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A common pathological hallmark of protein-conformational brain diseases is the formation of disease-specific protein aggregates. In Alzheimer’s disease, these are comprised of amyloid-β and Tau as opposed to α-synuclein in Parkinson’s disease and N-terminal fragments of mutant huntingtin in Huntington’s disease. Most aggregates also sequester molecular chaperones, a protein family that assists in the folding, refolding, stabilization, and processing of client proteins, including misfolded proteins in brain diseases. Molecular chaperone modulation has achieved remarkable therapeutic effects in some cellular and preclinical animal models of protein-conformational diseases. This has raised hope for chaperone-based strategies to combat these diseases. Here, we review briefly the functional diversity and medical significance of molecular chaperones, their therapeutic potential, and common and specific challenges towards clinical application.

Leucine-rich repeat kinase 2 (Lrrk2) has been implicated in the pathophysiology of Parkinson’s disease. Lrrk2 is expressed in diverse cells including neurons and dendritic cells (DCs). In DCs Lrrk2 was shown to up-regulate Na+/Ca2+-exchanger activity. The elimination of Ca2+ by Na+/Ca2+ -exchangers requires maintenance of the Na+ gradient by the Na+/K+ -ATPase. The present study thus explored whether Lrrk2 impacts on Na+/K+ -ATPase expression and function. To this end DCs were isolated from gene-targeted mice lacking Lrrk2 (Lrrk2−/−) and their wild-type littermates (Lrrk2+/+). Na+/K+ -ATPase activity was estimated from K+ induced, ouabain sensitive, current determined by whole cell patch clamp. Na+/K+ -ATPase α1 subunit transcript and protein levels were determined by RT-qPCR and flow cytometry. As a result, the K+ induced current was significantly smaller in Lrrk2−/− than in Lrrk2+/+ DCs and was completely abolished by ouabain (100 μM) in both genotypes. The K+ induced, ouabain sensitive, current in Lrrk2+/+ DCs was significantly blunted by Lrrk2 inhibitor GSK2578215A (1 μM, 24 hours). The Na+/K+ -ATPase α1 subunit transcript and protein levels were significantly lower in Lrrk2−/− than in Lrrk2+/+ DCs and significantly decreased by Lrrk2 inhibitor GSK2578215A (1 μM, 24 hours). In conclusion, Lrrk2 is a powerful regulator of Na+/K+ -ATPase expression and activity in dendritic cells.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

In the version of this article initially published, the catalog numbers for BoNT A and B were given in the Methods section as T0195 and T5644; the correct numbers are B8776 and B6403. The error has been corrected in the HTML and PDF versions of the article.

The role of alpha-synuclein in pathogenesis of familial and idiopathic forms of Parkinson's disease, and other human disorders known as alpha-synucleinopathies, is well established. In contrast, the involvement of two other members of the synuclein family, beta-synuclein and gamma-synuclein, in the development and progression of neurodegeneration is poorly studied. However, there is a growing body of evidence that alpha-synuclein and beta-synuclein have opposite neuropathophysiological effects. Unlike alpha-synuclein, overexpressed beta-synuclein does not cause pathological changes in the nervous system of transgenic mice and even ameliorates the pathology caused by overexpressed alpha-synuclein. To assess the consequences of excess expression of the third family member, gamma-synuclein, on the nervous system we generated transgenic mice expressing high levels of mouse gamma-synuclein under control of Thy-1 promoter. These animals develop severe age- and transgene dose-dependent neuropathology, motor deficits and die prematurely. Histopathological changes include aggregation of gamma-synuclein, accumulation of various inclusions in neuronal cell bodies and processes, and astrogliosis. These changes are seen throughout the nervous system but are most prominent in the spinal cord where they lead to loss of spinal motor neurons. Our data suggest that down-regulation of small heat shock protein HSPB1 and disintegration of neurofilament network play a role in motor neurons dysfunction and death. These findings demonstrate that gamma-synuclein can be involved in neuropathophysiological changes and the death of susceptible neurons suggesting the necessity of further investigations of the potential role of this synuclein in disease.

In ischemic stroke, cytosolic death pathways are activated in injured neurons destined to die. Neuronal injury is modulated by cell surface receptors, among which the tumor necrosis factor receptor family obtained particular interest. Cytokine response modifier A (CrmA) is a cowpox virus-derived caspase inhibitor, which interferes with the so-called death-inducing signaling complex, thereby blocking receptor-mediated apoptosis. To elucidate CrmA's therapeutic potential in ischemic stroke, we characterized a transgenic mouse line expressing CrmA under a Thy1 promoter, which we subjected to intraluminal middle cerebral artery (MCA) occlusion. Using in situ hybridization histochemistry and Western blots, we show that the crmA gene integrated into chromosome 8 of the mouse genome, CrmA being expressed in the cerebral cortex and striatum. Although robustly expressed, transgenic CrmA did not influence ischemic injury, both when relatively long-lasting (90 min) and mild (30 min) MCA occlusions were imposed. As such, neither infarct volume, brain swelling or neurological deficits following 90-min ischemia, nor disseminated neuronal injury or caspase-3 activation following 30-min ischemia were influenced by CrmA. Our data argue against a therapeutic effect of CrmA in ischemic stroke.

Broad evidence indicates that modulation of the glutamatergic system could be an efficient way to achieve antidepressant activity. Metabotropic glutamate receptor (mGlu receptor) ligands seem to be promising agents to treat several central nervous system disorders, including psychiatric ones.The aim of our study was to investigate potential antidepressant-like activity of the first, selective, and bio-available mGlu7 receptor agonist, AMN082 (N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride), in wild-type (WT) and mGlu7 receptor knock-out (KO) mice.The forced swim test (FST) and the tail suspension test (TST) in mice were used to assess antidepressant-like activity of AMN082.We found that AMN082, administered IP, induced a dose-dependent decrease in the immobility time of WT animals in the FST and TST, suggesting antidepressant-like potency of an mGlu7 receptor agonist. Moreover, AMN082 did not change the behaviour of mGlu7 receptor KO mice compared to WT littermates in the TST, while imipramine, used as a reference control, significantly reduced their immobility, indicating an mGlu7 receptor-dependent mechanism of the antidepressant-like activity of AMN082. However, at high doses, AMN082 significantly decreased spontaneous locomotor activity of both mGlu7 receptor KO mice and WT control animals, suggesting off-target activity of AMN082 resulting in hypo-locomotion.These results strongly suggest that activation of the mGlu7 receptor elicits antidepressant-like effects.

The neuronal ceroid lipofuscinoses, collectively called NCLs, are rare and fatal lysosomal storage diseases that mainly affect children. Due to the fact that NCLs are both rare and heterogeneous (mutations in thirteen different genes) significant gaps exist in both preclinical and clinical research. Altogether, these gaps are major hurdles to bring therapies to patients while the need for new therapies is urgent to help them and their families. To define gaps and discuss solutions, a round table discussion involving teams and different stake holders took place during the 14th International Conference on Neuronal Ceroid Lipofuscinoses (Batten Disease) in Cordóba, Argentina. Topics covered by the teams and their leaders (in parentheses) included basic and translational research gaps with regard to large animal models (I. Tammen, D.N. Palmer), human NCL pathology and access to human tissue (J.D. Cooper, H.H. Goebel), rare NCLs (S. Hofman, I. Noher), links of NCLs to other diseases (F.M. Platt), gaps between clinic and clinical trials (H. Adams, A. Schulz), international collaborative efforts working towards a cure (S.E. Mole, H. Band) perspectives on palliative care from patient organizations (M. Frazier, A. West), and issues NCL researchers face when progressing to independent career in academia (M. Bond). Thoughts presented by the team leaders include previously unpublished opinions and information on the lack of understanding of disease pathomechanisms, gene function, assays for drug discovery and target validation, natural history of disease, and biomarkers for monitoring disease progression and treatment effects. This article is not intended to review the NCL literature. It includes personal opinions of the authors and it provides the reader with a summary of gaps discussed and solutions proposed by the teams. This article is part of a Special Issue entitled: Current Research on the Neuronal Ceroid Lipofuscinoses (Batten Disease).

Metabotropic GABAB receptors predominantly function as heterodimers of GABAB(1) and GABAB(2) subunits, but GABAB(1) can also form functional receptors in the absence of GABAB(2). Mice lacking the GABAB(1) subunit have altered behavioural responses in tests for anxiety and depression. In these studies, we investigated anxiety and depression in GABAB(2)-deficient mice. We compared the effects directly with that of genetic deletion of the GABAB(1) receptor subunit. Both GABAB(1) and GABAB(2)-deficient mice were found to be more anxious than wild type in the light-dark box paradigm. In contrast, these mice exhibited an antidepressant-like behaviour in the forced swim test. Taken together, these data suggest that heterodimeric GABAB(1,2) receptors are required for the normal regulation of emotional behaviour.

The role of metabotropic glutamate (mGlu) receptors in long-term potentiation (LTP) in the hippocampus is controversial. In the present study, we have used mice in which the mGlu1, mGlu5 or mGlu7 receptor has been deleted, by homologous recombination, to study the role of these receptor subtypes in LTP at CA1 synapses. We investigated the effects of the knockouts on both LTP and the molecular switch, a form of metaplasticity that renders LTP insensitive to the actions of the mGlu receptor antagonist MCPG ((S)-alpha-methyl-4-carboxyphenylglycine). We find that LTP is readily induced in the three knockouts and in an mGlu1 and mGlu5 double knockout. In addition, the molecular switch operates normally in either the mGlu1 or mGlu7 knockout. In contrast, the molecular switch is completely non-functional in the mGlu5 knockout, such that MCPG invariably blocks the induction of additional LTP in an input where LTP has already been induced. The effect of the mGlu5 receptor knockout was replicated in wildtype mouse slices perfused with the specific mGlu5 receptor antagonist MPEP (2-methyl-6-(phenylethynyl)-pyridine). In addition, the mGlu5 selective agonist CHPG ((RS)-2-chloro-5-hydroxyphenylglycine) sets the molecular switch. These data demonstrate that the operation of the molecular switch requires activation of mGlu5 receptors.

Gene expression analysis implicates an increasing number of novel genes in the brain as potential targets for the treatment of neurological and psychiatric disorders. Frequently, these genes are ubiquitously expressed in the brain and, thus, may contribute to a pathophysiological state through actions in several brain nuclei. Current strategies employing genetically modified animals for in vivo validation of such targets are time-consuming and often limited by developmental adaptations. Somatic gene manipulation using viral-mediated RNA interference (RNAi) has emerged recently, although restricting the target validation to specific brain nuclei. We investigated whether nonviral infusion of short interfering RNA (siRNA) into the ventricular system would enable a sequence-specific gene knockdown. The temporality and extent of siRNA-induced down-regulation were analyzed by targeting a transgene, EGFP, in mice overexpressing EGFP. Extensive knockdown of EGFP was observed, especially in regions adjacent or dorsoventrally and mediolaterally distant to the infusion site (dorsal third ventricle), with lesser knockdown in more distal regions. We challenged our RNAi approach to generate a specific knockdown of an endogenous gene, encoding the dopamine transporter (DAT) in regions (ventral midbrain) far distal to the infusion site. DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein in the brain and also elicited a temporal hyperlocomotor response similar to that (but delayed) obtained upon infusion of GBR-12909, a pharmacologically selective DAT inhibitor. Application of this nonviral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Balb/c GABA(B(1))(-/-) mice develop complex epileptiform activity, including spontaneous and audiogenic generalized seizures, 6-8 weeks after birth. The neuronal systems involved in these epilepsies have not been identified yet. Because the hippocampus is critically involved in epileptiform activity, we now investigated whether this brain region exhibits seizure-related alterations. Using semi-quantitative immunohistochemistry, we studied the temporal and cellular hippocampal expression pattern of two seizure-sensitive calcium-binding proteins, calbindin-D-28k and calretinin, in GABA(B(1))(-/-) mice. One month after birth, before the onset of overt epileptiform activity, wild-type (WT) and GABA(B(1))(-/-) mice exhibit comparable expression profiles for the two calcium-binding proteins. Three months after birth, once the epileptic phenotype is established, we observe clear alterations in the expression of calcium-binding proteins in the dentate gyrus area. GABA(B(1))(-/-) mice exhibit a 50% decline in the staining intensity of calbindin-D-28k expressing neurons and a 70% increase in the number of calretinin-positive neurons when compared to WT littermates. Six months after birth, the down-regulation of calbindin-D-28k protein is even more pronounced, while the calretinin expression in GABA(B(1))(-/-) mice reverts to the pattern seen in WT littermates. Our data demonstrate that the absence of functional GABA(B) receptors causes epileptiform activity through a mechanism that crucially involves dentate gyrus granule cells, and that this pathological activity is accompanied by adaptive changes.

Metabotropic glutamate receptors (mGluRs), and in particular the mGluR group III receptors (subtypes 4, 6, 7, 8) are known to play a role in synaptic plasticity and learning. Here, we report the effect of mGluR7 gene ablation in different learning paradigms. In the acoustic startle response (ASR), no differences were seen between knockout (KO) mice and wildtype (WT) littermates in parameters including prepulse inhibition and habituation. In an open field test, no differences were seen between genotypes in motor activity, exploratory behaviour, and fearful behaviour. In a T-maze reinforced alternation working memory (WM) task, again no difference was seen between groups. However, when increasing the demands on working-memory in a 4-arm and 8-arm maze task, KO mice committed more WM errors than WT littermates thereby uncovering a highly significant difference between the two groups that persisted every day for the whole 9 days of the experiment. In a 4-arm maze with 2 arms baited, KO and wildtype mice committed the same number of LTM errors, whereas KOs committed more WM errors. Altogether, these findings suggest that a lack of mGluR7 mainly impairs short-term working but not long-term memory performance while having no effect on sensorimotor processing, non-associative learning, motor activity and spatial orientation. The effects on WM are task-dependent and become apparent in more complex but not simple learning tasks. We discuss how mGluR7 could influence WM.

GABABreceptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABABreceptors are usually only observed after coexpression of GABAB(1)and GABAB(2)subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1)is responsible for binding of GABA, whereas GABAB(2)is necessary for surface trafficking and G-protein coupling. Consistent with thesein vitroobservations, the GABAB(1)subunit is also essential for all GABABsignalingin vivo. Mice lacking the GABAB(1)subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABABagonists. However, GABAB(1)exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1)could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1)has the potential to signal without GABAB(2)in neurons. We show that GABAB(2)-/-mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/-mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2)receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/-mice, we still detect atypical electrophysiological GABABresponses in hippocampal slices of GABAB(2)-/-mice. Furthermore, in the absence of GABAB(2), the GABAB(1)protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2)with GABAB(1)is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABABreceptors exist in neurons that naturally lack GABAB(2)subunits.

Alpha-synuclein (alphaSN) brain pathology is a conspicuous feature of several neurodegenerative diseases. These include prevalent conditions such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), and the Lewy body variant of Alzheimer's disease (LBVAD), as well as rarer conditions including multiple systems atrophy (MSA), and neurodegeneration with brain iron accumulation type-1 (NBIA-1). Common in these diseases, some referred to as alpha-synucleinopathies, are microscopic proteinaceous insoluble inclusions in neurons and glia that are composed largely of fibrillar aggregates of alphaSN. This molecular form of alphaSN contrasts sharply with normal alphaSN, which is an abundant soluble presynaptic protein in brain neurons. alphaSN is a highly conserved protein in vertebrates and only seven of its 140 amino acids differ between human and mouse. Flies lack an alphaSN gene. Implicated in neurotoxicity are two alphaSN mutants (A53T and A30P) that cause extremely rare familial forms of PD, alphaSN fibrils and protofibrils, soluble protein complexes of alphaSN with 14-3-3 protein, and phosphorylated, nitrosylated, and ubiquitylated alphaSN species. Unlike rare forms of fPD caused by mutations in alphaSN, disease mechanisms in most alpha-synucleinopathies implicate wildtype alphaSN and seem to converge around oxidative damage and impairments in protein catabolism. It is not known whether these causalities involve alphaSN from the beginning, but defects in the handling of this protein seem to contribute to disease progression because accumulation of toxic alphaSN forms damage neurons. Here, we summarize the main structural features of alphaSN and its functions, and discuss the molecular alphaSN species implicated in human disease and transgenic animal models of alpha-synucleinopathy in fly and rodents.

Glutamatergic neurotransmission has been strongly implicated in the pathophysiology of affective disorders, such as major depression and anxiety. Of all glutamate receptors, the role of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8) in such disorders is the least investigated because of the lack of specific pharmacological tools. To this end, we examined the behavioural profiles of mice with a targeted deletion of the gene for mGluR7 (mGluR7 - / - ) in animal models of depression and anxiety. mGluR7 - / - mice were compared with wild-type (mGluR7 + / + ) littermates and showed substantially less behavioural immobility in both the forced swim test and the tail suspension test. Both behavioural paradigms are widely used to predict antidepressant-like activity. Further, mGluR7 - / - mice displayed anxiolytic activity in four different behavioural tests, i.e. the light-dark box, the elevated plus maze, the staircase test, and the stress-induced hyperthermia test, while their cognitive performance was normal in the passive avoidance paradigm. Analysis of locomotor activity in a novel environment demonstrated that mGluR7 - / - mice were slightly more active in the initial minutes following placement in the chamber only. Together, these data suggest that mGluR7 may play a pivotal role in mechanisms that regulate behavioural responses to aversive states. Therefore, drugs acting at mGluR7 may provide novel treatments for psychiatric disorders such as depression and anxiety.

Gene variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with susceptibility to Parkinson's disease (PD). Besides brain and periphery, LRRK2 is expressed in various immune cells including dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. However, the function of LRRK2 in the immune system is still incompletely understood. Here, Ca(2+)-signaling was analyzed in DCs isolated from gene-targeted mice lacking lrrk2 (Lrrk2(-/-)) and their wild-type littermates (Lrrk2(+/+)). According to Western blotting, Lrrk2 was expressed in Lrrk2(+/+) DCs but not in Lrrk2(-/-)DCs. Cytosolic Ca(2+) levels ([Ca(2+)]i) were determined utilizing Fura-2 fluorescence and whole cell currents to decipher electrogenic transport. The increase of [Ca(2+)]i following inhibition of sarcoendoplasmatic Ca(2+)-ATPase with thapsigargin (1 µM) in the absence of extracellular Ca(2+) (Ca(2+)-release) and the increase of [Ca(2+)]i following subsequent readdition of extracellular Ca(2+) (SOCE) were both significantly larger in Lrrk2(-/-) than in Lrrk2(+/+) DCs. The augmented increase of [Ca(2+)]i could have been due to impaired Ca(2+) extrusion by K(+)-independent (NCX) and/or K(+)-dependent (NCKX) Na(+)/Ca(2+)-exchanger activity, which was thus determined from the increase of [Ca(2+)]i, (Δ[Ca(2+)]i), and current following abrupt replacement of Na(+) containing (130 mM) and Ca(2+) free (0 mM) extracellular perfusate by Na(+) free (0 mM) and Ca(2+) containing (2 mM) extracellular perfusate. As a result, both slope and peak of Δ[Ca(2+)]i as well as Na(+)/Ca(2+) exchanger-induced current were significantly lower in Lrrk2(-/-) than in Lrrk2(+/+) DCs. A 6 or 24 hour treatment with the LRRK2 inhibitor GSK2578215A (1 µM) significantly decreased NCX1 and NCKX1 transcript levels, significantly blunted Na(+)/Ca(2+)-exchanger activity, and significantly augmented the increase of [Ca(2+)]i following Ca(2+)-release and SOCE. In conclusion, the present observations disclose a completely novel functional significance of LRRK2, i.e., the up-regulation of Na(+)/Ca(2+) exchanger transcription and activity leading to attenuation of Ca(2+)-signals in DCs.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

GABA(B) (gamma-aminobutyric acid type B) receptors are important for keeping neuronal excitability under control. Cloned GABA(B) receptors do not show the expected pharmacological diversity of native receptors and it is unknown whether they contribute to pre- as well as postsynaptic functions. Here, we demonstrate that Balb/c mice lacking the GABA(B(1)) subunit are viable, exhibit spontaneous seizures, hyperalgesia, hyperlocomotor activity, and memory impairment. Upon GABA(B) agonist application, null mutant mice show neither the typical muscle relaxation, hypothermia, or delta EEG waves. These behavioral findings are paralleled by a loss of all biochemical and electrophysiological GABA(B) responses in null mutant mice. This demonstrates that GABA(B(1)) is an essential component of pre- and postsynaptic GABA(B) receptors and casts doubt on the existence of proposed receptor subtypes.

Mutations in the alpha-synuclein (alphaSYN) gene are associated with rare cases of familial Parkinson's disease, and alphaSYN is a major component of Lewy bodies and Lewy neurites. Here we have investigated the localization of wild-type and mutant [A30P]alphaSYN as well as betaSYN at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic colocalization of alphaSYN and betaSYN in human and mouse brain. In a sucrose gradient equilibrium centrifugation assay, a portion of betaSYN floated into lower density fractions, which also contained the synaptic vesicle marker synaptophysin. Likewise, wild-type and [A30P]alphaSYN were found in floating fractions. Subcellular fractionation of mouse brain revealed that both alphaSYN and betaSYN were present in synaptosomes. In contrast to synaptophysin, betaSYN and alphaSYN were recovered from the soluble fraction upon lysis of the synaptosomes. Synaptic colocalization of alphaSYN and betaSYN was directly visualized by confocal microscopy of double-stained human brain sections. The Parkinson's disease-associated human mutant [A30P]alphaSYN was found to colocalize with betaSYN and synaptophysin in synapses of transgenic mouse brain. However, in addition to their normal presynaptic localization, transgenic wild-type and [A30P]alphaSYN abnormally accumulated in neuronal cell bodies and neurites throughout the brain. Thus, mutant [A30P]alphaSYN does not fail to be transported to synapses, but its transgenic overexpression apparently leads to abnormal cellular accumulations.

In an attempt to obtain suitable in vivo models for optimizing new tumor therapy strategies for intestinal adenocarcinomas, carcinoembryonic antigen (CEA) promoter/SV40 T antigen gene constructs have been used to generate transgenic mice. One transgenic line (L5496), which contains a 424-bp CEA promoter/SV40 T antigen transgene, exclusively developed multi-focal carcinomas in the pyloric region of the stomach in 100% of the offspring. Tumors were already observable in 37-day-old animals as dysplastic cell foci within the mucosal layer. In 50-day-old mice, the tumor mass was mainly restricted to the mucosa with invasive growth into the submucosal tissue. The animals became moribund at 100-130 days of age due to blockage of the pylorus. At this time, the tumor had penetrated into the duodenum and had invaded all tissue layers within the stomach. In contrast to most other stomach tumor models, this one perfectly matches the development of the most common stomach cancers found in humans. Furthermore, after crossing these mice with mice that are transgenic for the human CEA gene, the double transgenic offspring revealed expression of CEA in the resulting tumors. Thus, as well as being a model for studying gastric carcinoma development and prevention, this system should provide a useful preclinical model for CEA-targeted gastric tumor therapy.

Metabotropic glutamate receptors (mGluRs) consist of eight different subtypes and exert their effects on second messengers and ion channels via G-proteins. The function of individual mGluR subtypes in the CNS, however, largely remains to be clarified. We examined the fear response of freezing after electric shock in wild-type and mGluR7−/−knockout littermates. Wild-type mice displayed freezing immediately after and 1 d after footshock. In comparison, mGluR7−/−knockout mice showed significantly reduced levels in both immediate postshock and delayed freezing responses. However, the knockout mice exhibited no abnormalities in pain sensitivity and locomotor activity. To further examine amygdala-dependent behavior, we performed conditioned taste aversion (CTA) experiments. In wild-type mice, the administration of saccharin followed by intraperitoneal injection of the malaise-inducing agent LiCl resulted in an association between saccharin and LiCl. This association caused strong CTA toward saccharin. In contrast, mGluR7−/−knockout mice failed to associate between the taste and the negative reinforcer in CTA experiments. Again, the knockout mice showed no abnormalities in taste preference and in the sensitivity to LiCl toxicity. These results indicate that mGluR7 deficiency causes an impairment of two distinct amygdala-dependent behavioral paradigms. Immunohistochemical and immunoelectron-microscopic analyses showed that mGluR7 is highly expressed in amygdala and preferentially localized at the presynaptic axon terminals of glutamatergic neurons. Together, these findings strongly suggest that mGluR7 is involved in neural processes subserving amygdala-dependent averse responses.

The distributions of two alternative splicing variants of metabotropic glutamate receptor mGluR7, mGluR7a and mGluR7b, were examined immunohistochemically in the rat and mouse by using variant-specific antibodies raised against C-terminal portions of rat mGluR7a and human mGluR7b. Many regions throughout the central nervous system (CNS) showed mGluR7-like immunoreactivities (LI). The distribution patterns of mGluR7-LI in the rat were substantially the same as those in the mouse, although some species differences were observed in a few regions. Intense mGluR7a-LI was seen in the main and accessory olfactory bulbs, anterior olfactory nucleus, islands of Calleja, superficial layers of the olfactory tubercle, piriform cortex and entorhinal cortex, periamygdaloid cortex, amygdalohippocampal area, hippocampus, layer I of the neocortical regions, globus pallidus, superficial layers of the superior colliculus, locus coeruleus, and superficial layers of the medullary and spinal dorsal horns. The distribution of mGluR7b was more restricted. It was intense in the islands of Calleja, substantia innominata, hippocampus, ventral pallidum, and globus pallidus. The medial habenular nucleus also showed intense mGluR7a-LI in the rat but not in the mouse. For both mGluR7a- and mGluR7b-LI, localization in the active zones of presynaptic axon terminals was confirmed electron microscopically at synapses of both the asymmetrical and symmetrical types. It is noteworthy that mGluR7a-LI is seen preferentially in relay nuclei of the sensory pathways and that both mGluR7a- and mGluR7b-LI are observed not only in presumed glutamatergic axon terminals, but also in non-glutamatergic axon terminals including presumed inhibitory ones. Thus, mGluR7 may play roles not only as an autoreceptor in glutamatergic axon terminals, but also as a presynaptic heteroreceptor in non-glutamatergic axon terminals in various CNS regions.

The nonspecific cross-reacting antigen-95 (NCA-95/CD66b), is a member of the human carcinoembryonic antigen (CEA) family encoded by the CGM6 gene that is exclusively expressed in neutrophils and eosinophils. No murine counterpart is known to exist. We have analyzed a cosmid containing the complete CGM6 gene. The coding sequence is contained within six exons spanning a 16.5 kb region. The main transcriptional start site was mapped to a tight cluster between nucleotides -95 and -101 relative to the translational start site. As with other members of the CEA gene family, no typical TATA or CAAT-box sequences were found in the CGM6 gene. Transgenic mice were established with the cosmid insert. CD66b expression is first seen in the fetal liver on day 12.5 of mouse embryonic development, and it first appears in the bone marrow at day 17.5. Northern blot analysis showed that CD66b transcripts are confined to the bone marrow of adult mice, whereas immunohistochemistry also showed CD66b-positive granulocytes in the spleen, thymus, and lungs. FACScan analyses of bone marrow and spleen cells showed CD66b expression to be exclusive to granulocytes. Thus, all the elements necessary for regulating granulocyte-specific expression are present within this cosmid clone. These mice could provide a model for transplantation and for inflammation studies using CD66b as a granulocyte-specific marker.

HDAC4 histone deacetylase is found to associate with huntingtin in a polyQ-length dependent manner. Reduction of HDAC4 levels in mouse models of Huntington's disease (HD) delays cytoplasmic aggregation in the brain and improves the molecular pathology of HD, providing a potential new therapeutic target., Histone deacetylase (HDAC) 4 is a transcriptional repressor that contains a glutamine-rich domain. We hypothesised that it may be involved in the molecular pathogenesis of Huntington's disease (HD), a protein-folding neurodegenerative disorder caused by an aggregation-prone polyglutamine expansion in the huntingtin protein. We found that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and co-localises with cytoplasmic inclusions. We show that HDAC4 reduction delayed cytoplasmic aggregate formation, restored Bdnf transcript levels, and rescued neuronal and cortico-striatal synaptic function in HD mouse models. This was accompanied by an improvement in motor coordination, neurological phenotypes, and increased lifespan. Surprisingly, HDAC4 reduction had no effect on global transcriptional dysfunction and did not modulate nuclear huntingtin aggregation. Our results define a crucial role for the cytoplasmic aggregation process in the molecular pathology of HD. HDAC4 reduction presents a novel strategy for targeting huntingtin aggregation, which may be amenable to small-molecule therapeutics., Author Summary Huntington's disease (HD) is a late-onset neurodegenerative disorder caused by protein-folding defects in the huntingtin protein. Mutations in huntingtin can result in extra-long tracts of the amino acid glutamine, resulting in aberrant interactions with other proteins and also causing huntingtin proteins to self-associate and -aggregate. The pathology of HD is therefore associated with nuclear and cytoplasmic aggregates. HDAC4 is a histone deacetylase protein traditionally associated with roles in transcription repression. The HDAC4 protein contains a glutamine-rich domain and in this work we find that HDAC4 associates with huntingtin in a polyglutamine-length-dependent manner and that these proteins co-localise in cytoplasmic inclusions. Importantly, reducing HDAC4 levels delays cytoplasmic aggregate formation and rescues neuronal and cortico-striatal synaptic function in mouse models of HD. In addition, we observe improvements in motor coordination and neurological phenotypes, as well as increased lifespan in these mice. Nuclear huntingin aggregates or transcription regulation, however, remained unaffected when HDAC4 levels were reduced to enable these effects. Our results thus provide valuable insight into separating cytoplasmic and nuclear pathologies, and define a crucial role for cytoplasmic aggregations in HD progression. HDAC4 reduction presents a novel strategy for alleviating the toxicity of huntingtin protein aggregation, thereby influencing the molecular pathology of Huntington's disease. As there are currently no disease-modifying therapeutics available for Huntington's disease, we hope that this HDAC4-mediated regulation may be amenable to small-molecule therapeutics.

Glutamate transmission and synaptic plasticity in the amygdala are essential for the learning and expression of conditioned fear. Glutamate activates both ionotropic glutamate receptors and eight subtypes of metabotropic glutamate receptors (mGlu1-8). In the present study, we investigated the roles of mGlu7 and mGlu8 in amygdala-dependent behavior and synaptic plasticity. We show that ablation of mGlu7 but not mGlu8 attenuates long-term potentiation (LTP) at thalamo-lateral amygdala (LA) synapses where a strong association between LTP and learning has been demonstrated. mGlu7-deficient mice express a general deficit in conditioned fear whereas mGlu8-deficient mice show a dramatic reduction in contextual fear. The mGlu7 agonist AMN082 reduced thalamo-LA LTP and intra-amygdala administration blocked conditioned fear learning. In contrast, the mGlu8 agonist DCPG decreased synaptic transmission but not LTP at thalamo-LA synapses. Intra-amygdala DCPG selectively reduced the expression of contextual fear but did not affect the acquisition and expression of cued fear. Taken together, these data revealed very different roles for mGlu7 and mGlu8 in amygdala synaptic transmission, fear learning and its expression. These receptors seem promising targets for treating anxiety disorders with different underlying pathologies with exaggerated fear learning (mGlu7) or contextual fear (mGlu8).

Our knowledge regarding the molecular pathophysiology underlying anxiety disorders remains incomplete. Increasing evidence points to a role of glutamate in anxiety. The group III metabotropic glutamate receptors (mGlu4, mGlu6, mGlu7 and mGlu8 receptors) remain the least investigated glutamate receptor subtypes partially due to a delay in the development of specific pharmacological tools. Early work using knockout animals and pharmacological tools aimed at investigating the role of mGlu7 receptor in the pathophysiology of anxiety disorders has yielded exciting yet not always consistent results. To further investigate the role this receptor plays in anxiety-like behaviour, we knocked down mGlu7 receptor mRNA levels in the adult mouse brain using siRNA delivered via an osmotic minipump. This reduced anxiety-like behaviour in the light-dark box coupled with an attenuation of stress-induced hyperthermia (SIH) and a reduction of the acoustic startle response (ASRs) in the fear-potentiated startle paradigm (FPS). These effects on anxiety-like behaviour were independent of any impairment of locomotor activity and surprisingly, no behavioural changes were observed in the forced swim test (FST), which is in contrast to mGlu7 receptor knockout animals. Furthermore, the previously reported epilepsy-prone phenotype seen in mGlu7 receptor knockout animals was not observed following siRNA-induced knockdown of the receptor. These data suggest targeting mGlu7 receptors with selective antagonist drugs may be an effective and safe strategy for the treatment of anxiety disorders.

Huntington's disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.

The group III metabotropic glutamate (mGlu) receptors mGlu7 and mGlu8 are receiving increased attention as potential novel therapeutic targets for anxiety disorders. The effects mediated by these receptors appear to result from a complex interplay of facilitatory and inhibitory actions at different brain sites in the anxiety/fear circuits. To better understand the effect of mGlu7 and mGlu8 receptors on extinction of contextual fear and their critical sites of action in the fear networks, we focused on the amygdala. Direct injection into the basolateral complex of the amygdala of the mGlu7 receptor agonist AMN082 facilitated extinction, whereas the mGlu8 receptor agonist (S)-3,4-DCPG sustained freezing during the extinction acquisition trial. We also determined at the ultrastructural level the synaptic distribution of these receptors in the basal nucleus (BA) and intercalated cell clusters (ITCs) of the amygdala. Both areas are thought to exert key roles in fear extinction. We demonstrate that mGlu7 and mGlu8 receptors are located in different presynaptic terminals forming both asymmetric and symmetric synapses, and that they preferentially target neurons expressing mGlu1α receptors mostly located around ITCs. In addition we show that mGlu7 and mGlu8 receptors were segregated to different inputs to a significant extent. In particular, mGlu7a receptors were primarily onto glutamatergic afferents arising from the BA or midline thalamic nuclei, but not the medial prefrontal cortex (mPFC), as revealed by combined anterograde tracing and pre-embedding electron microscopy. On the other hand, mGlu8a showed a more restricted distribution in the BA and appeared absent from thalamic, mPFC and intrinsic inputs. This segregation of mGlu7 and mGlu8 receptors in different neuronal pathways of the fear circuit might explain the distinct effects on fear extinction training observed with mGlu7 and mGlu8 receptor agonists. This article is part of a Special Issue entitled ‘Metabotropic Glutamate Receptors’., Highlights ► The mGlu7 receptor agonist AMN082 facilitated extinction of contextual fear. ► The mGlu8 receptor agonist DCPG exerted an anxiogenic-effect amygdala mediated. ► mGlu7a and mGlu8a receptors segregate mostly to different inputs in the BA. ► Inputs innervating large ITC neurons are enriched in mGlu7a and mGlu8a receptors. ► Thalamic and BA inputs, but not mPFC, to the amygdala possess mGlu7a receptors.

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species.

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson's disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy.

BACKGROUND: Aggregation and misfolded alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of alpha-synuclein and thus be beneficial in alpha-synucleinopathies. METHODOLOGY/PRINCIPAL FINDINGS: We co-overexpressed human A53T point-mutated alpha-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the alpha-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in alpha-synuclein oligomers/aggregates, truncations and phosphorylation levels and alpha-synuclein localization was unchanged in immunostainings. CONCLUSION/SIGNIFICANCE: Overexpressing HSP70 in a mouse model of alpha-synucleinopathy did not lower the toxic load of alpha-synuclein species and had no beneficial effect on alpha-synuclein-related motor deficits.

To dissect mechanisms that co-ordinate specific events in thymopoiesis we have characterized alterations in thymic structure and function caused by expression of a transgene. This gene encodes SV40Tag and is specifically expressed in a subset of thymic epithelial (TE) cells around birth. As a result the number of immortal TE cells increases, thymic mass increases (up to 3 g), and thymopoiesis is expanded. The latter is reflected by a approximately 100-fold increase of the major thymocyte subsets and increased peripheral T cell counts. Grossly hyperplastic thymi retain many but not all morphological features of a normal thymus. Also in grafts, SV40Tag+ TE cells steer expansion (up to 8 g) and organize a tissue with mainly cortex-like features that includes mainly SV40Tag+ TE cells, thymocytes, and macrophages. To investigate expression of specialized gene functions in the immortal TE cells, a cell line was derived. The Epi-A1 cell line expresses the genes for major histocompatibility complex class I and II, Thy-1, interleukin (IL)-6, IL-7, macrophage-colony-stimulating factor, and transforming growth factor-beta 3. Most importantly, Epi-A1 cells also express the IL-4 receptor and the c-kit ligand (KL), a factor that, in concert with commitment factors, channels progenitors into hemopoietic lineages. The expression of low constitutive levels of KL mRNA does not require IL-4, but KL mRNA levels are increased dramatically in response to IL-4. Since constitutive expression of KL mRNA in vivo is restricted to a small subset of TE cells in the thymus, our findings reveal a novel specific interaction between thymocytes and a specialized subset of TE cells.

We describe the anxiolytic-like effects of the first, selective metabotropic G-protein-coupled glutamate 7 (mGlu7) receptor agonist, N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082), as measured in the modified stress-induced hyperthermia (SIH) and the four-plate tests. Administration of AMN082 (3-6 mg/kg intraperitoneally) to Swiss mice produced anxiolytic-like effects in the modified SIH and four-plate tests. Moreover, it was ineffective as an anxiolytic in the SIH test in mGlu7 receptor knockout mice as compared with wild-type C57BL/6J littermate controls. In contrast, diazepam (1.25-5 mg/kg) significantly reduced SIH in both the wild-type and knockout animals. The anxiolytic-like effect of AMN082 in the SIH paradigm was abolished by pretreatment with flumazenil (10 mg/kg intraperitoneally). This indicates an involvement of gamma-aminobutyric acid-ergic neurotransmission in AMN's anxiolytic actions. The results indicate that activation of the mGlu7 receptor produces anxiolytic-like effects via the modulation of the gamma-aminobutyric acid system.

Metabotropic glutamate receptor 7 (mGluR7) is expressed in brain regions implicated in emotional learning and working memory, and previous behavioral experiments indicated contributions of mGluR7 to various complex behaviors. In the present study, we investigated the specific effects of mGluR7 deletion on a variety of conditioning paradigms that model crucial neurocognitive and psychopathological behavioral phenomena. Null-mutant mGluR7(-/-) mice displayed defects during scheduled appetitive conditioning, acquisition and extinction of appetitive odor conditioning, extinction of response suppression-based conditioned emotional responding (CER), acquisition of discriminative CER, and contextual fear conditioning. mGluR7(-/-) animals were slower to acquire the association between a conditioned stimulus and a positive or negative reinforcer, but eventually reached similar performance levels to their wildtype littermates. Notably, extinction learning of conditioned responses was slower in mGluR7(-/-) compared to wildtype animals. The observed delays in the acquisition of complicated stimulus associations across conditioning procedures may suggest a critical role for mGluR7 in neurocognitive functions and psychopathology.

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.

Pavlovian fear conditioning, a simple form of associative learning, is thought to involve the induction of associative, NMDA receptor-dependent long-term potentiation (LTP) in the lateral amygdala. Using a combined genetic and electrophysiological approach, we show here that lack of a specific GABA(B) receptor subtype, GABA(B(1a,2)), unmasks a nonassociative, NMDA receptor-independent form of presynaptic LTP at cortico-amygdala afferents. Moreover, the level of presynaptic GABA(B(1a,2)) receptor activation, and hence the balance between associative and nonassociative forms of LTP, can be dynamically modulated by local inhibitory activity. At the behavioral level, genetic loss of GABA(B(1a)) results in a generalization of conditioned fear to nonconditioned stimuli. Our findings indicate that presynaptic inhibition through GABA(B(1a,2)) receptors serves as an activity-dependent constraint on the induction of homosynaptic plasticity, which may be important to prevent the generalization of conditioned fear.

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N , N ′-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTPγS binding (EC 50 -values, 64-290 nM) with agonist efficacies comparable with those of l -2-amino-4-phosphonobutyrate (L-AP 4 ) and superior to those of l -glutamate. AMN082 (≤10 μM) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.

Regulation of neurotransmission via group-III metabotropic glutamate receptors (mGluR4, -6, -7, and -8) has recently been implicated in the pathophysiology of affective disorders, such as major depression and anxiety. For instance, mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) showed antidepressant and anxiolytic-like effects in a variety of stress-related paradigms, including the forced swim stress and the stress-induced hyperthermia tests. Deletion of mGluR7 reduces also amygdala- and hippocampus-dependent conditioned fear and aversion responses. Since the hypothalamic-pituitary-adrenal (HPA) axis regulates the stress response we investigate whether parameters of the HPA axis at the levels of selected mRNA transcripts and endocrine hormones are altered in mGluR7-deficient mice. Over all, mGluR7-/- mice showed only moderately lower serum levels of corticosterone and ACTH compared with mGluR7+/+ mice. More strikingly however, we found strong evidence for upregulated glucocorticoid receptor (GR)-dependent feedback suppression of the HPA axis in mice with mGluR7 deficiency: (i) mRNA transcripts of GR were significantly upregulated in the hippocampus of mGluR7-/- animals, (ii) similar increases were seen with 5-HT1A receptor transcripts, which are thought to be directly controlled by the transcription factor GR and finally (iii) mGluR7-/- mice showed elevated sensitivity to dexamethasone-induced suppression of serum corticosterone when compared with mGluR7+/+ animals. These results indicate that mGluR7 deficiency causes dysregulation of HPA axis parameters, which may account, at least in part, for the phenotype of mGluR7-/- mice in animal models for anxiety and depression. In addition, we present evidence that protein levels of brain-derived neurotrophic factor are also elevated in the hippocampus of mGluR7-/- mice, which we discuss in the context of the antidepressant-like phenotype found in those animals. We conclude that genetic ablation of mGluR7 in mice interferes at multiple sites in the neuronal circuitry and molecular pathways implicated in affective disorders.

Metabotropic glutamate receptors (mGluRs) are known to play a role in synaptic plasticity and learning. We have previously shown that mGluR7 deletion in mice produces a selective working memory (WM) impairment, while other types of memory such as reference memory remain unaffected. Since WM has been associated with Theta activity (6-12 Hz) in EEGs, and since EEG abnormalities have been observed in these mice before, we studied the effect of mGluR7 gene ablation on EEG activity in the hippocampus, in particular in the Theta range, during performance of a WM task. In an eight-arm maze with four arms baited, mGluR7 knock-out (KO) and wild-type mice committed the same number of reference memory errors, whereas KOs committed more WM errors. While performing the task, KO mice showed substantially higher Theta amplitudes, and the ratio of Theta to overall EEG power was much increased. No change was seen in the Delta (0-5 Hz), or Gamma (30-40 Hz) EEG bands compared with controls. When recording EEGs during periods of rest in the home cages, no difference was seen between groups. These findings suggest that mGluR7 is important for modulation and control of Theta activity. Since only WM was affected, and only the Theta range of EEG activity was altered, these results show a correlation between Theta rhythm and WM performance, and therefore support the concept that Theta activity in the hippocampus is involved in WM storage.

Type B γ-aminobutyric acid receptor (GABA B R) is a G protein-coupled receptor that regulates neurotransmitter release and neuronal excitability throughout the brain. In various neurons, GABA B Rs are concentrated at excitatory synapses. Although these receptors are assumed to respond to GABA spillover from neighboring inhibitory synapses, their function is not fully understood. Here we show a previously undescribed function of GABA B R exerted independent of GABA. In cerebellar Purkinje cells, interaction of GABA B R with extracellular Ca 2+ ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{Ca}}_{{\mathrm{o}}}^{2+}\end{equation*}\end{document} ) leads to a constitutive increase in the glutamate sensitivity of metabotropic glutamate receptor 1 (mGluR1). mGluR1 sensitization is clearly mediated by GABA B R because it is absent in GABA B R1 subunit-knockout cells. However, the mGluR1 sensitization does not require G i/o proteins that mediate the GABA B R's classical functions. Moreover, coimmunoprecipitation reveals complex formation between GABA B R and mGluR1 in the cerebellum. These findings demonstrate that GABA B R can act as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{Ca}}_{{\mathrm{o}}}^{2+}\end{equation*}\end{document} -dependent cofactors to enhance neuronal metabotropic glutamate signaling.

Although there is much evidence for a role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the pathophysiology of anxiety and depression, the role of GABA(B) receptors in behavioral processes related to these disorders has not yet been fully established. GABA(B) receptors are G-protein-coupled receptors, which act as functional heterodimers made up of GABA(B(1)) and GABA(B(2)) subunits. Using recently generated GABA(B(1)) -/- mice, which lack functional GABA(B) receptors, and pharmacological tools we assessed the role of GABA(B) receptors in anxiety- and antidepressant-related behaviors. In the light-dark box, GABA(B(1)) -/- mice were more anxious than their wild-type littermates (less time spent in the light; reduced number of transitions). GABA(B(1)) -/- mice were also more anxious in the staircase test. Conversely, acute and chronic treatment with GS39783, a novel GABA(B) receptor positive modulator, decreased anxiety in the light-dark box and elevated zero maze tests for anxiety. On the other hand, GABA(B(1)) -/- mice had decreased immobility (antidepressant-like behavior) in the forced swim test (FST). These behavioral effects are unrelated to alterations in locomotor activity. In confirmation of the genetic data, acute and chronic treatment with CGP56433A, a selective GABA(B) receptor antagonist, also decreased immobility in the FST, whereas GS39783 did not alter this behavior. Taken together, these data suggest that positive modulation of the GABA(B) receptor may serve as a novel therapeutic strategy for the development of anxiolytics, whereas GABA(B) receptor antagonism may serve as a basis for the generation of novel antidepressants.

gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.

Background Global cerebral ischemia is associated with delayed neuronal death. Given the role of caspases in apoptosis, caspase inhibitors may provide neuronal protection after cardiac arrest. To this end, the authors generated a transgenic rat line expressing baculovirus p35, a broad-spectrum caspase inhibitor, in central neurons. Its effects were evaluated on neuronal cell death and outcome after global cerebral ischemia. Methods Global cerebral ischemia was induced by cardiocirculatory arrest. After 6 min, animals were resuscitated by controlled ventilation, extrathoracic cardiac massage, epinephrine, and electrical countershocks. Neuronal death was assessed after 7 days by histologic evaluation of the hippocampal cornu ammonis 1 sector. Postischemic outcome was assessed by determination of overall survival and according to neurologic deficit scores 24 h, 3 days, and 7 days after resuscitation. Results The rate of 7-day survival after cardiac arrest for the transgenic rats (85%) was significantly higher than that for the nontransgenic controls (52%; P < 0.05). However, no differences were observed either in the number of terminal deoxynucleotidyltransferase-mediated d-uracil triphosphate-biotin nick end-labeling-positive cells or viable neurons in the cornu ammonis 1 sector or in the neurologic deficit score when comparing surviving transgenic and nontransgenic rats. These findings suggest that neuronal apoptosis after cardiac arrest is not primarily initiated by activation of caspases. Conclusion Expression of baculovirus p35 can improve survival after cardiac arrest in rats, but the mode and site of action remain to be elucidated.

Glutamatergic neurotransmission has been strongly implicated in the pathophysiology of affective disorders, such as major depression and anxiety. Of all glutamate receptors, the role of group III metabotropic glutamate receptors (mGluR4, mGluR6, mGluR7, mGluR8) in such disorders is the least investigated because of the lack of specific pharmacological tools. To this end, we examined the behavioural profiles of mice with a targeted deletion of the gene for mGluR7 (mGluR7-/-) in animal models of depression and anxiety. mGluR7-/- mice were compared with wild-type (mGluR7+/+) littermates and showed substantially less behavioural immobility in both the forced swim test and the tail suspension test. Both behavioural paradigms are widely used to predict antidepressant-like activity. Further, mGluR7-/- mice displayed anxiolytic activity in four different behavioural tests, i.e. the light-dark box, the elevated plus maze, the staircase test, and the stress-induced hyperthermia test, while their cognitive performance was normal in the passive avoidance paradigm. Analysis of locomotor activity in a novel environment demonstrated that mGluR7-/- mice were slightly more active in the initial minutes following placement in the chamber only. Together, these data suggest that mGluR7 may play a pivotal role in mechanisms that regulate behavioural responses to aversive states. Therefore, drugs acting at mGluR7 may provide novel treatments for psychiatric disorders such as depression and anxiety.

Parkin is an E3 ligase that plays an important role in the ubiquitin/proteosome pathway responsible for protein degradation events. Mutations in parkin result in a loss-of-function and lead to Parkinson's disease, a progressive neurological disorder of movement. Presumably, this occurs due to the toxic build-up of proteins that are no longer effectively cleared/degraded by the parkin-dependent ubiqutin/proteosome pathway. To date, three types of proteins have been shown to interact with parkin. Firstly, the E2 ubiquitin conjugating proteins called UbcH7 and UbcH8 interact with parkin. Secondly, putative substrates interacting with parkin include a synaptic vesicle associated GTPase named CDCrel-1; a G protein-coupled receptor named Pael; a novel from of alpha-synuclein; and an alpha-synuclein interacting protein synphilin-1. Thirdly and more recently, a PDZ domain containing scaffolding protein CASK/Lin2 has been shown to interact with the PDZ binding motif of parkin. A network of PDZ-interacting proteins has potential to form a complex web of molecules that surround parkin and regulate its subcellular localisation and function.