The impact of Disrupted-in-Schizophrenia 1 (DISC1) on the dopaminergic system: a systematic review.

Disrupted-in-Schizophrenia 1 (DISC1) is a gene known as a risk factor for mental illnesses possibly associated with dopamine impairments. DISC1 is a scaffold protein interacting with proteins involved in the dopamine system. Here we summarise the impact of DISC1 disruption on the dopamine system in animal models, considering its effects on presynaptic dopaminergic function (tyrosine hydroxylase levels, dopamine transporter levels, dopamine levels at baseline and after amphetamine administration) and postsynaptic dopaminergic function (dopamine D1 and D2 receptor levels, dopamine receptor-binding potential and locomotor activity after amphetamine administration). Our findings show that many but not all DISC1 models display (1) increased locomotion after amphetamine administration, (2) increased dopamine levels after amphetamine administration in the nucleus accumbens, and (3) inconsistent basal dopamine levels, dopamine receptor levels and binding potentials. There is also limited evidence for decreased tyrosine hydroxylase levels in the frontal cortex and increased dopamine transporter levels in the striatum but not nucleus accumbens, but these conclusions warrant further replication. The main dopaminergic findings are seen across different DISC1 models, providing convergent evidence that DISC1 has a role in regulating dopaminergic function. These results implicate dopaminergic dysregulation as a mechanism underlying the increased rate of schizophrenia seen in DISC1 variant carriers, and provide insights into how DISC1, and potentially DISC1-interacting proteins such as AKT and GSK-3, could be used as novel therapeutic targets for schizophrenia.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits.

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Immunization with DISC1 protein in an animal model of ADHD influences behavior and excitatory amino acids in prefrontal cortex and striatum.

The Disrupted-in-schizophrenia 1 (DISC1) gene is involved in vulnerability to neuropsychiatric disorders. Naples high-excitability (NHE) rat model neuropsychiatric problems characterized by an unbalanced mesocortical dopamine system. Here, we assessed behavioral and neurochemical effects of immunization against multimeric rat DISC1 protein in adult NHE rats, an animal model of attention-deficit hyperactivity disorder and their Random-Bred (NRB) controls. Males of both lines received subcutaneous injections of vehicle (PB), adjuvant only (AD) or recombinant rat DISC1 protein purified from E. coli, suspended in AD (anti-DISC1) at age of 30, 45 and 60 postnatal days (pnd). At 75 pnd, the rats were exposed to a Làt maze and 2 days later to an Olton eight-arm radial maze, and horizontal (HA) and vertical activities (VA) were monitored. Non-selective (NSA) and selective spatial attention (SSA) were monitored in the Làt and in the Olton maze by duration of rearings and working memory, respectively. Post mortem neurochemistry in the prefrontal cortex (PFc), dorsal (DS) and ventral (VS) striatum of L-Glutamate, L-Aspartate and L-Leucine was performed. All immunized rats showed a clear humoral IgM (but not IgG) immune response against the immunogen, indicating that immunological self-tolerance to DISC1 can be overcome by immunization. NHE rats exhibited a higher unspecific IgM response to adjuvant, indicating an immunological abnormality. The sole anti-DISC1 immunization-specific behavioral in the NHE rats was an increased horizontal activity in the Làt maze. Adjuvant treatment increased vertical activity in both lines, but in the NRB controls it increased rearing and decreased horizontal activity. Liquid chromatography/tandem mass spectrometry analysis of soluble or membrane-trapped neurotransmitters aspartate, glutamate and leucine revealed increased soluble aspartate levels in the ventral striatum of NRB controls after anti-DISC1 immunization. Immune activation by adjuvant independent of simultaneous DISC1 immunization led to other specific changes in NHE and control NRB rats. In DISC1-immunized NHE rats, horizontal activity in Lat maze correlated with membrane-trapped glutamate in PFc and in the NRB rats, duration of rearing in Olton maze correlated with membrane-trapped glutamate in PFc and aspartate in dorsal striatum. In addition to non-specific immune activation (by AD), the postnatal anti-DISC1 immune treatment led to behavioral changes related to mechanisms of activity and attention and had influenced amino acids and synaptic markers in striatum and neocortex in the adult NHE as well as control animals.

DISC1 regulates trafficking and processing of APP and Aß generation.

We report the novel regulation of proteolytic processing of amyloid precursor protein (APP) by DISC1, a major risk factor for psychiatric illnesses, such as depression and schizophrenia. RNAi knockdown of DISC1 in mature primary cortical neurons led to a significant increase in the levels of intracellular α-C-terminal fragment of APP (APP-CTFα) and the corresponding N-terminal-secreted ectodomain product sAPPα. DISC1 knockdown also elicited a significant decrease in the levels of amyloid beta (Aß)42 and Aß40. These aberrant proteolytic events were successfully rescued by co-expression of wild-type DISC1, but not by mutant DISC1 lacking the amino acids required for the interaction with APP, suggesting that APP-DISC1 protein interactions are crucial for the regulation of the C-terminal proteolysis. In a genetically engineered model in which a major full-length DISC1 isoform is depleted, consistent changes in APP processing were seen: an increase in APP-CTFα and decrease in Aß42 and Aß40 levels. Finally, we found that knockdown of DISC1 increased the expression of APP at the cell surface and decreased its internalization. The presented DISC1 mechanism of APP proteolytic processing and Aß peptide generation, which is central to Alzheimer's disease pathology, suggests a novel interface between neurological and psychiatric conditions.

BRI2 ectodomain affects Aß42 fibrillation and tau truncation in human neuroblastoma cells.

Alzheimer's disease (AD) is pathologically characterized by the presence of misfolded proteins such as amyloid beta (Aß) in senile plaques, and hyperphosphorylated tau and truncated tau in neurofibrillary tangles (NFT). The BRI2 protein inhibits Aß aggregation via its BRICHOS domain and regulates critical proteins involved in initiating the amyloid cascade, which has been hypothesized to be central in AD pathogenesis. We recently detected the deposition of BRI2 ectodomain associated with Aß plaques and concomitant changes in its processing enzymes in early stages of AD. Here, we aimed to investigate the effects of recombinant BRI2 ectodomain (rBRI276-266) on Aß aggregation and on important molecular pathways involved in early stages of AD, including the unfolded protein response (UPR), phosphorylation and truncation of tau, as well as apoptosis. We found that rBRI276-266 delays Aß fibril formation, although less efficiently than the BRI2 BRICHOS domain (BRI2 residues 113-231). In human neuroblastoma SH-SY5Y cells, rBRI276-266 slightly decreased cell viability and increased up to two-fold the Bax/Bcl-2 ratio and the subsequent activity of caspases 3 and 9, indicating activation of apoptosis. rBRI276-266 upregulated the chaperone BiP but did not modify the mRNA expression of other UPR markers (CHOP and Xbp-1). Strikingly, rBRI276-266 induced the activation of GSK3ß but not the phosphorylation of tau. However, exposure to rBRI276-266 significantly induced the truncation of tau, indicating that BRI2 ectodomain can contribute to NFT formation. Since BRI2 can also regulate the metabolism of Aß, the current data suggests that BRI2 ectodomain is a potential nexus between Aß, tau pathology and neurodegeneration.

Intranasal dopamine treatment reinstates object-place memory in aged rats.

Following oral or IV administration, dopamine (DA) cannot cross the blood-brain barrier to a significant extent, but can enter the brain when administered via the nasal passages. Intranasal administration of DA was shown to increase extracellular DA in the striatum, to have antidepressant action and to improve attention and working memory in rats. Here we show that aged (22-24 months old) rats are deficient in an object-place learning task, but that this learning/memory is intact and comparable with that of adult rats upon pre-trial administration of 0.3 mg/kg DA gel into the nasal passages. This result raises the possibility of the therapeutic application of intranasal DA treatment for age-related cognitive disorders.

Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease.

Prion diseases in humans and animals are invariably fatal. Prions are composed of a disease-causing isoform (PrP(Sc)) of the normal host prion protein (PrP(C)) and replicate by stimulating the conversion of PrP(C) into nascent PrP(Sc). We report here that tricyclic derivatives of acridine and phenothiazine exhibit half-maximal inhibition of PrP(Sc) formation at effective concentrations (EC(50)) between 0.3 microM and 3 microM in cultured cells chronically infected with prions. The EC(50) for chlorpromazine was 3 microM, whereas quinacrine was 10 times more potent. A variety of 9-substituted, acridine-based analogues of quinacrine were synthesized, which demonstrated variable antiprion potencies similar to those of chlorpromazine and emphasized the importance of the side chain in mediating the inhibition of PrP(Sc) formation. Thus, our studies show that tricyclic compounds with an aliphatic side chain at the middle ring moiety constitute a new class of antiprion reagents. Because quinacrine and chlorpromazine have been used in humans for many years as antimalarial and antipsychotic drugs, respectively, and are known to pass the blood-brain barrier, we suggest that they are immediate candidates for the treatment of Creutzfeldt-Jakob disease and other prion diseases.

Dose-dependent effects of endotoxin on human sleep.

The role of the central nervous system in the host response to infection and inflammation and modulation of these responses by the hypothalamic-pituitary-adrenal system are well established. In animals, activation of host defense mechanisms increases non-rapid eye movement (NREM) sleep amount and intensity, which, in turn, are thought to support host defense, or the body's ability to defend itself against challenges to its immune system. In humans, the evidence is conflicting. Therefore, we investigated the effects of three placebo-controlled doses of endotoxin on host response, including nocturnal sleep in healthy volunteers. Administered before nocturnal sleep onset, endotoxin dose dependently increased rectal temperature, heart rate, and the plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-1 receptor antagonist, IL-6, and cortisol. The lowest dose reliably increased circulating levels of cytokines and soluble cytokine receptors, but it did not affect rectal temperature, heart rate, or cortisol. This subtle host defense activation increased deep NREM sleep amount, often referred to as slow-wave sleep (stages 3 and 4), and intensity (delta power). Conversely, the highest dose of endotoxin disrupted sleep. Whereas it is well established that the endocrine and thermoregulatory systems are very sensitive to endotoxin, this study shows that human sleep-wake behavior is even more sensitive to activation of host defense mechanisms.

Monoclonal antibodies specific for the native, disease-associated isoform of the prion protein.

Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. When considering presenting an antigen to a mouse with the goal of obtaining specific monoclonal antibodies against a misfolded or aggregated form of a host protein, it is desirable to increase the definition of a subtle conformational difference. This can be achieved by immunizing an antigen knockout mouse that has not developed self-tolerance against the respective antigen. Furthermore, if conformational isoforms and/or oligomeric forms of a protein sequence are understood to exist in an equilibrium, high and pure amounts of recombinant protein may increase the likelihood that a particular population of protein conformation passes an antigenic threshold necessary to start an immunogenic response. Pulling out the monoclonal antibodies by correct screening is essential. Screening against the pure misfolded or aggregated protein is often complicated by its poor solubility and hence the ability to immobilize. In the present case, immobilization of disease-associated PrP on nitrocellulose had been established as a conformation-sensitive screening method, allowing to "freeze" PrP in its distinguishable, disease-associated conformation. We are cautious to generalize conclusions of how to assess the generation of monoclonal antibodies against these particular protein isoforms to other diseases of protein misfolding and/or aggregation, but ultimately the present approach may inspire respective experiments.

Prion (PrPSc)-specific epitope defined by a monoclonal antibody.

Prions are infectious particles causing transmissible spongiform encephalopathies (TSEs). They consist, at least in part, of an isoform (PrPSc) of the ubiquitous cellular prion protein (PrPC). Conformational differences between PrPC and PrPSc are evident from increased beta-sheet content and protease resistance in PrPSc. Here we describe a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of PrP. Such an antibody has been long sought as it should be invaluable for characterizing the infectious particle as well as for diagnosis of TSEs such as bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (CJD) in humans. 15B3 specifically precipitates bovine, murine or human PrPSc, but not PrPC, suggesting that it recognizes an epitope common to prions from different species. Using immobilized synthetic peptides, we mapped three polypeptide segments in PrP as the 15B3 epitope. In the NMR structure of recombinant mouse PrP, segments 2 and 3 of the 15B3 epitope are near neighbours in space, and segment 1 is located in a different part of the molecule. We discuss models for the PrPSc-specific epitope that ensure close spatial proximity of all three 15B3 segments, either by intermolecular contacts in oligomeric forms of the prion protein or by intramolecular rearrangement.

Recombinant full-length murine prion protein, mPrP(23-231): purification and spectroscopic characterization.

The cellular prion protein of the mouse, mPrP(C), consists of 208 amino acids (residues 23-231). It contains a carboxy-terminal domain, mPrP(121-231), which represents an autonomous folding unit with three alpha-helices and a two-stranded antiparallel beta-sheet. We expressed the complete amino acid sequence of the prion protein, mPrP(23-231), in the cytoplasm of Escherichia coli. mPrP(23-231) was solubilized from inclusion bodies by 8 M urea, oxidatively refolded and purified to homogeneity by conventional chromatographic techniques. Comparison of near-UV circular dichroism, fluorescence and one-dimensional 1H-NMR spectra of mPrP(23-231) and mPrP(121-231) shows that the amino-terminal segment 23-120, which includes the five characteristic octapeptide repeats, does not contribute measurably to the manifestation of three-dimensional structure as detected by these techniques, indicating that the residues 121-231 might be the only polypeptide segment of PrP(C) with a defined three-dimensional structure.

Diurnal variations in the human host response to endotoxin.

To investigate diurnal variations in the host response to endotoxin, Salmonella abortus equi endotoxin (0.8 ng/kg) was given intravenously to healthy men in a placebo-controlled design at 0900 or 1900 h. The time course of rectal temperature and the plasma levels of tumor necrosis factor- alpha (TNF-alpha), interleukin-6 (IL-6), adrenocorticotropic hormone (ACTH), and cortisol were monitored for 11 h following the injections. The time of day did not affect the endotoxin-induced increase in plasma TNF-alpha or IL-6. However, subjects who received endotoxin in the evening, when endogenous glucocorticoid levels were low, showed about twice the increases in rectal temperature and plasma ACTH and cortisol levels as those who received endotoxin in the morning, when endogenous glucocorticoid levels were high. These results demonstrate diurnal variations in the human susceptibility to endotoxin that may be due to a suppression of the biologic effects of TNF-alpha and IL-6 by endogenous glucocorticoids.

Effects of repeated administration of granulocyte colony-stimulating factor (G-CSF) on neutrophil counts, plasma cytokine, and cytokine receptor levels.

Granulocyte colony-stimulating factor (G-CSF) induces the release of mature neutrophils from storage pools and stimulates differentiation of myeloid progenitor cells. Recently, it has been shown that a standard dose of G-CSF induces immunophenotypical and functional changes of neutrophils that persist for 1 week. The authors investigated the effects of 300 micrograms of G-CSF administered subcutaneously twice, 1 week apart, to 10 healthy volunteers, on neutrophil and monocyte counts and on the plasma levels of interleukin 1 receptor antagonist (IL-1ra), tumour necrosis factor-alpha (TNF-alpha), soluble TNF receptor (sTNF-r) p55 and p75. G-CSF on both occasions increased neutrophil and monocyte counts and the plasma levels of all cytokines and cytokine receptors measured. All parameters assessed had returned to baseline prior to the second administration of G-CSF. The increases in neutrophil counts and plasma IL-1ra levels were larger following the second injection, whereas the increase in plasma sTNF-r p75 levels was smaller. G-CSF induced increases in monocyte counts, TNF-alpha and sTNF-R p55 levels did not differ between the first and second injection. Our finding of an increased response of healthy humans to a second G-CSF challenge 1 week following a first one may be due to long-lasting increases in storage pool neutrophils.

Influence of endotoxin on daytime sleep in humans.

Administration of endotoxin in the evening has been shown to transiently suppress rapid eye movement (REM) and to promote non-REM sleep in humans. In a single-blind placebo-controlled crossover design, we assessed the effects of Salmonella abortus equi endotoxin administered intravenously in the morning on the primary host response and on daytime sleep by use of a multiple napping protocol in healthy volunteers. The extent of the host response achieved by 0.8 ng of endotoxin per kg of body weight given at 0900 h was comparable to that previously reported to result from the administration of 0.4 ng/kg at 1900 h. However, sleep was only slightly influenced. Endotoxin reduced the amount of REM sleep and increased REM latency. Non-REM sleep amount in the first nap, although not significantly changed, correlated negatively with the individual peak levels of interleukin-6 (r = -0.73, P < 0.05). Subjective tiredness, sleep onset latency, total sleep time, and the amounts of slow-wave and non-REM sleep were not affected by endotoxin throughout the entire experiment. Spectral analysis of the electroencephalogram obtained during non-REM sleep yielded no condition differences. We conclude that endotoxin administration in the morning to healthy volunteers, while activating the host defense to the same extent as a lower dose that has been reported to promote non-REM sleep when given in the evening, does not affect non-REM sleep. REM sleep suppression is, to date, the most consistently reported effect of endotoxin on human sleep.

Prion function and dysfunction: a structure-based scenario.

Prion diseases are transmissible, neurodegenerative disorders associated with as yet incompletely defined isoforms of a cellular protein termed prion protein (PrP). We have now identified in PrP structural information compatible with nucleotide- and nucleic acid-binding. As such, PrP contains a putative nicotinamide adenine dinucleotide (NADH)-binding site. Moreover, the PrP octarepeats reveal homology to the nucleic acid-binding and strand-annealing octarepeats of mammalian heterogeneous ribonucleoprotein (RNP) A1. Therefore, PrP may have NADH-dependent oxidoreductase activity as well as A1-like functions such as nucleic acid annealing and splicing. Moreover, we propose that infectious prions are propagated through a dynamic molecular symbiosis between a ribozyme-like nucleic acid and a conformational isomer of the RNP-like prion protein. Thus, our model has important implications for the understanding and treatment of prion diseases.

Effects of granulocyte colony-stimulating factor on plasma cytokine and cytokine receptor levels and on the in vivo host response to endotoxin in healthy men.

We investigated the effects of granulocyte colony-stimulating factor (G-CSF) on cytokine and cytokine receptor plasma levels and on the in vivo host response to Salmonella abortus equi endotoxin in healthy males. Twenty volunteers received 0.8 ng/kg endotoxin and saline intravenously 1 week apart in randomized order. Twelve hours before both experiments, 10 of these subjects were pretreated with 300 micrograms G-CSF subcutaneously. G-CSF itself increased granulocyte and monocyte counts and the plasma levels of tumor necrosis factor-alpha (TNF-alpha), soluble TNF receptors (sTNF-R) p55, and p75 and interleukin-1 receptor antagonist (IL-1ra). G-CSF did not influence plasma IL-1 beta and IL-6 levels. In the G-CSF-pretreated subjects endotoxin-induced surges in rectal temperature and in the plasma levels of TNF-alpha plasma levels were about 50% increased, and surges in the plasma levels of both sTNF-Rs and IL-1ra were about twice as high as in the control group. Endotoxin-induced increases in IL-6, cortisol, and heart rate were not modified by G-CSF pretreatment. Endotoxin administration induced a transient 50% reduction in leukocyte counts in the G-CSF-pretreated subjects that was not seen in the control group. We conclude that a single stand dose of G-CSF increases the plasma levels of cytokines and cytokine receptors and considerably modifies the host response of healthy humans to a low dose of endotoxin.

A co-evolutionary theory of sleep.

Based on recent experimental evidence, a novel theory of sleep function and regulation is advanced, stating that sleep primarily evolved to protect the brain against a wakefulness-dependent increase in the permeability of the blood-brain barrier. A restitutional mechanism for the blood-brain barrier had to co-evolve against the omnipresent gut-derived bacterial cell wall constituents, because these and their elicited cellular responses increase blood-brain barrier permeability and potentially harm nervous tissue. Thus, in order to develop a highly organized cerebral structure, an immune-like response specific for the brain co-evolved during the phylogeny of the symbiosis between animals and gut bacteria to control the detrimental effects of bacterial cell wall constituents. In the course of further evolution, the sleep-associated 'controlled inflammatory state' of the brain employed the growth-factor activities of locally activated cytokines to enforce cerebral development and the maintenance of cognitive functions.

Influence of host defense activation on sleep in humans.

Despite considerable progress in our understanding of the phenomenology of sleep and wakefulness, their regulation and peculiar functions are poorly understood. Recent animal research has revealed considerable evidence for interactions between host defense and sleep. Therefore, it has been hypothesized that host response mediators, mainly cytokines like interleukin-1 (IL-1), are involved in physiological sleep regulation. Furthermore, it has been suggested that sleep, and non rapid eye movement (NREM) sleep in particular, has an immuno-supportive function. In humans, sleep-host defense interactions are just starting to be understood. There is quite good evidence that some viral diseases cause excessive sleepiness. Other infectious diseases induce, however, serious disturbances of the distribution of sleep and wakefulness rather than excessive sleep. In addition, some disorders with excessive sleep, daytime fatigue or disturbed night sleep as prominent symptoms are thought to involve, at least in part, immuno-pathophysiological mechanisms. Experimental settings have only recently been used to elucidate host defense-sleep interactions in humans. The effects of endotoxin, a cell-wall lipopolysaccharide of gram-negative bacteria, on sleep have been tested in different settings in healthy volunteers. Endotoxin transiently suppresses rapid eye movement (REM) sleep independently of the time of the day of administration. Only low doses, given in the evening, promote NREM sleep. Electorencephalogram (EEG) power in higher frequency bands is enhanced during NREM sleep, whereas delta activity is not affected. In rats and rabbits, on the other hand, the effects of endotoxin and of the mediators of its activity on REM sleep are variable. Enhanced NREM sleep is a common finding and most pronounced during the active part of the nycthemeron and, in general, EEG delta activity is augmented. In view of these species differences, hypotheses regarding the underlying mechanisms and the biological significance of host defense-sleep interactions, primarily derived from the results of animal studies, may not entirely fit human physiology. They should therefore be re-evaluated and probably modified, through the use of additional experimental approaches in humans.