Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

GlialCAM/MLC1 modulates LRRC8/VRAC currents in an indirect manner: Implications for megalencephalic leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM genes. Previous work indicated that chloride currents mediated by the volume-regulated anion channel (VRAC) and ClC-2 channels were affected in astrocytes deficient in either Mlc1 or Glialcam. ClC-2 forms a ternary complex with GlialCAM and MLC1. LRRC8 proteins have been identified recently as the molecular components of VRAC, but the relationship between MLC and LRRC8 proteins is unknown. Here, we first demonstrate that LRRC8 and MLC1 are functionally linked, as MLC1 cannot potentiate VRAC currents when LRRC8A, the main subunit of VRAC, is knocked down. We determine that LRRC8A and MLC1 do not co-localize or interact and, in Xenopus oocytes, MLC1 does not potentiate LRRC8-mediated VRAC currents, indicating that VRAC modulation in astrocytes by MLC1 may be indirect. Investigating the mechanism of modulation, we find that a lack of MLC1 does not influence either mRNA or total and plasma membrane protein levels of LRRC8A; and neither does it affect LRRC8A subcellular localization. In agreement with recent results that indicated that overexpression of MLC1 decreases the phosphorylation of extracellular signal-regulated kinases (ERK), we find that astrocytes lacking MLC1 show an increase in ERK phosphorylation. In astrocytes with reduced or increased levels of MLC1 we observe changes in the phosphorylation state of the VRAC subunit LRRC8C. Our results thus reinforce previous suggestions that indicated that GlialCAM/MLC1 might modify signal transduction pathways that influence the activity of different proteins, such as VRAC.

Megalencephalic leukoencephalopathy with subcortical cysts: A personal biochemical retrospective.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy characterized by dysfunction of the role of glial cells in controlling brain fluid and ion homeostasis. Patients affected by MLC present macrocephaly, cysts and white matter vacuolation, which lead to motor and cognitive impairments. To date, there is no treatment for MLC, only supportive care. MLC is caused by mutations in the MLC1 and GLIALCAM genes. MLC1 is a membrane protein with low identity to the Kv1.1 potassium channel and GlialCAM belongs to an adhesion molecule family. Both proteins form a complex with an as-yet-unknown function that is expressed mainly in the astrocytes surrounding the blood-brain barrier and in Bergmann glia. GlialCAM also acts as an auxiliary subunit of the chloride channel ClC-2, thus regulating its localization at cell-cell junctions and modifying its functional properties by affecting the common gate of ClC-2. Recent studies in Mlc1-, GlialCAM- and Clcn2-knockout mice or Mlc1-knockout zebrafish have provided fresh insight into the pathophysiology of MLC and further details about the molecular interactions between these three proteins. Additional studies have shown that GlialCAM/MLC1 also regulates other ion channels (TRPV4, VRAC) or transporters (Na+/K+-ATPase) in a not-understood manner. Furthermore, it has been shown that GlialCAM/MLC1 may influence signal transduction mechanisms, thereby affecting other proteins not related with transport such as the EGF receptor. Here, we offer a personal biochemical retrospective of the work that has been performed to gain knowledge of the pathophysiology of MLC, and we discuss future strategies that may be used to identify therapeutic solutions for MLC patients.

Depolarization causes the formation of a ternary complex between GlialCAM, MLC1 and ClC-2 in astrocytes: implications in megalencephalic leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.

Memory Improvement in the AßPP/PS1 Mouse Model of Familial Alzheimer's Disease Induced by Carbamylated-Erythropoietin is Accompanied by Modulation of Synaptic Genes.

Neuroprotection of erythropoietin (EPO) following long-term administration is hampered by the associated undesirable effects on hematopoiesis and body weight. For this reason, we tested carbamylated-EPO (CEPO), which has no effect on erythropoiesis, and compared it with EPO in the AßPP/PS1 mouse model of familial Alzheimer's disease. Groups of 5-month old wild type (WT) and transgenic mice received chronic treatment consisting of CEPO (2,500 or 5,000 UI/kg) or EPO (2,500 U I/kg) 3 days/week for 4 weeks. Memory at the end of treatment was assessed with the object recognition test. Microarray analysis and quantitative-PCR were used for gene expression studies. No alterations in erythropoiesis were observed in CEPO-treated WT and AßPP/PS1 transgenic mice. EPO and CEPO improved memory in AßPP/PS1 animals. However, only EPO decreased amyloid-ß (Aß)plaque burden and soluble Aß(40). Microarray analysis of gene expression revealed a limited number of common genes modulated by EPO and CEPO. CEPO but not EPO significantly increased gene expression of dopamine receptors 1 and 2, and adenosine receptor 2a, and significantly down-regulated adrenergic receptor 1D and gastrin releasing peptide. CEPO treatment resulted in higher protein levels of dopamine receptors 1 and 2 in WT and AßPP/PS1 animals, whereas the adenosine receptor 2a was reduced in WT animals. The present results suggest that the improved behavior observed in AßPP/PS1 transgenic mice after CEPO treatment may be mediated, at least in part, by the observed modulation of the expression of molecules involved in neurotransmission.

Discovery of chiral cyclopropyl dihydro-alkylthio-benzyl-oxopyrimidine (S-DABO) derivatives as potent HIV-1 reverse transcriptase inhibitors with high activity against clinically relevant mutants.

The role played by stereochemistry in the C2-substituent (left part) on the S-DABO scaffold for anti-HIV-1 activity has been investigated for the first time. A series of S-DABO analogues, where the double bond in the C2-substituent is replaced by an enantiopure isosteric cyclopropyl moiety, has been synthesized, leading to the identification of a potent lead compound endowed with picomolar activity against RT (wt) and nanomolar activity against selected drug-resistant mutants. Molecular modeling calculation, enzymatic studies, and surface plasmon resonance experiments allowed us to rationalize the biological behavior of the synthesized compounds, which act as mixed-type inhibitors of HIV-1 RT K103N, with a preferential association to the enzyme-substrate complex. Taken together, our data show that the right combination of stereochemistry on the left and right parts (C6-substituent) of the S-DABO scaffold plays a key role in the inhibition of both wild-type and drug-resistant enzymes, especially the K103N mutant.

Anti-HIV activity and resistance profile of the CXC chemokine receptor 4 antagonist POL3026.

We have studied the mechanism of action of Arg(*)-Arg-Nal(2)-Cys(1x)-Tyr-Gln-Lys-(d-Pro)-Pro-Tyr-Arg-Cit-Cys(1x)-Arg-Gly-(d-Pro)(*) (POL3026), a novel specific beta-hairpin mimetic CXC chemokine receptor (CXCR)4 antagonist. POL3026 specifically blocked the binding of anti-CXCR4 monoclonal antibody 12G5 and the intracellular Ca(2+) signal induced by CXC chemokine ligand 12. POL3026 consistently blocked the replication of human immunodeficiency virus (HIV), including a wide panel of X4 and dualtropic strains and subtypes in several culture models, with 50% effective concentrations (EC(50)) at the subnanomolar range, making POL3026 the most potent CXCR4 antagonist described to date. However, 1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)-resistant and stromal cell-derived factor-1alpha-resistant HIV-1 strains were cross-resistant to POL3026. Time of addition experiments and a multiparametric evaluation of HIV envelope function in the presence of test compounds confirmed the activity of POL3026 at an early step of virus replication: interaction with the coreceptor. Generation of HIV-1 resistance to POL3026 led to the selection of viruses 12- and 25-fold less sensitive and with mutations in gp120, including the V3 loop region. However, POL3026 prevented the emergence of CXCR4-using variants from an R5 HIV-1 strain that may occur in the presence of anti-HIV agents targeting CC chemokine receptor 5.

HIV-1 escape to CCR5 coreceptor antagonism through selection of CXCR4-using variants in vitro.

BACKGROUND: HIV-1 coreceptor switch from CCR5 to CXCR4 is associated with disease progression and AIDS. Selection of resistant HIV-1 to CCR5 agents in cell culture has often occurred in the absence of coreceptor switch. With CCR5 antagonists currently in clinical trials, their impact on coreceptor use is still in doubt. METHODS: Six R5 HIV-1 strains were passaged in lymphoid cells expressing high CXCR4 and low CCR5, in the absence or presence of CCR5 inhibitors (TAK-779, mAb 2D7 and CCL5). AMD3100, zidovudine and lamivudine were used as controls. Phenotype and genotype changes as well as virus coreceptor use were evaluated. RESULTS: In the absence of drug pressure, three out of six strains expanded their coreceptor use to CXCR4 at different times, suggesting that not all virus strains had the capacity to do so. Lowering the replication rate with a suboptimal concentration of different anti-HIV agents (reverse transcriptase inhibitors or CCR5 agents) delayed coreceptor switch. However, virus breakthrough was observed earlier in the presence of CCR5-targeting agents than in presence of reverse transcriptase inhibitors and was associated with a change in sensitivity to TAK-779 or AMD3100, virus coreceptor expansion to CXCR4 and changes in the V3 loop region of gp120. CONCLUSION: Our results suggest that HIV-1 may escape CCR5 drug pressure through coreceptor switch. Experimental conditions strongly determine the outcome of CCR5 drug pressure in cell culture. A cell culture model of the evolution of HIV-1 coreceptor use may be relevant to assess the propensity of clinical isolates to develop resistance through coreceptor change.

Multiparametric assay to screen and dissect the mode of action of anti-human immunodeficiency virus envelope drugs.

A flow cytometry-based assay was used to simultaneously quantify X4 and R5 human immunodeficiency virus (HIV) envelope-mediated cell-to-cell viral transfer, cell death, and cell-to-cell fusion. In this assay, different anti-HIV envelope drugs showed characteristic inhibitory profiles for each measured parameter, allowing for the rapid identification of the mode of action of active compounds.