Reduced cortical neurotransmitter receptor complex levels in fetal Down syndrome brain.

In this study, cortical receptor complex levels were determined in fetal Down syndrome (DS, trisomy 21) brain. Frontal cortices were obtained from individuals with DS (19th-22nd week of gestation) and controls. Membrane proteins were extracted, assayed on blue native gels and immunoblotted with brain receptor antibodies. Levels of a D1R-containing complex were markedly decreased in male and female cortices of DS individuals. Females with DS had significant reductions of nicotinic acetylcholine receptors α4 and α7, NMDA receptor GluN1 and AMPA receptor GluA1- and GluA3-containing receptor complexes. Levels of other brain receptor complexes (5-hydroxytryptamine 1A, GluA2 and GluR4 receptor-containing complexes) were comparable between the groups of females. Levels of GluA2- and GluA3-containing complexes were significantly increased in males. Decreased levels of D1R complexes in both sexes, along with the significant reduction of α4, α7-containing receptor complexes observed in females, may explain the brain deficits and impaired cognition observed in DS.

Changes of several brain receptor complexes in the cerebral cortex of patients with Alzheimer disease: probable new potential pharmaceutical targets.

Although Alzheimer disease (AD) has been linked to defects in major brain receptors, studies thus far have been limited to the determination of receptor subunits or specific ligand binding studies. However, the availability of current technology enables the determination and quantification of brain receptor complexes. Thus, we examined levels of native receptor complexes in the brains of patients with AD. Cortical tissue was obtained from control subjects (n = 12 females and 12 males) and patients with AD (n = 12 females and 12 males) within a 3-h postmortem time period. The tissues were kept frozen until further biochemical analyses. Membrane proteins were extracted and subsequently enriched by ultracentrifugation using a sucrose gradient. Membrane proteins were then electrophoresed onto native gels and immunoblotted using antibodies against individual brain receptors. We found that the levels were comparable for complexes containing GluR2, GluR3 and GluR4 as well as 5-HT1A. Moreover, the levels of complexes containing muscarinic AChR M1, NR1 and GluR1 were significantly increased in male patients with AD. Nicotinic AChRs 4 and 7 as well as dopaminergic receptors D1 and D2 were also increased in males and females with AD. These findings reveal a pattern of altered receptor complex levels that may contribute to the deterioration of the concerted activity of these receptors and thus result in cognitive deficits observed in patients with AD. It should be emphasised that receptor complexes function as working units rather than individual subunits. Thus, the receptor deficits identified may be relevant for the design of experimental therapies. Therefore, specific pharmacological modulation of these receptors is within the pharmaceutical repertoire.

Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B.

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.

Connected neurons in multiple neocortical areas, comprising parallel circuits, encode essential information for visual shape learning.

Neocortical areas comprised of multiple neuronal circuits which are encoded with innumerable advanced cognitive tasks. Studies focused on neuronal network and synaptic plasticity has hypothesized that every specific neuron and the circuit process the explicit essential information for the specific tasks. However, the structure of these circuits and the involved critical neurons remain to be elucidated. Considering our previous studies, showing the specificity of rat postrhinal cortex comprising specific neuronal circuit for encoding both the learning and recall of shape discrimination through a fast neurotransmitter release from the transduced neurons, here we have demonstrated that postsynaptic neurons in two distinct areas, perirhinal cortex and the ventral temporal association areas are required for the specific visual shape discriminations learning. The constitutively active PKC was delivered into neuronal cells in postrhinal cortex, and the animals were allowed to learn the new shape discriminations, and then the silencing siRNA was delivered into postsynaptic neurons in either perirhinal cortex or ventral temporal association areas, using a novel technology for gene transfer into connected neurons. We observed that expression of the siRNA caused the deficits in visual performance, via blocking the activity in the neurons, as displayed by activity-dependent gene imaging, and also subsequently obstructed the activation of specific signaling pathways required for further learning, and dendritic protein synthesis and CREB. Thus, ratifying the conclusion that the two parallel circuits are both required for the visual shape discrimination learning.

The ratio of 1/3 linoleic acid to alpha linolenic acid is optimal for oligodendrogenesis of embryonic neural stem cells.

During neural development, embryonic neural stem cells (eNSCs) differentiate toward glial, oligodendrocytic, and neuronal cells. Dysregulation of polyunsaturated fatty acids (PUFAs) induce a wide range of neurological and developmental disorders. In this study, we investigated the effect of various concentrations and ratios of linoleic acid (LA) and alpha linolenic acid (ALA), which belong respectively to omega-6 and omega-3 PUFAs, on the proliferation and differentiation of eNSCs.Results showed that low (25 and 50µM) or high (100 and 200µM) concentrations of ALA, but not LA, and the ratio of 1:3 of LA/ALA significantly increased neurospheres size, frequency and cell numbers, in comparison to controls. Moreover, low or high concentrations of ALA, but not LA, and different ratios of LA/ALA resulted in a significant increase in mRNA expression levels of Notch1, Hes1 and Ki-67, and the differentiation of eNSCs toward astrocytes (GFAP) and oligodendrocytes (MBP), but not neurons (ß-III Tubulin), with the highest increase being for LA/ALA ratio of 1:3, in comparison to controls. These results demonstrate the importance of higher concentrations of ALA in enhancing proliferation and differentiation of eNSCs, which could be used in diet to help preventing neurodevelopmental syndromes, cognitive decline during aging, and various psychiatric disorders.

Formation of GABAA receptor complexes containing α1 and α5 subunits is paralleling a multiple T-maze learning task in mice.

There is limited information on the role of GABA type A receptors (GABAARs) containing α1, α5 and γ2 subunits in learning and memory. Here, we assessed the possible role of such receptors in spatial learning using the multiple T-maze (MTM) paradigm. C57BL/6J mice were trained in the MTM which induced elevated levels of α1 and α5 subunit-containing hippocampal GABAAR complexes. Moreover, spatial learning evoked a significant increase in the colocalization of α1 and α5 subunits in both, CA1 and dentate gyrus regions of the hippocampus suggesting the formation of complexes containing both subunits. Additionally, the presence of α1, α5 and γ2 subunits in high molecular weight GABAARs was detected and significant correlation in the level of α1-containing complexes with those containing α5 and γ2 subunits was demonstrated. Accordingly, α1 deficiency led to decreased levels of γ2 subunit-containing complexes, however, had no effect on α5-containing ones. On the other hand, α1 knockout mice showed impaired performance in the MTM correlating with increased levels of α5 subunit-containing GABAARs in comparison to trained floxed control animals which quickly learned the task. Taken together, these results suggest that α1, α5 and γ2-containing hippocampal GABAAR complexes play an essential role in spatial learning and memory in which targeted disruption of the α1 subunit produces profound deficits.

In silico comparison of Iranian HIV -1 envelop glycoprotein with five nearby countries.

HIV-1 envelope (env) glycoprotein mediates an important role in entry of the virus into the susceptible target cells. As env glycoprotein of HIV-1 is highly variable in the different geographical regions, in the present study, different properties of this protein in Iran are compared with five nearby countries. The sequences of HIV-1 env glycoproteins of Iran, Afghanistan, Russia, Turkey, Pakistan and Saudi Arabia databases were collected from databases. Amino acid composition and physical and chemical properties of the proteins from these countries were studied using Protparam and COPid tools. Receiver-operating characteristic (ROC) curve analysis and Support Vector Machine (SVM) were used to evaluate association between the properties of HIV-1 env glycoprotein of Iran with five nearby countries. The results verify that amino acid composition and four physical and chemical properties (molecular weight, isoelectric point, Aliphatic Index, and grand average of hydropathicity) of HIV-1 env protein in Iran and Russia were not significantly different. In conclusion, the results indicate that in silico techniques provide valuable information for comparing HIV-1 envelop glycoprotein in different geographical locations.

Mouse hippocampal GABAB1 but not GABAB2 subunit-containing receptor complex levels are paralleling retrieval in the multiple-T-maze.

GABAB receptors are heterodimeric G-protein coupled receptors known to be involved in learning and memory. Although a role for GABAB receptors in cognitive processes is evident, there is no information on hippocampal GABAB receptor complexes in a multiple T maze (MTM) task, a robust paradigm for evaluation of spatial learning. Trained or untrained (yoked control) C57BL/6J male mice (n = 10/group) were subjected to the MTM task and sacrificed 6 h following their performance. Hippocampi were taken, membrane proteins extracted and run on blue native PAGE followed by immunoblotting with specific antibodies against GABAB1, GABAB1a, and GABAB2. Immunoprecipitation with subsequent mass spectrometric identification of co-precipitates was carried out to show if GABAB1 and GABAB2 as well as other interacting proteins co-precipitate. An antibody shift assay (ASA) and a proximity ligation assay (PLA) were also used to see if the two GABAB subunits are present in the receptor complex. Single bands were observed on Western blots, each representing GABAB1, GABAB1a, or GABAB2 at an apparent molecular weight of approximately 100 kDa. Subsequently, densitometric analysis revealed that levels of GABAB1 and GABAB1a but not GABAB2- containing receptor complexes were significantly higher in trained than untrained groups. Immunoprecipitation followed by mass spectrometric studies confirmed the presence of GABAB1, GABAB2, calcium calmodulin kinases I and II, GluA1 and GluA2 as constituents of the complex. ASA and PLA also showed the presence of the two subunits of GABAB receptor within the complex. It is shown that increased levels of GABAB1 subunit-containing complexes are paralleling performance in a land maze.

Identification of new phosphorylation sites of AMPA receptors in the rat hippocampus--A resource for neuroscience research.

PURPOSE: AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) are glutamate-gated ion channels that mediate the majority of fast excitatory synaptic transmissions in the mammalian brain. A series of phosphorylation sites have been predicted or identified and knowledge on phosphorylations is mandatory for understanding receptor biology and functions. EXPERIMENTAL DESIGN: Immunoprecipitation from extracted hippocampal rat proteins was carried out using an antibody against the AMPAR GluA1 subunit, followed by identification of GluA1 and binding partners by MS. Bands from SDS-PAGE were picked, peptides were generated by trypsin and chymotrypsin digestion and identified by MS/MS (LTQ Orbitrap Velos). RESULTS: Using Mascot as a search engine, phosphorylation sites S506, S645, S720, S849, S863, S895, T858, Y228, Y419, and T734 were found on GluA1; S357, S513, S656, S727, T243, T420, T741, Y 143, Y301,Y426 on GluA2; S301, S516, S657, S732, T222, and T746 were observed on GluA3; and S514, S653 was phosphorylated on GluA4. CONCLUSIONS AND CLINICAL RELEVANCE: A series of additional protein modifications were observed and in particular, tyrosine and tryptophan nitrations on GluA1 were detected that may raise questions on additional regulation mechanisms for AMPARs in addition to phosphorylations. The findings are relevant for interpretation of previous work and design of future studies using AMPAR serving as a resource for neuroscience research and indeed, phosphorylations and PTMs per se would have to be respected when neuropathological and neurological disorders are being studied.

Dorsal hippocampal brain receptor complexes linked to the protein synthesis-dependent late phase (LTP) in the rat.

In order to link major brain receptor complex levels to in vivo electrically induced LTP, a bipolar stimulation electrode was chronically implanted into the perforant path, while two monopolar recording electrodes were implanted into the dentate gyrus of the dorsal hippocampus. The recording electrode was measuring extracellular excitatory postsynaptic potentials, while the other one measured population spikes. Immunoblotting of native receptor proteins was carried out in the DH based upon blue-native gel electrophoresis and immunoprecipitation followed by mass spectrometrical identification of the NR1-GluA1-GluA2 complex was used to provide evidence for complex formation. The induction of LTP in DH was proven and NMDA receptor complex levels containing NR1, GluA1, GluA2 and GluA3 were modulated by LTP induction. The LTP-associated changes of receptor complex levels may indicate concerted action, interaction and represent a pattern of major brain receptor complexes in the DH following electrical induction of LTP in the rat.

Dietary magnesium restriction reduces amygdala-hypothalamic GluN1 receptor complex levels in mice.

Reduced daily intake of magnesium (Mg(2+)) is suggested to contribute to depression. Indeed, preclinical studies show dietary magnesium restriction (MgR) elicits enhanced depression-like behaviour establishing a causal relationship. Amongst other mechanisms, Mg(2+) gates the activity of N-methyl-D-asparte (NMDA) receptors; however, it is not known whether reduced dietary Mg(2+) intake can indeed affect brain NMDA receptor complexes. Thus, the aim of the current study was to reveal whether MgR induces changes in brain NMDA receptor subunit composition that would indicate altered NMDA receptor regulation. The results revealed that enhanced depression-like behaviour elicited by MgR was associated with reduced amygdala-hypothalamic protein levels of GluN1-containing NMDA complexes. No change in GluN1 mRNA levels was observed indicating posttranslational changes were induced by dietary Mg(2+) restriction. To reveal possible protein interaction partners, GluN1 immunoprecipitation and proximity ligation assays were carried out revealing the expected GluN1 subunit association with GluN2A, GluN2B, but also novel interactions with GluA1, GluA2 in addition to known downstream signalling proteins. Chronic paroxetine treatment in MgR mice normalized enhanced depression-like behaviour, but did not alter protein levels of GluN1-containing NMDA receptors, indicating targets downstream of the NMDA receptor. Collectively, present data demonstrate that dietary MgR alters brain levels of GluN1-containing NMDA receptor complexes, containing GluN2A, GluN2B, AMPA receptors GluA1, GluA2 and several protein kinases. These data indicate that the modulation of dietary Mg(2+) intake may alter the function and signalling of this receptor complex indicating its involvement in the enhanced depression-like behaviour elicited by MgR.

Hippocampal levels of GluR1 and GluR2 complexes are modulated by training in the Multiple T-maze in C57BL/6J mice.

A series of studies has shown the importance of AMPA-type glutamate receptors (AMPARs) for memory formation. The aim of the current study was to show whether GluR1 and GluR2 complexes rather than subunits in mouse hippocampus were involved in training in the multiple T-Maze (MTM). C57BL/6J mice were trained in the MTM and compared to yoked controls. 6 h following the completion of the fourth day training, mice were euthanized, hippocampi were taken and proteins extracted, run on blue native gels with subsequent immunoblotting with antibodies against mouse GluR1 and GluR2. On blue-native western blotting, GluR1 protein was represented by a single band at the apparent molecular weight of about 480 kDa probably indicating a tetrameric assembly. GluR2 protein was represented by a single band between apparent molecular weights of 480 and 720 kDa indicating a homo- or heteropolymer probably with other AMPAR or regulatory subunits. In mice trained in the MTM, protein levels for GluR1 were significantly increased while GluR2 levels were significantly decreased. On two-dimensional (2D) gel electrophoresis, the presence of GluR1 and GluR2 were identified by mass spectrometry, and 2D immunoblotting revealed several expression forms of these receptor subunits. Findings unequivocally show that GluR1 and GluR2 complexes are linked to training in the MTM in C57BL/6J mice. These results may not only form the basis for studying receptor complexes rather than receptor subunits in memory formation or mechanisms of potential cognitive enhancers but represent a tool for investigations into pharmacological studies including the use of glutamate receptor agonists and antagonists.

Hippocampal AMPA-type receptor complexes containing GluR3 and GluR4 are paralleling training in the Multiple T-Maze.

Although it is well-known that AMPA receptors are involved in spatial learning and memory, published data on GluR3 and GluR4 are limited. Moreover, there is no information about GluR3 and GluR4 receptor complex levels in spatial memory training. It was therefore the aim of the study to determine the above-mentioned receptor levels following training in the Multiple T-Maze (MTM). Results from the MTM and hippocampal membrane proteins from C57BL/6J mice were taken from an own previous study and GluR3 and GluR4 receptor complexes were run on blue native gel electrophoresis followed by immunoblotting and quantification of bands. Subsequently, GluR3 and GluR4 were identified under denaturing conditions from two-dimensional gels by mass spectrometry (nano-LC-ESI-MS/MS). Hippocampal levels of GluR3 containing complexes (apparent molecular weight between 480 and 720) were decreased while GluR4 containing complexes (apparent molecular weight between 480 and 720) were increased. GluR4 complex levels in trained mice were correlating with latency and speed. Mass spectrometry unambiguously identified the two receptor subunits. The findings show that GluR3 and GluR4 may have different functions in the processes of spatial memory training in the MTM and indeed, different neurobiological functions of the two receptor subunits have been already reported. GluR3 and GluR4 receptor complex rather than subunit levels are paralleling training in the MTM and GluR4 complex levels were even linked to memory training, which may be of relevance for understanding molecular memory processes, interpretation of previous work or for design of future AMPA receptor studies.

NMDA-complexes linked to spatial memory performance in the Barnes maze in CD1 mice.

The N-methyl-d-aspartic acid receptor (NMDAR) is a well-documented key element in the formation of several memories including spatial, olfactory and contextual memory. Although receptor subunits have been linked to memory formation, data on the involvement of the NMDAR complexes is limited. In previous work CD1 mice were trained in the Barnes maze, a low-stress landmaze, and yoked controls were serving as controls. Hippocampal samples from this behavioural study were taken for comparing NMDAR complexes. Hippocampi were taken and stored until analysis at -80 °C. Membrane proteins were extracted from hippocampi using an ultracentrifugation step and applied on Blue Native gels that in turn were used for immunoblotting with antibodies against subunits NR1, NR2A and NR2B. The subunit content of the complexes was determined by denaturing two-dimensional gel electrophoresis and subsequent immunoblotting. An NMDAR complex with an apparent molecular weight between between 146 and 242 kDa, probably representing an NR1 dimer was the only complex that was significantly different between trained and yoked animals. A series of NMDAR complexes containing modulatory subunits NR2A or NR2B or both were detected. All complexes contained the NR1 subunit. The NR1 dimer complex level, increased in memory formation, may be directly or indirectly involved in the process of spatial memory formation in the CD1 mouse. The results are enabling and challenging further NMDAR studies, both, at the pharmacological and molecular level. Moreover, several NMDAR complexes in the CD1 mouse were shown to be mainly heteropolymers of subunits NR1, NR2A and NR2B, although other recently described subunits were not tested due to unavailability of specific antibodies. Determination of native receptor complexes rather than individual subunits is mandatory and provides the molecular basis for understanding mechanisms of spatial memory.