Spinophilin Levels Research Articles

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life.

Loss of precuneus dendritic spines immunopositive for spinophilin is related to cognitive impairment in early Alzheimer's disease

Precuneus (PreC) cortex is affected with amyloid plaques early in Alzheimer's disease (AD), and this pathology may be associated with alterations in PreC synapses and cognitive impairment. We quantified the spinophilin-immunoreactive (ir) dendritic spine density and the intensity of spinophilin immunofluorescence, the latter as a measure of relative protein levels of spinophilin, in PreC lamina III from 33 subjects with clinical diagnoses of no cognitive impairment (NCI), mild cognitive impairment (MCI), mild-moderate AD (mAD), or severe AD (sAD). Both measures of spinophilin were lower in mAD and sAD compared with NCI. The MCI group had higher protein levels of spinophilin compared with mAD and sAD, and higher spinophilin-ir dendritic spine density compared with sAD. Lower spinophilin-ir dendritic spine density and relative protein levels of spinophilin were associated with greater amyloid beta (Aβ) plaque burden, detected with a derivative of Pittsburgh compound-B (6-CN-PiB), and worse cognitive performance. Clinical onset of AD is marked by the loss of PreC spinophilin-ir dendritic spines that is related to Aβ pathology and may contribute to cognitive symptoms early in the disease.

TFEB Overexpression in the P301S Model of Tauopathy Mitigates Increased PHF1 Levels and Lipofuscin Puncta and Rescues Memory Deficits.

Transcription factor EB (TFEB) was recently shown to be a master regulator of autophagy lysosome pathway. Here, we successfully generated and characterized transgenic mice overexpressing flag-TFEB. Enhanced autophagy in the flag-TFEB transgenic mice was confirmed by an increase in the cellular autophagy markers, as determined by both immunoblots and transmission electron microscopy. Surprisingly, in the flag-TFEB mice we observed increased activity of senescence-associated β-galactosidase by ∼66% of neurons in the cortex (p < 0.001) and 73% of neurons in the hippocampus (p < 0.001). More importantly, flag-TFEB expression remarkably reduced the levels of paired-helical filament (PHF)-tau from 372% in the P301S model of tauopathy to 171% (p < 0.001) in the cortex, and from 436% to 212% (p < 0.001) in the hippocampus. Significantly, reduced levels of NeuN in the cortex (38%, p < 0.001) and hippocampus (25%, p < 0.05) of P301S mice were almost completely restored to WT levels in the P301S/flag-TFEB double-transgenic mice. Also, levels of spinophilin in both the cortex (p < 0.001) and hippocampus (p < 0.001) were restored almost to WT levels. Most importantly, the age-associated lipofuscin granules, which are generally presumed to be nondegradable, were reduced by 57% (p < 0.001) in the cortex and by 55% (p < 0.001) in the hippocampus in the double-transgenic mice. Finally, TFEB overexpression in the P301S mice markedly reversed learning deficits in terms of spatial memory (Barnes maze), as well as working and reference memories (T maze). These data suggest that the overexpression of TFEB can reliably enhance autophagy in vivo, reduce levels of PHF-tau, and thereby reverse the deposition of lipofuscin granules and memory deficits.

Depressive-like phenotype induced by AAV-mediated overexpression of human α-synuclein in midbrain dopaminergic neurons

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague–Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic–pituitary–adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to alterations in proteins involved in synaptic plasticity.

Low spinophilin expression enhances aggressive biological behavior of breast cancer.

Spinophilin, a putative tumor suppressor gene, has been shown to be involved in the pathogenesis of certain types of cancer, but its role has never been systematically explored in breast cancer. In this study, we determined for the first time the expression pattern of spinophilin in human breast cancer molecular subtypes (n = 489) and correlated it with survival (n = 921). We stably reduced spinophilin expression in breast cancer cells and measured effects on cellular growth, apoptosis, anchorage-independent growth, migration, invasion and self-renewal capacity in vitro and metastases formation in vivo. Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells. Spinophilin expression was significantly lower in basal-like breast cancer (p<0.001) and an independent poor prognostic factor in breast cancer patients (hazard ratio = 1.93, 95% confidence interval: 1.24 -3.03; p = 0.004) A reduction of spinophilin levels increased cellular growth in breast cancer cells (p<0.05), without influencing activation of apoptosis. Anchorage-independent growth, migration and self-renewal capacity in vitro and metastatic potential in vivo were also significantly increased in spinophilin-silenced cells (p<0.05). Finally, we identified several differentially expressed genes in spinophilin-silenced cells. According to our data, low levels of spinophilin are associated with aggressive behavior of breast cancer.

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.

RanBP9 overexpression reduces dendritic arbor and spine density

RanBP9 is a multi-domain scaffolding protein known to integrate extracellular signaling with intracellular targets. We previously demonstrated that RanBP9 enhances Aβ generation and amyloid plaque burden which results in loss of specific pre- and postsynaptic proteins in vivo in a transgenic mouse model. Additionally, we showed that the levels of spinophilin, a marker of dendritic spines were inversely proportional to the RanBP9 protein levels within the synaptosomes isolated from AD brains. In the present study, we found reduced dendritic intersections within the layer 6 pyramidal neurons of the cortex as well as the hippocampus of RanBP9 transgenic mice compared to age-matched wild-type (WT) controls at 12 months of age but not at 6months. Similarly, the dendritic spine numbers were reduced in the cortex at only 12 months of age by 30% (p<0.01), but not at 6months. In the hippocampus also the spine densities were reduced at 12 months of age (38%, p<0.01) in the RanBP9 transgenic mice. Interestingly, the levels of phosphorylated form of cofilin, an actin binding protein that plays crucial role in the regulation of spine numbers were significantly decreased in the cortical synaptosomes at only 12months of age by 26% (p<0.01). In the hippocampal synaptosomes, the decrease in cofilin levels were 36% (p<0.01) at 12 months of age. Thus dendritic arbor and spine density were directly correlated to the levels of phosphorylated form of cofilin in the RanBP9 transgenic mice. Similarly, cortical synaptosomes showed a 20% (p<0.01) reduction in the levels of spinophilin in the RanBP9 transgenic mice. These results provided the physical basis for the loss of synaptic proteins by RanBP9 and most importantly it also explains the impaired spatial learning and memory skills previously observed in the RanBP9 transgenic mice.

RanBP9 aggravates synaptic damage in the mouse brain and is inversely correlated to spinophilin levels in Alzheimer’s brain synaptosomes

We previously demonstrated that overexpression of RanBP9 led to enhanced Aβ generation in a variety of cell lines and primary neuronal cultures, and subsequently, we confirmed increased amyloid plaque burden in a mouse model of Alzheimer's disease (AD). In the present study, we found striking reduction of spinophilin protein levels when RanBP9 is overexpressed. At 12 months of age, we found spinophilin levels reduced by 70% (P<0.001) in the cortex of APΔE9/RanBP9 mice compared with that in wild-type (WT) controls. In the hippocampus, the spinophilin levels were reduced by 45% (P<0.01) in the APΔE9/RanBP9 mice. Spinophilin immunoreactivity was also reduced by 22% (P<0.01) and 12% (P<0.05) in the cortex of APΔE9/RanBP9 and APΔE9 mice, respectively. In the hippocampus, the reductions were 27% (P<0.001) and 14% (P<0.001) in the APΔE9/RanBP9 and APΔE9 mice, respectively. However, in the cerebellum, spinophilin levels were not altered in either APΔE9 or APΔE9/RanBP9 mice. Additionally, synaptosomal functional integrity was reduced under basal conditions by 39% (P<0.001) in the APΔE9/RanBP9 mice and ∼23% (P<0.001) in the APΔE9 mice compared with that in WT controls. Under ATP- and KCl-stimulated conditions, we observed higher mitochondrial activity in the WT and APΔE9 mice, but lower in the APΔE9/RanBP9 mice. Significantly, we confirmed the inverse relationship between RanBP9-N60 and spinophilin in the synaptosomes of Alzheimer's brains. More importantly, both APΔE9 and APΔE9/RanBP9 mice showed impaired learning and memory skills compared to WT controls. These data suggest that RanBP9 might play a crucial role in the loss of spines and synapses in AD.

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.

Spinophilin acts as a tumor suppressor by regulating Rb phosphorylation

The scaffold protein Spinophilin (SPN) is a regulatory subunit of phosphatase1a located at 17q21.33. This region is frequently associated with microsatellite instability and LOH containing a relatively high density of known tumor suppressor genes, including BRCA1. Several linkage studies have suggested the existence of an unknown tumor suppressor gene distal to BRCA1. Spn may be this gene, but the mechanism through which this gene makes its contribution to cancer has not been described. In this study, we aimed to determine how loss of Spn may contribute to tumorigenesis. We explored the contribution of SPN to PP1a-mediated Rb regulation. We found that the loss of Spn downregulated PPP1CA and PP1a activity, resulting in a high level of phosphorylated Rb and increased ARF and p53 activity. However, in the absence of p53, reduced levels of SPN enhanced the tumorigenic potential of the cells. Furthermore, the ectopic expression of SPN in human tumor cells greatly reduced cell growth. Taken together, our results demonstrate that the loss of Spn induces a proliferative response by increasing Rb phosphorylation, which, in turn, activates p53, thereby neutralizing the proliferative response. We suggest that Spn may be the tumor suppressor gene located at 17q21.33 acting through Rb regulation.

Methamphetamine enhances paced mating behaviors and neuroplasticity in the medial amygdala of female rats

Methamphetamine (METH) is a psychomotor stimulant strongly associated with increases in sexual drive and behavior in women and men. Even though men and women are equally as likely to be addicted to or use METH, studies of sexual behavior often focus on male users. The paucity in studies examining the effect of METH in women is of great concern, when one considers the high correlation with sexually transmitted diseases such as HIV/AIDS and unplanned pregnancies. In fact, why METH so profoundly increases sexual drive is unknown. We have demonstrated that repeated exposure to METH enhances both receptivity and proceptivity in hormonally primed female rats. The current study examined whether a repeated exposure to METH enhanced female-initiated sexual behaviors in hormonally primed rats. In a paced mating paradigm, METH treatment significantly decreased the female's return latency following a mount (57%) and an ejaculation (44%), and the likelihood to leave the male following an intromission (37%) compared to controls. The METH-induced changes in paced mating behavior were accompanied by a 60% increase in spinophilin levels in the medial amygdala following hormonal priming and METH treatment. Taken together, these findings suggest that METH increases female sexual motivation and behavior in the rat potentially via changes in the neural substrate that require repeated exposure to the drug.

A sensitizing d‐amphetamine regimen induces long‐lasting spinophilin protein upregulation in the rat striatum and limbic forebrain

Structural studies have shown that chronic regimens of psychostimulants increase dendritic spine number in the rat striatum. The present study used Western blotting and radioimmunocytochemistry to examine psychostimulant-induced changes in the levels of spinophilin, a protein found abundantly in dendritic spines. Spinophilin determinations were conducted in striatum as well as several other subcortical regions implicated in psychostimulant-induced neuroplasticity. Rats received an escalating (1-8 mg/kg) dosing regimen of d-amphetamine (twice daily, i.p.) for 5 weeks, were tested for locomotor sensitization, and were killed 28 days later. This amphetamine dosing regimen was found to induce a significant sensitization of locomotor activity in these animals. Using both Western blotting and radioimmunocytochemistry, spinophilin protein was found to be upregulated in the striatum of amphetamine-treated rats. In addition, radioimmunocytochemistry revealed that spinophilin was increased in the septum, hippocampus, amygdala and the cingulate cortex, and was unchanged in sensorimotor cortices. Because it binds to F-actin and protein phosphatase-1, spinophilin has been proposed as a protein linking synaptic transmission to changes in spine morphology. Radioimmunocytochemistry for spinophilin provides a novel approach to identification of brain regions whose neurons undergo dendritic change after chronic exposure to drugs of abuse.

Identification of prostaglandin E2 receptors mediating perinatal masculinization of adult sex behavior and neuroanatomical correlates

Prostaglandin E2 (PGE2) mediates the organization of male rat sexual behavior and medial preoptic area (MPOA) neuroanatomy during a sensitive perinatal window. PGE2 is up-regulated in response to estradiol, and initiates a two-fold increase in dendritic spines densities on neurons. All the four receptors for PGE2 and EP1-4 are present in developing POA, a critical region controlling male sexual behavior. Previous studies explored that EP receptors are involved in PGE2-induction of neonatal levels of spinophilin protein, a surrogate marker for dendritic spine formation, but did not assess behavioral masculinization. Here, we used two approaches, suppression of EP receptor expression with antisense oligonucleotides and activation of EP receptors with selective agonists, to test which receptors are necessary and sufficient, respectively, for the effects of PGE2 on behavior and neuronal morphology. In female rats, neonatal treatment with antisense oligonucleotides against EP2 or EP4 but not EP1 or EP3 completely prevented the expression of adult behavior organized by PGE2 exposure. The effects of ONO-DI-004, ONO-AE-259-01, ONO-AE-248, and ONO-AE1-329 (EP1-4 agonists respectively) were equivalent to PGE2 treatment, which suggests activating any EP receptor neonatally suffices in masculinizing sex behavior. When given alone, not all EP agonists increased neonatal POA spinophilin levels; yet giving each agonist neonatally increased adult levels. Moreover, adult spinophilin levels significantly correlated with two measures of male sexual behavior. The body of evidence suggests that EP2 and EP4 are both necessary and sufficient for PGE2-induced masculinization of sex behavior, whereas EP1 and EP3 provide redundant roles.

Sex Differences in Neurotransmission Parameters in Response to Repeated Mild Restraint Stress Exposures in Intact Male, Female and Ovariectomised Female Rats

The present study determined whether a repeated mild restraint stress exposure would differentially alter neuronal activity in male and female rats to gain insights into neurobiological substrates involved in sex differences in stress-induced behavioural responses. In our first set of experiments, we used Western blot analysis to determine whether alterations in several synaptic proteins were elicited by the repeated stress treatment. We found bidirectional changes in synaptophysin levels in female cerebral cortex and hippocampus that diverged between intact and ovariectomised females. There were persistent elevations in spinophilin levels in the male, but not female, hippocampus following the repeated mild restraint stress exposure. By contrast, levels of the NMDA receptor scaffolding protein, PSD-95, were altered only in intact female cerebral cortex and ovariectomised female hippocampus. We next used immunohistochemical evaluation of Fos expression as a marker for neuronal activation. We found significant increases in Fos immunoreactivity in all sex conditions across multiple brain regions in response to the repeated mild stress. Fos protein induction was greatest in the frontal cortex, piriform cortex and amygdala, with the degree of induction varying by sex condition. Fos induction was dramatically higher in amygdala and piriform cortex only in intact females following repeated stress compared to a single restraint stress exposure, suggestive of sensitisation rather than habituation. By contrast, the frontal cortex of intact and ovariectomised females showed habituation to the repeated stressor. Males displayed modest sensitisation in both the frontal cortex and dentate gyrus with no changes in other brain areas. Taken together, these findings show that exposure to a mild repeated stress results in sex differences in synaptic adaptations and patterns of brain activation that likely contribute to observed sex differences in stress-induced behaviours. This approach provides valuable insights into interactions between the hormonal milieu and responses to a repeated mild stress, and further supports the importance of considering hormonal status in treatment of stress-related disorders.

Glutamate AMPA/kainate receptors, not GABAA receptors, mediate estradiol‐induced sex differences in the hypothalamus

Sex differences in brain morphology underlie physiological and behavioral differences between males and females. During the critical perinatal period for sexual differentiation in the rat, gonadal steroids act in a regionally specific manner to alter neuronal morphology. Using Golgi-Cox impregnation, we examined several parameters of neuronal morphology in postnatal day 2 (PN2) rats. We found that in the ventromedial nucleus of the hypothalamus (VMN) and in areas just dorsal and just lateral to the VMN that there was a sex difference in total dendritic spine number (males greater) that was abolished by treating female neonates with exogenous testosterone. Dendritic branching was similarly sexually differentiated and hormonally modulated in the VMN and dorsal to the VMN. We then used spinophilin, a protein that positively correlates with the amount of dendritic spines, to investigate the mechanisms underlying these sex differences. Estradiol, which mediates most aspects of masculinization and is the aromatized product of testosterone, increased spinophilin levels in female PN2 rats to that of males. Muscimol, an agonist at GABA(A) receptors, did not affect spinophilin protein levels in either male or female neonates. Kainic acid, an agonist at glutamatergic AMPA/kainate receptors, mimicked the effect of estradiol in females. Antagonizing AMPA/kainate receptors with NBQX prevented the estradiol-induced increase in spinophilin in females but did not affect spinophilin level in males.

Dendritic pruning of the medial amygdala during pubertal development of the male Syrian hamster

The medial amygdala (Me), a brain region essential for mating behavior, changes in size during puberty. In pre-, mid-, and late pubertal (21, 35, and 49 days of age) male Syrian hamsters, we examined neuronal structure in Me and protein levels of spinophilin and synaptophysin in the amygdaloid complex for evidence of synaptic plasticity coincident with behavioral and physiological development. Body weight, testes weight, and testosterone levels increased during puberty. Mounting behavior, including ectopic, nonintromittive, and intromittive mounts, also increased. Neuronal structure in the posterodorsal medial amygdala (MePD) was assessed in Golgi-impregnated neurons. Pruning occurred during puberty in the number of dendrites emanating from the cell body and in terminal dendritic spine densities. Approximately half of all MePD neurons analyzed had an axon emanating from a dendrite rather than the cell body. However, prepubertal males were more likely to have the axon emanating from a higher order dendritic segment (secondary or tertiary) than were mid- and late pubertal males. Finally, protein levels in the amygdaloid complex varied with pubertal age. Spinophilin decreased, while synaptophysin and GAPDH protein levels increased. These results suggest that puberty is a period of dramatic synaptic plasticity in Me. Specifically, pruning of dendrites and spines, in combination with axonal changes, is likely to modify the afferent influences and electrophysiological properties of Me neurons. Because the Me is an integral component of a social behavior neural network, these changes may be related not only to sexual behavior, but also to other behaviors that mature during puberty, including aggressive, risk-taking, fear-related, and parental behaviors.

Dopamine receptor signaling molecules are altered in elderly schizophrenic cortex

Alterations of molecules that mediate dopaminergic signal transduction have been found in schizophrenia, supporting the hypothesis of altered dopaminergic neurotransmission in this illness. To further explore this hypothesis, the authors measured transcript expression of three proteins involved in dopamine (DA) signaling in postmortem dorsolateral prefrontal and anterior cingulate cortex of elderly schizophrenic subjects and a comparison group. The transcript encoding calcyon, a protein that potentiates crosstalk between D1 DA receptors and Gq/11-linked receptors, was increased in schizophrenic prefrontal and cingulate cortex by 25%. Transcript levels of spinophilin, a protein enriched in dendritic spines that modulates excitatory neurotransmission, were increased 22% in dorsolateral prefrontal cortex but were unchanged in anterior cingulate cortex in schizophrenia. Levels of DARPP-32 mRNA, a downstream effector of dopaminergic neurotransmission, were similar in both groups for both cortical groups. These alterations in spinophilin and calcyon mRNA levels in schizophrenic prefrontal and cingulate cortex provide further evidence of altered dopaminergic neurotransmission in this illness.

Antipsychotic treatment induces alterations in dendrite- and spine-associated proteins in dopamine-rich areas of the primate cerebral cortex

Background: Mounting evidence indicates that long-term treatment with antipsychotic medications can alter the morphology and connectivity of cellular processes in the cerebral cortex. The cytoskeleton plays an essential role in the maintenance of cellular morphology and is subject to regulation by intracellular pathways associated with neurotransmitter receptors targeted by antipsychotic drugs. Methods: We have examined whether chronic treatment with the antipsychotic drug haloperidol interferes with phosphorylation state and tissue levels of a major dendritic cytoskeleton–stabilizing agent, microtubule-associated protein 2 (MAP2), as well as levels of the dendritic spine–associated protein spinophilin and the synaptic vesicle–associated protein synaptophysin in various regions of the cerebral cortex of rhesus monkeys. Results: Among the cortical areas examined, the prefrontal, orbital, cingulate, motor, and entorhinal cortices displayed significant decreases in levels of spinophilin, and with the exception of the motor cortex, each of these regions also exhibited increases in the phosphorylation of MAP2. No changes were observed in either spinophilin levels or MAP2 phosphorylation in the primary visual cortex. Also, no statistically significant changes were found in tissue levels of MAP2 or synaptophysin in any of the cortical regions examined. Conclusions: Our findings demonstrate that long-term haloperidol exposure alters neuronal cytoskeleton– and spine–associated proteins, particularly in dopamine-rich regions of the primate cerebral cortex, many of which have been implicated in the psychopathology of schizophrenia. The ability of haloperidol to regulate cytoskeletal proteins should be considered in evaluating the mechanisms of both its palliative actions and its side effects.

Clone 13-2, a novel gene transiently expressed in granule cells undergoing migration and synaptogenesis

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of nigral dopaminergic neurons and by the presence of aggregates containing α-synuclein called Lewy bodies. Viral vector-induced overexpression of α-synuclein in dopaminergic neurons represents a model of PD which recapitulates disease progression better than commonly used neurotoxin models. Previous studies using this model have reported motor and cognitive impairments, whereas depression, mood and anxiety phenotypes are less described. To investigate these psychiatric phenotypes, Sprague–Dawley rats received bilateral injections of a recombinant adeno-associated virus (AAV) vector expressing human α-synuclein or GFP into the substantia nigra pars compacta. Behavior was assessed at two timepoints: 3 and 8 weeks post-injection. We report that nigral α-synuclein overexpression led to a pronounced nigral dopaminergic cell loss accompanied by a smaller cell loss in the ventral tegmental area, and to a decreased striatal density of dopaminergic fibers. The AAV-α-synuclein group exhibited modest, but significant motor impairments 8 weeks after vector administration. The AAV-α-synuclein group displayed depressive-like behavior in the forced swim test after 3 weeks, and reduced sucrose preference at week 8. At both timepoints, overexpression of α-synuclein was linked to a hyperactive hypothalamic–pituitary–adrenal (HPA) axis regulation of corticosterone. The depressive-like phenotype was also correlated with decreased nigral brain-derived neurotrophic factor and spinophilin levels, and with decreased striatal levels of the activity-regulated cytoskeleton-associated protein. This study demonstrates that AAV-mediated α-synuclein overexpression in dopamine neurons is not only useful to model motor impairments of PD, but also depression. This study also provides evidence that depression in experimental Parkinsonism is correlated to dysregulation of the HPA axis and to alterations in proteins involved in synaptic plasticity.