Biomedical subjects

A I Levey

Publications and source records attributed to A I Levey.

At least 19 recordsLinked to original sources

PubMed2001

Muscarinic receptor M(2) in cat visual cortex: laminar distribution, relationship to gamma-aminobutyric acidergic neurons, and effect of cingulate lesions.

Acetylcholine can have diverse effects on visual cortical neurons as a result of variations in postsynaptic receptor subtypes as well as the types of neurons and subcellular sites targeted. This study examines the cellular basis for cholinergic activation in visual cortex via M(2) type muscarinic receptors in gamma-aminobutyric acid (GABA)-ergic and non-GABAergic cells, using immunocytochemical techniques. At light microscopic resolution, M(2) immunoreactivity (-ir) was seen in all layers except area and sublayer specific bands in layer 4. Subcellularly, M(2)-ir occurred in both dendrites and terminals that form symmetric and asymmetric junctions. Layers 5 and 6 were characterized by axosomatic contacts that displayed labeling in the presynaptic component, and layer 6 displayed perikaryal postsynaptic staining, suggesting that corticofugal output neurons may be modulated particularly strongly via M(2). Infragranular layers differed from the supragranular layers in that more labeled profiles were axonal than dendritic, indicating a dominant presynaptic effect by acetylcholine via M(2) there. Unilateral cingulate cortex cuts caused reduction of cholinergic and noradrenergic fibers in the lesioned hemisphere at light microscopic resolution; at electron microscopic resolution, the synapse density and axonal M(2) labeling were reduced, suggesting that M(2) was localized presynaptically on extrathalamic modulatory inputs. Dual labeling with GABA in visual cortex layer 5 showed that half of M(2)-labeled dendrites originated from GABAergic neurons. Given that only one-fifth of all cortical dendritic profiles are GABAergic, this prevalence of dual labeling indicates an enrichment of M(2) within GABAergic dendrites and, thus, implicates abundant postsynaptic action on GABAergic neurons via M(2). In contrast, only one-tenth of M(2)-labeled terminals originated from GABAergic neurons, suggesting that the presynaptic action of acetylcholine via M(2) receptors would be more selective for non-GABAergic terminals.

Animals↗

PubMed2001

Apolipoprotein E and age at onset of Alzheimer's disease in African American patients.

The authors examined whether the APOE-epsilon4 allele is associated with an earlier age at onset of AD in 71 African American patients with probable AD. The authors found a linear dose effect in which each copy of the epsilon4 allele was associated with a 3.6-year earlier onset of AD, indicating a dose-dependent relationship between APOE-epsilon4 and age at onset of AD in African Americans.

Age Factors↗

PubMed2001

Presenilin 1 independently regulates beta-catenin stability and transcriptional activity.

Presenilin 1 (PS1) regulates beta-catenin stability; however, published data regarding the direction of the effect are contradictory. We examined the effects of wild-type and mutant forms of PS1 on the membrane, cytoplasmic, nuclear, and signaling pools of endogenous and exogenous beta-catenin by immunofluorescence microscopy, subcellular fractionation, and in a transcription assay. We found that PS1 destabilizes the cytoplasmic and nuclear pools of beta-catenin when stabilized by Wnt or Dvl but not when stabilized at lower levels of the Wnt pathway. The PS1 mutants examined were less able to reduce the stability of beta-catenin. PS1 also inhibited the transcriptional activity of endogenous beta-catenin, and the PS1 mutants were again less inhibitory at the level of Dvl but showed a different pattern of inhibition toward transcription below Dvl. The transcriptional activity of exogenously expressed wild-type beta-catenin and two mutants, DeltaN89beta-catenin and DeltaSTbeta-catenin, were also inhibited by wild-type and mutant PS1. We conclude that PS1 negatively regulates the stability and transcriptional activity of beta-catenin at different levels in the Wnt pathway, that the effect on transcriptional activity appears to be independent of the GSK-3beta mediated degradation of beta-catenin, and that mutations in PS1 differentially affect the stability and transcriptional activity of beta-catenin.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Rab5-dependent trafficking of the m4 muscarinic acetylcholine receptor to the plasma membrane, early endosomes, and multivesicular bodies.

The m4 subtype of muscarinic acetylcholine receptor regulates many physiological processes and is a novel therapeutic target for neurologic and psychiatric disorders. However, little is known about m4 regulation because of the lack of pharmacologically selective ligands. A crucial component of G protein-coupled receptor regulation is intracellular trafficking. We thus used subtype-specific antibodies and quantitative immunocytochemistry to characterize the intracellular trafficking of m4. We show that following carbachol stimulation, m4 co-localizes with transferrin, and the selective marker of early endosomes, EEA1. In addition, m4 intracellular localization depends on Rab5 activity. The dominant negative Rab5S34N inhibits m4 endocytosis initially following carbachol stimulation, and reduces the size of m4 containing vesicles. The constitutively active Rab5Q79L enhances m4 intracellular distribution, even in unstimulated cells. Rab5Q79L also produces strikingly enlarged vacuoles, which by electron microscopy contain internal vesicles, suggesting that they are multivesicular bodies. m4 localizes both to the perimeter and interior of these vacuoles. In contrast, transferrin localizes only to the vacuole perimeter, demonstrating divergence of m4 trafficking from the pathway followed by constitutively endocytosed transferrin. We thus suggest a novel model by which multivesicular bodies sort G protein-coupled receptors from a transferrin-positive recycling pathway to a nonrecycling, possibly degradative pathway.

Animals↗

PubMed2001

Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein.

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.

Alanine↗

PubMed2001

Muscarinic m1 and m2 receptor proteins in local circuit and projection neurons of the primate striatum: anatomical evidence for cholinergic modulation of glutamatergic prefronto-striatal pathways.

The cellular and subcellular localization of muscarinic receptor proteins m1 and m2 was examined in the neostriatum of macaque monkeys by using light and electron microscopic immunocytochemical techniques. Double-labeling immunocytochemistry revealed m1 receptors in calbindin-D28k--positive medium spiny projection neurons. Muscarinic m1 labeling was dramatically more intense in the striatal matrix compartment in juvenile monkeys but more intense in striosomes in the adult caudate, suggesting that m1 expression undergoes a developmental age-dependent change. Ultrastructurally, m1 receptors were predominantly localized in asymmetric synapse-forming spines, indicating that these spines receive extrastriatal excitatory afferents. The association of m1-positive spines with lesion-induced degenerating prefronto-striatal axon terminals demonstrated that these afferents originate in part from the prefrontal cortex. The synaptic localization of m1 in these spines indicates a role of m1 in the modulation of excitatory neurotransmission. To a lesser extent, m1 was present in symmetric synapses, where it may also modulate inhibitory neurotransmission originating from local striatal neurons or the substantia nigra. Conversely, m2/choline acetyltransferase (ChAT) double labeling revealed that m2-positive neurons corresponded to large aspiny cholinergic interneurons and ultrastructurally, that the majority of m2 labeled axons formed symmetric synapses. The remarkable segregation of the m1 and m2 receptor proteins to projection and local circuit neurons suggests a functional segregation of m1 and m2 mediated cholinergic actions in the striatum: m1 receptors modulate extrinsic glutamatergic and monoaminergic afferents and intrinsic GABAergic afferents onto projection neurons, whereas m2 receptors regulate acetylcholine release from axons of cholinergic interneurons.

Acetylcholine↗

PubMed2001

Ionotropic and metabotropic GABA and glutamate receptors in primate basal ganglia.

The functions of glutamate and GABA in the CNS are mediated by ionotropic and metabotropic, G protein-coupled, receptors. Both receptor families are widely expressed in basal ganglia structures in primates and nonprimates. The recent development of highly specific antibodies and/or cDNA probes allowed the better characterization of the cellular localization of various GABA and glutamate receptor subtypes in the primate basal ganglia. Furthermore, the use of high resolution immunogold techniques at the electron microscopic level led to major breakthroughs in our understanding of the subsynaptic and subcellular localization of these receptors in primates. In this review, we will provide a detailed account of the current knowledge of the localization of these receptors in the basal ganglia of humans and monkeys.

Animals↗

PubMed2001

Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1.

PDZ domain-containing proteins play an important role in the targeting and localization of synaptic membrane proteins. Here, we report an interaction between the PDZ domain-containing protein PICK1 and monoamine neurotransmitter transporters in vitro and in vivo. In dopaminergic neurons, PICK1 colocalizes with the dopamine transporter (DAT) and forms a stable protein complex. Coexpression of PICK1 with DAT in mammalian cells and neurons in culture results in colocalization of the two proteins in a cluster pattern and an enhancement of DAT uptake activity through an increase in the number of plasma membrane DAT. Deletion of the PDZ binding site at the carboxyl terminus of DAT abolishes its association with PICK1 and impairs the localization of the transporter in neurons. These findings indicate a role for PDZ-mediated protein interactions in the localization, expression, and function of monoamine transporters.

Animals↗

PubMed2001

The regulation of presenilin-1 by nerve growth factor.

Presenilin-1 (PS1) protein concentration is linked to neuronal development and to the pathogenesis of Alzheimer's disease, yet little is known about the biological factors and mechanisms that control cellular levels of PS1 protein. As PS1 levels are highest in the developing brain, we tested whether neurotrophin-induced differentiation influences PS1 expression using neuronotypic pheochromocytoma (PC12) cells. Treatment of PC12 cells with nerve growth factor (NGF) caused approximately 60-75% increases in the steady-state levels of endogenous PS1 N- and C-terminal fragments. PS1 protein accumulation was dose-responsive to NGF and required the presence of the TrkA NGF receptor tyrosine kinase. NGF also induced PS1 fragment accumulation in cultured explants of rat dorsal root ganglia. Quantitative northern blot analysis using PC12 cultures indicated that NGF did not increase steady-state PS1 mRNA levels. However, pulse-chase experiments indicated that NGF slowed the degradation rate of endogenous PS1 fragments, increasing the half-life from t(1/2) @22.5 to @25.0 h. This increase in half-life was insufficient to account for the approximately 60-75% increase in PS1 fragment levels measured in NGF-treated cells. Thus, NGF may regulate PS1 protein concentration in NGF-responsive cells by a complex mechanism that increases PS1 fragment production independent of holoprotein synthesis.

Animals↗

PubMed2000

Functional downregulation of GluR2 in piriform cortex of kindled animals.

We have previously shown kindling-induced downregulation of the AMPA receptor GluR2 subunit in piriform cortex, as measured by Western blotting. In the present studies, we performed whole-cell patch clamp analysis of AMPA receptor-mediated currents from kindled and control animals to determine if the downregulation observed previously had any functional significance. These experiments were done in the absence and presence of N-hydroxyphenylpropanoyl spermine (HPPS), a polyamine that blocks currents through AMPA receptors lacking GluR2. We report that AMPA receptor-mediated currents recorded from piriform cortex layer II pyramidal cells in slices from animals kindled to 10 fully generalized seizures were blocked by HPPS. In contrast, application of HPPS had no effect on current amplitude in control animals, or in animals that had not been fully kindled. Western blotting revealed that decreases in GluR2 were seen in animals that had experienced at least one fully generalized seizure, but were not observed at earlier stages of kindling development. The increased polyamine sensitivity of AMPA receptor-mediated currents in kindled animals is consistent with the hypothesis that kindling induces formation of AMPA receptors that lack GluR2 in piriform cortex pyramidal cells. It has been demonstrated that polyamine sensitivity is directly correlated with the calcium permeability of the AMPA receptor, suggesting that kindling results in the formation of AMPA receptors that are calcium-permeable. Increases in intracellular calcium through these receptors could act as a second messenger and play a role in the initiation of long-term changes that contribute to the pathogenesis of kindling-induced epilepsy.

Animals↗

PubMed2000

Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus.

The subthalamic nucleus (STN) is a key nucleus in the basal ganglia motor circuit that provides the major glutamatergic excitatory input to the basal ganglia output nuclei. The STN plays an important role in normal motor function, as well as in pathological conditions such as Parkinson's disease (PD) and related disorders. Development of a complete understanding of the roles of the STN in motor control and the pathophysiological changes in STN that underlie PD will require a detailed understanding of the mechanisms involved in regulation of excitability of STN neurons. Here, we report that activation of group I metabotropic glutamate receptors (mGluRs) induces a direct excitation of STN neurons that is characterized by depolarization, increased firing frequency, and increased burst-firing activity. In addition, activation of group I mGluRs induces a selective potentiation of NMDA-evoked currents. Immunohistochemical studies at the light and electron microscopic levels indicate that both subtypes of group I mGluRs (mGluR1a and mGluR5) are localized postsynaptically in the dendrites of STN neurons. Interestingly, pharmacological studies suggest that each of the mGluR-mediated effects is attributable to activation of mGluR5, not mGluR1, despite the presence of both subtypes in STN neurons. These results suggest that mGluR5 may play an important role in the net excitatory drive to the STN from glutamatergic afferents. Furthermore, these studies raise the exciting possibility that selective ligands for mGluR5 may provide a novel approach for the treatment of a variety of movement disorders that involve changes in STN activity.

Animals↗

PubMed2000

Cortical inputs to m2-immunoreactive striatal interneurons in rat and monkey.

Previous anatomical studies have been unsuccessful in demonstrating significant cortical inputs to cholinergic and somatostatinergic striatal interneurons in rats. On the other hand, electrophysiological studies have shown that cortical stimulation induces monosynaptic EPSPs in cholinergic interneurons. It has been proposed that the negative anatomical findings might have been the result of incomplete labeling of distal dendrites. In the present study, we reinvestigated this issue using m2 muscarinic receptor antibodies as a selective marker for cholinergic and somatostatinergic interneurons in the striatum. This was combined with injections of either the anterograde tracer biotinylated dextran amine (BDA) in the monkey prefrontal cortex or aspiration lesion of the sensorimotor cortex in rats. The results showed that, in both species, a small percentage (1-2%) of cortical terminals make asymmetric synaptic contacts with m2-immunoreactive interneurons in the striatum. Interestingly, the majority of these synapses are onto small dendritic spines or spine-like appendages, as opposed to dendritic shafts and/or cell bodies. Thus, m2-containing striatal interneurons do receive direct cortical inputs and can, therefore, integrate and modulate cortical information flow through the striatum. Although the density of cortical terminals in contact with individual striatal interneurons is likely to be relatively low compared to the massive cortical input to projection neurons, both cholinergic and somatostatinergic interneurons display intrinsic properties that allow even small and distal inputs to influence their overall state of neuronal activity.

Animals↗

PubMed2000

Phosphorylation of the rat vesicular acetylcholine transporter.

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.

Acetylcholine↗

PubMed2000

Activation of group II metabotropic glutamate receptors inhibits synaptic excitation of the substantia Nigra pars reticulata.

Loss of nigrostriatal dopaminergic neurons in Parkinson's disease (PD) leads to increased activity of glutamatergic neurons in the subthalamic nucleus (STN). Recent studies reveal that the resultant increase in STN-induced excitation of basal ganglia output nuclei is responsible for the disabling motor impairment characteristic of PD. On the basis of this, it is possible that any manipulation that reduces activity at excitatory STN synapses onto basal ganglia output nuclei could be useful in the treatment of PD. We now report that group II metabotropic glutamate receptors (mGluRs) are presynaptically localized on STN terminals and that activation of these receptors inhibits excitatory transmission at STN synapses. In agreement with the hypothesis that this could provide a therapeutic benefit in PD, a selective agonist of group II mGluRs induces a dramatic reversal of catalepsy in a rat model of PD. These results raise the exciting possibility that selective agonists of group II mGluRs could provide an entirely new approach to the treatment of PD. These novel therapeutic agents would provide a noninvasive pharmacological treatment that does not involve the manipulation of dopaminergic systems, thus avoiding the problems associated with current therapies.

Animals↗

PubMed2000

Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus.

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.

Animals↗

PubMed2000

Molecules of the brain.

Progress against a range of brain disorders is being sustained by the use of genetic research techniques to identify specific molecules involved in brain disease, and by the realization that the identified molecules may disclose novel therapeutic targets. Both strategies are illustrated by recent insights and interventions in Alzheimer's disease and Parkinson's disease.

Alzheimer Disease↗

PubMed2000

LIM kinase 1 accumulates in presynaptic terminals during synapse maturation.

LIM kinase 1 (LIMK1) is a cytoplasmic protein kinase that is highly expressed in neurons. In transfected cells, LIMK1 binds to the cytoplasmic tail of neuregulins and regulates the breakdown of actin filaments. To identify potential functions of LIMK1 in vivo, we have determined the subcellular distribution of LIMK1 protein within neurons of the rat by using immunomicroscopy. At neuromuscular synapses in the adult hindlimb, LIMK1 was concentrated in the presynaptic terminal. However, little LIMK1 immunoreactivity was detected at neuromuscular synapses before the 2nd week after birth, and most motoneuron terminals were not strongly LIMK1 immunoreactive until the 3rd week after birth. Thus, LIMK1 accumulation at neuromuscular synapses coincided with their maturation. In contrast, SV2, like many other presynaptic terminal proteins, can be readily detected at neuromuscular synapses in the embryo. Similar to its late accumulation at developing synapses, LIMK1 accumulation at regenerating neuromuscular synapses occurred long after these synapses first formed. In the adult ventral spinal cord, LIMK1 was concentrated in a subset of presynaptic terminals. LIMK1 gradually accumulated at spinal cord synapses postnatally, reaching adult levels only after P14. This study is the first to implicate LIMK1 in the function of presynaptic terminals. The concentration of LIMK1 in adult, but not nascent, presynaptic terminals suggests a role for this kinase in regulating the structural or functional characteristics of mature synapses.

Animals↗

PubMed2000

Dopamine D(5) receptor immunolocalization in rat and monkey brain.

Dopamine D(5) receptor localization has been difficult because even the most specific ligands cannot distinguish between molecular subtypes of the D(1)-like receptor subfamily. Antifusion protein rabbit polyclonal antibodies directed against the C-terminus of human D(5) receptor were therefore developed for immunolocalization of the D(5) receptor protein in brain. The antibodies were characterized by immunoblot analysis and immunoprecipitation and used for light microscopic immunocytochemistry in rat and monkey brain. Affinity purified D(5) antibodies were specific for D(5) fusion protein as well as cloned and native D(5) receptor on Western blots, and D(5) antisera specifically immunoprecipitated solubilized, cloned D(5) receptor. Regional distribution of D(5) receptor immunoreactivity was consistent across species and correlated well with D(5) mRNA distribution previously reported in monkey brain. Immunoreactivity was widespread and tended to label perikarya and proximal dendrites of neurons in cerebral cortex, basal ganglia, basal forebrain, hippocampus, diencephalon, brainstem, and cerebellum. Neuropil was immunoreactive in olfactory bulb, islands of Calleja, cerebral cortex, superior colliculus, and molecular layer of cerebellum. The distribution of D(5) in brain was clearly different from that of other dopamine receptor subtypes, including D(1), the other member of the D(1)-like receptor subfamily. This unique distribution corroborates the idea that the D(5) receptor subtype has a distinct role in dopamine neurotransmission.

Animals↗