Marcus V. Gomez

The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.

There are three isoforms of the inositol 1,4,5- trisphosphate receptor (InsP3R), each of which has a distinct effect on Ca2+ signaling. However, it is not known whether each isoform similarly plays a distinct role in the activation of Ca2+-mediated events. To investigate this question, we examined the effects of each InsP3R isoform on transmission of Ca2+ signals to mitochondria and induction of apoptosis. Each isoform was selectively silenced using isoform-specific small interfering RNA in Chinese hamster ovary cells, which express all three InsP3R isoforms. ATP-induced cytosolic Ca2+ signaling patterns were altered, regardless of which isoform was silenced, but in a different fashion depending on the isoform. ATP also induced Ca2+ signals in mitochondria, which were inhibited more effectively by silencing the type III InsP3R than by silencing either the type I or type II isoform. The type III isoform also co-localized most strongly with mitochondria. When apoptosis was induced by activation of either the extrinsic or intrinsic apoptotic pathway, induction was reduced most effectively by silencing the type III InsP3R. These findings provide evidence that the type III isoform of the InsP3R plays a special role in induction of apoptosis by preferentially transmitting Ca2+ signals into mitochondria.

Mice Deficient for the Vesicular Acetylcholine Transporter Are Myasthenic and Have Deficits in Object and Social Recognition

An important step for cholinergic transmission involves the vesicular storage of acetylcholine (ACh), a process mediated by the vesicular acetylcholine transporter (VAChT). In order to understand the physiological roles of the VAChT, we developed a genetically altered strain of mice with reduced expression of this transporter. Heterozygous and homozygous VAChT knockdown mice have a 45% and 65% decrease in VAChT protein expression, respectively. VAChT deficiency alters synaptic vesicle filling and affects ACh release. Whereas VAChT homozygous mutant mice demonstrate major neuromuscular deficits, VAChT heterozygous mice appear normal in that respect and could be used for analysis of central cholinergic function. Behavioral analyses revealed that aversive learning and memory are not altered in mutant mice; however, performance in cognitive tasks involving object and social recognition is severely impaired. These observations suggest a critical role of VAChT in the regulation of ACh release and physiological functions in the peripheral and central nervous system.

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrPc, a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrPc is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrPc, leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrPc trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrPcaccumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrPc but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrPc internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrPc is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrPcis delivered to classic endosomes after internalization.

Phoneutria nigriventer venom: a cocktail of toxins that affect ion channels.

Abstract1. We review the pharmacological actions of toxins present in the venom of the aggressive spider Phoneutria nigriventer.2. This venom is rich in toxins that affect ion channels and neurotransmitter release. Voltage-gated sodium, calcium, and potassium channels have been described as the main targets of these toxins.3. In addition to these classical actions Phoneutria toxins have also been shown to affect glutamate transporter.4. It is expected that molecular genetics in addition to biochemical, biophysical and pharmacological approaches will help to further define Phoneutria toxins and their mechanisms of action in the near future.

c-Met Must Translocate to the Nucleus to Initiate Calcium Signals

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca2+ signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin β1, and formation of Ca2+ signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.

Regulation of acetylcholine synthesis and storage

Acetylcholine is one of the major modulators of brain functions and it is the main neurotransmitter at the peripheral nervous system. Modulation of acetylcholine release is crucial for nervous system function. Moreover, dysfunction of cholinergic transmission has been linked to a number of pathological conditions. In this manuscript, we review the cellular mechanisms involved with regulation of acetylcholine synthesis and storage. We focus on how phosphorylation of key cholinergic proteins can participate in the physiological regulation of cholinergic nerve-endings.

EFFECT OF SCORPION VENOM, TITYUSTOXIN, ON THE RELEASE OF ACETYLCHOLINE FROM INCUBATED SLICES OF RAT BRAIN

Abstract— Purified tityustoxin (TsTX) from the venom of the scorpion, Tityus serrulatus, when incubated in vitro with slices of rat cerebral cortex, increased the amount of free ace‐tylcholine (ACh) in the incubation medium and, simultaneously, reduced the amount of bound ACh in the slices. The effect was optimal at pH 7.4 and was dependent upon time of incubation, an energy source and the concentration of toxin. Tityustoxin increased the synthesis of ACh, but this effect seemed to be related to an increase in the release of ACh. The effect of the TsTX was independent of the concentration of K+ ion but was dependent on the presence of Na+ and Ca2+ in the incubation medium. Hexamethonium and hemicholinium reduced the effect of tityustoxin, but cocaine had no effect on the release of ACh stimulated by the TsTX. Tetrodotoxin blocked completely the stimulation caused by the tityustoxin. We suggest that probably both tityustoxin and tetrodotoxin exert different and antagonistic effects competing in the Na+ channels.

The hemicholinium-3 sensitive high affinity choline transporter is internalized by clathrin-mediated endocytosis and is present in endosomes and synaptic vesicles.

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.

Endocytosis of Prion Protein Is Required for ERK1/2 Signaling Induced by Stress-Inducible Protein 1

The secreted cochaperone STI1 triggers activation of protein kinase A (PKA) and ERK1/2 signaling by interacting with the cellular prion (PrPC) at the cell surface, resulting in neuroprotection and increased neuritogenesis. Here, we investigated whether STI1 triggers PrPC trafficking and tested whether this process controls PrPC-dependent signaling. We found that STI1, but not a STI1 mutant unable to bind PrPC, induced PrPC endocytosis. STI1-induced signaling did not occur in cells devoid of endogenous PrPC; however, heterologous expression of PrPC reconstituted both PKA and ERK1/2 activation. In contrast, a PrPC mutant lacking endocytic activity was unable to promote ERK1/2 activation induced by STI1, whereas it reconstituted PKA activity in the same condition, suggesting a key role of endocytosis in the former process. The activation of ERK1/2 by STI1 was transient and appeared to depend on the interaction of the two proteins at the cell surface or shortly after internalization. Moreover, inhibition of dynamin activity by expression of a dominant-negative mutant caused the accumulation and colocalization of these proteins at the plasma membrane, suggesting that both proteins use a dynamin-dependent internalization pathway. These results show that PrPC endocytosis is a necessary step to modulate STI1-dependent ERK1/2 signaling involved in neuritogenesis.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

ABSTRACT The vesicular acetylcholine (ACh) transporter (VAChT) mediates ACh storage by synaptic vesicles. However, the VAChT-independent release of ACh is believed to be important during development. Here we generated VAChT knockout mice and tested the physiological relevance of the VAChT-independent release of ACh. Homozygous VAChT knockout mice died shortly after birth, indicating that VAChT-mediated storage of ACh is essential for life. Indeed, synaptosomes obtained from brains of homozygous knockouts were incapable of releasing ACh in response to depolarization. Surprisingly, electrophysiological recordings at the skeletal-neuromuscular junction show that VAChT knockout mice present spontaneous miniature end-plate potentials with reduced amplitude and frequency, which are likely the result of a passive transport of ACh into synaptic vesicles. Interestingly, VAChT knockouts exhibit substantial increases in amounts of choline acetyltransferase, high-affinity choline transporter, and ACh. However, the development of the neuromuscular junction in these mice is severely affected. Mutant VAChT mice show increases in motoneuron and nerve terminal numbers. End plates are large, nerves exhibit abnormal sprouting, and muscle is necrotic. The abnormalities are similar to those of mice that cannot synthesize ACh due to a lack of choline acetyltransferase. Our results indicate that VAChT is essential to the normal development of motor neurons and the release of ACh.