Sandra Schäble

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.

Neurokinin3 receptor as a target to predict and improve learning and memory in the aged organism

Significance Cognitive decline during aging impairs life quality and may lead to dementia. It is associated with a dysfunction of the brain acetylcholinergic system. Here we demonstrate that pharmacological stimulation of neurokinin3 receptors improves learning and memory in aged rats by enhancing acetylcholinergic function in the brain. In a human association study we show that a single-nucleotide polymorphism in the neurokinin3-receptor–coding gene TACR3 can predict learning and memory in elderly patients with cognitive impairments and their hippocampus volume. These findings suggest the neurokinin3 receptor as a potential biomarker and treatment target for cognitive enhancement in the elderly. Impaired learning and memory performance is often found in aging as an early sign of dementia. It is associated with neuronal loss and reduced functioning of cholinergic networks. Here we present evidence that the neurokinin3 receptors (NK3-R) and their influence on acetylcholine (ACh) release may represent a crucial mechanism that underlies age-related deficits in learning and memory. Repeated pharmacological stimulation of NK3-R in aged rats was found to improve learning in the water maze and in object-place recognition. This treatment also enhanced in vivo acetylcholinergic activity in the frontal cortex, hippocampus, and amygdala but reduced NK3-R mRNA expression in the hippocampus. Furthermore, NK3-R agonism incurred a significantly higher increase in ACh levels in aged animals that showed superior learning than in those that were most deficient in learning. Our findings suggest that the induced activation of ACh, rather than basal ACh activity, is associated with superior learning in the aged. To test whether natural variation in NK3-R function also determines learning and memory performance in aged humans, we investigated 209 elderly patients with cognitive impairments. We found that of the 15 analyzed single single-nucleotide ploymorphism (SNPs) of the NK3-R–coding gene, TACR3, the rs2765 SNP predicted the degree of impairment of learning and memory in these patients. This relationship could be partially explained by a reduced right hippocampus volume in a subsample of 111 tested dementia patients. These data indicate the NK3-R as an important target to predict and improve learning and memory performance in the aged organism.

Amyloid-β dimers in the absence of plaque pathology impair learning and synaptic plasticity.

Despite amyloid plaques, consisting of insoluble, aggregated amyloid-β peptides, being a defining feature of Alzheimers disease, their significance has been challenged due to controversial findings regarding the correlation of cognitive impairment in Alzheimers disease with plaque load. The amyloid cascade hypothesis defines soluble amyloid-β oligomers, consisting of multiple amyloid-β monomers, as precursors of insoluble amyloid-β plaques. Dissecting the biological effects of single amyloid-β oligomers, for example of amyloid-β dimers, an abundant amyloid-β oligomer associated with clinical progression of Alzheimers disease, has been difficult due to the inability to control the kinetics of amyloid-β multimerization. For investigating the biological effects of amyloid-β dimers, we stabilized amyloid-β dimers by an intermolecular disulphide bridge via a cysteine mutation in the amyloid-β peptide (Aβ-S8C) of the amyloid precursor protein. This construct was expressed as a recombinant protein in cells and in a novel transgenic mouse, termed tgDimer mouse. This mouse formed constant levels of highly synaptotoxic soluble amyloid-β dimers, but not monomers, amyloid-β plaques or insoluble amyloid-β during its lifespan. Accordingly, neither signs of neuroinflammation, tau hyperphosphorylation or cell death were observed. Nevertheless, these tgDimer mice did exhibit deficits in hippocampal long-term potentiation and age-related impairments in learning and memory, similar to what was observed in classical Alzheimers disease mouse models. Although the amyloid-β dimers were unable to initiate the formation of insoluble amyloid-β aggregates in tgDimer mice, after crossbreeding tgDimer mice with the CRND8 mouse, an amyloid-β plaque generating mouse model, Aβ-S8C dimers were sequestered into amyloid-β plaques, suggesting that amyloid-β plaques incorporate neurotoxic amyloid-β dimers that by themselves are unable to self-assemble. Our results suggest that within the fine interplay between different amyloid-β species, amyloid-β dimer neurotoxic signalling, in the absence of amyloid-β plaque pathology, may be involved in causing early deficits in synaptic plasticity, learning and memory that accompany Alzheimers disease.

Neurokinin3-R agonism in aged rats has anxiolytic-, antidepressant-, and promnestic-like effects and stimulates ACh release in frontal cortex, amygdala and hippocampus

Neurokinin-3 receptors (NK(3)-R) are localized in brain regions which have been implicated in processes governing learning and memory as well as emotionality. The effects of acute subcutaneous (s.c.) senktide (0.2 and 0.4 mg/kg), a NK(3)-R agonist, were tested in aged (23-25 month old) Wistar rats: (a) in an episodic-like memory test, using an object discrimination task (this is the first study to test for deficits in episodic-like memory in aged rats, since appropriate tests have only recently became available); (b) on parameters of anxiety in an open field test, (c) on indices of depression in the forced swimming test and (d) on the activity of cholinergic neurons of the basal forebrain, using in vivo microdialysis and HPLC. Neither the saline-, nor senktide-treated aged animals, exhibited episodic-like memory. However, the senktide-, but not the vehicle-treated group, exhibited object memory for spatial displacement, a component of episodic memory. Senktide injection also had anxiolytic- and antidepressant-like effects. Furthermore, the active doses of senktide on behavior increased ACh levels in the frontal cortex, amygdala and hippocampus, suggesting a relationship between its cholinergic and behavioral actions. The results indicate cholinergic modulation by the NK(3)-R in conjunction with a role in the processing of memory and emotional responses in the aged rat.

Effects of intranasally applied dopamine on behavioral asymmetries in rats with unilateral 6-hydroxydopamine lesions of the nigro-striatal tract

Due to its lipophobic properties, dopamine is unable to cross the blood-brain barrier following systemic application. However, recently it has been demonstrated that, when applied directly via the nasal passages in the rat, dopamine exerts neurochemical and behavioural action, including increases of dopamine in striatal subregions, antidepressive-like action, and increased behavioral activity. These effects could potentially be mediated by exogenous dopamine acting as a direct agonist at postsynaptic dopamine receptors. However, it is also possible that intranasally applied dopamine acts indirectly via the modulation of the activity of dopaminergic cell bodies. To approach this question, the present study used rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal tract, as these lesions lead to pharmacologically stimulated behavioural asymmetries which are specific for direct and indirect dopamine agonists. We found that 7 days of repeated treatment with intranasal dopamine induced a sensitization of the turning response to amphetamine, but not to apomorphine. Furthermore, intranasal dopamine dose-dependently increased the use of the forepaw ipsilateral to the 6-OHDA-lesioned side of the brain. These results suggest that intranasally administered dopamine acts via an indirect mechanism of action, putatively by increasing the release of endogenous dopamine in the brain.

The NK3 receptor agonist senktide ameliorates scopolamine-induced deficits in memory for object, place and temporal order

Senktide, a potent neurokinin-3 receptor (NK3-R) agonist, increases acetylcholine (ACh) release in the striatum, the prefrontal cortex (Schäble et al., 2011), the amygdala and hippocampus, presumably via postsynaptic mechanisms. A promnestic action of NK3-R agonists has been described in a variety of learning/memory tasks. The memory-enhancing effects of NK3-R agonists and their activating influence on ACh suggest a possible role of the NK3-R in learning and memory via cholinergic modulation. Deterioration of the cholinergic system in the basal forebrain has been associated with learning and memory deficits and cholinergic agents have promnestic effects in a variety of learning paradigms. The anticholinergic drug, scopolamine, a muscarinic ACh receptor antagonist, incurs deficits in a variety of learning tasks and provides a useful tool to investigate the role of the cholinergic systems in mechanisms underlying learning and memory. The aim of this study was to ascertain the effect of the NK3-R agonist, senktide, in the scopolamine-induced deficit model. We hypothesized that senktide treatment would attenuate scopolamine-induced (subcutaneous--s.c. 0.75 mg/kg) memory impairment in three novelty preference paradigms based on spontaneous object exploration: namely object recognition, object-place recognition and object recognition for temporal order. Administration of senktide reversed the scopolamine-induced memory deficits by re-establishing object recognition (s.c. 0.2 mg/kg), object-place recognition (0.2 and 0.4 mg/kg), as well as object recognition for temporal order (0.4 mg/kg) in adult Wistar rats. These results indicate memory enhancing effects of senktide in animals subjected to scopolamine-induced memory impairments and indicate that the promnestic action of NK3-R agonists is mediated by muscarinic cholinergic mechanisms.

Peripheral DISC1 protein levels as a trait marker for schizophrenia and modulating effects of nicotine.

The Disrupted-in-Schizophrenia 1 (DISC1) protein plays a key role in behavioral control and vulnerability for mental illnesses, including schizophrenia. In this study we asked whether peripheral DISC1 protein levels in lymphocytes of patients diagnosed with schizophrenia can serve as a trait marker for the disease. Since a prominent comorbidity of schizophrenia patients is nicotine abuse or addiction, we also examined modulation of lymphocyte DISC1 protein levels in smokers, as well as the relationship between nicotine and DISC1 solubility status. We show decreased DISC1 levels in patients diagnosed with schizophrenia independent of smoking, indicating its potential use as a trait marker of this disease. In addition, lymphocytic DISC1 protein levels were decreased in smoking, mentally healthy individuals but not to the degree of overriding the trait level. Since DISC1 protein has been reported to exist in different solubility states in the brain, we also investigated DISC1 protein solubility in brains of rats treated with nicotine. Sub-chronic treatment with progressively increasing doses of nicotine from 0.25mg/kg to 1mg/kg for 15 days led to a decrease of insoluble DISC1 in the medial prefrontal cortex. Our results demonstrate that DISC1 protein levels in human lymphocytes are correlated with the diagnosis of schizophrenia independent of smoking and thus present a potential biomarker. Reduced DISC1 protein levels in lymphocytes of healthy individuals exposed to nicotine suggest that peripheral DISC1 could have potential for monitoring the effects of psychoactive substances.

Neurokinin-2 receptor antagonism in medial septum influences temporal-order memory for objects and forebrain cholinergic activity

UNLABELLED In the mammalian brain the neurokinin NK(2) receptors are predominantly located in the hippocampus, thalamus, septum and frontal cortex. It has been shown that administration of the NK(2) receptor agonist, neurokinin A (NKA), into the medial septum of rats increases extracellular levels of acetylcholine (ACh) in the hippocampus and that NK(2) receptor antagonism blocks this increase. Therefore, given the prominent role of hippocampal ACh in information processing, we hypothesized that NK(2) receptor antagonism in the medial septum would negatively affect learning and memory via its influence on the cholinergic neurons of the basal forebrain. We investigated the action of local application of the peptidic NK(2) receptor antagonist, Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH (1, 10 and 100pmol), into the medial septum on object memory for temporal order and spatial location using an object novelty paradigm. By means of in vivo microdialysis and HPLC analyses, we also examined the influence of NK(2) receptor antagonism in the medial septum on ACh in major cholinergic projection areas of the basal forebrain, namely, hippocampus, frontal cortex and amygdala. RESULTS Injection of vehicle alone into the medial septum impaired memory for temporal order and spatial location of objects. Application of 1pmol of the NK(2) receptor antagonist partially reversed this deficit by reinstating memory for temporal order. Injection of 10pmol of the NK(2) receptor antagonist into the medial septum decreased levels of ACh in the hippocampus (at 30min post-injection), and frontal cortex (at 30 and 80min post-injection) in comparison to vehicle. However, this apparent decrease was the result of the blockade of a saline-induced increase in ACh levels.

Cognitive training during infancy and adolescence accelerates adult associative learning: Critical impact of age, stimulus contingency and training intensity

A growing body of evidence supports the hypothesis that juvenile cognitive training shapes neural networks and behavior, and thereby determines the adults capacity for learning and memory. In particular, we have shown that infant rats, even though they do not develop an active avoidance strategy in a two-way active avoidance task, show as adults accelerated learning in the same learning task. This indicates that a memory trace was formed in the infant rats, which most likely is recruited during adult training. To identify the learning conditions, which are essential prerequisites to form this memory trace in infancy or adolescence, we investigated the critical impact of: (i) age, (ii) CS-UCS contingency, and (iii) pre-training intensity on this facilitating effect. We observed: (i) an age-dependent improvement of avoidance learning, (ii) that the beneficial impact of infant or adolescent pre-training on adult learning increases with the age at pre-training, (iii) that CS-UCS contingency during infant pre-training was most efficient to accelerate adult learning, (iv) that pre-training intensity (i.e. number of pre-training trials) was positively correlated with the pre-training induced acceleration of adult learning, and (v) that infant rats, compared to adolescent rats, need a higher training intensity to show learning improvement as adults. These results indicate that infant rats develop a goal-oriented escape strategy, which during adult training is replaced by an avoidance strategy, facilitated by the recruitment of the CS-UCS association, which has been learned during infant training. Based on these results the future challenge will be to identify the specific contribution of prefronto-limbic circuits in infant and adult learning in relation to their functional maturation.

Neurokinin2‐R in medial septum regulate hippocampal and amygdalar ACh release induced by intraseptal application of neurokinins A and B

The neurokinin receptors (NK‐R), NK2‐ and NK3‐R, have been implicated in behavioral processes, but apparently in opposite ways: while NK2‐R agonism disrupts memory and has anxiogenic‐like action, NK3‐R agonists facilitate memory and display anxiolytic‐like effects. Systemic application of NK2‐R antagonists block the release of acetylcholine (ACh) in the hippocampus, which is induced by intraseptal administration of the NK2‐R ligand, neurokinin A (NKA). We investigated the effects of medial septal injection of NKA and a preferred ligand of NK3‐R, neurokinin B (NKB), on the activity of cholinergic neurons of the basal forebrain and assessed the role of the medial septal NK2‐R in the control of extracellular ACh levels in cholinergic projection areas. ACh was dialysed in the frontal cortex, amygdala and hippocampus of anesthetized animals and was analysed by HPLC‐EC. ACh levels in hippocampus and amygdala, but not in frontal cortex were increased after intraseptal injection of either NKA or NKB (0.1, 1, 10 μM). Application of the nonpeptidic NK2‐R antagonist, saredutant SR48968 (1, 10, 100 pM), followed by NKA (1 μM) or NKB (10 μM) injection into the medial septum, blocked the ACh increase in hippocampus and amygdala. These results indicate that medial septal NK2‐R have an important role in mediating ACh release, for one, via the septal‐hippocampal cholinergic projection and, secondly, via direct or indirect route to the amygdala, but not frontal cortex. They also support the hypothesis that hippocampal cholinergic neurotransmission controls amygdala function suggesting that this interaction is regulated via NK2‐R in the medial septum.