Herman van der Putten

Neuropathology in Mice Expressing Human α-Synuclein

The presynaptic protein α-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinsons disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimers disease. α-Synuclein accumulates in Lewy bodies and Lewy neurites, and two missense mutations (A53T and A30P) in the α-synuclein gene are genetically linked to rare familial forms of Parkinsons disease. Under control of mouse Thy1 regulatory sequences, expression of A53T mutant human α-synuclein in the nervous system of transgenic mice generated animals with neuronal α-synucleinopathy, features strikingly similar to those observed in human brains with Lewy pathology, neuronal degeneration, and motor defects, despite a lack of transgene expression in dopaminergic neurons of the substantia nigra pars compacta. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions in several muscles examined, suggesting that α-synuclein interfered with a universal mechanism of synapse maintenance. Thy1 transgene expression of wild-type human α-synuclein resulted in similar pathological changes, thus supporting a central role for mutant and wild-type α-synuclein in familial and idiotypic forms of diseases with neuronal α-synucleinopathy and Lewy pathology. These mouse models provide a means to address fundamental aspects of α-synucleinopathy and test therapeutic strategies.

Epilepsy, Hyperalgesia, Impaired Memory, and Loss of Pre- and Postsynaptic GABA B Responses in Mice Lacking GABA B(1)

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.

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.

Genetic and Pharmacological Evidence of a Role for GABA B Receptors in the Modulation of Anxiety- and Antidepressant-Like Behavior

Although there is much evidence for a role of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) in the pathophysiology of anxiety and depression, the role of GABAB receptors in behavioral processes related to these disorders has not yet been fully established. GABAB receptors are G-protein-coupled receptors, which act as functional heterodimers made up of GABAB(1) and GABAB(2) subunits. Using recently generated GABAB(1) −/− mice, which lack functional GABAB receptors, and pharmacological tools we assessed the role of GABAB receptors in anxiety- and antidepressant-related behaviors. In the light–dark box, GABAB(1) −/− mice were more anxious than their wild-type littermates (less time spent in the light; reduced number of transitions). GABAB(1) −/− mice were also more anxious in the staircase test. Conversely, acute and chronic treatment with GS39783, a novel GABAB receptor positive modulator, decreased anxiety in the light–dark box and elevated zero maze tests for anxiety. On the other hand, GABAB(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 GABAB 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 GABAB receptor may serve as a novel therapeutic strategy for the development of anxiolytics, whereas GABAB receptor antagonism may serve as a basis for the generation of novel antidepressants.

Immunohistochemical localization of metabotropic glutamate receptors, mGluR7a and mGluR7b, in the central nervous system of the adult rat and mouse: A light and electron microscopic study

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. J. Comp. Neurol. 393:332–352, 1998.

Antidepressant and anxiolytic-like effects in mice lacking the group III metabotropic glutamate receptor mGluR7

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.

Part II: α-synuclein and its molecular pathophysiological role in neurodegenerative disease

α-Synuclein (αSN) 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 α-synucleinopathies, are microscopic proteinaceous insoluble inclusions in neurons and glia that are composed largely of fibrillar aggregates of αSN. This molecular form of αSN contrasts sharply with normal αSN, which is an abundant soluble presynaptic protein in brain neurons. αSN is a highly conserved protein in vertebrates and only seven of its 140 amino acids differ between human and mouse. Flies lack an αSN gene. Implicated in neurotoxicity are two αSN mutants (A53T and A30P) that cause extremely rare familial forms of PD, αSN fibrils and protofibrils, soluble protein complexes of αSN with 14-3-3 protein, and phosphorylated, nitrosylated, and ubiquitylated αSN species. Unlike rare forms of fPD caused by mutations in αSN, disease mechanisms in most α-synucleinopathies implicate wildtype αSN and seem to converge around oxidative damage and impairments in protein catabolism. It is not known whether these causalities involve αSN from the beginning, but defects in the handling of this protein seem to contribute to disease progression because accumulation of toxic αSN forms damage neurons. Here, we summarize the main structural features of αSN and its functions, and discuss the molecular αSN species implicated in human disease and transgenic animal models of α-synucleinopathy in fly and rodents.

Redistribution of GABAB(1) Protein and Atypical GABAB Responses in GABAB(2)-Deficient Mice

GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors 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 these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. 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 GABAB responses 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 GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.

Generalization of amygdala LTP and conditioned fear in the absence of presynaptic inhibition

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 GABAB receptor subtype, GABAB(1a,2), unmasks a nonassociative, NMDA receptor–independent form of presynaptic LTP at cortico-amygdala afferents. Moreover, the level of presynaptic GABAB(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 GABAB(1a) results in a generalization of conditioned fear to nonconditioned stimuli. Our findings indicate that presynaptic inhibition through GABAB(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.

Specific γ‐hydroxybutyrate‐binding sites but loss of pharmacological effects of γ‐hydroxybutyrate in GABAB(1)‐deficient mice

γ‐Hydroxybutyrate (GHB), a metabolite of γ‐aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. γ‐Hydroxybutyrate and its prodrug, γ‐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 GTPγ[35S] responses in brain membrane preparations from wild‐type but not GABAB(1)−/− mice. The GTPγ[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 GTPγ[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.