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1.  Channel-mediated astrocytic glutamate modulates hippocampal synaptic plasticity by activating postsynaptic NMDA receptors 
Molecular Brain  2015;8:7.
Background
Activation of G protein coupled receptor (GPCR) in astrocytes leads to Ca2+-dependent glutamate release via Bestrophin 1 (Best1) channel. Whether receptor-mediated glutamate release from astrocytes can regulate synaptic plasticity remains to be fully understood.
Results
We show here that Best1-mediated astrocytic glutamate activates the synaptic N-methyl-D-aspartate receptor (NMDAR) and modulates NMDAR-dependent synaptic plasticity. Our data show that activation of the protease-activated receptor 1 (PAR1) in hippocampal CA1 astrocytes elevates the glutamate concentration at Schaffer collateral-CA1 (SC-CA1) synapses, resulting in activation of GluN2A-containing NMDARs and NMDAR-dependent potentiation of synaptic responses. Furthermore, the threshold for inducing NMDAR-dependent long-term potentiation (LTP) is lowered when astrocytic glutamate release accompanied LTP induction, suggesting that astrocytic glutamate is significant in modulating synaptic plasticity.
Conclusions
Our results provide direct evidence for the physiological importance of channel-mediated astrocytic glutamate in modulating neural circuit functions.
doi:10.1186/s13041-015-0097-y
PMCID: PMC4320468  PMID: 25645137
Astrocytes; Bestrophin 1; Ca2+-activated anion channel; Synaptic plasticity; Glutamate; NMDA receptor; LTP; PAR1
2.  Evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer’s disease 
Molecular Brain  2015;8:6.
Background
In Alzheimer’s disease synapse loss precedes neuronal loss and correlates best with impaired memory formation. However, the mechanisms underlying synaptic degeneration in Alzheimer’s disease are not well known. Further, it is unclear why synapses in AD cerebellum are protected from degeneration. Our recent work on the cyclin-dependent kinase 5 activator p25 suggested that expression of the multifunctional presynaptic molecule cysteine string protein alpha (CSPalpha) may be affected in Alzheimer’s disease.
Results
Using western blots and immunohistochemistry, we found that CSPalpha expression is reduced in hippocampus and superior temporal gyrus in Alzheimer’s disease. Reduced CSPalpha expression occurred before synaptophysin levels drop, suggesting that it contributes to the initial stages of synaptic degeneration. Surprisingly, we also found that CSPalpha expression is upregulated in cerebellum in Alzheimer’s disease. This CSPalpha upregulation reached the same level as in young, healthy cerebellum. We tested the idea whether CSPalpha upregulation might be neuroprotective, using htau mice, a model of tauopathy that expresses the entire wild-type human tau gene in the absence of mouse tau. In htau mice CSPalpha expression was found to be elevated at times when neuronal loss did not occur.
Conclusion
Our findings provide evidence that the presynaptic vesicle protein CSPalpha is a key player in synaptic degeneration and protection in Alzheimer’s disease. In the forebrain CSPalpha expression is reduced early in the disease and this may contribute to the initial stages of synaptic degeneration. In the cerebellum CSPalpha expression is upregulated to young, healthy levels and this may protect cerebellar synapses and neurons to survive. Accordingly, CSPalpha upregulation also occurs in a mouse model of tauopathy only at time when neuronal loss does not take place.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-015-0096-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-015-0096-z
PMCID: PMC4314762  PMID: 25631211
Alzheimer’s disease; Cerebellum; Cysteine string protein; Hippocampus; Synapses; Neuroprotection
3.  Up-regulation of neural and cell cycle-related microRNAs in brain of amyotrophic lateral sclerosis mice at late disease stage 
Molecular Brain  2015;8:5.
Background
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease.
Results
Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.
Conclusions
Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-015-0095-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-015-0095-0
PMCID: PMC4318136  PMID: 25626686
G93A-SOD1 mice; microRNAs; Neural stem progenitor cells
4.  N-methyl-D-aspartate receptors mediate activity-dependent down-regulation of potassium channel genes during the expression of homeostatic intrinsic plasticity 
Molecular Brain  2015;8:4.
Background
Homeostatic intrinsic plasticity encompasses the mechanisms by which neurons stabilize their excitability in response to prolonged and destabilizing changes in global activity. However, the milieu of molecular players responsible for these regulatory mechanisms is largely unknown.
Results
Using whole-cell patch clamp recording and unbiased gene expression profiling in rat dissociated hippocampal neurons cultured at high density, we demonstrate here that chronic activity blockade induced by the sodium channel blocker tetrodotoxin leads to a homeostatic increase in action potential firing and down-regulation of potassium channel genes. In addition, chronic activity blockade reduces total potassium current, as well as protein expression and current of voltage-gated Kv1 and Kv7 potassium channels, which are critical regulators of action potential firing. Importantly, inhibition of N-Methyl-D-Aspartate receptors alone mimics the effects of tetrodotoxin, including the elevation in firing frequency and reduction of potassium channel gene expression and current driven by activity blockade, whereas inhibition of L-type voltage-gated calcium channels has no effect.
Conclusions
Collectively, our data suggest that homeostatic intrinsic plasticity induced by chronic activity blockade is accomplished in part by decreased calcium influx through N-Methyl-D-Aspartate receptors and subsequent transcriptional down-regulation of potassium channel genes.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-015-0094-1) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-015-0094-1
PMCID: PMC4333247  PMID: 25599691
Homeostatic intrinsic plasticity; Potassium channel; NMDA receptor; Action potential; Hippocampus
5.  Heterogeneity of tremor mechanisms assessed by tremor-related cortical potential in mice 
Molecular Brain  2015;8:3.
Background
Identifying a neural circuit mechanism that is differentially involved in tremor would aid in the diagnosis and cure of such cases. Here, we demonstrate that tremor-related cortical potential (TRCP) is differentially expressed in two different mouse models of tremor.
Results
Hybrid tremor analysis of harmaline-induced and genetic tremor in mice revealed that two authentic tremor frequencies for each type of tremor were conserved and showed an opposite dependence on CaV3.1 T-type Ca2+ channels. Electroencephalogram recordings revealed that α1−/−;α1G-/- mice double-null for the GABA receptor α1 subunit (Gabra1) and CaV3.1 T-type Ca2+ channels (Cacna1g), in which the tremor caused by the absence of Gabra1 is potentiated by the absence of Cacna1g, showed a coherent TRCP that exhibited an onset that preceded the initiation of behavioral tremor by 3 ms. However, harmaline-induced tremor, which is known to be abolished by α1G−/−, showed no TRCP.
Conclusions
Our results demonstrate that the α1−/−;α1G−/− double-knockout tremor model is useful for studying cortical mechanisms of tremor.
doi:10.1186/s13041-015-0093-2
PMCID: PMC4304607  PMID: 25588467
Tremor mechanism; Cortical rhythm; Harmaline; T-type Ca2+ channels
6.  High expression of long intervening non-coding RNA OLMALINC in the human cortical white matter is associated with regulation of oligodendrocyte maturation 
Molecular Brain  2015;8:2.
Background
Long intervening non-coding RNAs (lincRNAs) are a recently discovered subclass of non-coding RNAs. LincRNAs are expressed across the mammalian genome and contribute to the pervasive transcription phenomenon. They display a tissue-specific and species-specific mode of expression and are present abundantly in the brain.
Results
Here, we report the expression patterns of oligodendrocyte maturation-associated long intervening non-coding RNA (OLMALINC), which is highly expressed in the white matter (WM) of the human frontal cortex compared to the grey matter (GM) and peripheral tissues. Moreover, we identified a novel isoform of OLMALINC that was also up-regulated in the WM. RNA-interference (RNAi) knockdown of OLMALINC in oligodendrocytes, which are the major cell type in the WM, caused significant changes in the expression of genes regulating cytostructure, cell activation and membrane signaling. Gene ontology enrichment analysis revealed that over 10% of the top 25 up- and down-regulated genes were involved in oligodendrocyte maturation. RNAi experiments in neuronal cells resulted in the perturbation of genes controlling cell proliferation. Furthermore, we identified a novel cis-natural antisense non-coding RNA, which we named OLMALINC-AS, which maps to the first exon of the dominant isoform of OLMALINC.
Conclusions
Our study has demonstrated for the first time that a primate-specific lincRNA regulates the expression of genes critical to human oligodendrocyte maturation, which in turn might be regulated by an antisense counterpart.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0091-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0091-9
PMCID: PMC4302521  PMID: 25575711
Long intervening non-coding RNA; OLMALINC; Human brain; Frontal cortex; White and grey matter; Antisense RNA
7.  Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression 
Molecular Brain  2015;8:1.
Background
Regulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop.
Results
LTD in the cerebellum of Ptprr−/− mice is strongly impeded, in vitro and in vivo. Comparison of basal phospho-MAPK levels between wild-type and PTPRR deficient cerebellar slices revealed increased levels in mutants. This high basal phospho-MAPK level attenuated further increases in phospho-MAPK during chemical induction of LTD, essentially disrupting the positive feedback loop and preventing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation and endocytosis.
Conclusions
Our findings indicate an important role for PTPRR in maintaining low basal MAPK activity in Purkinje cells. This creates an optimal ‘window’ to boost MAPK activity following signals that induce LTD, which can then propagate through feed-forward signals to cause AMPAR internalization and LTD.
doi:10.1186/s13041-014-0092-8
PMCID: PMC4304614  PMID: 25571783
Cerebellum; ERK; Long-term synaptic plasticity; Knock-out mice; LTD; PTPBR7; PTP-SL
8.  Nlrx1 regulates neuronal cell death 
Molecular Brain  2014;7(1):90.
Background
Regulation of cell death during neurodegeneration is one of the key factors that play a role in the speed at which a disease progresses. Out of several cellular pathways responsible for this progression, necrosis and apoptosis are situated on the opposite spectrum of cell death regulation. Necrosis produces an environment that promotes inflammation and cytotoxicity and apoptosis is a highly organized process that maintains tissue homeostasis. A recently discovered protein, Nlrx1, regulates inflammatory and cell death responses during infection.
Findings
Using transfections of N2A cell line, we demonstrate that Nlrx1 redirects cells away from necrosis and towards an apoptotic pathway following rotenone treatments. In addition, Nlrx1 promotes DRP1 phosphorylation and increases mitochondrial fission.
Conclusion
Our results suggest a novel molecular pathway for regulating mitochondrial dynamics and neuronal death. Nlrx1 may play an important role in neurodegenerative diseases, where necrosis is a prominent factor.
doi:10.1186/s13041-014-0090-x
PMCID: PMC4302421  PMID: 25540124
Nlrx1; Cells death; Necrosis; Apoptosis
9.  Genome-wide screen for modifiers of Na+/K+ATPase alleles identifies critical genetic loci 
Molecular Brain  2014;7(1):89.
Background
Mutations affecting the Na+/ K+ATPase (a.k.a. the sodium-potassium pump) genes cause conditional locomotor phenotypes in flies and three distinct complex neurological diseases in humans. More than 50 mutations have been identified affecting the human ATP1A2 and ATP1A3 genes that are known to cause rapid-onset Dystonia Parkinsonism, familial hemiplegic migraine, alternating hemiplegia of childhood, and variants of familial hemiplegic migraine with neurological complications including seizures and various mood disorders. In flies, mutations affecting the ATPalpha gene have dramatic phenotypes including altered longevity, neural dysfunction, neurodegeneration, myodegeneration, and striking locomotor impairment. Locomotor defects can manifest as conditional bang-sensitive (BS) or temperature-sensitive (TS) paralysis: phenotypes well-suited for genetic screening.
Results
We performed a genome-wide deficiency screen using three distinct missense alleles of ATPalpha and conditional locomotor function assays to identify novel modifier loci. A secondary screen confirmed allele-specificity of the interactions and many of the interactions were mapped to single genes and subsequently validated. We successfully identified 64 modifier loci and used classical mutations and RNAi to confirm 50 single gene interactions. The genes identified include those with known function, several with unknown function or that were otherwise uncharacterized, and many loci with no described association with locomotor or Na+/K+ ATPase function.
Conclusions
We used an unbiased genome-wide screen to find regions of the genome containing elements important for genetic modulation of ATPalpha dysfunction. We have identified many critical regions and narrowed several of these to single genes. These data demonstrate there are many loci capable of modifying ATPalpha dysfunction, which may provide the basis for modifying migraine, locomotor and seizure dysfunction in animals.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0089-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0089-3
PMCID: PMC4302446  PMID: 25476251
Drosophila melanogaster; ATPalpha; Sodium pump; Temperature-sensitive paralysis; Conditional paralysis; Seizure; Migrane; Screen; Genome-wide; Seizure suppressor
10.  Human post-mortem synapse proteome integrity screening for proteomic studies of postsynaptic complexes 
Molecular Brain  2014;7(1):88.
Background
Synapses are fundamental components of brain circuits and are disrupted in over 100 neurological and psychiatric diseases. The synapse proteome is physically organized into multiprotein complexes and polygenic mutations converge on postsynaptic complexes in schizophrenia, autism and intellectual disability. Directly characterising human synapses and their multiprotein complexes from post-mortem tissue is essential to understanding disease mechanisms. However, multiprotein complexes have not been directly isolated from human synapses and the feasibility of their isolation from post-mortem tissue is unknown.
Results
Here we establish a screening assay and criteria to identify post-mortem brain samples containing well-preserved synapse proteomes, revealing that neocortex samples are best preserved. We also develop a rapid method for the isolation of synapse proteomes from human brain, allowing large numbers of post-mortem samples to be processed in a short time frame. We perform the first purification and proteomic mass spectrometry analysis of MAGUK Associated Signalling Complexes (MASC) from neurosurgical and post-mortem tissue and find genetic evidence for their involvement in over seventy human brain diseases.
Conclusions
We have demonstrated that synaptic proteome integrity can be rapidly assessed from human post-mortem brain samples prior to its analysis with sophisticated proteomic methods. We have also shown that proteomics of synapse multiprotein complexes from well preserved post-mortem tissue is possible, obtaining structures highly similar to those isolated from biopsy tissue. Finally we have shown that MASC from human synapses are involved with over seventy brain disorders. These findings should have wide application in understanding the synaptic basis of psychiatric and other mental disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0088-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0088-4
PMCID: PMC4271336  PMID: 25429717
Synapse; Proteomics; Mass spectrometry; Supercomplex; Post-mortem brain; MAGUK; Psychiatric disorder
11.  Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms 
Molecular Brain  2014;7(1):85.
Background
As the primary immune cells of the central nervous system, microglia contribute to development, homeostasis, and plasticity of the central nervous system, in addition to their well characterized roles in the foreign body and inflammatory responses. Increasingly, inappropriate activation of microglia is being reported as a component of inflammation in neurodegenerative and neuropsychiatric disorders. The statin class of cholesterol-lowering drugs have been observed to have anti-inflammatory and protective effects in both neurodegenerative diseases and ischemic stroke, and are suggested to act by attenuating microglial activity.
Results
We sought to investigate the effects of simvastatin treatment on the secretory profile and phagocytic activity of primary cultured rat microglia, and to dissect the mechanism of action of simvastatin on microglial activity. Simvastatin treatment altered the release of cytokines and trophic factors from microglia, including interleukin-1-β, tumour necrosis factor-α, and brain derived neurotrophic factor in a cholesterol-dependent manner. Conversely, simvastatin inhibited phagocytosis in microglia in a cholesterol-independent manner.
Conclusions
The disparity in cholesterol dependence of cytokine release and phagocytosis suggests the two effects occur through distinct molecular mechanisms. These two pathways may provide an opportunity for further refinement of pharmacotherapies for neuroinflammatory, neurodegenerative, and neuropsychiatric disorders.
doi:10.1186/s13041-014-0085-7
PMCID: PMC4247600  PMID: 25424483
Inflammation; Cholesterol; Mevalonate; Phagocytosis
12.  Ex vivo 1H nuclear magnetic resonance spectroscopy reveals systematic alterations in cerebral metabolites as the key pathogenetic mechanism of bilirubin encephalopathy 
Molecular Brain  2014;7(1):87.
Background
Bilirubin encephalopathy (BE) is a severe neurologic sequelae induced by hyperbilirubinemia in newborns. However, the pathogenetic mechanisms underlying the clinical syndromes of BE remain ambiguous. Ex vivo1H nuclear magnetic resonance (NMR) spectroscopy was used to measure changes in the concentrations of cerebral metabolites in various brain areas of newborn 9-day-old rats subjected to bilirubin to explore the related mechanisms of BE.
Results
When measured 0.5 hr after injection of bilirubin, levels of the amino acid neurotransmitters glutamate (Glu), glutamine (Gln), and γ-aminobutyric acid (GABA) in hippocampus and occipital cortex significantly decreased, by contrast, levels of aspartate (Asp) considerably increased. In the cerebellum, Glu and Gln levels significantly decreased, while GABA, and Asp levels showed no significant differences. In BE 24 hr rats, all of the metabolic changes observed returned to normal in the hippocampus and occipital cortex; however, levels of Glu, Gln, GABA, and glycine significantly increased in the cerebellum.
Conclusions
These metabolic changes for the neurotransmitters are mostly likely the result of a shift in the steady-state equilibrium of the Gln-Glu-GABA metabolic cycle between astrocytes and neurons, in a region-specific manner. Changes in energy metabolism and the tricarboxylic acid cycle may also be involved in the pathogenesis of BE.
doi:10.1186/s13041-014-0087-5
PMCID: PMC4252999  PMID: 25424547
Bilirubin encephalopathy; Nuclear magnetic resonance spectroscopy; Metabonomics; Gln-Glu-GABA cycle
13.  TWIK-1 contributes to the intrinsic excitability of dentate granule cells in mouse hippocampus 
Molecular Brain  2014;7(1):80.
Background
Two-pore domain K+ (K2P) channels have been shown to modulate neuronal excitability. However, physiological function of TWIK-1, the first identified member of the mammalian K2P channel family, in neuronal cells is largely unknown.
Results
We found that TWIK-1 proteins were expressed and localized mainly in the soma and proximal dendrites of dentate gyrus granule cells (DGGCs) rather than in distal dendrites or mossy fibers. Gene silencing demonstrates that the outwardly rectifying K+ current density was reduced in TWIK-1-deficient granule cells. TWIK-1 deficiency caused a depolarizing shift in the resting membrane potential (RMP) of DGGCs and enhanced their firing rate in response to depolarizing current injections. Through perforant path stimulation, TWIK-1-deficient granule cells showed altered signal input-output properties with larger EPSP amplitude values and increased spiking compared to control DGGCs. In addition, supra-maximal perforant path stimulation evoked a graded burst discharge in 44% of TWIK-1-deficient cells, which implies impairment of EPSP-spike coupling.
Conclusions
These results showed that TWIK-1 is functionally expressed in DGGCs and contributes to the intrinsic excitability of these cells. The TWIK-1 channel is involved in establishing the RMP of DGGCs; it attenuates sub-threshold depolarization of the cells during neuronal activity, and contributes to EPSP-spike coupling in perforant path-to-granule cell synaptic transmission.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0080-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0080-z
PMCID: PMC4240835  PMID: 25406588
K2P channel; TWIK-1; Intrinsic excitability; Dentate gyrus granule cell
14.  CNS axon regeneration inhibitors stimulate an immediate early gene response via MAP kinase-SRF signaling 
Molecular Brain  2014;7(1):86.
Background
CNS axon regeneration inhibitors such as Nogo and CSPGs (Chondroitin Sulfate Proteoglycans) are major extrinsic factors limiting outgrowth of severed nerve fibers. However, knowledge on intracellular signaling cascades and gene expression programs activated by these inhibitors in neurons is sparse. Herein we studied intracellular signaling cascades activated by total myelin, Nogo and CSPGs in primary mouse CNS neurons.
Results
Total myelin, Nogo and CSPGs stimulated gene expression activity of the serum response factor (SRF), a central gene regulator of immediate early (IEG) and actin cytoskeletal gene transcription. As demonstrated by pharmacological interference, SRF-mediated IEG activation by myelin, Nogo or CSPGs depended on MAP kinase, to a lesser extent on Rho-GTPase but not on PKA signaling. Stimulation of neurons with all three axon growth inhibitors activated the MAP kinase ERK. In addition to ERK activation, myelin activated the IEG c-Fos, an important checkpoint of neuronal survival vs. apoptosis. Employing Srf deficient neurons revealed that myelin-induced IEG activation requires SRF. This suggests an SRF function in mediating neuronal signaling evoked by axon regeneration associated inhibitors. Besides being a signaling target of axon growth inhibitors, we show that constitutively-active SRF-VP16 can be employed to circumvent neurite growth inhibition imposed by myelin, Nogo and CSPGs.
Conclusion
In sum, our data demonstrate that axon regeneration inhibitors such as Nogo trigger gene expression programs including an SRF-dependent IEG response via MAP kinases and Rho-GTPases.
doi:10.1186/s13041-014-0086-6
PMCID: PMC4243276  PMID: 25406759
SRF; Immediate early gene; Axon; Regeneration; MAP kinase; Neuron; Myelin; c-Fos
15.  Anatomo-proteomic characterization of human basal ganglia: focus on striatum and globus pallidus 
Molecular Brain  2014;7(1):83.
Background
The basal ganglia (BG) are a complex network of subcortical nuclei involved in the coordination and integration of the motor activity. Although these independent anatomical structures are functionally related, the proteome present in each isolated nucleus remains largely unexplored. In order to analyse the BG proteome in a large-scale format, we used a multi-dimensional fractionation approach which combines isolation of anatomically-defined nuclei, and protein/peptide chromatographic fractionation strategies coupled to mass spectrometry.
Results
Using this workflow, we have obtained a proteomic expression profile across striatum and globus pallidus structures among which 1681 proteins were located in caudate nucleus (CN), 1329 in putamen, 1419 in medial globus pallidus (GPi), and 1480 in lateral globus pallidus (GPe), establishing a BG reference proteome to a depth of 2979 unique proteins. Protein interactome mapping highlighted significant clustering of common proteins in striatal and pallidal structures, contributing to oxidative phosphorylation, protein degradation and neurotrophin signalling pathways. In silico analyses emphasized specific pathways represented in striatal and pallidal structures highlighting 5-hydroxytryptamine degradation, synaptic vesicle trafficking, and dopamine, metabotropic glutamate and muscarinic acetylcholine receptor pathways. Additional bioinformatic analyses also revealed that: i) nearly 4% of identified proteins have been previously associated to neurodegenerative syndromes, ii) 11% of protein set tends to localize to synaptic terminal, and iii) 20% of identified proteins were also localized in cerebrospinal fluid (CSF).
Conclusions
Overall, the anatomo-proteomic profiling of BG complements the anatomical atlas of the human brain transcriptome, increasing our knowledge about the molecular basis of the BG and the etiology of the movement disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0083-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0083-9
PMCID: PMC4236423  PMID: 25406675
Basal ganglia; Striatum; Globus pallidus; Proteomics; Mass spectrometry; Bioinformatics
16.  Cdk5/p35 functions as a crucial regulator of spatial learning and memory 
Molecular Brain  2014;7(1):82.
Background
Cyclin-dependent kinase 5 (Cdk5), which is activated by binding to p35 or p39, is involved in synaptic plasticity and affects learning and memory formation. In Cdk5 knockout (KO) mice and p35 KO mice, brain development is severely impaired because neuronal migration is impaired and lamination is disrupted. To avoid these developmental confounders, we generated inducible CreER-p35 conditional (cKO) mice to study the role of Cdk5/p35 in higher brain function.
Results
CreER-p35 cKO mice exhibited spatial learning and memory impairments and reduced anxiety-like behavior. These phenotypes resulted from a decrease in the dendritic spine density of CA1 pyramidal neurons and defective long-term depression induction in the hippocampus.
Conclusions
Taken together, our findings reveal that Cdk5/p35 regulates spatial learning and memory, implicating Cdk5/p35 as a therapeutic target in neurological disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0082-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0082-x
PMCID: PMC4239319  PMID: 25404232
Spatial learning; Memory; Kinase; Synaptic plasticity; Hippocampus
17.  Calretinin-positive L5a pyramidal neurons in the development of the paralemniscal pathway in the barrel cortex 
Molecular Brain  2014;7(1):84.
Background
The rodent barrel cortex has been established as an ideal model for studying the development and plasticity of a neuronal circuit. The barrel cortex consists of barrel and septa columns, which receive various input signals through distinct pathways. The lemniscal pathway transmits whisker-specific signals to homologous barrel columns, and the paralemniscal pathway transmits multi-whisker signals to both barrel and septa columns. The integration of information from both lemniscal and paralemniscal pathways in the barrel cortex is critical for precise object recognition. As the main target of the posterior medial nucleus (POm) in the paralemniscal pathway, layer 5a (L5a) pyramidal neurons are involved in both barrel and septa circuits and are considered an important site of information integration. However, information on L5a neurons is very limited. This study aims to explore the cellular features of L5a neurons and to provide a morphological basis for studying their roles in the development of the paralemniscal pathway and in information integration.
Results
1. We found that the calcium-binding protein calretinin (CR) is dynamically expressed in L5a excitatory pyramidal neurons of the barrel cortex, and L5a neurons form a unique serrated pattern similar to the distributions of their presynaptic POm axon terminals.
2. Infraorbital nerve transection disrupts this unique alignment, indicating that it is input dependent.
3. The formation of the L5a neuronal alignment develops synchronously with barrels, which suggests that the lemniscal and paralemniscal pathways may interact with each other to regulate pattern formation and refinement in the barrel cortex.
4. CR is specifically expressed in the paralemniscal pathway, and CR deletion disrupts the unique L5a neuronal pattern, which indicates that CR may be required for the development of the paralemniscal pathway.
Conclusions
Our results demonstrate that L5a neurons form a unique, input-dependent serrated alignment during the development of cortical barrels and that CR may play an important role in the development of the paralemniscal pathway. Our data provide a morphological basis for studying the role of L5a pyramidal neurons in information integration within the lemniscal and paralemniscal pathways.
doi:10.1186/s13041-014-0084-8
PMCID: PMC4246560  PMID: 25404384
Calretinin; L5a pyramidal neuron; Paralemniscal pathway; Posterior medial nucleus; Barrel cortex
18.  Lack of evidence of the interaction of the Aβ peptide with the Wnt signaling cascade in Drosophila models of Alzheimer’s disease 
Molecular Brain  2014;7(1):81.
Background
Alzheimer’s disease (AD) is the leading form of dementia worldwide. The Aβ-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aβ impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aβ.
Findings
Expression of the human Aβ42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aβ42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aβ42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila.
Conclusions
Our data confirm that the life span is a useful readout of Aβ42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aβ42 toxicity and might serve as a drug target against AD.
doi:10.1186/s13041-014-0081-y
PMCID: PMC4232725  PMID: 25387847
Alzheimer’s disease; Aβ peptide; Drosophila; Wnt signaling
19.  The planar cell polarity protein Vangl2 bidirectionally regulates dendritic branching in cultured hippocampal neurons 
Molecular Brain  2014;7(1):79.
Background
Van Gogh-like (Vangl) 2 is a planar cell polarity (PCP) protein that regulates the induction of polarized cellular and tissue morphology during animal development. In the nervous system, the core PCP signaling proteins have been identified to regulate neuronal maturation. In axonal growth cones, the antagonistic interaction of PCP components makes the tips of filopodia sensitive to guidance cues. However, the molecular mechanism by which the PCP signaling regulates spine and dendritic development remains obscure.
Findings
Here we explored the finding that a loss of function of Vangl2 results in a significant reduction in spine density and complexity of dendritic branching. In spite of a previous report, in which the Vangl2 C-terminal TSV motif was shown to be required for the interaction with PSD-95 and the C-terminal intracellular domain was shown to associate with N-cadherin, overexpression of deletion mutants (Vangl2-∆TSV and Vangl2-∆C) had little effect on spine density. However, when an N-terminal region deletion mutant was overexpressed, spine density was slightly down-regulated. Intriguingly, the deletion mutants had a more potent effect on dendritic branching, such that the deletion of the N-terminal region reduced dendritic branching, whereas deletion of the C-terminal region increased it.
Conclusions
Based on these results, Vangl2, a core PCP signaling pathway component, appears to have a functional role in neural complex formation. Especially in the case of dendritic branching, Vangl2 serves as a molecular hub to regulate neural morphology in opposite directions.
doi:10.1186/s13041-014-0079-5
PMCID: PMC4234848  PMID: 25387771
Planar cell polarity signal; Van Gogh-like protein; Dendritic spine; Sholl analysis
20.  Adenylyl cyclase-5 in the dorsal striatum function as a molecular switch for the generation of behavioral preferences for cue-directed food choices 
Molecular Brain  2014;7(1):77.
Background
Behavioral choices in habits and innate behaviors occur automatically in the absence of conscious selection. These behaviors are not easily modified by learning. Similar types of behaviors also occur in various mental illnesses including drug addiction, obsessive-compulsive disorder, schizophrenia, and autism. However, underlying mechanisms are not clearly understood. In the present study, we investigated the molecular mechanisms regulating unconditioned preferred behaviors in food-choices.
Results
Mice lacking adenylyl cyclase-5 (AC5 KO mice), which is preferentially expressed in the dorsal striatum, consumed food pellets nearly one after another in cages. AC5 KO mice showed aversive behaviors to bitter tasting quinine, but they compulsively chose quinine-containing AC5 KO-pellets over fresh pellets. The unusual food-choice behaviors in AC5 KO mice were due to the gain of behavioral preferences for food pellets containing an olfactory cue, which wild-type mice normally ignored. Such food-choice behaviors in AC5 KO mice disappeared when whiskers were trimmed. Conversely, whisker trimming in wildtype mice induced behavioral preferences for AC5 KO food pellets, indicating that preferred food-choices were not learned through prior experience. Both AC5 KO mice and wildtype mice with trimmed whiskers had increased glutamatergic input from the barrel cortex into the dorsal striatum, resulting in an increase in the mGluR1-dependent signaling cascade. The siRNA-mediated inhibition of mGluR1 in the dorsal striatum in AC5 KO mice and wildtype mice with trimmed whiskers abolished preferred choices for AC5 KO food pellets, whereas siRNA-mediated inhibition of mGluR3 glutamate receptors in the dorsal striatum in wildtype mice induced behavioral preferences for AC5 KO food pellets, thus mimicking AC5 KO phenotypes.
Conclusions
Our results show that the gain and loss of behavioral preferences for a specific cue-directed option were regulated by specific cellular factors in the dorsal striatum, such that the preferred food choices were switched on when either the mGluR3-AC5 pathway was inactive or the mGluR1 pathway was active, whereas the preferred food-choices were switched off when mGluR1 or its downstream pathway was suppressed. These results identify the AC5 and mGluR system in the dorsal striatum as molecular on/off switches to direct decisions on behavioral preferences for cue-oriented options.
doi:10.1186/s13041-014-0077-7
PMCID: PMC4233066  PMID: 25378213
AC5; mGluRs; Preferences; Choices; Dorsal striatum; Sensory integration
21.  Effects of PI3Kγ overexpression in the hippocampus on synaptic plasticity and spatial learning 
Molecular Brain  2014;7(1):78.
Previous studies have shown that a family of phosphoinositide 3-kinases (PI3Ks) plays pivotal roles in the brain; in particular, we previously reported that knockout of the γ isoform of PI3K (PI3Kγ) in mice impaired synaptic plasticity and reduced behavioral flexibility. To further examine the role of PI3Kγ in synaptic plasticity and hippocampus-dependent behavioral tasks we overexpressed p110γ, the catalytic subunit of PI3Kγ, in the hippocampal CA1 region. We found that the overexpression of p110γ impairs NMDA receptor-dependent long-term depression (LTD) and hippocampus-dependent spatial learning in the Morris water maze (MWM) task. In contrast, long-term potentiation (LTP) and contextual fear memory were not affected by p110γ overexpression. These results, together with the previous knockout study, suggest that a critical level of PI3Kγ in the hippocampus is required for successful induction of LTD and normal learning.
doi:10.1186/s13041-014-0078-6
PMCID: PMC4226891  PMID: 25373491
22.  Postsynaptic insertion of AMPA receptor onto cortical pyramidal neurons in the anterior cingulate cortex after peripheral nerve injury 
Molecular Brain  2014;7(1):76.
Long-term potentiation (LTP) is the key cellular mechanism for physiological learning and pathological chronic pain. Postsynaptic accumulation of AMPA receptor (AMPAR) GluA1 plays an important role for injury-related cortical LTP. However, there is no direct evidence for postsynaptic GluA1 insertion or accumulation after peripheral injury. Here we report nerve injury increased the postsynaptic expression of AMPAR GluA1 in pyramidal neurons in the layer V of the anterior cingulate cortex (ACC), including the corticospinal projecting neurons. Electrophysiological recordings show that potentiation of postsynaptic responses was reversed by Ca2+ permeable AMPAR antagonist NASPM. Finally, behavioral studies show that microinjection of NASPM into the ACC inhibited behavioral sensitization caused by nerve injury. Our findings provide direct evidence that peripheral nerve injury induces postsynaptic GluA1 accumulation in cingulate cortical neurons, and inhibits postsynaptic GluA1 accumulation which may serve as a novel target for treating neuropathic pain.
doi:10.1186/s13041-014-0076-8
PMCID: PMC4221704  PMID: 25359681
23.  Cortical parvalbumin and somatostatin GABA neurons express distinct endogenous modulators of nicotinic acetylcholine receptors 
Molecular Brain  2014;7(1):75.
Background
Inhibition from GABAergic interneurons in brain circuits is a critical component of cognitive function. This inhibition is regulated through a diverse network of neuromodulation. A number of recent studies suggest that one of the major regulators of interneuron function is nicotinic acetylcholinergic transmission and dysregulation of both systems is common in psychiatric conditions. However, how nicotinic modulation impacts specific subpopulations of diverse GABAergic interneurons remains in question. One potential way of conferring specificity to the convergence of GABAergic and nicotinic signaling is through the expression of a unique family of nicotinic acetycholine receptor modulators, the Lynx family. The present study sought to identify members of the Lynx family enriched in cortical interneurons and to elucidate subpopulations of GABAergic neurons that express unique nicotinic modulators.
Results
We utilize double fluorescence in situ hybridization to examine the interneuronal expression of the Lynx family in adult mouse visual cortex. We find that two of the Lynx family members, Lynx1 and Lypd6, are enriched in interneuron populations in cortex. Nearly all parvalbumin interneurons express Lynx1 but we did not detect Lypd6 in this population. Conversely, in somatostatin interneurons Lypd6 was found in a subset localized to deep cortical layers but no somatostatin neurons show detectable levels of Lynx1. Using a combination of genetic and viral manipulations we further show that a subpopulation of deep-layer cortico-cortical long-range somatostatin neurons also express Lypd6.
Conclusions
This work shows that distinct subpopulations of GABAergic interneurons express unique Lynx family members. The pattern of expression of Lynx family members within interneurons places them in a unique position to potentially regulate the convergence of GABAergic and nicotinic systems, dysfunction of which are characteristic of psychiatric disorders.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0075-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0075-9
PMCID: PMC4228157  PMID: 25359633
GABA; Parvalbumin; Somatostatin; Lynx family; Lynx1; Lypd6; Nicotinic acetylcholine receptor; Visual cortex; Mouse
24.  Behavioral characterization of mice overexpressing human dysbindin-1 
Molecular Brain  2014;7(1):74.
Background
The dysbindin-1 gene (DTNBP1: dystrobrevin binding protein 1) is a promising schizophrenia susceptibility gene, known to localize almost exclusively to neurons in the brain, and participates in the regulation of neurotransmitter release, membrane-surface receptor expression, and synaptic plasticity. Sandy mice, with spontaneous Dtnbp1 deletion, display behavioral abnormalities relevant to symptoms of schizophrenia. However, it remains unknown if dysbindin-1 gain-of-function is beneficial or detrimental.
Results
To answer this question and gain further insight into the pathophysiology and therapeutic potential of dysbindin-1, we developed transgenic mice expressing human DTNBP1 (Dys1A-Tg) and analyzed their behavioral phenotypes. Dys1A-Tg mice were born viable in the expected Mendelian ratios, apparently normal and fertile. Primary screening of behavior and function showed a marginal change in limb grasping in Dys1A-Tg mice. In addition, Dys1A-Tg mice exhibited increased hyperlocomotion after methamphetamine injection. Transcriptomic analysis identified several up- and down-regulated genes, including the immediate-early genes Arc and Egr2, in the prefrontal cortex of Dys1A-Tg mice.
Conclusions
The present findings in Dys1A-Tg mice support the role of dysbindin-1 in psychiatric disorders. The fact that either overexpression (Dys1A-Tg) or underexpression (Sandy) of dysbindin-1 leads to behavioral alterations in mice highlights the functional importance of dysbindin-1 in vivo.
doi:10.1186/s13041-014-0074-x
PMCID: PMC4201722  PMID: 25298178
Dysbindin; DTNBP1; Dystrobrevin binding protein 1; Psychiatric disorder; Schizophrenia; Transgenic mice; Behavior; Methamphetamine; Phencyclidine; Immediate-early gene
25.  In vivo evidence of pathogenicity of VPS35 mutations in the Drosophila 
Molecular Brain  2014;7(1):73.
Mutations of VPS35, a component of the retromer complex have been associated with late onset familial Parkinson’s disease. The D620N mutation in VPS35 appears to be most prevalent, however, P316S was found in two cases within the same family and a control, whereas L774M was identified in 6 cases and 1 control. In vivo evidence of their pathogenicity is lacking. Here we investigated the in vivo effects of P316S, D620N and L774M using Drosophila as a model. We generated transgenic human VPS35-expressing mutations and demonstrated that VPS35 D620N transgenic flies led to late-onset loss of TH-positive DA neurons, poor mobility, shortened lifespans and increased sensitivity to rotenone, a PD-linked environmental toxin, with some of these phenotypes observed for P316S but not in L774M transgenic flies. We conclude that D620N and to a smaller extent P316S are associated with pathogenicity in PD.
Electronic supplementary material
The online version of this article (doi:10.1186/s13041-014-0073-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13041-014-0073-y
PMCID: PMC4193144  PMID: 25288323
Parkinson’s disease; Drosophila; VPS35; Retromer complex

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