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1.
PLoS Genet ; 17(12): e1009938, 2021 12.
Article in English | MEDLINE | ID: mdl-34914708

ABSTRACT

Choline is an essential component of Acetylcholine (ACh) biosynthesis pathway which requires high-affinity Choline transporter (ChT) for its uptake into the presynaptic terminals of cholinergic neurons. Previously, we had reported a predominant expression of ChT in memory processing and storing region of the Drosophila brain called mushroom bodies (MBs). It is unknown how ChT contributes to the functional principles of MB operation. Here, we demonstrate the role of ChT in Habituation, a non-associative form of learning. Odour driven habituation traces are laid down in ChT dependent manner in antennal lobes (AL), projection neurons (PNs), and MBs. We observed that reduced habituation due to knock-down of ChT in MBs causes hypersensitivity towards odour, suggesting that ChT also regulates incoming stimulus suppression. Importantly, we show for the first time that ChT is not unique to cholinergic neurons but is also required in inhibitory GABAergic neurons to drive habituation behaviour. Our results support a model in which ChT regulates both habituation and incoming stimuli through multiple circuit loci via an interplay between excitatory and inhibitory neurons. Strikingly, the lack of ChT in MBs shows characteristics similar to the major reported features of Autism spectrum disorders (ASD), including attenuated habituation, sensory hypersensitivity as well as defective GABAergic signalling. Our data establish the role of ChT in habituation and suggest that its dysfunction may contribute to neuropsychiatric disorders like ASD.


Subject(s)
Acetylcholine/genetics , Membrane Transport Proteins/genetics , Mushroom Bodies/metabolism , Olfactory Bulb/metabolism , Smell/genetics , Acetylcholine/metabolism , Animals , Autism Spectrum Disorder/genetics , Autism Spectrum Disorder/metabolism , Cholinergic Neurons/metabolism , Cholinergic Neurons/physiology , Drosophila melanogaster/genetics , Drosophila melanogaster/physiology , GABAergic Neurons/metabolism , Larva/genetics , Larva/physiology , Learning , Memory/physiology , Mushroom Bodies/physiology , Odorants/analysis , Presynaptic Terminals/metabolism , Signal Transduction/genetics , Smell/physiology
2.
Dev Biol ; 446(1): 80-93, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30529058

ABSTRACT

Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/ß core (α/ßc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/ßc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Membrane Transport Proteins/metabolism , Mushroom Bodies/metabolism , Neuromuscular Junction/metabolism , Neurons/metabolism , Animals , Animals, Genetically Modified , Cholinergic Neurons/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Gene Expression Regulation, Developmental , Larva/genetics , Larva/growth & development , Larva/metabolism , Membrane Transport Proteins/genetics , Mushroom Bodies/cytology , Mushroom Bodies/growth & development , Neuromuscular Junction/genetics , Neuromuscular Junction/growth & development , Pupa/genetics , Pupa/growth & development , Pupa/metabolism , RNA Interference , Synaptic Transmission/genetics , Synaptic Transmission/physiology
3.
Traffic ; 13(7): 979-91, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22486887

ABSTRACT

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.


Subject(s)
Axonal Transport/physiology , Choline O-Acetyltransferase/metabolism , Drosophila Proteins/metabolism , Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Animals , Animals, Genetically Modified , Axonal Transport/genetics , Carrier Proteins/metabolism , Cholinergic Neurons/enzymology , Cholinergic Neurons/metabolism , Drosophila/enzymology , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/chemistry , Fluorescence Recovery After Photobleaching , Kinesins/chemistry , Larva/enzymology , Larva/metabolism , Microtubule-Associated Proteins/chemistry , Protein Interaction Domains and Motifs , Synapses/enzymology , Synapses/metabolism
4.
J Neurosci ; 30(26): 8974-83, 2010 Jun 30.
Article in English | MEDLINE | ID: mdl-20592218

ABSTRACT

The two proteases beta-secretase and gamma-secretase generate the amyloid beta peptide and are drug targets for Alzheimer's disease. Here we tested the possibility of targeting the cellular environment of beta-secretase cleavage instead of the beta-secretase enzyme itself. beta-Secretase has an acidic pH optimum and cleaves the amyloid precursor protein in the acidic endosomes. We identified two drugs, bepridil and amiodarone, that are weak bases and are in clinical use as calcium antagonists. Independently of their calcium-blocking activity, both compounds mildly raised the membrane-proximal, endosomal pH and inhibited beta-secretase cleavage at therapeutically achievable concentrations in cultured cells, in primary neurons, and in vivo in guinea pigs. This shows that an alkalinization of the cellular environment could be a novel therapeutic strategy to inhibit beta-secretase. Surprisingly, bepridil and amiodarone also modulated gamma-secretase cleavage independently of endosomal alkalinization. Thus, both compounds act as dual modulators that simultaneously target beta- and gamma-secretase through distinct molecular mechanisms. In addition to Alzheimer's disease, compounds with dual properties may also be useful for drug development targeting other membrane proteins.


Subject(s)
Amiodarone/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Bepridil/pharmacology , Enzyme Inhibitors/pharmacology , Alzheimer Disease/drug therapy , Alzheimer Disease/enzymology , Amiodarone/chemistry , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/blood , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Bepridil/chemistry , Brain/drug effects , Brain/enzymology , Brain/metabolism , Cell Line , Cells, Cultured , Enzyme Inhibitors/chemistry , Female , Guinea Pigs , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Mice , Mice, Transgenic , Neurons/drug effects , Neurons/enzymology , Neurons/metabolism , Protease Nexins , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism
5.
J Neurochem ; 102(4): 1264-75, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17763532

ABSTRACT

Consecutive cleavages of amyloid precursor protein (APP) generate APP intracellular domain (AICD). Its cellular function is still unclear. In this study, we investigated the functional role of AICD in cellular Ca(2+) homeostasis. We could confirm previous observations that endoplasmic reticulum Ca(2+) stores contain less calcium in cells with reduced APP gamma-secretase cleavage products, increased AICD degradation, reduced AICD expression or in cells lacking APP. In addition, we observed an enhanced resting cytosolic calcium concentration under conditions where AICD is decreased or missing. In view of the reciprocal effects of Ca(2+) on mitochondria and of mitochondria on Ca(2+) homeostasis, we analysed further the cellular ATP content and the mitochondrial membrane potential. We observed a reduced ATP content and a mitochondrial hyperpolarisation in cells with reduced amounts of AICD. Blockade of mitochondrial oxidative phosphorylation chain in control cells lead to similar alterations as in cells lacking AICD. On the other hand, substrates of Complex II rescued the alteration in Ca(2+) homeostasis in cells lacking AICD. Based on these observations, our findings indicate that alterations observed in endoplasmic reticulum Ca(2+) storage in cells with reduced amounts of AICD are reciprocally linked to mitochondrial bioenergetic function.


Subject(s)
Adenosine Triphosphate/metabolism , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Calcium/metabolism , Homeostasis/physiology , Amyloid beta-Protein Precursor/deficiency , Analysis of Variance , Animals , Animals, Newborn , Cells, Cultured , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Homeostasis/drug effects , Homeostasis/genetics , Humans , Indoles/pharmacology , Membrane Potential, Mitochondrial/physiology , Mice , Mice, Knockout , Mutation/physiology , Protein Structure, Tertiary/physiology , Time Factors , Triglycerides/pharmacology , gamma-Aminobutyric Acid/analogs & derivatives , gamma-Aminobutyric Acid/pharmacology
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