RESUMO
N-Methyl-D-aspartate receptors (NMDARs) are highly expressed in brain and play important roles in neurodevelopment and various neuropathologic conditions. Here, we describe a new phenotype in an individual associated with a novel de novo deleterious variant in GRIN1 (c.1595C>A, p.Pro532His). The clinical phenotype is characterized with developmental encephalopathy, striking stimulus-sensitive myoclonus, and frontal lobe and frontal white matter hypoplasia, with no apparent seizures detected. NMDARs that contained the P532H within the glycine-binding domain of GluN1 with either the GluN2A or GluN2B subunits were evaluated for changes in their pharmacological and biophysical properties, which surprisingly revealed only modest changes in glycine potency but a significant decrease in glutamate potency, an increase in sensitivity to endogenous zinc inhibition, a decrease in response to maximally effective concentrations of agonists, a shortened synaptic-like response time course, a decreased channel open probability, and a reduced receptor cell surface expression. Molecule dynamics simulations suggested that the variant can lead to additional interactions across the dimer interface in the agonist-binding domains, resulting in a more open GluN2 agonist-binding domain cleft, which was also confirmed by single-molecule fluorescence resonance energy transfer measurements. Based on the functional deficits identified, several positive modulators were evaluated to explore potential rescue pharmacology.
RESUMO
Zn2+ has been shown to have a wide range of modulatory effects on neuronal AMPARs. However, the mechanism of modulation is largely unknown. Here we show that Zn2+ inhibits GluA2(Q) homomeric receptors in an activity- and voltage-dependent manner, indicating a pore block mechanism. The rate of inhibition is slow, in the hundreds of milliseconds at millimolar Zn2+ concentrations; hence, the inhibition is only observed in the residual nondesensitizing currents. Consequently, the inhibition is higher for GluA2 receptors in complex with auxiliary subunits γ2 and γ8 where the residual activation is larger. The extent of inhibition is also dependent on charge at site 607, the site that undergoes RNA editing in GluA2 subunits replacing glutamine to arginine, with the percent inhibition being lower and IC50 being higher for the edited GluA2(R) relative to unedited GluA2(Q) and to GluA2(Q607E), a mutation observed in the genetic screen of a patient exhibiting developmental delays. We also show that Zn2+ inhibition is significant during rapid repetitive activity with pulses of millimolar concentrations of glutamate in both receptors expressed in HEK cells as well as in native receptors in cortical neurons of C57BL/6J mice of either sex, indicating a physiological relevance of this inhibition.SIGNIFICANCE STATEMENT Zn2+ is present along with glutamate in synaptic vesicles and coreleased during synaptic transmission, modulating the postsynaptic ionotropic glutamate receptors. While Zn2+ inhibition of the NMDA subtype of the ionotropic glutamate receptors is well characterized, the mechanism of modulation of the AMPA subtype is much less known. Here we have systematically studied Zn2+ inhibition of AMPARs by varying calcium permeability, auxiliary subunits, and activation levels and show that Zn2+ inhibits AMPARs in an activity-dependent manner, opening up this pathway as a means to pharmacologically modulate the receptors.
Assuntos
Receptores de AMPA/antagonistas & inibidores , Zinco/farmacologia , Animais , Córtex Cerebral/metabolismo , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Edição de RNA , Receptores de AMPA/genética , TransfecçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that has been associated with the deposition of aggregates of superoxide dismutase 1 (SOD1). Effective therapeutics against SOD1 fibrillation is still an area of active research. Herein, we demonstrate the potential of two naturally occurring flavonoids (quercetin and baicalein) to inhibit fibrillation of wild-type SOD1 with the aid of a series of biophysical techniques. Our seeding experiments reveal that both of these flavonoids significantly affect the fibril elongation. Interestingly, our ThT binding assay, TEM, and SDS-PAGE experiments suggest that these flavonoids also disintegrate the fibrils into shorter fragments but do not completely depolymerize them into monomers. Binding parameters obtained from the analysis of UV-vis spectra suggest that these flavonoids bind moderately to native SOD1 dimer and have different binding sites. Docking of these flavonoids with a non-native monomer, non-native trimer, and oligomer derived from the 11-residue segment of SOD1 indicates that both quercetin and baicalein can bind to these species and thus can arrest the elongation of fibrils by blocking the fibrillar core regions on the intermediate species formed during aggregation of SOD1. MTT assay data revealed that both the flavonoids reduced the cytotoxicity of SOD1 fibrils. Experimental data also show the antiamyloidogenic potential of both flavonoids against A4V SOD1 mutant fibrillation. Thus, our findings may provide a direction for designing effective therapeutic agents against ALS which can act as promising antiamyloidogenic and fibril destabilizing agents.
Assuntos
Amiloide/efeitos dos fármacos , Flavanonas/farmacologia , Quercetina/farmacologia , Superóxido Dismutase-1/metabolismo , Amiloide/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Flavanonas/metabolismo , Humanos , Mutação/efeitos dos fármacos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Quercetina/metabolismo , Superóxido Dismutase/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/efeitos dos fármacosRESUMO
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons. Unfortunately, effective therapeutics against this disease is still not available. Almost 20% of familial ALS (fALS) is suggested to be associated with pathological deposition of superoxide dismutase (SOD1). Evidences suggest that SOD1-containing pathological inclusions in ALS exhibit amyloid like properties. An effective strategy to combat ALS may be to inhibit amyloid formation of SOD1 using small molecules. In the present study, we observed the fibrillation of one of the premature forms of SOD1 (SOD1 with reduced disulfide) in the presence of curcumin. Using ThT binding assay, AFM, TEM images and FTIR, we demonstrate that curcumin inhibits the DTT-induced fibrillation of SOD1 and favors the formation of smaller and disordered aggregates of SOD1. The enhancement in curcumin fluorescence on the addition of oligomers and pre-fibrillar aggregates of SOD1 suggests binding of these species to curcumin. Docking studies indicate that putative binding site of curcumin may be the amyloidogenic regions of SOD1. Further, there is a significant increase in SOD1 mediated toxicity in the regime of pre-fibrillar and fibrillar aggregates which is not evident in curcumin containing samples. All these data suggest that curcumin reduces toxicity by binding to the amyloidogenic regions of the species on the aggregation pathway and blocking the formation of the toxic species. Nanoparticles of curcumin with higher aqueous solubility show similar aggregation control as that of curcumin bulk. This suggests a potential role for curcumin in the treatment of ALS.