Cerebral dopamine neurotrophic factor protects and repairs dopamine neurons by novel mechanism

Midbrain dopamine neurons deteriorate in Parkinson's disease (PD) that is a progressive neurodegenerative movement disorder. No cure is available that would stop the dopaminergic decline or restore function of injured neurons in PD. Neurotrophic factors (NTFs), e.g., glial cell line-derived neurotrophic factor (GDNF) are small, secreted proteins that promote neuron survival during mammalian development and regulate adult neuronal plasticity, and they are studied as potential therapeutic agents for the treatment of neurodegenerative diseases. However, results from clinical trials of GDNF and related NTF neurturin (NRTN) in PD have been modest so far. In this review, we focus on cerebral dopamine neurotrophic factor (CDNF), an unconventional neurotrophic protein. CDNF delivered to the brain parenchyma protects and restores dopamine neurons in animal models of PD. In a recent Phase I-II clinical trial CDNF was found safe and well tolerated. CDNF deletion in mice led to age-dependent functional changes in the brain dopaminergic system and loss of enteric neurons resulting in slower gastrointestinal motility. These defects in Cdnf(-/-) mice intriguingly resemble deficiencies observed in early stage PD. Different from classical NTFs, CDNF can function both as an extracellular trophic factor and as an intracellular, endoplasmic reticulum (ER) luminal protein that protects neurons and other cell types against ER stress. Similarly to the homologous mesencephalic astrocyte-derived neurotrophic factor (MANF), CDNF is able to regulate ER stress-induced unfolded protein response (UPR) signaling and promote protein homeostasis in the ER. Since ER stress is thought to be one of the pathophysiological mechanisms contributing to the dopaminergic degeneration in PD, CDNF, and its small-molecule derivatives that are under development may provide useful tools for experimental medicine and future therapies for the treatment of PD and other neurodegenerative protein-misfolding diseases.Peer reviewe

Uusi CDNF/MANF proteiiniperhe : Molekyylirakenne, ilmentyminen ja neurotroofinen aktiivisuus

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.Hermokasvutekijät ovat solun erittämiä proteiineja, jotka edistävät neuronien eloonjäämistä, kontaktien muodostumista ja säätelevät hermosolujen toimintaa. Edullisten ominaisuuksien vuoksi hermokasvutekijöitä on tutkittu mahdollisina lääkkeinä hermoston rappeumasairauksiin. Parkinsonin tauti on etenevä liikesairaus, jossa keskiaivojen mustatumakkeen dopamiinineuronit vähitellen tuhoutuvat. Tautiin ei ole olemassa hoitoa, joka pysäyttäisi neuronien tuhoutumisen tai edistäisi niiden toiminnan palautumista. Ryhmämme tutkii hermokasvutekijöitä, joista voisi olla hyötyä myös hermoston rappeumatautien hoidossa tulevaisuudessa. Tässä työssä kuvattiin uusi conserved dopamine neurotrophic factor (CDNF)-proteiini, joka on läheistä sukua aiemmin kuvatulle MANF-proteiinille. Selkärankaisten CDNF ja MANF, sekä selkärangattomien eläinten, kuten banaanikärpäsen ja sukkulamadon MANF muodostavat evoluutiossa varhain syntyneen proteiiniperheen. Osoitimme, että nisäkkäiden CDNF ja MANF ovat solusta erittyviä proteiineja, jotka ilmentyvät laajasti aivokudoksessa. Ne ilmentyvät paitsi keskiaivoissa, myös muissa aivojen osissa ja useissa hermoston ulkopuolisissa kudoksissa. Työssä tuotimme CDNF-proteiinia ja testasimme sen vaikutusta kokeellisessa Parkinsonin taudin mallissa rotilla. Rotille aiheutettiin 6-hydroksidopamiini (6-OHDA)-toksiinilla Parkinsonin tautia muistuttava vaurio. Osoitimme, että CDNF estää dopamiinineuronien tuhoutumisen, kun CDNF injisoidaan rotan aivoihin ennen toksiinia. Toisessa koesarjassa, jossa CDNF injisoitiin aivoihin neljä viikkoa toksiinivaurion jälkeen, CDNF korjasi dopamiinineuronien toimintaa ja rottien liikehäiriötä. Jälkimmäinen koeasetelma muistuttaa tilannetta Parkinson-potilailla, kun jo olemassa olevaa vauriota pyritään korjaamaan lääkehoidolla. Kokeiden perusteella CDNF saattaa olla hyödyllinen Parkinsonin taudin hoidossa. Työssä selvitimme myös CDNF- ja MANF-proteiinien molekyylirakenteen, josta on apua tutkittaessa näiden proteiinien toimintamekanismeja solussa. Rakenteen perusteella saatiin selville, että CDNF ja MANF sitoutuvat lipideihin ja mahdollisesti solumembraaneihin. CDNF:n neurotroofinen vaikutus saattaa välittyä soluun membraani-interaktion kautta. Rakenne sisältää myös rikkisillan, jonka kaltainen löytyy tietyiltä solun endoplasmisessa retikulumissa (ER:ssa) toimivilta entsyymeiltä, jotka auttavat proteiineja laskostumaan oikein. Tämä tukee muiden tutkimusryhmien raportteja, joissa osoitetaan MANF-proteiinin edistävän viljeltyjen solujen eloonjäämistä stressitilanteissa kun ER:n toiminta ja proteiinieritys ovat häiriintyneet. Rakennemallin perusteella CDNF ja MANF saattavat edistää proteiinien laskostumista ER:ssa, ja näin suojata soluja stressitilanteissa. Tutkimuksen perusteella CDNF ja MANF saattavat olla hyödyllisiä neurodegeneratiivisten sairauksien hoidossa. Jatkotutkimuksin on tarkoitus selvittää millaiset molekyylimekanismit toimivat CDNF/MANF-proteiinien soluja suojaavan ja solujen toimintaa edistävän aktiivisuuden taustalla

CDNF and MANF in the brain dopamine system and their potential as treatment for Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disease characterized by gradual loss of midbrain dopamine neurons, leading to impaired motor function. Preclinical studies have indicated cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) to be potential therapeutic molecules for the treatment of PD. CDNF was proven to be safe and well tolerated when tested in Phase I-II clinical trials in PD patients. Neuroprotective and neurorestorative effects of CDNF and MANF were demonstrated in animal models of PD, where they promoted the survival of dopamine neurons and improved motor function. However, biological roles of endogenous CDNF and MANF proteins in the midbrain dopamine system have been less clear. In addition to extracellular trophic activities, CDNF/MANF proteins function intracellularly in the endoplasmic reticulum (ER), where they modulate protein homeostasis and protect cells against ER stress by regulating the unfolded protein response (UPR). Here, our aim is to give an overview of the biology of endogenous CDNF and MANF in the brain dopamine system. We will discuss recent studies on CDNF and MANF knockout animal models, and effects of CDNF and MANF in preclinical models of PD. To elucidate possible roles of CDNF and MANF in human biology, we will review CDNF and MANF tissue expression patterns and regulation of CDNF/MANF levels in human diseases. Finally, we will discuss novel findings related to the molecular mechanism of CDNF and MANF action in ER stress, UPR, and inflammation, all of which are mechanisms potentially involved in the pathophysiology of PD

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Is Highly Expressed in Mouse Tissues With Metabolic Function

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Reduction of FENO by tap water and carbonated water mouthwashes : magnitude and time course

Fractional exhaled nitric oxide (F-ENO) assesses eosinophilic inflammation of the airways, but F-ENO values are also influenced by oral nitric oxide (NO). The aim of this pilot study was to measure F-ENO and compare the effect of two different mouthwashes on F-ENO and analyse the duration of the effect. F-ENO was measured in 12 randomized volunteers (healthy or asthmatic subjects) with a NIOX VERO (R) analyser at an expiratory flow rate of 50mL/s. After a baseline measurement, a mouthwash was performed either with tap water or carbonated water and was measured during 20min in 2min intervals. The procedure was repeated with the other mouthwash. We found that both mouthwashes reduced F-ENO immediately at the beginning compared to the baseline (pPeer reviewe

Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration

Copyright © 2023, The Author(s), under exclusive licence to Springer Nature America, Inc. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.Age-related decline in skeletal muscle regenerative capacity is multifactorial, yet the contribution of immune dysfunction to regenerative failure is unknown. Macrophages are essential for effective debris clearance and muscle stem cell activity during muscle regeneration, but the regulatory mechanisms governing macrophage function during muscle repair are largely unexplored. Here, we uncover a new mechanism of immune modulation operating during skeletal muscle regeneration that is disrupted in aged animals and relies on the regulation of macrophage function. The immune modulator mesencephalic astrocyte-derived neurotrophic factor (MANF) is induced following muscle injury in young mice but not in aged animals, and its expression is essential for regenerative success. Regenerative impairments in aged muscle are associated with defects in the repair-associated myeloid response similar to those found in MANF-deficient models and could be improved through MANF delivery. We propose that restoring MANF levels is a viable strategy to improve myeloid response and regenerative capacity in aged muscle.This work was supported by EMBO (IG4448 to P.S.V.) and FCT (PTDC/MED-OUT/8010/2020 and EXPL/MED-OUT/1601/2021 to P.S.V. and J.N.). P.S.V. was supported by ‘la caixa’ Foundation Junior Leader Fellowship (LCF/BQ/PI19/11690006). J.N. was supported by an assistant research contract from FCT (2021.03843.CEECIND). P.L. was supported by the Academy of Finland (grant 343299) and by the Jane and Aatos Erkko Foundation.info:eu-repo/semantics/publishedVersio

CDNF Interacts with ER Chaperones and Requires UPR Sensors to Promote Neuronal Survival

Cerebral dopamine neurotrophic factor (CDNF) is a neurotrophic factor that has beneficial effects on dopamine neurons in both in vitro and in vivo models of Parkinson’s disease (PD). CDNF was recently tested in phase I-II clinical trials for the treatment of PD, but the mechanisms underlying its neuroprotective properties are still poorly understood, although studies have suggested its role in the regulation of endoplasmic reticulum (ER) homeostasis and the unfolded protein response (UPR). The aim of this study was to investigate the mechanism of action of CDNF through analyzing the involvement of UPR signaling in its anti-apoptotic function. We used tunicamycin to induce ER stress in mice in vivo and used cultured primary neurons and found that CDNF expression is regulated by ER stress in vivo and that the involvement of UPR pathways is important for the neuroprotective function of CDNF. Moreover, we used AP-MS and BiFC to perform the first interactome screening for CDNF and report novel binding partners of CDNF. These findings allowed us to hypothesize that CDNF protects neurons from ER-stress-inducing agents by modulating UPR signaling towards cell survival outcomes

Characterization of CDNF-Secreting ARPE-19 Cell Clones for Encapsulated Cell Therapy

Cerebral Dopamine Neurotrophic Factor (CDNF) shows beneficial effects in rodent models of Parkinson?s and Alzheimer?s disease. The brain is a challenging target for protein therapy due to its exclusive blood?brain barrier. Hence, the therapeutic protein should be delivered directly to the brain parenchyma. Implantation of encapsulated mammalian cells that constantly secrete CDNF is a potential approach for targeted and long-term protein delivery to the brain. In this study, we generated several CDNF-secreting cell clones derived from human retinal pigment epithelial cell line ARPE-19, and studied CDNF secretion from the clones maintained as monolayers and in polymeric microcapsules. The secretion of wild type (wt) CDNF transgene was low and the majority of the produced protein remained intracellular, locating mainly to the endoplasmic reticulum (ER). The secretion of wtCDNF decreased to even lower levels when the clones were in a non-dividing state, as in the microcapsules. Both codon optimization and deletion of the putative ER-retrieval signal (four last amino acids: KTEL) improved CDNF secretion. More importantly, the secretion of KTEL-deleted CDNF remained constant in the non-dividing clones. Thus, cells expressing KTEL-deleted CDNF, in contrast to wtCDNF, can be considered for cell encapsulation applications if the KTEL-deleted CDNF is proven to be biologically active in vivo.Peer reviewe

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.Peer reviewe