Biology subjects

Karelson, M.

Publications and source records attributed to Karelson, M..

8 recordsLinked to original sources

bioRxiv2024

CDNF rescues human iPSCs-derived dopamine neurons through direct binding to unfolded protein response sensors PERK and IRE1α

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional trophic factor that protects dopamine neurons in cellular and animal models of Parkinsons disease (PD). CDNF was safe and well tolerated in phase 1 clinical trials for PD treatment, and currently, its peptide analogue is under investigation in phase 1 clinical trials for PD. Despite prominent neuroprotective and neurorestorative activity, the receptors and exact mechanism of CDNF functioning have been obscure. Intracellularly acting CDNF exerts cytoprotection by attenuating endoplasmic reticulum (ER) stress and unfolded protein response (UPR). We demonstrated that this activity occurs through the direct binding of CDNF to ER transmembrane UPR sensors PERK and IRE1 for purified proteins and in cells. We identified CDNF mutants deficient for binding to UPR sensors. CDNF binding to PERK and IRE1 appeared to be crucial for the survival of mouse dopamine neurons in culture. Importantly for clinical translation, CDNF rescues human induced pluripotent stem cell-derived dopamine neurons and promotes their regeneration. CDNF binding to UPR sensors alleviated terminal UPR and promoted neurite outgrowth of human dopamine neurons through direct binding to PERK and IRE1. CDNF binding to BiP was dispensable for the neuroprotective and neurorestorative activity of CDNF. Therefore, CDNF, or small molecules mimicking its binding to UPR sensors and acting selectively on dopamine neurons with activated UPR, are promising drug candidates for PD treatment.

neuroscience↗

bioRxiv2024

USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells

Ubiquitin specific protease-14 (USP14) is critical for controlling protein homeostasis disturbed in human disorders like Parkinso[n]s disease (PD). Here we investigated the role of USP14 in regulating proteasome and autophagy pathways, and their influence on -synuclein (-syn) degradation. Data showed that -syn and phosphorylated serine129 -syn (pS129 -syn) were elevated in USP14 gene-deleted SH-SY5Y dopaminergic cells with concomitant reduction in proteasome activity. Inhibition of proteasomes using MG132 particularly elevated pS129 -syn in these cells, but the levels were not influenced by inhibiting autophagy using chloroquine. In contrast, autophagy and the CLEAR (Coordinated Lysosomal Expression and Regulation) pathways were elevated in USP14 lacking cells with an upregulation of the transcription factor TFEB. USP14-ablated cells also exhibited increases in reactive oxidative species (ROS) and elongation of mitochondria. The addition of N-Acetylcysteine amide (NACA) to counteract oxidative stress, reduced pS129 -syn and -syn levels in USP14 deficient cells. Phospho-proteomic analyses revealed that USP14 is phosphorylated at S143 affecting its function and structure as shown by molecular modeling, and protein interaction studies. Re-expression of wild-type and the phospho-mimetic S143D-USP14 mutant decreased ROS, pS129 -syn, and -syn in USP14 lacking cells. These results demonstrate that pS129 -syn levels are sensitive to oxidative stress in SH-SY5Y dopaminergic cells. USP14 by stimulating the proteasome activity and reducing oxidative stress is a promising factor for targeting -syn and its pathogenic variants in PD.

neuroscience↗

bioRxiv2024

Effect of RNA m6A methyltransferase activation by a low molecular weight compound on anxiety- and depression-related behaviours, monoamine neurochemistry and striatal gene expression in the rat

Modification of mRNA by methylation is involved in post-transcriptional regulation of gene expression by affecting the splicing, transport, stability and translation of mRNA. Methylation of adenosine at N6 (m6A) is the most common and most important cellular modification occurring in the mRNA of eukaryotes. Evidence that m6A mRNA methylation is involved in regulation of stress response and that its dysregulation may contribute to the pathogenesis of neuropsychiatric disorders is accumulating. We have examined the acute and subchronic (up to 18 days once per day intraperitoneally) effect of the first METTL3/METTL14 activator compound CHMA1004 (methyl-piperazine-2-carboxylate) at two doses (1 and 5 mg/kg) in male and female rats. CHMA1004 had a profound locomotor activating and anxiolytic-like profile in open field and elevated zero-maze tests. In female rats sucrose consumption and swimming in Porsolts test were increased. Nevertheless, CHMA1004 did not exhibit strong psychostimulant-like properties: CHMA1004 had no effect on 50-kHz ultrasonic vocalizations except that it reduced the baseline difference between male and female animals, and acute drug treatment had no effect on extracellular dopamine levels in striatum. Subchronic CHMA1004 altered ex vivo catecholamine levels in several brain regions. RNA sequencing of female rat striata after subchronic CHMA1004 treatment revealed changes in the expression of a number of genes linked to dopamine neuron viability, neurodegeneration, depression, anxiety and stress response. Conclusively, the first-in-class METTL3/METTL14 activator compound CHMA1004 increased locomotor activity and elicited anxiolytic-like effects after systemic administration, demonstrating tha pharmacological activation of RNA m6A methylation has potential for neuropsychiatric drug development.

pharmacology and toxicology↗

bioRxiv2023

Small molecule activation of m6A mRNA methylation as a novel approach for neuroprotection

N6-Methyladenosine (m6A) is the most common mRNA base modification in eukaryotes. Methylation of adenosine residues to m6A contributes to the regulation of splicing, transport, stability, and translation of mRNA and two main classes of enzymes regulate it. The formation of m6A is catalysed by a methyltransferase complex containing methyltransferase-like 3 (METTL3), METTL14, and Wilms tumour 1-associated protein (WTAP) as well as monomeric METTL16. Demethylation of m6A is catalysed by the fat mass and obesity-associated protein FTO and the RNA demethylase AlkB homolog 5 (ALKBH5). The m6A mRNA methylation dysregulation occurs in the nervous system and in Parkinsons disease (PD), but it remains poorly studied. Moreover, the role of m6A mRNA methylation in neuronal survival, neuroprotection, and neuroregeneration is unclear. We have earlier used high-throughput virtual screening of large compound libraries and identified four unique small-molecule ligands that activate m6A mRNA methylation by binding to the METTL3/14/WTAP complex and enhancing the binding of the methylation substrate SAM to nanomolar concentrations. Following this, we now discovered that two methyltransferase activators at 10 nM concentrations supported the survival and protected dopamine (DA) neurons in culture in growth factor deprivation and 6-hydroxydopamine (6-OHDA) neurotoxin models. In contrast, METTL3/14 inhibitor STM2457 triggered death of DA neurons. For clinical translation we also tested the most efficient compound C4 on induced pluripotent stem cell-derived human DA neurons and in animal model of Parkinsons disease (PD). C4 compound protected human DA neurons from 6-OHDA-induced cell death and increased neurite outgrowth and the number of processes demonstrating that it has both neuroprotective and neurorestorative properties. METTL3/14 activator C4 improved motor behaviour and protected DA neurons and their fibres faster and much more efficiently than GDNF in the rat 6-OHDA model of PD. These are the first specific activators of METTL3/14/WTAP and first demonstration that m6A regulators can protect and regenerate neurons. These data demonstrate that m6A mRNA methylation is a novel pathway regulating neuronal survival and regeneration.

neuroscience↗

bioRxiv2023

Novel RNA m6A methyltransferase METTL16 inhibitors

The overexpression of RNA 6-N-methyladenosine (m6A) methyltransferase METTL16 has oncogenic role in the case of several cancer types, including leukemia, but efficient small-molecule inhibitors are not available. Initially identified by high-throughput virtual screening of the ZINC15 database in vivo subset, but then confirmed by measuring catalytic activity, two nanomolar-active METTL16 inhibitors, compounds 1 (IC50 = 25.82 {+/-} 17.19 nM) and 2 (IC50 = 60.91 {+/-} 2.75 nM) were found. The inhibitory activity of the compounds was measured using the m6A antibody-based ELISA assay. We also present the results on the effect of these inhibitors on the viability of promyeloblast HL-60 and lymphoblast CCRF-CEM leukemia cell lines. In unstressed growth conditions, both identified METTL16 inhibitors reduced the viability of HL-60 cells by up to 40%. The effect on the viability of CCRF-CEM cells was smaller with no dose dependency observed. In parallel, the level of the m6A as compared to unmodified adenosine in the HL-60 cell mRNAs was significantly reduced by the inhibitor 1. Collectively, we herein demonstrate novel METTL16 inhibitors that exert tumor cell-lineage-selective antiproliferative effects.

pharmacology and toxicology↗

bioRxiv2021

Small-molecule inhibitors of the RNA m6A demethylase FTO potently support the survival of dopamine neurons

The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signalling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 M (compound 2) and Kd = 337 nM; IC50 = 28.9 M (compound 3). Importantly, the treatment of mouse midbrain dopamine neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, these inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The compounds 2 and 3 protected dopamine neurons with greater potency than our recently developed alkylation repair homolog protein 5 (AlkBH5) m6A demethylase inhibitors. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study, can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases.

neuroscience↗

bioRxiv2020

MANF regulates unfolded protein response and neuronal survival through its ER-located receptor IRE1α

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in numerous cell types in vitro and in models of neurodegeneration and diabetes in vivo. So far, the exact mode of its action has remained elusive and plasma membrane or ER-located receptors of MANF have not been identified. We have found that MANF can directly interact with transmembrane unfolded protein response (UPR) receptor IRE1 and compete with the major ER chaperone BiP (GRP78) for the interaction with IRE1. With lower affinities MANF can also interact with other UPR receptors, PERK and ATF6. Using molecular modeling and mutagenesis analysis, we have identified the exact structural MANF regions involved in its binding to the luminal domain of IRE1. MANF attenuates UPR signaling by decreasing IRE1 oligomerization and IRE1 phosphorylation. MANF mutant deficient in IRE1 binding cannot regulate IRE1 oligomerization and fails to protect neurons from ER stress induced death. Importantly, we found that MANF-IRE1 interaction is also crucial for the survival promoting action of MANF for dopamine neurons in an animal model of Parkinsons disease. Our data reveal a novel mechanism of IRE1 regulation during ER stress and demonstrate the intracellular mode of action of MANF as a modulator of UPR and neuronal cell survival through the direct interaction with IRE1 and regulation of its activity. Furthermore, our data explain why MANF in contrast to other growth factors has no effects on naive cells and rescues only ER stressed or injured cells.

cell biology↗

bioRxiv2019

GDNF receptor agonist supports dopamine neurons in vitro and protects their function in animal model of Parkinson's disease.

Motor symptoms of Parkinsons disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently no cure for this disease is available. Existing drugs alleviate PD symptoms, but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but its clinical use is complicated by pharmacokinetic properties. In the present study we demonstrate the ability of a small molecule agonist of GDNF receptor RET to support the survival of cultured dopamine neurons only when they express GDNF receptors. In addition, BT13 activates intracellular signaling cascades in vivo, stimulates release of dopamine and protect the function of dopaminergic neurons in a 6-hydroxydopamine (6-OHDA) rat model of PD. In contrast to GDNF, BT13 is able to penetrate through the blood-brain-barrier. Thus, BT13 serves as an excellent tool compound for the development of novel disease-modifying treatments against PD.

neuroscience↗