CCR5 deficiency normalizes TIMP levels, working memory, and gamma oscillation power in APOE4 targeted replacement mice.

The APOE4 allele increases the risk for Alzheimer's disease (AD) in a dose-dependent manner and is also associated with cognitive decline in non-demented elderly controls. In mice with targeted gene replacement (TR) of murine APOE with human APOE3 or APOE4, the latter show reduced neuronal dendritic complexity and impaired learning. APOE4 TR mice also show reduced gamma oscillation power, a neuronal population activity which is important to learning and memory. Published work has shown that brain extracellular matrix (ECM) can reduce neuroplasticity as well as gamma power, while attenuation of ECM can instead enhance this endpoint. In the present study we examine human cerebrospinal fluid (CSF) samples from APOE3 and APOE4 individuals and brain lysates from APOE3 and APOE4 TR mice for levels of ECM effectors that can increase matrix deposition and restrict neuroplasticity. We find that CCL5, a molecule linked to ECM deposition in liver and kidney, is increased in CSF samples from APOE4 individuals. Levels of tissue inhibitor of metalloproteinases (TIMPs), which inhibit the activity of ECM-degrading enzymes, are also increased in APOE4 CSF as well as astrocyte supernatants brain lysates from APOE4 TR mice. Importantly, as compared to APOE4/wild-type heterozygotes, APOE4/CCR5 knockout heterozygotes show reduced TIMP levels and enhanced EEG gamma power. The latter also show improved learning and memory, suggesting that the CCR5/CCL5 axis could represent a therapeutic target for APOE4 individuals.

cAMP is an allosteric modulator of DNA-binding specificity in the cAMP receptor protein from Mycobacterium tuberculosis.

Allosteric proteins with multiple subunits and ligand-binding sites are central in regulating biological signals. The cAMP receptor protein from Mycobacterium tuberculosis (CRPMTB) is a global regulator of transcription composed of two identical subunits, each one harboring structurally conserved cAMP- and DNA-binding sites. The mechanisms by which these four binding sites are allosterically coupled in CRPMTB remain unclear. Here, we investigate the binding mechanism between CRPMTB and cAMP, and the linkage between cAMP and DNA interactions. Using calorimetric and fluorescence-based assays, we find that cAMP binding is entropically driven and displays negative cooperativity. Fluorescence anisotropy experiments show that apo-CRPMTB forms high-order CRPMTB-DNA oligomers through interactions with nonspecific DNA sequences or preformed CRPMTB-DNA complexes. Moreover, we find that cAMP prevents and reverses the formation of CRPMTB-DNA oligomers, reduces the affinity of CRPMTB for nonspecific DNA sequences, and stabilizes a 1-to-1 CRPMTB-DNA complex, but does not increase the affinity for DNA like in the canonical CRP from Escherichia coli (CRPEcoli). DNA-binding assays as a function of cAMP concentration indicate that one cAMP molecule per homodimer dissociates high-order CRPMTB-DNA oligomers into 1-to-1 complexes. These cAMP-mediated allosteric effects are lost in the double-mutant L47P/E178K found in CRP from Mycobacterium bovis Bacille Calmette-Guérin (CRPBCG). The functional behavior, thermodynamic stability, and dimerization constant of CRPBCG are not due to additive effects of L47P and E178K, indicating long-range interactions between these two sites. Altogether, we provide a previously undescribed archetype of cAMP-mediated allosteric regulation that differs from CRPEcoli, illustrating that structural homology does not imply allosteric homology.

Expression and secretion of apoE isoforms in astrocytes and microglia during inflammation.

Neuroinflammation is a common feature in neurodegenerative diseases, modulated by the Alzheimer's disease risk factor, apolipoprotein E (APOE). In the brain, apoE protein is synthesized by astrocytes and microglia. We examined primary cultures of astrocytes and microglia from human APOE (E2, E3, and E4) targeted-replacement mice. Astrocytes secreted two species of apoE, whereas cellular apoE consisted of only one. Both forms of secreted astrocytic apoE were bound during glycoprotein isolation, and enzymatic removal of glycans produced a convergence of the two forms of apoE to a single form; thus, the two species of astrocyte-secreted apoE are differentially glycosylated. Microglia released only a single species of apoE, while cellular apoE consisted of two forms; the secreted apoE and one of the two forms of cellular apoE were glycosylated. We treated the primary glia with either endogenous (TNFα) or exogenous (LPS) pro-inflammatory stimuli. While LPS had no effect on astrocytic apoE, APOE2, and APOE3 microglia increased release of apoE; APOE4 microglia showed no effect. APOE4 microglia showed higher baseline secretion of TNFα compared to APOE2 and APOE3 microglia. TNFα treatment reduced the secretion and cellular expression of apoE only in APOE4 astrocytes. The patterns of apoE species produced by astrocytes and microglia were not affected by inflammation. No changes in APOE mRNA were observed in astrocytes after both treatments. Together, our data demonstrate that astrocytes and microglia differentially express and secrete glycosylated forms of apoE and that APOE4 astrocytes and microglia are deficient in immunomodulation compared to APOE2 and APOE3.

ApoE Lipidation as a Therapeutic Target in Alzheimer's Disease.

Apolipoprotein E (APOE) is the major cholesterol carrier in the brain, affecting various normal cellular processes including neuronal growth, repair and remodeling of membranes, synaptogenesis, clearance and degradation of amyloid ß (Aß) and neuroinflammation. In humans, the APOE gene has three common allelic variants, termed E2, E3, and E4. APOE4 is considered the strongest genetic risk factor for Alzheimer's disease (AD), whereas APOE2 is neuroprotective. To perform its normal functions, apoE must be secreted and properly lipidated, a process influenced by the structural differences associated with apoE isoforms. Here we highlight the importance of lipidated apoE as well as the APOE-lipidation targeted therapeutic approaches that have the potential to correct or prevent neurodegeneration. Many of these approaches have been validated using diverse cellular and animal models. Overall, there is great potential to improve the lipidated state of apoE with the goal of ameliorating APOE-associated central nervous system impairments.

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery.

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals.

Glial- and Neuronal-Specific Expression of CCL5 mRNA in the Rat Brain.

Chemokine (C-C motif) ligand 5 (CCL5) belongs to a group of chemokines that play a role in the peripheral immune system, mostly as chemoattractant molecules, and mediate tactile allodynia. In the central nervous system (CNS), CCL5 and its receptors have multiple functions, including promoting neuroinflammation, insulin signaling, neuromodulator of synaptic activity and neuroprotection against a variety of neurotoxins. Evidence has also suggested that this chemokine may regulate opioid response. The multifunctional profile of CCL5 might correlate with its ability to bind different chemokine receptors, as well as with its unique cellular expression. In this work, we have used fluorescence in situ hybridization combined with immunohistochemistry to examine the expression profile of CCL5 mRNA in the adult rat brain and provide evidence of its cellular localization. We have observed that the highest expression of CCL5 mRNA occurs in all major fiber tracts, including the corpus callosum, anterior commissure, and cerebral peduncle. In these tracts, CCL5 mRNA was localized in oligodendrocytes, astrocytes and microglia. Astrocytic and microglial expression was also evident in several brain areas including the cerebral cortex, caudate/putamen, hippocampus, and thalamus. Furthermore, using a specific neuronal marker, we observed CCL5 mRNA expression in discrete layers of the cortex and hippocampus. Interestingly, in the midbrain, CCL5 mRNA co-localized with tyrosine hydroxylase (TH) positive cells of the ventral tegmental area, suggesting that CCL5 might be expressed by a subset of dopaminergic neurons of the mesolimbic system. The expression of CCL5 mRNA and protein, together with its receptors, in selected brain cell populations proposes that this chemokine could be involved in neuronal/glial communication.

Ethanol-mediated facilitation of AMPA receptor function in the dorsomedial striatum: implications for alcohol drinking behavior.

We found previously that acute ex vivo as well as repeated cycles of in vivo ethanol exposure and withdrawal, including excessive voluntary consumption of ethanol, produces a long-lasting increase in the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in the dorsomedial striatum (DMS) of rats (Wang et al., 2010a). Activation of NMDARs is required for the induction of long-term potentiation (LTP) of AMPA receptor (AMPAR)-mediated synaptic response. We therefore examined whether the ethanol-mediated upregulation of NMDAR activity alters the induction of LTP in the DMS. We found that ex vivo acute exposure of striatal slices to, and withdrawal from, ethanol facilitates the induction of LTP in DMS neurons, which is abolished by the inhibition of NR2B-NMDARs. We also report that repeated systemic administration of ethanol causes an NR2B-NMDAR-dependent facilitation of LTP in the DMS. LTP is mediated by the insertion of AMPAR subunits into the synaptic membrane, and we found that repeated systemic administration of ethanol, as well as cycles of excessive ethanol consumption and withdrawal, produced a long-lasting increase in synaptic localization of the GluR1 and GluR2 subunits of AMPARs in the DMS. Importantly, we report that inhibition of AMPARs in the DMS attenuates operant self-administration of ethanol, but not of sucrose. Together, our data suggest that aberrant synaptic plasticity in the DMS induced by repeated cycles of ethanol exposure and withdrawal contributes to the molecular mechanisms underlying the development and/or maintenance of excessive ethanol consumption.

Ethanol-induced increase in Fyn kinase activity in the dorsomedial striatum is associated with subcellular redistribution of protein tyrosine phosphatase α.

In vivo exposure of rodents to ethanol leads to a long-lasting increase in Fyn kinase activity in the dorsomedial striatum (DMS). In this study, we set out to identify a molecular mechanism that contributes to the enhancement of Fyn activity in response to ethanol in the DMS. Protein tyrosine phosphatase α (PTPα) positively regulates the activity of Fyn, and we found that repeated systemic administration or binge drinking of ethanol results in an increase in the synaptic localization of PTPα in the DMS, the same site where Fyn resides. We also demonstrate that binge drinking of ethanol leads to an increase in Fyn activity and to the co-localization of Fyn and PTPα in lipid rafts in the DMS. Finally, we show that the level of tyrosine phosphorylated (and thus active) PTPα in the synaptic fractions is increased in response to contingent or non-contingent exposure of rats to ethanol. Together, our results suggest that the redistribution of PTPα in the DMS into compartments where Fyn resides is a potential mechanism by which the activity of the kinase is increased upon ethanol exposure. Such neuroadaptations could be part of a mechanism that leads to the development of excessive ethanol consumption.

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum.

We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS). We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits. In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.

Quantification of RNA editing of the serotonin 2C receptor (5-HT(2C)R) ex vivo.

The 5-HT(2C)R receptor (5-HT(2C)R) exerts tonic and phasic inhibitory influence over brain circuitry, and dysregulation of this influence contributes to the neurochemical underpinnings in the etiology of a variety of neuropsychiatric disorders including addiction, depression, and schizophrenia. A strategically important regulator of the 5-HT(2C)R function and protein diversity is mRNA editing, a type of posttranscriptional modification that alters codon identity and thus the translation of distinct, though closely related, isoforms of 5-HT(2C)R from a single, original transcript. The 5-HT(2C)R mRNA can be edited at five closely spaced sites, altering the identity of up to three amino acids in the predicted second intracellular loop of the receptor to modulate receptor:G-protein coupling and constitutive activity. Methods to study changes in mRNA editing based upon direct DNA sequencing are both time and labor intensive. To streamline the acquisition of mRNA editing data and improve quantification, we have adapted real-time reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantify mRNA editing in 5-HT(2C)R transcripts by utilizing TaqMan® probes modified with a minor groove binder (MGB). The method is very sensitive, detecting as little as 10⁻¹8 g (1 attogram) of standard cDNA template and can discriminate closely related 5-HT(2C)R mRNA edited isoforms. This technique expands the breadth of available quantification methods for mRNA editing and is particularly useful for the ex vivo analyses of mRNA editing of the 5-HT(2C)R by allowing the rapid collection of data on large numbers of tissue samples. In addition, the general technique can be adapted easily to investigate edited mRNA from other genes, thus facilitating the development of a broader knowledge base of the physiological role of mRNA editing.

Long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum play a crucial role in alcohol consumption and relapse.

A growing number of studies suggest that the development of compulsive drug seeking and taking depends on dorsostriatal mechanisms. We previously observed that ex vivo acute exposure of the dorsal striatum to, and withdrawal from, alcohol induces long-term facilitation (LTF) of the activity of NR2B-containing NMDA receptors (NR2B-NMDARs) in a mechanism that requires the Src family protein tyrosine kinase (PTK), Fyn (Wang et al., 2007). In the present study, we first compared alcohol's actions in rat dorsomedial (DMS) and the dorsolateral (DLS) subregions of the striatum, which differ in their anatomical connectivity and function. We found that alcohol-mediated induction of LTF of NR2B-NMDAR activity is centered in the DMS. Next, we tested whether in vivo exposure of rats to alcohol leads to long-term adaptations of the NMDAR system in the DMS. We observed that repeated daily administration of alcohol results in a long-lasting increase in the activity of the NR2B-NMDARs in the DMS. The same procedure leads to a prolonged activation of Fyn, increased NR2B phosphorylation, and membrane localization of the subunit. Importantly, similar electrophysiological and biochemical modifications were observed in the DMS of rats that consumed large quantities of alcohol. Finally, we show that inhibition of NR2B-NMDARs or Src family PTKs in the DMS, but not in the DLS, significantly decreases operant self-administration of alcohol and reduces alcohol-priming-induced reinstatement of alcohol seeking. Our results suggest that the upregulation of NR2B-NMDAR activity within the DMS by alcohol contributes to the maladaptive synaptic changes that lead to excessive alcohol intake and relapse.

Serotonin 5-HT2C receptor protein expression is enriched in synaptosomal and post-synaptic compartments of rat cortex.

The action of serotonin (5-HT) at the 5-HT(2C) receptor (5-HT(2C)R) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5-HT(2C)R protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5-HT(2C)R in synaptosomes and the post-synaptic density, an electron-dense thickening specialized for post-synaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, post-synaptic density), and controls (peptide neutralization, 5-HT(2C)R stably-expressing cells), we established the selectivity of two commercially available 5-HT(2C)R antibodies and employed the antibodies in western blot and immunoprecipitation studies of prefrontal cortex (PFC) and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5-HT(2C)R in post-synaptic density-enriched fractions from both PFC and motor cortex. Co-immunoprecipitation studies revealed the presence of post-synaptic density-95 within the 5-HT(2C)R protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5-HT(2C)R is localized within the post-synaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.

An innovative real-time PCR method to measure changes in RNA editing of the serotonin 2C receptor (5-HT(2C)R) in brain.

The serotonin 2C receptor (5-HT(2C)R) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT(2C)R is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT(2C)R RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT(2C)R isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT(2C)R RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT(2C)R transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT(2C)R profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT(2C)R mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.