Early deficits in an in vitro striatal microcircuit model carrying the Parkinson's GBA-N370S mutation.

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.

Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons.

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.

Histaminergic Control of Corticostriatal Synaptic Plasticity during Early Postnatal Development.

A reduction in the synthesis of the neuromodulator histamine has been associated with Tourette's syndrome and obsessive-compulsive disorder. Symptoms of these disorders are thought to arise from a dysfunction or aberrant development ofcorticostriatal circuits. Here, we investigated how histamine affects developing corticostriatal circuits, both acutely and longer-term, during the first postnatal weeks, using patch-clamp and field recordings in mouse brain slices (C57Bl/6, male and female). Immunohistochemistry for histamine-containing axons reveals striatal histaminergic innervation by the second postnatal week, and qRT-PCR shows transcripts for H1, H2, and H3 histamine receptors in striatum from the first postnatal week onwards, with pronounced developmental increases in H3 receptor expression. Whole-cell patch-clamp recordings of striatal spiny projection neurons and histamine superfusion demonstrates expression of functional histamine receptors from the first postnatal week onwards, with histamine having diverse effects on their electrical properties, including depolarization of the membrane potential while simultaneously decreasing action potential output. Striatal field recordings and electrical stimulation of corticostriatal afferents revealed that histamine, acting at H3 receptors, negatively modulates corticostriatal synaptic transmission from the first postnatal week onwards. Last, we investigated effects of histamine on longer-term changes at developing corticostriatal synapses and show that histamine facilitates NMDA receptor-dependent LTP via H3 receptors during the second postnatal week, but inhibits synaptic plasticity at later developmental stages. Together, these results show that histamine acutely modulates developing striatal neurons and synapses and controls longer-term changes in developing corticostriatal circuits, thus providing insight into the possible etiology underlying neurodevelopmental disorders resulting from histamine dysregulation.SIGNIFICANCE STATEMENT Monogenic causes of neurologic disorders, although rare, can provide opportunities to both study and understand the brain. For example, a nonsense mutation in the coding gene for the histamine-synthesizing enzyme has been associated with Tourette's syndrome and obsessive-compulsive disorder, and dysfunction of corticostriatal circuits. Nevertheless, the etiology of these neurodevelopmental disorders and histamine's role in the development of corticostriatal circuits have remained understudied. Here we show that histamine is an active neuromodulator during the earliest periods of postnatal life and acts at developing striatal neurons and synapses. Crucially, we show that histamine permits NMDA receptor-dependent corticostriatal synaptic plasticity during an early critical period of postnatal development, which suggests that genetic or environmental perturbations of histamine levels can impact striatal development.

Histamine H3 receptor activation reduces the impairment in prepulse inhibition (PPI) of the acoustic startle response and Akt phosphorylation induced by MK-801 (dizocilpine), antagonist at N-Methyl-d-Aspartate (NMDA) receptors.

We have investigated the effect of the local activation of histamine H3 receptors (H3Rs) in the rat prefrontal cortex (PFCx) on the impairment of pre-pulse inhibition (PPI) of the startle response induced by the systemic administration of MK-801, antagonist at glutamate N-Methyl-d-Aspartate (NMDA) receptors, and the possible functional interaction between H3Rs and MK-801 on PFCx dopaminergic transmission. Infusion of the H3R agonist RAMH (19.8 ng/1 µl) into the PFCx reduced or prevented the inhibition by MK-801 (0.15 mg/kg, ip) of PPI evoked by different auditory stimulus intensities (5, 10 and 15 dB), and the RAMH effect was blocked by the H3R antagonist/inverse agonist ciproxifan (30.6 ng/1 µl). MK-801 inhibited [3H]-dopamine uptake (-45.4 ±â€¯2.1%) and release (-32.8 ±â€¯2.6%) in PFCx synaptosomes or slices, respectively, and molecular modeling indicated that MK-801 binds to and blocks the rat and human dopamine transporters. However, H3R activation had no effect on the inhibitory action of MK-801 on dopamine uptake and release. In PFCx slices, MK-801 and the activation of H3Rs or dopamine D1 receptors (D1Rs) stimulated ERK-1/2 and Akt phosphorylation. The co-activation of D1Rs and H3Rs prevented ERK-1/2 and Akt phosphorylation, and H3R activation or D1R blockade prevented the effect of MK-801. In ex vivo experiments, the intracortical infusion of the D1R agonist SKF-81297 (37 ng/1 µl) or the H3R agonist RAMH increased Akt phosphorylation, prevented by D1R/H3R co-activation. These results indicate that MK-801 enhances dopaminergic transmission in the PFCx, and that H3R activation counteracts the post-synaptic actions of dopamine.

Monoamines and their Derivatives on GPCRs: Potential Therapy for Alzheimer's Disease.

Albeit cholinergic depletion remains the key event in Alzheimer's Disease (AD), recent information describes stronger links between monoamines (trace amines, catecholamines, histamine, serotonin, and melatonin) and AD than those known in the past century. Therefore, new drug design strategies focus efforts to translate the scope on these topics and to offer new drugs which can be applied as therapeutic tools in AD. In the present work, we reviewed the state-of-art regarding genetic, neuropathology and neurochemistry of AD involving monoamine systems. Then, we compiled the effects of monoamines found in the brain of mammals as well as the reported effects of their derivatives and some structure-activity relationships. Recent derivatives have triggered exciting effects and pharmacokinetic properties in both murine models and humans. In some cases, the mechanism of action is clear, essentially through the interaction on G-protein-coupled receptors as revised in this manuscript. Additional mechanisms are inhibition of enzymes for their biotransformation, regulation of free-radicals in the central nervous system and others for the effects on Tau phosphorylation or amyloid-beta accumulation. All these data make the monoamines and their derivatives attractive potential elements for AD therapy.

Clobenpropit, a histamine H3 receptor antagonist/inverse agonist, inhibits [3H]-dopamine uptake by human neuroblastoma SH-SY5Y cells and rat brain synaptosomes.

BACKGROUND: Clobenpropit, a potent antagonist/inverse agonist at the histamine H3 receptor (H3R), reduced the cytotoxic action of 6-hydroxydopamine (6-OHDA) in neuroblastoma SH-SY5Y cells transfected with the human H3R. We therefore set out to study whether this effect involved a receptor-independent action on dopamine transport. METHODS: The uptake of [3H]-dopamine was assayed in SH-SY5Y cells and rat striatal or cerebro-cortical isolated nerve terminals (synaptosomes). Clobenpropit binding to the human norepinephrine (NET) and dopamine (DAT) transporters was analyzed by molecular modeling. RESULTS: In SH-SY5Y cells, [3H]-dopamine uptake was inhibited by desipramine (selective NET inhibitor), GBR-12909 (selective DAT inhibitor), and fluoxetine (selective inhibitor of the serotonin transporter, SERT) with IC50 values 37, 537, and 2800nM, respectively. The potency rank order indicates that [3H]-dopamine uptake is primarily performed by NET. Clobenpropit inhibited [3H]-dopamine uptake (maximum inhibition 82.7±2.8%, IC50 490nM), and the effect was reproduced by the H3R antagonist/inverse agonist iodophenpropit, but not by the agonists R-α-methylhistamine and immepip or the antagonists/inverse agonists ciproxifan and A-331440. Clobenpropit also inhibited [3H]-dopamine uptake by rat striatal and cerebro-cortical synaptosomes (-54.6±11.3% and -46.3±9.6%, respectively, at 10µM). Modeling of the human NET and DAT obtained by homology from the crystal of Drosophila melanogaster DAT showed that clobenpropit can bind to a site also recognized in both transporters by nisoxetine, a potent NET inhibitor. CONCLUSION: These data indicate a direct inhibitory effect of clobenpropit on catecholamine transport.

Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum.

In the striatum, histamine H3 receptors (H3Rs) are co-expressed with adenosine A2A receptors (A2ARs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H3Rs and A2ARs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A2AR302-Gαqi4 and wild-type H3Rs in transfected HEK-293T cells. H3R activation with the agonist RAMH resulted in Ca2+ mobilization (pEC50 7.31 ±â€¯0.23; maximal stimulation, Emax 449 ±â€¯25% of basal) indicative of receptor heterodimerization. Functional H3R-A2AR heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H3R activation decreased A2AR affinity for the agonist CGS-21680 (pKi values 8.10 ±â€¯0.04 and 7.70 ±â€¯0.04). Moreover, H3Rs and A2ARs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H3R-A2AR heteromer with possible physiological implications for the modulation of the intra-striatal transmission.

The Histamine H3 Receptor: Structure, Pharmacology, and Function.

Among the four G protein-coupled receptors (H1-H4) identified as mediators of the biologic effects of histamine, the H3 receptor (H3R) is distinguished for its almost exclusive expression in the nervous system and the large variety of isoforms generated by alternative splicing of the corresponding mRNA. Additionally, it exhibits dual functionality as autoreceptor and heteroreceptor, and this enables H3Rs to modulate the histaminergic and other neurotransmitter systems. The cloning of the H3R cDNA in 1999 by Lovenberg et al. allowed for detailed studies of its molecular aspects. In this work, we review the characteristics of the H3R, namely, its structure, constitutive activity, isoforms, signal transduction pathways, regional differences in expression and localization, selective agonists, antagonists and inverse agonists, dimerization with other neurotransmitter receptors, and the main presynaptic and postsynaptic effects resulting from its activation. The H3R has attracted interest as a potential drug target for the treatment of several important neurologic and psychiatric disorders, such as Alzheimer and Parkinson diseases, Gilles de la Tourette syndrome, and addiction.

Histamine H3 receptor activation counteracts adenosine A2A receptor-mediated enhancement of depolarization-evoked [3H]-GABA release from rat globus pallidus synaptosomes.

High levels of histamine H3 receptors (H3Rs) are found in the globus pallidus (GP), a neuronal nucleus in the basal ganglia involved in the control of motor behavior. By using rat GP isolated nerve terminals (synaptosomes), we studied whether H3R activation modified the previously reported enhancing action of adenosine A2A receptor (A2AR) stimulation on depolarization-evoked [(3)H]-GABA release. At 3 and 10 nM, the A2AR agonist CGS-21680 enhanced [(3)H]-GABA release induced by high K(+) (20 mM) and the effect of 3 nM CGS-21680 was prevented by the A2AR antagonist ZM-241385 (100 nM). The presence of presynaptic H3Rs was confirmed by the specific binding of N-α-[methyl-(3)H]-histamine to membranes from GP synaptosomes (maximum binding, Bmax, 1327 ± 79 fmol/mg protein; dissociation constant, Kd, 0.74 nM), which was inhibited by the H3R ligands immepip, clobenpropit, and A-331440 (inhibition constants, Ki, 0.28, 8.53, and 316 nM, respectively). Perfusion of synaptosomes with the H3R agonist immepip (100 nM) had no effect on K(+)-evoked [(3)H]-GABA release, but inhibited the stimulatory action of A2AR activation. In turn, the effect of immepip was blocked by the H3R antagonist clobenpropit, which had no significant effect of its own on K(+)-induced [(3)H]-GABA release. These data indicate that H3R activation selectively counteracts the facilitatory action of A2AR stimulation on GABA release from striato-pallidal projections.