Magda S. Santos

The Ancestry of Brazilian mtDNA Lineages

We have analyzed 247 Brazilian mtDNAs for hypervariable segment (HVS)-I and selected restriction fragment-length-polymorphism sites, to assess their ancestry in different continents. The total sample showed nearly equal amounts of Native American, African, and European matrilineal genetic contribution but with regional differences within Brazil. The mtDNA pool of present-day Brazilians clearly reflects the imprints of the early Portuguese colonization process (involving directional mating), as well as the recent immigrant waves (from Europe) of the last century. The subset of 99 mtDNAs from the southeastern region encompasses nearly all mtDNA haplogroups observed in the total Brazilian sample; for this regional subset, HVS-II was analyzed, providing, in particular, some novel details of the African mtDNA phylogeny.

Neuro-transmitters in the central nervous system & their implication in learning and memory processes.

This review article gives an overview of a number of central neuro-transmitters, which are essential for integrating many functions in the central nervous system (CNS), such as learning, memory, sleep cycle, body movement, hormone regulation and many others. Neurons use neuro-transmitters to communicate, and a great variety of molecules are known to fit the criteria to be classified as such. A process shared by all neuro-transmitters is their release by excocytosis, and we give an outline of the molecular events and protein complexes involved in this mechanism. Synthesis, transport, inactivation, and cellular signaling can be very diverse when different neuro-transmitters are compared, and these processes are described separately for each neuro-transmitter system. Here we focus on the most well known neuro-transmitters: acetyl-choline, catechol-amines (dopamine and nor-adrenalin), indole-amine (serotonin), glutamate, and gamma-amino-butyric acid (GABA). Glutamate is the major excitatory neuro-transmitter in the brain and its actions are counter-balanced by GABA, which is the major inhibitory substance in the CNS. A balance of neuronal transmission between these two neuro-transmitters is essential to normal brain function. Acetyl-choline, serotonin and catechol-amines have a more modulatory function in the brain, being involved in many neuronal circuits. Apart from summarizing the current knowledge about the synthesis, release and receptor signaling of these transmitters, some disease states due to alteration of their normal neuro-transmission are also described.

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex.

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 µ 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.

Novel strains of mice deficient for the vesicular acetylcholine transporter: insights on transcriptional regulation and control of locomotor behavior.

Defining the contribution of acetylcholine to specific behaviors has been challenging, mainly because of the difficulty in generating suitable animal models of cholinergic dysfunction. We have recently shown that, by targeting the vesicular acetylcholine transporter (VAChT) gene, it is possible to generate genetically modified mice with cholinergic deficiency. Here we describe novel VAChT mutant lines. VAChT gene is embedded within the first intron of the choline acetyltransferase (ChAT) gene, which provides a unique arrangement and regulation for these two genes. We generated a VAChT allele that is flanked by loxP sequences and carries the resistance cassette placed in a ChAT intronic region (FloxNeo allele). We show that mice with the FloxNeo allele exhibit differential VAChT expression in distinct neuronal populations. These mice show relatively intact VAChT expression in somatomotor cholinergic neurons, but pronounced decrease in other cholinergic neurons in the brain. VAChT mutant mice present preserved neuromuscular function, but altered brain cholinergic function and are hyperactive. Genetic removal of the resistance cassette rescues VAChT expression and the hyperactivity phenotype. These results suggest that release of ACh in the brain is normally required to “turn down” neuronal circuits controlling locomotion.

Trafficking of green fluorescent protein tagged-vesicular acetylcholine transporter to varicosities in a cholinergic cell line: VAChT traffic in living cells

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.

Structural requirements for steady-state localization of the vesicular acetylcholine transporter

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.

Visualization and trafficking of the vesicular acetylcholine transporter in living cholinergic cells

Abstract: 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.

Neuroinflammation and Neurodegeneration: Pinpointing Pathological and Pharmacological Targets

For many years, the brain had been regarded as an immune-privileged organ because of an old tenet which stated that no classical immune activation or inflammation could take place intrathecally. However, this theory has quickly changed with the advent of several studies demonstrating that the central nervous system (CNS) is in fact immunologically specialized [1, 2]. Neuroinflammation has been viewed as a double-edged sword: it not only is essential for the recovery from a number of conditions, but also may play detrimental roles in neurodegenerative processes. In such disorders inflammation can be set off by versatile triggers: protein aggregates, mediators released from injured neurons, accumulation of abnormally modified cellular components, and suppression of mechanisms that would normally control inflammatory processes, just to mention a few [3]. Given the increased life-expectancy, the incidence of neurodegenerative diseases is steadily rising. In light of this, research into this large segment of neuropsychiatry is a top priority around the globe, and one of the main areas of focus is to understand neuroinflammation that underlies, at least in part, the most common degenerative conditions of the brain: Alzheimers dementia, Parkinsons disease, amyotrophic lateral sclerosis, Huntingtons chorea, and many others [4–6]. By addressing intrathecal inflammation, some of these disorders could be prevented or even successfully treated. This special issue compiles original articles and reviews dissecting various pharmacological targets of inflammation that may serve as a springboard for opening innovative therapeutic avenues and could be germane to advanced research in neurodegenerative disorders. Antonio Carlos Pinheiro de Oliveira Eduardo Candelario-Jalil Bernd L. Fiebich Magda da Silva Santos Andras Palotas Helton Jose dos Reis

Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells

We regret that we must retract the article Visualization of the Vesicular Acetylcholine Transporter in Living Cholinergic Cells by M. S. Santos, J. Barbosa Jr., C. Kushmerick, M. V. Gomez, V. F. Prado, and M. A. M. Prado (J. Neurochem. 74, 2425‐2435, 2000). We have made an unintentional mistake in the construction of the enhanced green fluorescent protein (EGFP) vectors, and consequently the vesicular acetylcholine transporter (VAChT) and its truncated forms are incorrectly expressed. We have repeated the key experiments with proper constructs and have found that the expression pattern is clearly different from that reported in the article. The truncated form of VAChT, without the C‐terminal tail, presents a distinct pattern of expression when compared to VAChT, and we have found no evidence that the C‐terminal tail of VAChT is able to drive EGFP to varicosity sites. As a consequence of this problem, our earlier conclusions were incorrect. We apologize for this mistake and for any problems that we may have caused.