-
Fluorescence lifetime imaging of AMPA receptor endocytosis in living neurons: effects of Aβ and PP1.Frontiers in Molecular Neuroscience 2024The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the...
The relative amount of AMPA receptors expressed at the surface of neurons can be measured using superecliptic pHluorin (SEP) labeling at their N-terminus. However, the high signal variability resulting from protein overexpression in neurons and the low signal observed in intracellular vesicles make quantitative characterization of receptor trafficking difficult. Here, we establish a real-time live-cell assay of AMPAR trafficking based on fluorescence lifetime imaging (FLIM), which allows for simultaneous visualization of both surface and intracellular receptors. Using this assay, we found that elevating amyloid-beta (Aβ) levels leads to a strong increase in intracellular GluA1 and GluA2-containing receptors, indicating that Aβ triggers the endocytosis of these AMPARs. In APP/PS1 Alzheimer's disease model mouse neurons, FLIM revealed strikingly different AMPAR trafficking properties for GluA1- and GluA3-containing receptors, suggesting that chronic Aβ exposure triggered the loss of both surface and intracellular GluA3-containing receptors. Interestingly, overexpression of protein phosphatase 1 (PP1) also resulted in GluA1 endocytosis as well as depressed synaptic transmission, confirming the important role of phosphorylation in regulating AMPAR trafficking. This new approach allows for the quantitative measurement of extracellular pH, small changes in receptor trafficking, as well as simultaneous measurement of surface and internalized AMPARs in living neurons, and could therefore be applied to several different studies in the future.
PubMed: 38915938
DOI: 10.3389/fnmol.2024.1409401 -
Neuroscience Letters Jun 2024To investigate the precise mechanism of xenon (Xe), pharmacologically isolated AMPA/KA and NMDA receptor-mediated spontaneous (s) and evoked (e) excitatory postsynaptic...
To investigate the precise mechanism of xenon (Xe), pharmacologically isolated AMPA/KA and NMDA receptor-mediated spontaneous (s) and evoked (e) excitatory postsynaptic currents (s/eEPSC and s/eEPSC) were recorded from mechanically isolated single spinal sacral dorsal commissural nucleus (SDCN) neurons attached with glutamatergic nerve endings (boutons) using conventional whole-cell patch-clamp technique. We analysed kinetic properties of both s/eEPSC and s/eEPSC by focal single- and/or paired-pulse electrical stimulation to compare them. The s/eEPSC showed smaller amplitude, slower rise time, and slower 1/e decay time constant (τ) than those of s/eEPSC. We previously examined how Xe modulates s/eEPSC, therefore, examined the effects on s/eEPSC in the present study. Xe decreased the frequency and amplitude of sEPSC, and decreased the amplitude but increased the failure rate and paired-pulse ratio of eEPSC without affecting their τ. It was concluded that Xe might suppress NMDA receptor-mediated synaptic transmission via both presynaptic and postsynaptic mechanisms.
PubMed: 38914276
DOI: 10.1016/j.neulet.2024.137885 -
Proceedings of the National Academy of... Jun 2024MDGA (MAM domain containing glycosylphosphatidylinositol anchor) family proteins were previously identified as synaptic suppressive factors. However, various genetic...
MDGA (MAM domain containing glycosylphosphatidylinositol anchor) family proteins were previously identified as synaptic suppressive factors. However, various genetic manipulations have yielded often irreconcilable results, precluding precise evaluation of MDGA functions. Here, we found that, in cultured hippocampal neurons, conditional deletion of MDGA1 and MDGA2 causes specific alterations in synapse numbers, basal synaptic transmission, and synaptic strength at GABAergic and glutamatergic synapses, respectively. Moreover, MDGA2 deletion enhanced both N-methyl-D-aspartate (NMDA) receptor- and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated postsynaptic responses. Strikingly, ablation of both MDGA1 and MDGA2 abolished the effect of deleting individual MDGAs that is abrogated by chronic blockade of synaptic activity. Molecular replacement experiments further showed that MDGA1 requires the meprin/A5 protein/PTPmu (MAM) domain, whereas MDGA2 acts via neuroligin-dependent and/or MAM domain-dependent pathways to regulate distinct postsynaptic properties. Together, our data demonstrate that MDGA paralogs act as unique negative regulators of activity-dependent postsynaptic organization at distinct synapse types, and cooperatively contribute to adjustment of excitation-inhibition balance.
Topics: Animals; Synapses; Mice; Hippocampus; Synaptic Transmission; Neurons; Receptors, N-Methyl-D-Aspartate; Mice, Knockout; Receptors, AMPA; Cell Adhesion Molecules, Neuronal; Nerve Tissue Proteins; Membrane Proteins; Cells, Cultured
PubMed: 38900791
DOI: 10.1073/pnas.2322978121 -
Journal of Neurochemistry Jun 2024Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders....
Kelch-like family member 17 (KLHL17), an actin-associated adaptor protein, is linked to neurological disorders, including infantile spasms and autism spectrum disorders. The key morphological feature of Klhl17-deficient neurons is impaired dendritic spine enlargement, resulting in the amplitude of calcium events being increased. Our previous studies have indicated an involvement of F-actin and the spine apparatus in KLHL17-mediated dendritic spine enlargement. Here, we show that KLHL17 further employs different mechanisms to control the expression of two types of glutamate receptors, that is, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and kainate receptors (KARs), to regulate dendritic spine enlargement and calcium influx. We deployed proteomics to reveal that KLHL17 interacts with N-ethylmaleimide-sensitive fusion protein (NSF) in neurons, with this interaction of KLHL17 and NSF enhancing NSF protein levels. Consistent with the function of NSF in regulating the surface expression of AMPAR, Klhl17 deficiency limits the surface expression of AMPAR, but not its total protein levels. The NSF pathway also contributes to synaptic F-actin distribution and the dendritic spine enlargement mediated by KLHL17. KLHL17 is known to act as an adaptor mediating degradation of the KAR subunit GluK2 by the CUL3 ubiquitin ligase complex, and Klhl17 deficiency impairs activity-dependent degradation of GluK2. Herein, we further demonstrate that GluK2 is critical to the increased amplitude of calcium influx in Klhl17-deficient neurons. Moreover, GluK2 is also involved in KLHL17-regulated dendritic spine enlargement. Thus, our study reveals that KLHL17 controls AMPAR and KAR expression via at least two mechanisms, consequently regulating dendritic spine enlargement. The regulatory effects of KLHL17 on these two glutamate receptors likely contribute to neuronal features in patients suffering from certain neurological disorders.
PubMed: 38898681
DOI: 10.1111/jnc.16148 -
Neuropsychopharmacology : Official... Jun 2024Synaptic plasticity occurs via multiple mechanisms to regulate synaptic efficacy. Homeostatic and Hebbian plasticity are two such mechanisms by which neuronal synapses...
Synaptic plasticity occurs via multiple mechanisms to regulate synaptic efficacy. Homeostatic and Hebbian plasticity are two such mechanisms by which neuronal synapses can be altered. Although these two processes are mechanistically distinct, they converge on downstream regulation of AMPA receptor activity to modify glutamatergic neurotransmission. However, much remains to be explored regarding how these two prominent forms of plasticity interact. Ketamine, a rapidly acting antidepressant, increases glutamatergic transmission via pharmacologically-induced homeostatic plasticity. Here, we demonstrate that Hebbian plasticity mechanisms are still intact in synapses that have undergone homeostatic scaling by ketamine after either systemic injection or perfusion onto hippocampal brain slices. We also investigated this relationship in the context of stress induced by chronic exposure to corticosterone (CORT) to better model the circumstances under which ketamine may be used as an antidepressant. We found that CORT induced an anhedonia-like behavioral phenotype in mice but did not impair long-term potentiation (LTP) induction. Furthermore, corticosterone exposure does not impact the intersection of homeostatic and Hebbian plasticity mechanisms, as synapses from CORT-exposed mice also demonstrated intact ketamine-induced plasticity and LTP in succession. These results provide a mechanistic explanation for how ketamine used for the treatment of depression does not impair the integrity of learning and memory processes encoded by mechanisms such as LTP.
PubMed: 38898206
DOI: 10.1038/s41386-024-01895-2 -
International Journal of Molecular... May 2024Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disorder. While there are five FDA-approved drugs for treating this disease, each has only modest benefits. To design new and more effective therapies for ALS, particularly for sporadic ALS of unknown and diverse etiologies, we must identify key, convergent mechanisms of disease pathogenesis. This review focuses on the origin and effects of glutamate-mediated excitotoxicity in ALS (the cortical hyperexcitability hypothesis), in which increased glutamatergic signaling causes motor neurons to become hyperexcitable and eventually die. We characterize both primary and secondary contributions to excitotoxicity, referring to processes taking place at the synapse and within the cell, respectively. '' include upregulation of calcium-permeable AMPA receptors, dysfunction of the EAAT2 astrocytic glutamate transporter, increased release of glutamate from the presynaptic terminal, and reduced inhibition by cortical interneurons-all of which have been observed in ALS patients and model systems. '' include changes to mitochondrial morphology and function, increased production of reactive oxygen species, and endoplasmic reticulum (ER) stress. By identifying key targets in the excitotoxicity cascade, we emphasize the importance of this pathway in the pathogenesis of ALS and suggest that intervening in this pathway could be effective for developing therapies for this disease.
Topics: Amyotrophic Lateral Sclerosis; Humans; Glutamic Acid; Animals; Motor Neurons; Aging; Receptors, AMPA; Endoplasmic Reticulum Stress; Mitochondria; Excitatory Amino Acid Transporter 2; Astrocytes; Reactive Oxygen Species
PubMed: 38891774
DOI: 10.3390/ijms25115587 -
The Journal of Neuroscience : the... Jun 2024Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these...
Calcineurin inhibitors, such as cyclosporine and tacrolimus (FK506), are commonly used immunosuppressants for preserving transplanted organs and tissues. However, these drugs can cause severe and persistent pain. GluA2-lacking, calcium-permeable AMPA receptors (CP-AMPARs) are implicated in various neurological disorders, including neuropathic pain. It is unclear whether and how constitutive calcineurin, a Ca/calmodulin protein phosphatase, controls synaptic CP-AMPARs. In this study, we found that blocking CP-AMPARs with IEM-1460 markedly reduced the amplitude of AMPAR-EPSCs in excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2), but not in inhibitory neurons expressing vesicular GABA transporter, in the spinal cord of FK506-treated male and female mice. FK506 treatment also caused an inward rectification in the current-voltage relationship of AMPAR-EPSCs specifically in VGluT2 neurons. Intrathecal injection of IEM-1460 rapidly alleviated pain hypersensitivity in FK506-treated mice. Furthermore, FK506 treatment substantially increased physical interaction of α2δ-1 with GluA1 and GluA2 in the spinal cord and reduced GluA1/GluA2 heteromers in endoplasmic reticulum-enriched fractions of spinal cords. Correspondingly, inhibiting α2δ-1 with pregabalin, genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C-terminus peptide reversed inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons caused by FK506 treatment. In addition, CK2 inhibition reversed FK506 treatment-induced pain hypersensitivity, α2δ-1 interactions with GluA1 and GluA2, and inward rectification of AMPAR-EPSCs in spinal VGluT2 neurons. Thus, the increased prevalence of synaptic CP-AMPARs in spinal excitatory neurons plays a major role in calcineurin inhibitor-induced pain hypersensitivity. Calcineurin and CK2 antagonistically regulate postsynaptic CP-AMPARs through α2δ-1-mediated GluA1/GluA2 heteromeric assembly in the spinal dorsal horn. Clinically used calcineurin inhibitors can cause severe pain, known as calcineurin inhibitor-induced pain syndrome (CIPS). However, its underlying mechanisms remain elusive. This study shows for the first time that calcineurin inhibition caused cell type-specific expression of synaptic Ca-permeable AMPARs in spinal cord excitatory neurons. Blocking spinal Ca-permeable AMPARs reduced CIPS. Calcineurin inhibition potentiated the α2δ-1 (previously known as a calcium channel subunit) interaction with GluA1 and GluA2 subunits, disrupting their intracellular assembly in the spinal cord. Additionally, inhibiting spinal CK2 diminished α2δ-1-AMPAR interactions and synaptic Ca-permeable AMPARs augmented by calcineurin inhibitors. Thus, calcineurin and CK2 dynamically control AMPAR phenotypes in spinal excitatory neurons through α2δ-1-mediated GluA1/GluA2 assembly. Targeting α2δ-1 and CK2 are effective strategies for treating CIPS.
PubMed: 38886057
DOI: 10.1523/JNEUROSCI.0392-24.2024 -
Zhurnal Nevrologii I Psikhiatrii Imeni... 2024Depression is a leading cause of disability and reduced work capacity worldwide. The monoamine theory of the pathogenesis of depression has remained dominant for many... (Review)
Review
Depression is a leading cause of disability and reduced work capacity worldwide. The monoamine theory of the pathogenesis of depression has remained dominant for many decades, however, drugs developed on its basis have limited efficacy. Exploring alternative mechanisms underlying this pathology could illuminate new avenues for pharmacological intervention. Targeting glutamatergic pathways in the CNS, particularly through modulation of NMDA and AMPA receptors, demonstrates promising results. This review presents some existing drugs with glutamatergic activity and novel developments based on it to enhance the efficacy of pharmacotherapy for depressive disorders.
Topics: Humans; Receptors, N-Methyl-D-Aspartate; Receptors, AMPA; Depressive Disorder; Antidepressive Agents; Animals
PubMed: 38884426
DOI: 10.17116/jnevro202412405122 -
Molecular Autism Jun 2024Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and...
BACKGROUND
Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5 rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5 rats.
METHODS
To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology.
RESULTS
Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5 rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca permeable AMPA receptor mediated currents are unchanged in Cdkl5 rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses.
CONCLUSIONS
Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity.
LIMITATIONS
This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.
Topics: Animals; Long-Term Potentiation; Receptors, N-Methyl-D-Aspartate; Receptors, AMPA; Spasms, Infantile; Disease Models, Animal; Rats; Protein Serine-Threonine Kinases; Hippocampus; Pyramidal Cells; Male; CA1 Region, Hippocampal; Epileptic Syndromes; Genetic Diseases, X-Linked; Synapses; Excitatory Postsynaptic Potentials
PubMed: 38877552
DOI: 10.1186/s13229-024-00601-9 -
PCN Reports : Psychiatry and Clinical... Dec 2023The domain of psychiatric drug development is currently witnessing a notable transformation, with a paramount emphasis on targeting nonmonoamine receptors and exploring... (Review)
Review
The domain of psychiatric drug development is currently witnessing a notable transformation, with a paramount emphasis on targeting nonmonoamine receptors and exploring inventive mechanisms of action. This paper presents an overview of the ongoing advancements in antipsychotic and antidepressant drug development. Historically, antipsychotics predominantly targeted dopamine receptors, but there is now an escalating interest in drugs that act on alternative receptors, exemplified by the TAAR1 receptor. One noteworthy candidate is Ulotaront (SEP-363856), an agent acting as a TAAR1 agonist with 5-HT1A agonist activity, demonstrating promising outcomes in the treatment of schizophrenia, devoid of extrapyramidal symptoms or metabolic side-effects. Similarly, MIN-101 (Roluperidone) and KarXT are currently in development, with its focus on addressing the symptoms in schizophrenia. In the domain of antidepressants, novel therapeutic approaches have surfaced, such as Auvelity, a Food and Drug Administration (FDA)-approved NMDA receptor antagonist synergistically combined with Bupropion to enhance its effects. Another notable candidate is Zuranolone, operating as a GABA A receptor-positive allosteric modulator, showcasing efficacy in treating major depressive disorder (MDD) and postpartum depression. Additionally, TAK-653 (NBI-1065845) and MJI821 (Onfasprodil) have emerged as potential antidepressants targeting AMPA receptors and NMDA receptor 2B (NR2B) negative allosteric modulation, respectively. This paper underscores the transformative potential of these novel drug candidates in psychiatric treatment and their ability to address cases that were previously treatment-resistant. By focusing on nonmonoamine receptors and introducing innovative mechanisms, these drugs offer a promising prospect of improved outcomes for individuals suffering from schizophrenia and MDD. Thus, sustained attention and dedication to the development of such drugs are essential to augmenting the therapeutic options available for psychiatric patients.
PubMed: 38868733
DOI: 10.1002/pcn5.157