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Significant evidence suggests that misfolded alpha-synuclein (aSyn), a major component of Lewy bodies, propagates in a prion-like manner contributing to disease progression in Parkinson's disease (PD) and other synucleinopathies. In fact, timed inoculation of M83 hemizygous mice with recombinant human aSyn preformed fibrils (PFF) has shown symptomatic deficits after substantial spreading of pathogenic alpha-synuclein, as detected by markers for the phosphorylation of S129 of aSyn. However, whether accumulated toxicity impact human-relevant cognitive and structural neuroanatomical measures is not fully understood. Here we performed a single unilateral striatal PFF injection in M83 hemizygous mice, and using two assays with translational potential, ex vivo magnetic resonance imaging (MRI) and touchscreen testing, we examined the combined neuroanatomical and behavioral impact of aSyn propagation. In PFF-injected mice, we observed widespread atrophy in bilateral regions that project to or receive input from the injection site using MRI. We also identified early deficits in reversal learning prior to the emergence of motor symptoms. Our findings highlight a network of regions with related cellular correlates of pathology that follow the progression of aSyn spreading, and that affect brain areas relevant for reversal learning. Our experiments suggest that M83 hemizygous mice injected with human PFF provides a model to understand how misfolded aSyn affects human-relevant pre-clinical measures and suggest that these pre-clinical biomarkers could be used to detect early toxicity of aSyn and provide better translational measures between mice and human disease., (© 2023 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.)

Chaperone networks are dysregulated with aging, but whether compromised Hsp70/Hsp90 chaperone function disturbs neuronal resilience is unknown. Stress-inducible phosphoprotein 1 (STI1; STIP1; HOP) is a co-chaperone that simultaneously interacts with Hsp70 and Hsp90, but whose function in vivo remains poorly understood. We combined in-depth analysis of chaperone genes in human datasets, analysis of a neuronal cell line lacking STI1 and of a mouse line with a hypomorphic Stip1 allele to investigate the requirement for STI1 in aging. Our experiments revealed that dysfunctional STI1 activity compromised Hsp70/Hsp90 chaperone network and neuronal resilience. The levels of a set of Hsp90 co-chaperones and client proteins were selectively affected by reduced levels of STI1, suggesting that their stability depends on functional Hsp70/Hsp90 machinery. Analysis of human databases revealed a subset of co-chaperones, including STI1, whose loss of function is incompatible with life in mammals, albeit they are not essential in yeast. Importantly, mice expressing a hypomorphic STI1 allele presented spontaneous age-dependent hippocampal neurodegeneration and reduced hippocampal volume, with consequent spatial memory deficit. We suggest that impaired STI1 function compromises Hsp70/Hsp90 chaperone activity in mammals and can by itself cause age-dependent hippocampal neurodegeneration in mice. Cover Image for this issue: doi: 10.1111/jnc.14749., (© 2019 International Society for Neurochemistry.)

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrP C ) and activating α7 nicotinic acetylcholine receptors (α7nAChR). Given that α7nAChR activation has also been involved in neuroprotection in stroke, it is possible that STI1 can have direct actions on neurons to prevent deleterious consequences of ischemic insults. Here, we tested this hypothesis by exposing primary neuronal cultures to 1-h oxygen-glucose deprivation (OGD) and reperfusion and assessing signaling pathways activated by STI1/PrP C . Our results demonstrated that STI1 treatment significantly decreased apoptosis and cell death in mouse neurons submitted to OGD in a manner that was dependent on PrP C and α7nAChR, but also on the activin A receptor 1 (ALK2), which has emerged as a signaling partner of STI1. Interestingly, pharmacological inhibition of the ALK2 receptor prevented neuroprotection by STI1, while activation of ALK2 receptors by bone morphogenetic protein 4 (BMP4) either before or after OGD was effective in decreasing neuronal death induced by ischemia. We conclude that PrP C /STI1 engagement and its subsequent downstream signaling cascades involving α7nAChR as well as the ALK2 receptor may be activated in neurons by increased levels of STI1. This signaling pathway protects neurons from ischemic insults., (© 2017 International Society for Neurochemistry.)

Striatal cholinergic interneurons (CIN) are pivotal for the regulation of the striatal network. Acetylcholine (ACh) released by CIN is centrally involved in reward behavior as well as locomotor or cognitive functions. Recently, BAC transgenic mice expressing channelrhodopsin-2 (ChR2) protein under the control of the choline acetyltransferase (ChAT) promoter (ChAT-ChR2) and displaying almost 50 extra copies of the VAChT gene were used to dissect cholinergic circuit connectivity and function using optogenetic approaches. These mice display over-expression of the vesicular acetylcholine transporter (VAChT) and increased cholinergic tone. Consequently, ChAT-ChR2 mice are a valuable model to investigate hypercholinergic phenotypes. Previous experiments established that ChAT-ChR2 mice display an increased sensitivity to amphetamine induced-locomotor activity and stereotypes. In the present report, we analyzed the impact of VAChT over-expression in the striatum of ChAT-ChR2 mice. ChAT-ChR2 mice displayed increased locomotor sensitization in response to low dose of cocaine. In addition, we observed a dramatic remodeling of the morphology of CIN in ChAT-ChR2 transgenic mice. VAChT immunolabeling was markedly enhanced in the soma and terminal of CIN from ChAT-ChR2 mice as previously shown (Crittenden et al. 2014). Interestingly, the number of cholinergic varicosities was markedly reduced (-87%) whereas their size was significantly increased (+177%). Moreover, VAChT over-expression dramatically modified its trafficking along the somatodendritic and axonal arbor. These findings demonstrate that ChAT-ChR2 mice present major alterations of CIN neuronal morphology and increased behavioral sensitization to cocaine, supporting the notion that the increased levels of VAChT observed in these mice make them fundamentally different from wild-type mice., (© 2017 International Society for Neurochemistry.)

It is well established that neurons secrete neuropeptides and ATP with classical neurotransmitters; however, certain neuronal populations are also capable of releasing two classical neurotransmitters by a process named co-transmission. Although there has been progress in our understanding of the molecular mechanism underlying co-transmission, the individual regulation of neurotransmitter secretion and the functional significance of this neuronal 'bilingualism' is still unknown. Striatal cholinergic interneurons (CINs) have been shown to secrete glutamate (Glu) in addition to acetylcholine (ACh) and are recognized for their role in the regulation of striatal circuits and behavior. Our review highlights the recent research into identifying mechanisms that regulate the secretion and function of Glu and ACh released by CINs and the roles these neurons play in regulating dopamine secretion and striatal activity. In particular, we focus on how the transporters for ACh (VAChT) and Glu (VGLUT3) influence the storage of neurotransmitters in CINs. We further discuss how these individual neurotransmitters regulate striatal computation and distinct aspects of behavior that are regulated by the striatum. We suggest that understanding the distinct and complementary functional roles of these two neurotransmitters may prove beneficial in the development of therapies for Parkinson's disease and addiction. Overall, understanding how Glu and ACh secreted by CINs impacts striatal activity may provide insight into how different populations of 'bilingual' neurons are able to develop sophisticated regulation of their targets by interacting with multiple receptors but also by regulating each other's vesicular storage. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms., (© 2017 International Society for Neurochemistry.)

Postural instability and gait disturbances, common disabilities in the elderly and frequently present in Parkinson's disease (PD), have been suggested to be related to dysfunctional cholinergic signaling in the brainstem. We investigated how long-term loss of cholinergic signaling from mesopontine nuclei influence motor behaviors. We selectively eliminated the vesicular acetylcholine transporter (VAChT) in pedunculopontine and laterodorsal tegmental nuclei cholinergic neurons to generate mice with selective mesopontine cholinergic deficiency (VAChT E n1-Cre-flox/flox ). VAChT E n1-Cre-flox/flox mice did not show any gross health or neuromuscular abnormality on metabolic cages, wire-hang and grip-force tests. Young VAChT E n1-Cre-flox/flox mice (2-5 months-old) presented motor learning/coordination deficits on the rotarod; moved slower, and had smaller steps on the catwalk, but showed no difference in locomotor activity on the open field. Old VAChT E n1-Creflox/flox mice (13-16 months-old) showed more pronounced motor learning/balance deficits on the rotarod, and more pronounced balance deficits on the catwalk. Furthermore, old mutants moved faster than controls, but with similar step length. Additionally, old VAChT-deficient mice were hyperactive. These results suggest that dysfunction of cholinergic neurons from mesopontine nuclei, which is commonly seen in PD, has causal roles in motor functions. Prevention of mesopontine cholinergic failure may help to prevent/improve postural instability and falls in PD patients. Read the Editorial Highlight for this article on page 688., (© 2016 International Society for Neurochemistry.)

Prion protein (PrP(C) ), a glycosylphosphatidylinositol-anchored protein corrupted in prion diseases, has been shown recently to interact with group I metabotropic glutamate receptors (mGluRs). Moreover, both PrP(C) and mGluRs were proposed to function as putative receptors for β-amyloid in Alzheimer's disease. PrP(C) can be processed in neurons via α or β-cleavage to produce PrP(C) fragments that are neuroprotective or toxic, respectively. We found PrP(C) α-cleavage to be 2-3 times higher in the cortex of APPswe/PS1dE9 mice, a mouse model of Alzheimer's disease. A similar age-dependent increase was observed for PrP(C) β-cleavage. Moreover, we observed considerable age-dependent increase in cortical expression of mGluR1, but not mGluR5. Exposure of cortical neuronal cultures to β-amyloid oligomers upregulated mGluR1 and PrP(C) α-cleavage, while activation of group I mGluRs increased PrP(C) shedding from the membrane, likely due to increased levels of a disintegrin and metalloprotease10, a key disintegrin for PrP(C) shedding. Interestingly, a similar increase in a disintegrin and metalloprotease10 was detected in the cortex of 9-month-old APPswe/PS1dE9 animals. Our experiments reveal novel and complex processing of PrP(C) in connection with mGluR overexpression that seems to be triggered by β-amyloid peptides. Prion protein (PrP(C) ) and metabotropic glutamate receptors (mGluR) are implicated in Alzheimer's disease (AD). We found age-dependent increase in PrP(C) processing, ADAM10 and mGluR1 levels in AD mouse model. These changes could be reproduced in cultured cortical neurons treated with Aβ peptide. Our findings suggest that increased levels of Aβ can trigger compensatory responses that may affect neuronal toxicity., (© 2013 International Society for Neurochemistry.)

Cholinergic neurons are known to regulate striatal circuits; however, striatal-dependent physiological outcomes influenced by acetylcholine (ACh) are still poorly under;?>stood. Here, we used vesicular acetylcholine transporter (VAChT)(D2-Cre-flox/flox) mice, in which we selectively ablated the vesicular acetylcholine transporter in the striatum to dissect the specific roles of striatal ACh in metabolic homeostasis. We report that VAChT(D) (2-Cre-flox/flox) mice are lean at a young age and maintain this lean phenotype with time. The reduced body weight observed in these mutant mice is not attributable to reduced food intake or to a decrease in growth rate. In addition, changed activity could not completely explain the lean phenotype, as only young VAChT(D) (2-Cre-flox/flox) mice showed increased physical activity. Interestingly, VAChT(D) (2-Cre-flox/flox) mice show several metabolic changes, including increased plasma levels of insulin and leptin. They also show increased periods of wakefulness when compared with littermate controls. Taken together, our data suggest that striatal ACh has an important role in the modulation of metabolism and highlight the importance of striatum cholinergic tone in the regulation of energy expenditure. These new insights on how cholinergic neurons influence homeostasis open new avenues for the search of drug targets to treat obesity., (© 2012 International Society for Neurochemistry.)

Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation. However, potential cross-talk between these protein complexes and their role in peripheral neurons has never been addressed. To explore this issue, we investigated PrP(C)-mediated axonogenesis in peripheral neurons in response to STI1 and laminin-γ1 chain-derived peptide (Ln-γ1). STI1 and Ln-γ1 promoted robust axonogenesis in wild-type neurons, whereas no effect was observed in neurons from PrP(C) -null mice. PrP(C) binding to Ln-γ1 or STI1 led to an increase in intracellular Ca(2+) levels via distinct mechanisms: STI1 promoted extracellular Ca(2+) influx, and Ln-γ1 released calcium from intracellular stores. Both effects depend on phospholipase C activation, which is modulated by mGluR1/5 for Ln-γ1, but depends on, C-type transient receptor potential (TRPC) channels rather than α7nAChR for STI1. Treatment of neurons with suboptimal concentrations of both ligands led to synergistic actions on PrP(C)-mediated calcium response and axonogenesis. This effect was likely mediated by simultaneous binding of the two ligands to PrP(C). These results suggest a role for PrP(C) as an organizer of diverse multiprotein complexes, triggering specific signaling pathways and promoting axonogenesis in the peripheral nervous system., (© 2012 International Society for Neurochemistry.)

In Alzheimer's disease, the amyloid-β peptide (Aβ) interacts with distinct proteins at the cell surface to interfere with synaptic communication. Recent data have implicated the prion protein (PrP(C)) as a putative receptor for Aβ. We show here that Aβ oligomers signal in cells in a PrP(C)-dependent manner, as might be expected if Aβ oligomers use PrP(C) as a receptor. Immunofluorescence, flow cytometry and cell surface protein biotinylation experiments indicated that treatment with Aβ oligomers, but not monomers, increased the localization of PrP(C) at the cell surface in cell lines. These results were reproduced in hippocampal neuronal cultures by labeling cell surface PrP(C). In order to understand possible mechanisms involved with this effect of Aβ oligomers, we used live cell confocal and total internal reflection microscopy in cell lines. Aβ oligomers inhibited the constitutive endocytosis of PrP(C), but we also found that after Aβ oligomer-treatment PrP(C) formed more clusters at the cell surface, suggesting the possibility of multiple effects of Aβ oligomers. Our experiments show for the first time that Aβ oligomers signal in a PrP(C)-dependent way and that they can affect PrP(C) trafficking, increasing its localization at the cell surface., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Mammalian motor nerve terminals contain hundreds of thousands of synaptic vesicles, but only a fraction of these vesicles is immediately available for release, the remainder forming a reserve pool. The supply of vesicles is replenished through endocytosis, and newly formed vesicles are refilled with acetylcholine through a process that depends on the vesicular acetylcholine transporter (VAChT). During expression of short-term plasticity, quantal release can be increased, but it is unknown whether this reflects enhanced recruitment of vesicles from the reserve pool or rapid recycling. We examined spontaneous and evoked release of acetylcholine at endplates from genetically modified VAChT KD(HOM) mice that express approximately 30% of the normal level of VAChT to determine steps rate-limited by synaptic vesicle filling. Quantal content and quantal size were reduced in VAChT KD(HOM) mice compared with wild-type controls. Although high-frequency stimulation did not reduce quantal size further, the post-tetanic increase in end-plate potential amplitude or MEPP frequency was significantly smaller in VAChT KD(HOM) mice. This was the case even when tetanic depression was eliminated using an extracellular solution containing reduced Ca(2+) and raised Mg(2+). These results reveal the dependence of short-term plasticity on the level of VAChT expression and efficient synaptic vesicle filling.

Maintenance of acetylcholine (ACh) synthesis depends on the activity of the high-affinity choline transporter (CHT1), which is responsible for the reuptake of choline from the synaptic cleft into presynaptic neurons. In this review, we discuss the current understanding of mechanisms involved in the cellular trafficking of CHT1. CHT1 protein is mainly found in intracellular organelles, such as endosomal compartments and synaptic vesicles. The presence of CHT1 at the plasma membrane is limited by rapid endocytosis of the transporter in clathrin-coated pits in a mechanism dependent on a dileucine-like motif present in the carboxyl-terminal region of the transporter. The intracellular pool of CHT1 appears to constitute a reserve pool of transporters, important for maintenance of cholinergic neurotransmission. However, the physiological basis of the presence of CHT1 in intracellular organelles is not fully understood. Current knowledge about CHT1 indicates that stimulated and constitutive exocytosis, in addition to endocytosis, will have major consequences for regulating choline uptake. Future investigations of CHT1 trafficking should elucidate such regulatory mechanisms, which may aid in understanding the pathophysiology of diseases that affect cholinergic neurons, such as Alzheimer's disease.

The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481-490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481-490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.

Maintenance of acetylcholine synthesis depends on the effective functioning of a high-affinity sodium-dependent choline transporter (CHT1). Recent studies have shown that this transporter is predominantly localized inside the cell, unlike other neurotransmitter transporters, suggesting that the trafficking of CHT1 to and from the plasma membrane may play a crucial role in regulating choline uptake. Here we found that CHT1 is rapidly and constitutively internalized in clathrin-coated vesicles to Rab5-positive early endosomes. CHT1 internalization is controlled by an atypical carboxyl-terminal dileucine-like motif (L531, V532) which, upon replacement by alanine residues, blocks CHT1 internalization in both human embryonic kidney 293 cells and primary cortical neurons and results in both increased CHT1 cell surface expression and choline transport activity. Perturbation of clathrin-mediated endocytosis with dynamin-I K44A increases cell surface expression and transport activity to a similar extent as mutating the dileucine motif, suggesting that we have identified the motif responsible for constitutive CHT1 internalization. Based on the observation that the localization of CHT1 to the plasma membrane is transient, we propose that acetylcholine synthesis may be influenced by processes that lead to the attenuation of constitutive CHT1 endocytosis.

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.

Synthesis of acetylcholine depends on the plasma membrane uptake of choline by a high affinity choline transporter (CHT1). Choline uptake is regulated by nerve impulses and trafficking of an intracellular pool of CHT1 to the plasma membrane may be important for this regulation. We have generated a hemagglutinin (HA) epitope tagged CHT1 to investigate the organelles involved with intracellular trafficking of this protein. Expression of CHT1-HA in HEK 293 cells establishes Na+-dependent, hemicholinium-3 sensitive high-affinity choline transport activity. Confocal microscopy reveals that CHT1-HA is found predominantly in intracellular organelles in three different cell lines. Importantly, CHT1-HA seems to be continuously cycling between the plasma membrane and endocytic organelles via a constitutive clathrin-mediated endocytic pathway. In a neuronal cell line, CHT1-HA colocalizes with the early endocytic marker green fluorescent protein (GFP)-Rab 5 and with two markers of synaptic-like vesicles, VAMP-myc and GFP-VAChT, suggesting that in cultured cells CHT1 is present mainly in organelles of endocytic origin. Subcellular fractionation and immunoisolation of organelles from rat brain indicate that CHT1 is present in synaptic vesicles. We propose that intracellular CHT1 can be recruited during stimulation to increase choline uptake in nerve terminals.

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.

Synaptic vesicle proteins are suggested to travel from the trans-Golgi network to active zones via tubulovesicular organelles, but the participation of different populations of endosomes in trafficking remains a matter of debate. Therefore, we generated a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) and studied the localization of VAChT in organelles in the cell body and varicosities of living cholinergic cells. GFP-VAChT is distributed to both early and recycling endosomes in the cell body and is also observed to accumulate in endocytic organelles within varicosities of SN56 cells. GFP-VAChT positive organelles in varicosities are localized close to plasma membrane and are labeled with FM4-64 and GFP-Rab5, markers of endocytic vesicles and early endosomes, respectively. A GFP-VAChT mutant lacking a dileucine endocytosis motif (leucine residues 485 and 486 changed to alanine residues) accumulated at the plasma membrane in SN56 cells. This endocytosis-defective GFP-VAChT mutant is localized primarily at the somal plasma membrane and exhibits reduced neuritic targeting. Furthermore, the VAChT mutant did not accumulate in varicosities, as did VAChT. Our data suggest that clathrin-mediated internalization of VAChT to endosomes at the cell body might be involved in proper sorting and trafficking of VAChT to varicosities. We conclude that genesis of competent cholinergic secretory vesicles depends on multiple interactions of VAChT with endocytic proteins.

The present experiments investigated the trafficking of the vesicular acetylcholine transporter (VAChT) tagged with the enhanced green fluorescent protein (EGFP) in living cholinergic cells (SN56). The EGFP-VAChT chimera was located in endosomal-like compartments in the soma of SN56 cells, and it was also targeted to varicosities of neurites. In contrast, EGFP alone in cells was soluble in the cytoplasm. The C-terminal cytoplasmic tail of VAChT has been implicated in targeting of VAChT to synaptic vesicles; thus, we have examined the role of the C-terminal region in the trafficking to varicosities. A C-terminal fragment tagged with EGFP appeared to be selectively accumulated in varicosities when expressed in SN56 cells. Interestingly, the protein was not freely soluble in the cytosol, and it presented a punctate pattern of expression. However, EGFP-C terminus did not present this peculiar pattern of expression in a nonneuronal cell line (HEK 293). Moreover, the C-terminal region of VAChT did not seem to be essential for VAChT trafficking, as a construct that lacks the C-terminal tail was, similar to EGFP-VAChT, partially targeted to endocytic organelles in the soma and sorted to varicosities. These experiments visualize VAChT for the first time in living cells and suggest that there might be multiple signals that participate in trafficking of VAChT to sites of synaptic vesicle accumulation.

The expression and localization of the vesicular acetylcholine transporter in a septal cell line, SN56, were investigated. Immunoprecipitation and immunoblot analysis of postnuclear supernatants indicated that this cell line expresses reasonable amounts of the transporter. Immunofluorescence and confocal microscopy experiments showed that the vesicular transporter is present in varicosities and also in the cell body of differentiated cells. Varicosities have the potential to be functional sites of transmitter release because they responded to depolarization with calcium influx through voltage-gated calcium channels and expressed the synaptic proteins synaptotagmin, SV2, synaptophysin, and a subunit of P/Q calcium channels. In the soma of SN56 cells, the transporter immunoreactivity was similar to that for synaptotagmin, and it colocalized with synaptophysin, but it did not colocalize with SV2. Labeling for SV2 appeared prominently in a defined perinuclear structure, whereas the two former proteins were widely distributed in the soma, where several endocytic compartments could be identified with the vital dye FM4-64. These data suggest that distinct synaptic vesicle proteins exist in different subcellular compartments, and consequently they may follow distinct pathways in neurites before reaching sites of transmitter storage and release in SN56 cells.

GH3 cells present spontaneous Ca2+ action potentials and oscillations of intracellular Ca2+, which can be modified by altering the activity of K+ or Ca2+ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3-1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3-1 increases the frequency of Ca2+ oscillations, as do two blockers of potassium channels, 4-aminopyridine and charybdotoxin. Whole-cell patch clamp experiments show that Tx3-1 reversibly inhibits the A-type K+ current (I(A)) but does not block other K+ currents (delayed-rectifying, inward-rectifying, and large-conductance Ca2+-sensitive) or Ca2+ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3-1, which shows that Tx3-1 has no homology to other known blockers of K+ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3-1 is a selective inhibitor of I(A), which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3-1, we suggest that I(A) is an important determinant of the frequency of Ca2+ oscillations in unstimulated GH3 cells.

The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3H]acetylcholine ([3H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by alpha-PMA, an inactive phorbol ester. PMA did not alter the release of [3H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.