A primate-specific short GluN2A-NMDA receptor isoform is expressed in the human brain

Glutamate receptors of the N-methyl-D-aspartate (NMDA) family are coincident detectors of pre- and postsynaptic activity, allowing Ca2+ influx into neurons. These properties are central to neurological disease mechanisms and are proposed to be the basis of associative learning and memory. In addition to the well-characterised canonical GluN2A NMDAR isoform, large-scale open reading frames in human tissues had suggested the expression of a primate-specific short GluN2A isoform referred to as GluN2A-S. Here, we confirm the expression of both GluN2A transcripts in human and primate but not rodent brain tissue, and show that they are translated to two corresponding GluN2A proteins present in human brain. Furthermore, we demonstrate that recombinant GluN2A-S co-assembles with the obligatory NMDAR subunit GluN1 to form functional NMDA receptors. These findings suggest a more complex NMDAR repertoire in human brain than previously thought

Pathogenic tau does not drive activation of the unfolded protein response

The unfolded protein response (UPR) is commonly associated with a range of neurodegenerative diseases, and targeting UPR components has been suggested as a therapeutic strategy. The UPR surveys protein folding within the endoplasmic reticulum (ER). However, many of the misfolded proteins that accumulate in neurodegeneration are localized such that they do not directly cause ER triggers that activate this pathway. Here, using a transgenic mouse model and primary cell cultures, along with qPCR, immunoblotting and immunohistochemistry, we tested whether UPR is induced in in vivo andin vitro murine models of tauopathy that are based on expression of mutant tauP301L. We found no evidence for UPR in the rTg4510 mouse model in which mutant tau is transgenically expressed under control oftetracycline-controlled transactivator protein (tTA). This observation was supported by results from acute experiments in which neuronal cultures expressed mutant tau and accumulated misfolded cytoplasmic tau aggregates, but exhibited no UPR activation. These results suggest that the UPR is not induced as a response to tau misfolding and aggregation, despite clear evidence for progressive cellular dysfunction and degeneration. We propose that caution is needed when evaluating the implied significance of the UPR as a critical determinant across major neurodegenerative diseases

Cellular responses to halofuginone reveal a vulnerability of the GCN2 branch of the integrated stress response.

Funder: EC | FP7 | FP7 Ideas: European Research Council (IDEE‐CER); Id: http://dx.doi.org/10.13039/100011199; Grant(s): FP7/(2007‐2013)/ERC grant 309516Halofuginone (HF) is a phase 2 clinical compound that inhibits the glutamyl-prolyl-tRNA synthetase (EPRS) thereby inducing the integrated stress response (ISR). Here, we report that halofuginone indeed triggers the predicted canonical ISR adaptations, consisting of attenuation of protein synthesis and gene expression reprogramming. However, the former is surprisingly atypical and occurs to a similar magnitude in wild-type cells, cells lacking GCN2 and those incapable of phosphorylating eIF2α. Proline supplementation rescues the observed HF-induced changes indicating that they result from inhibition of EPRS. The failure of the GCN2-to-eIF2α pathway to elicit a measurable protective attenuation of translation initiation allows translation elongation defects to prevail upon HF treatment. Exploiting this vulnerability of the ISR, we show that cancer cells with increased proline dependency are more sensitive to halofuginone. This work reveals that the consequences of EPRS inhibition are more complex than anticipated and provides novel insights into ISR signaling, as well as a molecular framework to guide the targeted development of halofuginone as a therapeutic

Molecular investigation of the unfolded protein response in select human tauopathies

Background: Tauopathies are a group of neurodegenerative diseases associated with the accumulation of misfolded tau protein. The mechanisms underpinning tau-dependent proteinopathy remain to be elucidated. A protein quality control pathway within the endoplasmic reticulum, the unfolded protein response (UPR), has been suggested as a possible pathway modulating cellular responses in a range of neurodegenerative diseases, including those associated with misfolded cytosolic tau.Objective: In this study we investigated three different clinically defined tauopathies to establish whether these diseases are accompanied by the activation of UPR.Methods: We used PCR and western blotting to probe for the modulation of several reliable UPR markers in mRNA and proteins extracted from three distinct tauopathies: 20 brain samples from Alzheimer's disease patients, 11 from Pick's disease, and 10 from progressive supranuclear palsy. In each disease samples from these patients were compared with equal numbers of age-matched non-demented controls.Results: Our investigation showed that different markers of UPR are not changed at the late stage of any of the human tauopathies investigated. Interestingly, UPR signatures were often observed in non-demented controls.Conclusion: These data from late-stage human cortical tissue report an activation of UPR markers within the aged brain across all cohorts investigated and further support the emerging evidence that the accumulation of misfolded cytosolic tau does not drive a disease-associated activation of UPR.</p

Activation of the integrated stress response by inhibitors of its kinases

Abstract Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases

Activation of the integrated stress response by inhibitors of its kinases.

Acknowledgements: We thank Shashank Rai and LMB Animal Facility for help with animal experiments; Ketan Malhotra for help with protein purification; Stephen McLaughlin for help with SEC-MALS and thermal shift experiments; Alison Inglis, Olga Perisic and Roger Williams for GCN2 and eIF2α plasmids, as well as, recombinant GCN2 pseudokinase and kinase domains; Michel Goedert and Petra Gross for insightful comments on the manuscript; Emily Naden for proof-reading the paper. This work was supported by Medical Research Council (UK) grant MC_U105185860 and Wellcome Trust Principal Investigator Award 206367/Z/ 17/Z to A.B. M.S. was supported by Human Frontier Science Program (HFSP) LT000162/2021-L and European Molecular Biology Organization (EMBO) ALTF 698-2020.Funder: Medical Research Council MC_U105185860 Wellcome Trust 206367/Z/ 17/ZPhosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases

Activation of the integrated stress response by inhibitors of its kinases.

Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases

Activation of the integrated stress response by inhibitors of its kinases

Acknowledgements: We thank Shashank Rai and LMB Animal Facility for help with animal experiments; Ketan Malhotra for help with protein purification; Stephen McLaughlin for help with SEC-MALS and thermal shift experiments; Alison Inglis, Olga Perisic and Roger Williams for GCN2 and eIF2α plasmids, as well as, recombinant GCN2 pseudokinase and kinase domains; Michel Goedert and Petra Gross for insightful comments on the manuscript; Emily Naden for proof-reading the paper. This work was supported by Medical Research Council (UK) grant MC_U105185860 and Wellcome Trust Principal Investigator Award 206367/Z/ 17/Z to A.B. M.S. was supported by Human Frontier Science Program (HFSP) LT000162/2021-L and European Molecular Biology Organization (EMBO) ALTF 698-2020.Funder: Medical Research Council MC_U105185860 Wellcome Trust 206367/Z/ 17/ZPhosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases