to cognate G proteins is enhanced in spinophilin-null mice. Thus, brain preparations from spinophilin-null mice demonstrate enhanced guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of alpha(2A)AR in the guanine-nucleotide-sensitive higher affinity state compared with those from wild-type mice. These findings suggest that eliminating spinophilin expression in native tissues leads to an enhanced receptor/G protein coupling efficiency that contributes to sensitization of receptor mediated responses in vivo.

Enriched environment increases spinophilin mRNA expression and spinophilin immunoreactive dendritic spines in hippocampus and cortex

Housing rodents in an enriched environment (EE) induces structural and functional plasticity in the adult brain, including increased dendritic sprouting and number of dendritic spines. However, the molecular mechanisms behind EE-induced brain plasticity remain largely unknown. Circadian rhythm plays an important role in memory processing but the neurobiological mechanisms of how circadian rhythm affects memory and brain plasticity remain controversial. In the current study, we studied the expression of spinophilin, a protein highly enriched in dendritic spines and involved in spine morphology and synaptic plasticity, to examine the effects of EE and circadian rhythm in rats housed in EE for different periods of time. Spinophilin mRNA expression was studied by in situ hybridization and the density of spinophilin immunoreactive puncta was quantified after immunohistochemical staining. Compared to rats living in a deprived environment (DE), we found a transient increase in the density of spinophilin immunoreactive puncta in hippocampus and cortex after 1 week of EE housing and persistent elevations of spinophilin mRNA expression during 1-4 weeks of environmental enrichment. Increased spinophilin expression was found during the light phase of the diurnal cycle, but not the dark phase. Thus, enriched housing altered the diurnal variation in spinophilin mRNA expression, suggesting that circadian modulation is likely to be important for experience dependent plasticity. The current results suggest a possible role for spinophilin in neuronal plasticity induced by environmental enrichment, but further studies are needed to establish a cause-effect relation.

Hypnotic and Hypotensive Responses to the Alpha2‐adrenergic Agonists Are Enhanced and Accompanied by Increased G Protein Coupling to the Alpha2AAR in Spinophilin Null Mice

We have previously identified spinophilin (SP) as a critical regulator of alpha2 adrenergic receptor trafficking and signaling both in vitro and in vivo. Compared to wild type mice, mice lacking SP expression are more sensitive to alpha2‐agonist‐elicited sedation. In the present study, we assessed cardiovascular responses and hypnotic effects elicited by systemic administration of an alpha2‐agonist, UK14,304, in SP null mice. Our data demonstrate that mice lacking SP expression display dramatically enhanced and prolonged hypotensive, bradycardic, and hypnotic responses to alpha2AAR stimulation. These changes in sensitivity to alpha2AR agonist occur independent of any changes in alpha2AAR density or intrinsic affinity for UK14,304 in the brain of SP null mice when compared to wild type controls. In contrast, the data indicate that coupling of the alpha2AAR to cognate G proteins is enhanced in SP null mice, based on changes in guanine nucleotide regulation of UK14,304 binding and evidence of a larger fraction of alpha2AAR in the high affinity state in preparations from SP null mice when compared to those from wild type mice. This suggests that an enhanced receptor/G protein coupling efficiency in the absence of SP expression contribute to an increased responsiveness of alpha2AARs in SP null mice, indicating that SP may exert ongoing regulation of receptor‐G protein coupling in vivo.

A sensitizing d-amphetamine dose regimen induces long-lasting spinophilin and VGLUT1 protein upregulation in the rat diencephalon

Numerous studies in this lab and others have reported psychostimulant-induced alterations in both synaptic protein expression and synaptic density in striatum and prefrontal cortex. Recently we have shown that chronic d-amphetamine (d-AMPH) administration in rats increased synaptic protein expression in striatum and limbic brain regions including hippocampus, amygdala, septum, and paraventricular nucleus of the thalamus (PVT). Potential synaptic changes in thalamic nuclei are interesting since the thalamus serves as a gateway to cerebral cortex and a nodal point for basal ganglia influences. Therefore we sought to examine drug-induced differences in synaptic protein expression throughout the diencephalon. Rats received an escalating (1–8mg/kg) dosing regimen of d-AMPH for five weeks and were euthanized 28 days later. Radioimmunocytochemistry (RICC) revealed significant upregulation of both spinophilin and the vesicular glutamate transporter, VGLUT1, in PVT, mediodorsal (MD), and ventromedial (VM) thalamic nuclei as well as in lateral hypothalamus (LH) and habenula. Strong positive correlations were observed between VGLUT1 and spinophilin expression in PVT, medial habenula, MD, VM and LH of d-AMPH-treated rats. No significant d-AMPH effect was seen in sensorimotor cortices for either protein. Additionally, no significant differences in the general vesicular protein synaptophysin were observed for any brain region. These findings add to evidence suggesting that long-lasting stimulant-induced synaptic alterations are widespread but not ubiquitous. Moreover, they suggest that d-AMPH-induced synaptic changes may occur preferentially in excitatory synapses.

Estrus cyclicity of spinogenesis: underlying mechanisms

Hippocampal spine density varies with the estrus cycle. The cyclic change in estradiol levels in serum was hypothesized to underlie this phenomenon, since treatment of ovariectomized animals with estradiol induced an increase in spine density in hippocampal dendrites of rats, as compared to ovariectomized controls. In contrast, application of estradiol to hippocampal slice cultures did not promote spinogenesis. In addressing this discrepancy, we found that hippocampal neurons themselves are capable of synthesizing estradiol de novo. Estradiol synthesis can be suppressed by aromatase inhibitors and by knock-down of Steroid Acute Regulatory Protein (StAR) and enhanced by substrates of steroidogenesis. Expression of estrogen receptors (ERs) and synaptic proteins, synaptogenesis, and long-term potentiation (LTP) correlated positively with aromatase activity in hippocampal cultures without any difference between genders. All effects due to inhibition of aromatase activity were rescued by application of estradiol to the cultures. Most importantly, gonadotropin-releasing hormone (GnRH) increased estradiol synthesis dose-dependently via an aromatase-mediated mechanism and consistently increased spine synapse density and spinophilin expression. As a consequence, our data suggest that cyclic fluctuations in spine synapse density result from pulsative release of GnRH from the hypothalamus and its effect on hippocampal estradiol synthesis, rather than from varying levels of serum estradiol. This hypothesis is further supported by higher GnRH receptor (GnRH-R) density in the hippocampus than in the cortex and hypothalamus and the specificity of estrus cyclicity of spinogenesis in the hippocampus, as compared to the cortex.

Gene expression in the anterior cingulate cortex and amygdala of adolescent marmoset monkeys following parental separations in infancy

Early life adversities are risk factors for later mood and emotional disorders. Repeated separation of infant marmosets from their parents provides a validated primate model of depression vulnerability, producing in-vivo biochemical and behavioural effects indicative of persistently altered stress reactivity and mild anhedonia. Here we report the long-term effect (in adolescence) of this intervention on the expression of synaptophysin, GAP-43, VGluT1, VGAT, MAP-2, spinophilin, and 5-HT1A and 5-HT2A receptors, in the anterior cingulate cortex (ACC; supragenual and subgenual areas) and amygdala (lateral, basal and central nuclei). These genes and regions are implicated in the response to stress or in mood disorder. The profile of 5-HT1A receptor binding in ACC was affected by early deprivation, notably in the subgenual region, with a decrease in deep laminae but an increase in superficial laminae. Following early deprivation, spinophilin mRNA was reduced in subgenual ACC. In the amygdala, no significant effects of the manipulation were seen, but expression of several transcripts was sexually dimorphic. There were correlations between expression of some transcripts and in-vivo measurements. The results show that early deprivation in a non-human primate has a selective long-term effect on expression of genes in the ACC, particularly the subgenual area. The results differ from those reported in the hippocampus of the same animals, indicating the presence of limbic region-specific long-term molecular responses to early life stress.

Role of the Multidomain Protein Spinophilin in Blood Pressure and Cardiac Function Regulation

Spinophilin controls intensity/duration of G protein-coupled receptor signaling and thereby influences synaptic activity. We hypothesize that spinophilin affects blood pressure through central mechanisms. We measured blood pressure and heart rate in SPL-deficient (SPL(-/-)), heterozygous SPL-deficient (SPL(+/-)), and wild-type (SPL(+/+)) mice by telemetry combined with fast Fourier transformation. We also assessed peripheral vascular reactivity and performed echocardiography. SPL(-/-) had higher mean arterial pressure than SPL(+/-) and SPL(+/+) (121+/-2, 112+/-1, and 113+/-1 mm Hg). Heart rate was inversely related to spinophilin expression (SPL(-/-) 565+/-0.4, SPL(+/-) 541+/-5, SPL(+/+) 525+/-8 bpm). The blood pressure response to prazosin, trimethapane, and the heart rate response to metoprolol were stronger in SPL(-/-) than SPL(+/+) mice, whereas heart rate response to atropine was attenuated in SPL(-/-). Mesenteric artery vasoreactivity after angiotensin II, phenylephrine, and the thromboxane mimetic (U46619) as well as change in heart rate, stroke volume, and cardiac output after dobutamine were similar in SPL(-/-) and SPL(+/+). Baroreflex sensitivity was attenuated in SPL(-/-) compared with SPL(+/-) and SPL(+/+), which was confirmed by pharmacological testing. Heart rate variability parameters were attenuated in SPL(-/-) mice. We suggest that an increase in central sympathetic outflow participates in blood pressure and heart rate increases in SPL(-/-) mice. The elevated blood pressure in SPL(-/-) mice was associated with attenuated baroreflex sensitivity and decreased parasympathetic activity. Our study is the first to show a role for the spinophilin gene in blood pressure regulation.

Effects of repeated treatment of phencyclidine on cognition and gene expression in C57BL/6 mice

A number of studies indicate that glutamatergic N-methyl-D-aspartate (NMDA) neurotransmission is disturbed in schizophrenia partly based on the findings that NMDA receptor antagonists such as phencyclidine (PCP) can reproduce a schizophrenia-like syndrome in both humans and rodents. This study investigated whether repeated administration of low doses of PCP can induce cognitive dysfunctions in mice at doses which produce no sensorimotor disturbances. In addition, the effects on cognition were related to the expression of two genes, Arc and spinophilin, which have been related to neuronal plasticity and learning. Adult male C57Bl/6J mice received daily s.c. doses of PCP (0.5-2.0 mg/kg) or saline for 7 d. Testing was performed 24 h after the last day of treatment. Only the 2.0 mg/kg PCP dose produced a consistent impairment in spatial learning and working memory performed in the water-maze task without any apparent sensorimotor deficits. Importantly, the 2.0 mg/kg PCP dose produced no impairment in a non-spatial learning paradigm in the water-maze task. PCP treatment altered Arc mRNA levels in the hippocampus and retrosplenial agranular cortex while leaving the striatum and prefrontal cortex unaffected. The mRNA expression of spinophilin was down-regulated in striatum by repeated PCP treatment. These results demonstrate that repeated treatment with low doses of PCP in mice can produce specific cognitive deficits which are associated with alterations in gene expression in brain regions that appear to play a role in the pathophysiology of schizophrenia. These results suggest that the low-dose PCP model may have significant potential in characterizing the behavioural and molecular mechanisms underlying cognitive deficits seen in schizophrenia patients.

Brain-derived Neurotrophic Factor-Tropomyosin-related Kinase B Signaling Contributes to Activity-dependent Changes in Synaptic Proteins

The ability of synapses to undergo changes in structure and function in response to alterations of neuronal activity is an essential property of neural circuits. One way that this is achieved is through global changes in the molecular composition of the synapse; however, it is not clear how these changes are coupled to the dynamics of neuronal activity. Here we found that, in cultured rat cortical neurons, bidirectional changes of neuronal activity led to corresponding alterations in the expression of brain-derived neurotrophic factor (BDNF) and phosphorylation of its receptor tropomyosin-related kinase B (TrkB), as well as in the level of synaptic proteins. Exogenous BDNF reversed changes in synaptic proteins induced by chronic activity blockade, while inhibiting Trk kinase activity or depleting endogenous BDNF abolished the concentration changes induced by chronic activity elevation. Both tetrodotoxin and bicuculline had significant, but opposite, effects on synaptic protein ubiquitination in a time-dependent manner. Furthermore, exogenous BDNF was sufficient to increase ubiquitination of synaptic proteins, whereas scavenging endogenous BDNF or inhibiting Trk kinase activity prevented the ubiquitination of synaptic proteins induced by chronic elevation of neuronal activity. Inhibiting the proteasome or blocking protein polyubiquitination mimicked the effect of tetrodotoxin on the levels of synaptic proteins and canceled the effects of BDNF. Our study indicates that BDNF-TrkB signaling acts upstream of the ubiquitin proteasome system, linking neuronal activity to protein turnover at the synapse.

Activation of α2A adrenoceptors alters dendritic spine development and the expression of spinophilin in cultured cortical neurones

α2 adrenoceptors have been shown to regulate the development of dendrites in mammalian cortical neurones. In this study we have investigated how agonists of α2 adrenoceptors affect length and density of dendritic spines in cultured cortical neurones from C57/B6 mice. A twenty-four hour incubation of 14 day old cultured neurones with UK 14304, an α2-adrenoceptor agonist, resulted in a significant increase in the average length and density of dendritic spines. Furthermore, incubation of neurones with the selective α2A agonist guanfacine resulted in 1.2-fold increase in spine length and 1.8-fold increase in spine density. These effects were blocked by RX 821002 and BRL 44408, α2- and α2A-adrenoceptor antagonists, respectively. The observed changes in the density and length of dendritic spines were correlated with increased expression of spinophilin, a key cytoskeletal protein in the formation and maintenance of dendritic spines, and a decrease in the phosphorylation of spinophilin on serine residues. The latter finding points to a possible mechanism by which adrenoceptors may regulate spinophilin function in dendritic spine development and structure in cortical neurones in vitro.