Molecular basis of lithium action: integration of lithium-responsive signaling and gene expression networks.

The clinical efficacy of lithium in the prophylaxis of recurrent affective episodes in bipolar disorder is characterized by a lag in onset and remains for weeks to months after discontinuation. Thus, the long-term therapeutic effect of lithium likely requires reprogramming of gene expression. Protein kinase C and glycogen synthase kinase-3 signal transduction pathways are perturbed by chronic lithium at therapeutically relevant concentrations and have been implicated in modulating synaptic function in nerve terminals. These signaling pathways offer an opportunity to model critical signals for altering gene expression programs that underlie adaptive responses of neurons to long-term lithium exposure. While the precise physiological events critical for the clinical efficacy of lithium remain unknown, we propose that linking lithium-responsive genes as a regulatory network will provide a strategy to identify signature gene expression patterns that distinguish between therapeutic and nontherapeutic actions of lithium.

Transcriptional down-regulation of MARCKS gene expression in immortalized hippocampal cells by lithium.

The gene (Macs) for the mouse myristoylated alanine-rich C kinase substrate (MARCKS) encodes a prominent substrate for protein kinase C that has been implicated in processes requiring signal dependent changes in actin-membrane plasticity and cytoskeletal restructuring. We have previously demonstrated that MARCKS protein is significantly down-regulated in rat hippocampus and in an immortalized hippocampal cell line (HN33.dw) following long-term exposure to lithium at clinically relevant concentrations (1 mM). Our current studies have examined transcriptional and post-transcriptional events that may underlie the lithium-induced down-regulation of MARCKS protein in the cultured hippocampal cell model system. MARCKS mRNA and protein expression were found to be concomitantly down-regulated following exposure of the HN33.dw cells to chronic lithium. Whereas the stability of MARCKS mRNA remained unchanged in the presence of lithium, nuclear run-off assay indicated that the transcription of nascent MARCKS mRNA was significantly reduced (approximately 50%) in the cells that had been treated with lithium for 7 days. Transient transfection of HN33.dw cells with a mouse cloned Macs promoter (993-bp) showed that the Macs promoter activity was attenuated to the same extent after chronic (7-10 days), but not subacute (24 h), lithium exposure. The inhibition of the Macs promoter was found to be dependent upon the presence of a 280-bp promoter region between -993-bp and -713-bp relative to the translation start site, suggesting that this region is a potential lithium-responsive region of Macs promoter (LRR). Mutant promoter lacking the LRR not only did not respond to chronic lithium exposure but also had significantly reduced promoter activity, suggesting that chronic lithium exposure represses the transcriptional activity of activator(s) bound to the promoter. Taken together, our data indicate that transcriptional inhibition of the Macs gene underlies the lithium-induced down-regulation of MARCKS expression in the immortalized hippocampal cells.

Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain.

We recently identified conformational changes that occur upon phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison, A. S. (1999) J. Biol. Chem. 274, 36472-36478). These results implied that the phosphorylation site domain of MARCKS has two actin-binding sites. We now present evidence for the existence of two actin-binding sites that not only mutually compete but also specifically compete with the actin-binding proteins thymosin beta(4) and actobindin to bind to actin. The effects of substitution of alanine for phenylalanine within a repeated hexapeptide segment suggest that the noncharged region of the domain contributes to binding affinity, but the binding affinity of peptides corresponding to each binding site has a steep dependence on salt concentration, consistent with presumed electrostatic interactions between these polycationic peptides and the polyanionic N terminus of actin. Phosphorylation decreases the site-specific affinity by no more than 0.7 kcal/mol, which is less than the effect of alanine substitution. However, phosphorylation has a much greater effect than alanine substitution on the loss of actin filament cross-linking activity. These results are consistent with the hypothesis that the compact structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.

Signaling: cellular insights into the pathophysiology of bipolar disorder.

Clinical studies over the years have provided evidence that monoamine signaling and hypothalamic-pituitary-adrenal axis disruption are integral to the pathophysiology of bipolar disorder. A full understanding of the pathophysiology from a molecular to a systems level must await the identification of the susceptibility and protective genes driving the underlying neurobiology of bipolar disorder. Furthermore, the complexity of the unique biology of this affective disorder, which includes the predisposition to episodic and often progressive mood disturbance, and the dynamic nature of compensatory processes in the brain, coupled with limitations in experimental design, have hindered our progress to date. Imaging studies in patient populations have provided evidence of a role for anterior cingulate, amygdala, and prefrontal cortex in the pathophysiology of bipolar disorder. More recent research strategies designed to uncover the molecular mechanisms underlying our pharmacologic treatments and their interaction in the regulation of signal transduction as well as more advanced brain imaging studies remain promising approaches. This experimental strategy provides data derived from the physiologic response of the system in affected individuals and addresses the critical dynamic interaction with pharmacologic agents that effectively modify the clinical expression of the pathophysiology.

Differential regulation of primary protein kinase C substrate (MARCKS, MLP, GAP-43, RC3) mRNAs in the hippocampus during kainic acid-induced seizures and synaptic reorganization.

In the mature hippocampus, kainic acid seizures lead to excitotoxic cell death and synaptic reorganization in which granule cell axons (mossy fibers) form ectopic synapses on granule cell dendrites. In the present study, we examined the expression of four major, developmentally regulated protein kinase C (PKC) substrates (MARCKS, MLP, GAP-43, RC3), which have different subcellular and regional localizations in the hippocampus at several time points (6 hr, 12 hr, 18 hr, 24 hr, 48 hr, 5 days, or 15 days) following kainic acid seizures using in situ hybridization. Consistent with previous reports, following kainate seizures, GAP-43 mRNA expression exhibited a delayed and protracted elevation in the granule cell layer, which peaked at 24 hr, whereas expression in fields CA1 and CA3 remained relatively unchanged. Conversely, RC3 mRNA expression exhibited a delayed reduction in the granule cell layer that was maximal at 18 hr, as well as a reduction CA1 at 48 hr, whereas CA3 levels did not change. MARCKS mRNA expression in the granule cell layer and CA1 remained stable following kainate, although an elevation was observed in subfield CA3c at 12 hr. Similarly, MLP mRNA expression did not change in the granule cell layer or CA1 following kainate but exhibited a protracted elevation in subfields CA3b,c beginning at 6 hr post-kainate. Collectively these data demonstrate that different PKC substrate mRNAs exhibit unique expression profiles and regulation in the different cell fields of the mature hippocampus following kainic acid seizures and during subsequent synaptic reorganization. The expression profiles following kainate seizures bear resemblance to those observed during postnatal hippocampal development, which may indicate the recruitment of common regulatory mechanisms.

Myristoylation alters retinoic acid-induced down-regulation of MARCKS in immortalized hippocampal cells.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly conserved domains including a 5' myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the protein to the membrane but also play a novel role in modulating cellular levels of MARCKS protein in response to RA.

Posttraumatic stress disorder in the primary care medical setting.

Posttraumatic stress disorder (PTSD) is a prevalent disorder that adversely affects 2-5% of the general population. Little is known about PTSD in the primary care setting. The purpose of the present study was to evaluate the utility of a screening instrument for PTSD (the PCL-C) in primary care and to examine comorbidity, disability, and patterns of healthcare utilization among persons with PTSD in this setting. Adult, English-speaking patients attending for routine medical care (N=368) participated in a two-stage screening consisting of the administration of a self-report measure for posttraumatic stress disorder (the PCL-C) followed by a structured diagnostic interview. Current (1-month) prevalence of PTSD was determined, as were current comorbid disorders. Brief functional impairment and disability indices were administered, and healthcare utilization in the prior 6 months was ascertained. 11.8% (standard error 1.7%) of primary care attendees met diagnostic criteria for either full or partial PTSD. Comorbidity with major depression (61% of cases of PTSD) and generalized anxiety disorder (39%) was common, but less so with social phobia (17%) and panic disorder (6%). Substance use disorder comorbidity (22%) was also fairly common. Patients with PTSD reported significantly more functional impairment than patients without mental disorders. Patients with PTSD also made greater use of healthcare resources than not mentally ill patients. PTSD frequently is encountered in primary care, and is associated with considerable functional impairment and healthcare utilization. Comorbidity with other mood and anxiety disorders is extensive. It remains to be seen if greater awareness and more aggressive treatment of PTSD in primary care will lead to improved functioning and reduced (or more appropriate) healthcare utilization. These are topics for further study.

Facial motor neuron regeneration induces a unique spatial and temporal pattern of myristoylated alanine-rich C kinase substrate expression.

We have previously shown that the myristoylated alanine-rich C kinase substrate, a primary protein kinase C substrate in brain that binds and cross-links filamentous actin, is enriched in neuronal growth cones and is developmentally regulated in brain. Here we examined myristoylated alanine-rich C kinase substrate expression in the facial motor nucleus during axonal regeneration following facial nerve axotomy or facial nerve resection lesions, which impede regeneration, or following motor neuron degeneration induced by the retrograde neurotoxin ricin. For comparative purposes, the protein kinase C substrates myristoylated alanine-rich C kinase substrate-like protein and growth-associated protein-43 were examined in parallel. Myristoylated alanine-rich C kinase substrate messenger RNA exhibited a robust increase in both neurons and non-neuronal cells in the facial motor nucleus beginning four days after axotomy, peaked at seven days (2.5-fold), and declined back to baseline levels by 40 days. Myristoylated alanine-rich C kinase substrate protein similarly exhibited a twofold elevation in the facial motor nucleus determined four and 14 days post-axotomy. Following nerve resection, myristoylated alanine-rich C kinase substrate messenger RNA levels increased at seven days and returned to baseline levels by 40 days. Unlike myristoylated alanine-rich C kinase substrate messenger RNA, myristoylated alanine-rich C kinase substrate-like messenger RNA levels did not increase in the facial motor nucleus at any time point following nerve axotomy or resection, whereas growth-associated protein-43 messenger RNA exhibited a rapid (one day) and prolonged (40 days) elevation in facial motor nucleus neurons following either nerve axotomy or resection. Ricin-induced degeneration of facial motor neurons elevated myristoylated alanine-rich C kinase substrate and myristoylated alanine-rich C kinase substrate-like messenger RNAs in both microglia (lectin-positive) and astrocytes (glial fibrillary acidic protein-positive).Collectively, these data demonstrate that myristoylated alanine-rich C kinase substrate exhibits a unique expression profile in the facial motor nucleus following facial nerve lesions, and it is proposed that myristoylated alanine-rich C kinase substrate may serve to mediate actin-membrane cytoskeletal plasticity in both neurons and glial cells in response to protein kinaseC-mediated signaling during nerve regeneration and degeneration.

Overview of the mechanism of action of lithium in the brain: fifty-year update.

Since its discovery, lithium has been shown to act upon various neurotransmitter systems at multiple levels of signaling in the brain. Lithium, affecting each neurotransmitter system within complex interactive neuronal networks, is suggested to restore the balance among aberrant signaling pathways in critical regions of the brain. Recent molecular studies have revealed the action of lithium on signal transduction mechanisms, such as phosphoinositide hydrolysis, adenylyl cyclase, G protein, glycogen synthase kinase-3beta, protein kinase C, and its substrate myristoylated alanine-rich C kinase substrate. Such effects are thought to trigger long-term changes in neuronal signaling patterns that account for the prophylactic properties of lithium in the treatment of bipolar disorder. Through its effects on glycogen synthase kinase-3beta and protein kinase C, lithium may alter the level of phosphorylation of cytoskeletal proteins, which leads to neuroplastic changes associated with mood stabilization. Chronic lithium regulates transcriptional factors, which in turn may modulate the expression of a variety of genes that compensate for aberrant signaling associated with the pathophysiology of bipolar disorder. Future studies on long-term neuroplastic changes caused by lithium in the brain will set the stage for new drug-discovery opportunities.

Dimensionality of posttraumatic stress symptoms: a confirmatory factor analysis of DSM-IV symptom clusters and other symptom models.

Recent exploratory [Taylor, S., Kuch, K., Koch, W. J., Crockett, D. J., & Passey, G. (1998). The structure of posttraumatic stress symptoms. Journal of Abnormal Psychology, 107, 154-160.] and confirmatory [Buckley, T. C., Blanchard, E. B., & Hickling, E. J. (1998). A confirmatory factor analysis of posttraumatic stress symptoms. Behaviour Research and Therapy, 36, 1091-1099; King, D. W., Leskin, G. A., King, L. A., & Weathers, F. W. (1998). Confirmatory factor analysis of the clinician-administered PTSD scale: evidence for the dimensionality of posttraumatic stress disorder. Psychological Assessment, 10, 90-96.] factor analytic investigations suggest that the three symptom clusters of posttraumatic stress disorder (PTSD) as defined in the Diagnostic and Statistical Manual [4th ed.; DSM-IV; American Psychiatric Association (1994). Diagnostic and statistical manual of mental disorders (4th ed.). Washington, DC: Author.] may not provide the best conceptualization of symptom dimensionality. However, the alternative models have not been in agreement, nor have they been compared against each other or models based on the DSM-IV. The purpose of the present investigation was to test a series of dimensional models suggested by these recent factor analytic investigations and the DSM-IV. Using data collected with the PTSD Checklist--Civilian Version [Weathers, F. W., Litz, B. T., Huska, J. A., & Keane, T. M. (1994). PCL-C for DSM-IV. Boston: National Center for PTSD--Behavioral Science Division.] from 349 referrals to a primary care medical clinic, we used confirmatory factor analysis to evaluate a: (1) hierarchical four-factor model, (2) four-factor intercorrelated model, (3) hierarchical three-factor model, (4) three-factor intercorrelated model, and (5) hierarchical two-factor model. The hierarchical four-factor model (comprising four first-order factors corresponding to reexperiencing, avoidance, numbing, and hyperarousal all subsumed by a higher-order general factor) provided the best overall fit to the data; although, all models met some standards specified for good model fit. More research is needed to establish the dimensional nature of PTSD symptoms and to assess whether identified dimensions differ as a function of the trauma experience. Implications for assessment, diagnosis, and treatment are also discussed.

The nature of bipolar disorder.

The underlying pathophysiology of bipolar disorder is a continually evolving complexity of multilayer interacting and independent systems. The dearth of adequate preclinical or clinical models that incorporate the various features of the illness, i.e., acute and chronic, recurrent and episodic, and time-course and treatment-related variables, has made the consistency and interpretation of data difficult. Newer technologies and the availability of structurally and mechanistically distinct pharmacologic agents have expanded opportunities for experimental study. In addition to the well-known neurotransmitter systems that are disrupted in mood disorders, critical guanine nucleotide-binding protein (G protein)-coupled signaling pathways are implicated in modulating mood state. Regulation of gene expression and identification of factors regulating neuroplasticity and neurotrophic events in the central nervous system in bipolar disorder are 2 of the more recent approaches contributing to clarification of the pathophysiology and potential treatment options.

Phosphorylation-dependent conformational changes induce a switch in the actin-binding function of MARCKS.

Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) by protein kinase C eliminates actin filament cross-linking activity, but residual filament binding activity docks phosphorylated MARCKS on filamentous actin. The postulated actin-binding region of MARCKS, which includes a Ca(2+)-calmodulin-binding site, has been portrayed with alpha-helical structure, analogous to other calmodulin-binding domains. Previous speculation suggested that MARCKS may dimerize to form the two functional actin-binding sites requisite for cross-linking activity. Contrary to these hypotheses, we show that MARCKS peptide with actin-cross-linking activity has an extended structure in aqueous solution but assumes a more compact structure upon phosphorylation. We hypothesize that structural changes in the MARCKS peptide induced by phosphorylation create a dynamic structure that, on average, has only one actin-binding site. Moreover, independent of the state of phosphorylation, this peptide is monomeric rather than dimeric, implying that two distinct actin-binding sites are responsible for the actin-cross-linking activity of unphosphorylated MARCKS. These studies uniquely elucidate the mechanism by which phosphorylation of MARCKS induces structural changes and suggest how these structural changes determine biological activity.

Ziskind-Somerfeld Research Award. Protein kinase C signaling in the brain: molecular transduction of mood stabilization in the treatment of manic-depressive illness.

Understanding the biology of the pharmacological stabilization of mood will undoubtedly serve to provide significant insight into the pathophysiology of manic-depressive illness (MDI). Accumulating evidence from our laboratories and those of other researchers has identified the family of protein kinase C isozymes as a shared target in the brain for the long-term action of both lithium and valproate. In rats chronically treated with lithium, there is a reduction in the hippocampus of the expression of two protein kinase isozymes, alpha and epsilon, as well as a reduction in the expression of a major PKC substrate, MARCKS, which has been implicated in long-term neuroplastic events in the developing and adult brain. In addition, we have been investigating the down-stream impact of these mood stabilizers on another kinase system, GSK-3 beta and on the AP-1 family of transcription factors. Further studies have generated promising preliminary data in support of the antimanic action of tamoxifen, and antiestrogen that is also a PKC inhibitor. Future studies must address the therapeutic relevance of these protein targets in the brain using innovative strategies in both animal and clinical investigations to ultimately create opportunities for the discovery of the next generations of mood stabilizers for the treatment of MDI.

Differential changes in the phosphorylation of the protein kinase C substrates myristoylated alanine-rich C kinase substrate and growth-associated protein-43/B-50 following Schaffer collateral long-term potentiation and long-term depression.

Activation of protein kinase C (PKC) is one of the biochemical pathways thought to be activated during activity-dependent synaptic plasticity in the brain, and long-term potentiation (LTP) and long-term depression (LTD) are two of the most extensively studied models of synaptic plasticity. Here we have examined changes in the in situ phosphorylation level of two major PKC substrates, myristoylated alanine-rich C kinase substrate (MARCKS) and growth-associated protein (GAP)-43/B-50, after pharmacological stimulation or induction of LTP or LTD in the CA1 field of the hippocampus. We find that direct PKC activation with phorbol esters, K+-induced depolarization, and activation of metabotropic glutamate receptors increase the in situ phosphorylation of both MARCKS and GAP-43/B-50. The induction of LTP increased the in situ phosphorylation of both MARCKS and GAP-43/B-50 at 10 min following high-frequency stimulation, but only GAP-43/B-50 phosphorylation remained elevated 60 min after LTP induction. Furthermore, blockade of LTP induction with the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid prevented elevations in GAP-43/B-50 phosphorylation but did not prevent the elevation in MARCKS phosphorylation 10 min following LTP induction. The induction of LTD resulted in a reduction in GAP-43/B-50 phosphorylation but did not affect MARCKS phosphorylation. Together these findings show that activity-dependent synaptic plasticity elicits PKC-mediated phosphorylation of substrate proteins in a highly selective and coordinated manner and demonstrate the compartmentalization of PKC-substrate interactions. Key Words: Protein kinase C-Myristoylated alanine-rich C kinase substrate-Growth-associated protein-43-Long-term potentiation-Long-term depression-(RS)-alpha-Methyl-4-carboxyphenylglycine-D-2-Amino-5-ph osphonopentanoic acid-Glutamate.

Generation of aberrant sprouting in the adult rat brain by GAP-43 somatic gene transfer.

The expression of GAP-43 was modulated genetically in the adult rat nigrostriatal or septohippocampal pathway using recombinant adeno-associated virus (rAAV) vectors incorporating the neuron specific enolase (NSE) promoter and either a rat GAP-43 cDNA or the corresponding antisense sequence. Bicistronic expression of green fluorescent protein (GFP) enabled us to evaluate transduced neurons selectively. Single injections of rAAV into the substantia nigra pars compacta (SNc) transduced both dopaminergic and non-dopaminergic neurons stably for the 3-month duration of the study. Transduction with the GAP-43 vector in this region: (1) increased GAP-43 mRNA levels 2-fold compared to controls; (2) led to GAP-43 immunoreactivity in neuronal perikarya, axons, and dendrites that was not observed otherwise; and (3) resulted in GAP-43/ GFP-positive axons that were traced to the striatum where they formed clusters of aberrant nets. The GAP-43 antisense vector, in contrast, decreased neuropil GAP-43 immunoreactivity compared to controls in the SNc. In septum, injections of the GAP-43 expressing vector also caused aberrant clusters of GAP-43 labelled fibers in terminal fields, i.e., fornix and hippocampus, that were not observed in control tissues. It therefore appears that rAAV vectors provide a novel approach for modulating intraneuronal GAP-43 expression in the adult brain.