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Parkinson’s disease (PD) pathology involves progressive degeneration and death of vulnerable dopamine neurons in the substantia nigra. Extensive axonal arborization and distinct functions make this type of neurons particularly sensitive to homeostatic perturbations, such as protein misfolding and Ca2+ dysregulation. Endoplasmic reticulum (ER) is a cell compartment orchestrating protein synthesis and folding, as well as synthesis of lipids and maintenance of Ca2+ homeostasis in eukaryotic cells. When misfolded proteins start to accumulate in ER lumen the unfolded protein response (UPR) is activated. UPR is an adaptive signaling machinery aimed at relieving of protein folding load in the ER. When UPR is chronic, it can either boost neurodegeneration and apoptosis or cause neuronal dysfunctions. We have recently discovered that mesencephalic astrocyte-derived neurotrophic factor (MANF) exerts its prosurvival action in dopamine neurons and in an animal model of PD through the direct binding to UPR sensor inositol-requiring protein 1 alpha (IRE1α) and attenuation of UPR. In line with this, UPR targeting resulted in neuroprotection and neurorestoration in various preclinical animal models of PD. Therefore, growth factors (GFs), possessing both neurorestorative activity and restoration of protein folding capacity are attractive as drug candidates for PD treatment especially their blood-brain barrier penetrating analogs and small molecule mimetics. In this review, we discuss ER stress as a therapeutic target to treat PD; we summarize the existing preclinical data on the regulation of ER stress for PD treatment. In addition, we point out the crucial aspects for successful clinical translation of UPR-regulating GFs and new prospective in GFs-based treatments of PD, focusing on ER stress regulation.

The cerebral dopamine neurotrophic factor (CDNF) together with the mesencephalic astrocyte-derived neurotrophic factor (MANF) form a unique family of neurotrophic factors (NTFs) structurally and functionally different from other proteins with neurotrophic activity. CDNF has no receptors on the cell membrane, is localized mainly in the cavity of endoplasmic reticulum (ER), and its primary function is to regulate ER stress. In addition, CDNF is able to suppress inflammation and apoptosis. Due to its functions, CDNF has demonstrated outstanding protective and restorative properties in various models of neuropathology associated with ER stress, including Parkinson's disease (PD). That is why CDNF already passed clinical trials in patients with PD. However, despite the name, CDNF functions extend far beyond the dopamine system in the brain. In particular, there are data on participation of CDNF in the maturation and maintenance of other neurotransmitter systems, regulation of the processes of neuroplasticity and non-motor behavior. In the present review, we discuss the features of CDNF structure and functions, its protective and regenerative properties.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are endoplasmic reticulum (ER) luminal proteins that confer trophic activities in a wide range of tissues under diverse pathological conditions. Despite initially being classified as neurotrophic factors, neither protein structurally nor functionally resembles bona fide neurotrophic factors. Their highly homologous structures comprise a unique globular, saposin-like domain within the N-terminus joined by a flexible linker to a C-terminus containing a SAP-like domain, CXXC motif and an ER retention sequence. Neurotrophic factors exert effects by binding to cognate receptors in the plasma membrane; however, no cell surface receptors have been identified for MANF and CDNF. Both can act as unfolded protein response (UPR) genes that modulate the UPR and inflammatory processes. The trophic activity of MANF and CDNF extends beyond the central nervous system with MANF being crucial for the development of pancreatic β cells and both have trophic effects in a variety of diseases related to the liver, heart, skeletal tissue, kidney and peripheral nervous system. In this article, the unique features of MANF and CDNF, such as their structure and mechanisms of action related to ER stress and inflammation, will be reviewed. Recently identified interactions with lipids and membrane trafficking will also be described. Lastly, their function and therapeutic potential in different diseases including a recent clinical trial using CDNF to treat Parkinson's disease will be discussed. Collectively, this review will highlight MANF and CDNF as broad-acting trophic factors that regulate functions of the endoplasmic reticulum.

Overexpression of neurotrophic factors in nigral dopamine neurons is a promising approach to reverse neurodegeneration of the nigrostriatal dopamine system, a hallmark in Parkinson’s disease. The human cerebral dopamine neurotrophic factor (hCDNF) has recently emerged as a strong candidate for Parkinson’s disease therapy. This study shows that hCDNF expression in dopamine neurons using the neurotensin-polyplex nanoparticle system reverses 6-hydroxydopamine-induced morphological, biochemical, and behavioral alterations. Three independent electron microscopy techniques showed that the neurotensin-polyplex nanoparticles containing the hCDNF gene, ranging in size from 20 to 150 nm, enabled the expression of a secretable hCDNF in vitro. Their injection in the substantia nigra compacta on day 21 after the 6-hydroxydopamine lesion resulted in detectable hCDNF in dopamine neurons, whose levels remained constant throughout the study in the substantia nigra compacta and striatum. Compared with the lesioned group, tyrosine hydroxylase-positive (TH+) nigral cell population and TH+ fiber density rose in the substantia nigra compacta and striatum after hCDNF transfection. An increase in βIII-tubulin and growth-associated protein 43 phospho-S41 (GAP43p) followed TH+ cell recovery, as well as dopamine and its catabolite levels. Partial reversal (80%) of drug-activated circling behavior and full recovery of spontaneous motor and non-motor behavior were achieved. Brain-derived neurotrophic factor recovery in dopamine neurons that also occurred suggests its participation in the neurotrophic effects. These findings support the potential of nanoparticle-mediated hCDNF gene delivery to develop a disease-modifying treatment against Parkinson’s disease. The Institutional Animal Care and Use Committee of Centro de Investigación y de Estudios Avanzados approved our experimental procedures for animal use (authorization No. 162-15) on June 9, 2019.

Atypical antipsychotics (AAP) are used in the therapy of Parkinson’s disease (PD) for elimination of psychotic symptoms. As the brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF) play a crucial role in the PD treatment, the aim of our study was the investigation of the effects of chronic treatment with commonly used AAP, clozapine and quetiapine, on the motor behavior and the BDNF, GDNF and CDNF genes expression in the mouse brain in the PD model produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Clozapine and quetiapine (1 mg/kg, i.p.) were administered 48 hours after the last MPTP injection, and treatment continued for the following 16 days. Then animals were euthanized, substantia nigra (SN), striatum (St) and hippocampus (Hc) were extracted for RT-PCR and tyrosine hydroxylase (TH) western blot assessment. MPTP treatment led to 50% depletion in the TH protein level in the St. MPTP caused significant decrease in both BDNF and GDNF mRNA level in the Hc and St, respectively. At the same time, increase in the GDNF expression in the MPTP + clozapine group in the SN was found. MPTP caused dramatic decrease in the CDNF mRNA level in the SN with simultaneous increase in the St. Both clozapine and quetiapine decreased it to a normal level in the St. The effect of AAP clozapine and quetiapine on the GDNF and CDNF genes expression in the pharmacological model of PD has been shown for the first time.

Neurotrophic factors and exercise training are effective in the growth and survival of neuronal cells. These factors play a protective role against oxidative stress damage and have the same function as antioxidants. The purpose of this study was to investigate the effect of a session of endurance training with three different intensities on CDNF, SOD, and MDA levels of cerebral cortex in male rats. Thirty-two male Wistar rats (aged 20 weeks) were divided randomly into two control and training groups. The training group consisted of low-, moderate-, and high-intensity trainings. The training groups, after getting familiarization with the rodent treadmill, were dealt with an acute training session with three different intensities. The CDNF level of cerebral cortex was measured by ELISA assay, and the SOD and MDA levels of cerebral cortex by spectrophotometery. A significant difference was seen in the CDNF level between low- and high-intensity groups, as well as between high-intensity groups and control group (P = 0.001). The levels of SOD were increased significantly among all groups (except for control and low-intensity groups). The acute training with different intensities significantly prevents the increase in MDA level of cerebral cortex (P = 0.005). The result of this study shows that the physical exercise even in the short term can affect the protective factors and antioxidant system in neuronal cells. However, the benefits of high-intensity training were higher than others. Therefore, suggested that the role of acute exercise with different intensities should be carefully considered for the preconditioning against neuronal degenerative diseases.

Background CDNF (cerebral dopamine neurotrophic factor) belongs to a new family of neurotrophic factors that exert systemic beneficial effects beyond the brain. Little is known about the role of CDNF in the cardiac context. Herein we investigated the effects of CDNF under endoplasmic reticulum‐stress conditions using cardiomyocytes (humans and mice) and isolated rat hearts, as well as in rats subjected to ischemia/reperfusion (I/R). Methods and Results We showed that CDNF is secreted by cardiomyocytes stressed by thapsigargin and by isolated hearts subjected to I/R. Recombinant CDNF (exoCDNF) protected human and mouse cardiomyocytes against endoplasmic reticulum stress and restored the calcium transient. In isolated hearts subjected to I/R, exoCDNF avoided mitochondrial impairment and reduced the infarct area to 19% when administered before ischemia and to 25% when administered at the beginning of reperfusion, compared with an infarct area of 42% in the untreated I/R group. This protection was completely abrogated by AKT (protein kinase B) inhibitor. Heptapeptides containing the KDEL sequence, which binds to the KDEL‐R (KDEL receptor), abolished exoCDNF beneficial effects, suggesting the participation of KDEL‐R in this cardioprotection. CDNF administered intraperitoneally to rats decreased the infarct area in an in vivo model of I/R (from an infarct area of ≈44% in the I/R group to an infarct area of ≈27%). Moreover, a shorter version of CDNF, which lacks the last 4 residues (CDNF‐ΔKTEL) and thus allows CDNF binding to KDEL‐R, presented no cardioprotective activity in isolated hearts. Conclusions This is the first study to propose CDNF as a new cardiomyokine that induces cardioprotection via KDEL receptor binding and PI3K/AKT activation.

Huntington’s disease (HD) is a neurodegenerative disorder with a progressive loss of medium spiny neurons in the striatum and aggregation of mutant huntingtin in the striatal and cortical neurons. Currently, there are no rational therapies for the treatment of the disease. Cerebral dopamine neurotrophic factor (CDNF) is an endoplasmic reticulum (ER) located protein with neurotrophic factor (NTF) properties, protecting and restoring the function of dopaminergic neurons in animal models of PD more effectively than other NTFs. CDNF is currently in phase I–II clinical trials on PD patients. Here we have studied whether CDNF has beneficial effects on striatal neurons in in vitro and in vivo models of HD. CDNF was able to protect striatal neurons from quinolinic acid (QA)-induced cell death in vitro via increasing the IRE1α/XBP1 signalling pathway in the ER. A single intrastriatal CDNF injection protected against the deleterious effects of QA in a rat model of HD. CDNF improved motor coordination and decreased ataxia in QA-toxin treated rats, and stimulated the neurogenesis by increasing doublecortin (DCX)-positive and NeuN-positive cells in the striatum. These results show that CDNF positively affects striatal neuron viability reduced by QA and signifies CDNF as a promising drug candidate for the treatment of HD.

This study investigated the effects of the Cong Rong Shu Jing (CRSJ) compound on endoplasmic reticulum stress in a rat model of Parkinson's disease (PD). A total of 40 rats were subcutaneously injected with rotenone-sunflower oil emulsion into the back of the neck to establish a rat model of PD. These PD rats were randomly divided into low-, medium-, and high-dose groups (intragastric administration of 0.5, 1, and 2 g/kg CRSJ, respectively) and a model group (intragastric administration of the solvent; 10 rats per group). Furthermore, 10 rats each were attributed to the control and vehicle groups (both received intragastric administration of the CRSJ solvent, and the vehicle group were injected additionally with sunflower oil alone). A traction test was conducted two times, after the PD model establishment and after 14 days of CRSJ gavage. The numbers of tyrosine hydroxylase- (TH-) positive cells and the dopamine levels in the substantia nigra were assessed using immunohistochemistry and high-performance liquid chromatography, respectively. Western blotting detected the expression levels of α-synuclein, endoplasmic reticulum stress pathways-related proteins, cerebral dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway-related proteins. Compared with the model group, the number of TH-positive cells in the substantia nigra was increased in the CRSJ groups. The expression levels of α-synuclein and the endoplasmic reticulum stress pathways-associated proteins glucose regulatory protein 78, inositol-requiring enzyme 1, apoptosis signal-regulating kinase 1, phosphorylated c-Jun N-terminal kinase, and caspase-12 were reduced. However, CRSJ administration elevated the expression levels of the neurotrophic factors CDNF and MANF, as well as those of p-PI3K and p-AKT. The CRSJ compound can relieve endoplasmic reticulum stress in PD rats and exerts protective effects in this animal model. These effects may be related to increased expression of neurotrophic factors and activation of the PI3K/AKT pathway.

Methamphetamine (METH) exerts significant neurotoxicity in experimental animals and humans when taken at high doses or abused chronically. Long-term abusers have decreased dopamine levels, and they are more likely to develop Parkinson's disease (PD). To date, few medications are available to treat the METH-induced damage of neurons. Glial cell line-derived neurotrophic factor (GDNF) has been previously shown to reduce the dopamine-depleting effects of neurotoxic doses of METH. However, the effect of cerebral dopamine neurotrophic factor (CDNF), which has been reported to be more specific and efficient than GDNF in protecting dopaminergic neurons against 6-OHDA toxicity, in attenuating METH neurotoxicity has not been determined. Thus, the present study aimed to evaluate the neuroprotective effect of CDNF against METH-induced damage to the dopaminergic system in vitro and in vivo. In vitro, CDNF protein increased the survival rate and reduced the tyrosine hydroxylase (TH) loss of METH-treated PC12 cells. In vivo, METH was administered to rats following human CDNF overexpression mediated by the recombinant adeno-associated virus. Results demonstrated that CDNF overexpression in the brain could attenuate the METH-induced dopamine and TH loss in the striatum but could not lower METH-induced hyperthermia.

Cerebral dopamine neurotrophic factor (CDNF) is expressed in the brain and is neuroprotective. We have previously shown that CDNF is also expressed in the bowel and that its absence leads to degeneration and autophagy in the enteric nervous system (ENS), particularly in the submucosal plexus. We now demonstrate that enteric CDNF immunoreactivity is restricted to neurons (submucosal > myenteric) and is not seen in glia, interstitial cells of Cajal, or smooth muscle. Expression of CDNF, moreover, is essential for the normal development and survival of enteric dopaminergic neurons; thus, expression of the dopaminergic neuronal markers, dopamine, tyrosine hydroxylase, and dopamine transporter are deficient in the ileum of Cdnf -/- mice. The normal age-related decline in proportions of submucosal dopaminergic neurons is exacerbated in Cdnf -/- animals. The defect in Cdnf -/- animals is not dopamine-restricted; proportions of other submucosal neurons (NOS-, GABA-, and CGRP-expressing), are also deficient. The deficits in submucosal neurons are reflected functionally in delayed gastric emptying, slowed colonic motility, and prolonged total gastrointestinal transit. CDNF is expressed selectively in isolated enteric neural crest-derived cells (ENCDC), which also express the dopamine-related transcription factor Foxa2. Addition of CDNF to ENCDC promotes development of dopaminergic neurons; moreover, survival or these neurons becomes CDNF-dependent after exposure to bone morphogenetic protein 4. The effects of neither glial cell-derived neurotrophic factor (GDNF) nor serotonin are additive with CDNF. We suggest that CDNF plays a critical role in development and long-term maintenance of dopaminergic and other sets of submucosal neurons. This article is protected by copyright. All rights reserved.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) form a family of atypical growth factors discovered for their neuroprotective properties in the central nervous system (CNS) in animal models of neurodegenerative diseases. Although their mechanism of protective action still remains unclear, it has been suggested that both MANF and CDNF promote cell survival through regulating the unfolded protein response (UPR), thereby relieving endoplasmic reticulum (ER) stress. Recent studies identified MANF for its emerging roles in metabolic function, inflammation and pancreatic β-cells. We have found that MANF deletion from the pancreas and β-cells leads to postnatal depletion of β-cells and diabetes. Moreover, global MANF-deficiency in mice results in severe diabetes-independent growth retardation. As the expression pattern of MANF in mouse tissues has not been extensively studied, we set out to thoroughly investigate MANF expression in embryonic and adult mice using immunohistochemistry, histochemical X-gal staining, enzyme-linked immunosorbent assay (ELISA), and quantitative reverse transcription PCR (RT-qPCR). We found that MANF is highly expressed in brain neurons regulating energy homeostasis and appetite, as well as in hypothalamic nuclei producing hormones and neuropeptides important for different body functions. Strong expression of MANF was also observed in peripheral mouse tissues and cells with high secretory and metabolic function. These include pituitary gland and interestingly we found that the anterior pituitary gland is smaller in MANF-deficient mice compared to wild-type mice. Consequently, we found reduction in the number of growth hormone- and prolactin-producing cells. This combined with increased expression of UPR genes, reduced number of proliferating cells in the anterior pituitary and dysregulated expression of pituitary hormones might contribute to the severe growth defect seen in the MANF knockout mice. Moreover, in this study we compared MANF and CDNF levels in mouse tissues. Unlike MANF, CDNF protein levels are generally lower in mouse tissues, and the highest levels of CDNF was observed in the tissues with high-energy demands and oxidative roles, including heart, muscle, testis, and brown adipose tissue.

Background: Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) promote the survival of midbrain dopamine neurons in animal models of Parkinson's disease (PD). However, little is known about endogenous concentrations of MANF and CDNF in human PD patients, and their relation to PD pathogenesis. Our main objective was to study whether circulating concentrations of MANF and CDNF differ between PD patients and controls, and if they correlate with clinical parameters. Levels of circulating CDNF were studied for the first time. Methods: MANF and CDNF levels were measured from serum samples of 34 PD patients and 35 controls using validated in-lab-designed enzyme-linked immunosorbent assay (ELISAs). MANF and CDNF mRNA levels in whole blood samples of 60 PD patients and 30 controls were measured by quantitative real time polymerase chain reaction (qRT-PCR). MANF concentrations in different blood cell types were measured by ELISA. Results: Circulating MANF concentrations were significantly higher in PD patients compared to controls (P

Background Cerebral dopamine neurotrophic factor plays a critical role in repairing and maintaining healthy neurons in pathological conditions such as stroke. However, the association between cerebral dopamine neurotrophic factor expression and stroke has only recently been investigated in preclinical models and is rarely described in human studies. Objectives The aims of this were to examine neurological alterations mirrored in human blood platelet cerebral dopamine neurotrophic factor gene expression. Cerebral dopamine neurotrophic factor is expressed in both the central nervous system and peripheral blood. Blood platelets are often used to model neuronal behavior because they exhibit biochemical impairments similar to brain tissues of patients with neurological disorders. Methods RNA was isolated from platelets and cDNA was synthesized to quantify cerebral dopamine neurotrophic factor gene expression of 36 stroke patients compared to 72 healthy aged-matched controls through real-time PCR. Further grouping analyses of data with regard to age, sex, and medication history were performed. Results Cerebral dopamine neurotrophic factor gene expression was significantly reduced in stroke patients relative to control subjects (P = .013). Subsequent analysis revealed a significant difference in expression between males and females within the control group (P = .026). Decreased cerebral dopamine neurotrophic factor expression was only observed in male stroke patients compared to their sex-matched controls (P = .008). Grouping stroke patients based on their medication history did not significantly alter cerebral dopamine neurotrophic factor gene expression. Conclusions Further studies investigating cerebral dopamine neurotrophic factor expression could be directed towards the interplay of the central nervous system, hematopoietic derivatives, and utilizing cerebral dopamine neurotrophic factor as a therapeutic tool.

Neurotrophic factors (NTF) are a subgroup of growth factors that promote survival and differentiation of neurons. Due to their neuroprotective and neurorestorative properties, their therapeutic potential has been tested in various neurodegenerative diseases. Bioavailability of NTFs in the target tissue remains a major challenge for NTF-based therapies. Various intracerebral delivery approaches, both protein and gene transfer-based, have been tested with varying outcomes. Three growth factors, glial cell-line derived neurotrophic factor (GDNF), neurturin (NRTN) and platelet-derived growth factor (PDGF-BB) have been tested in clinical trials in Parkinson’s disease (PD) during the past 20 years. A new protein can now be added to this list, as cerebral dopamine neurotrophic factor (CDNF) has recently entered clinical trials. Despite their misleading names, CDNF, together with its closest relative mesencephalic astrocyte-derived neurotrophic factor (MANF), form a novel family of unconventional NTF that are both structurally and mechanistically distinct from other growth factors. CDNF and MANF are localized mainly to the lumen of endoplasmic reticulum (ER) and their primary function appears to be modulation of the unfolded protein response (UPR) pathway. Prolonged ER stress, via the UPR signaling pathways, contributes to the pathogenesis in a number of chronic degenerative diseases, and is an important target for therapeutic modulation. Intraputamenally administered recombinant human CDNF has shown robust neurorestorative effects in a number of small and large animal models of PD, and had a good safety profile in preclinical toxicology studies. Intermittent monthly bilateral intraputamenal infusions of CDNF are currently being tested in a randomized placebo-controlled phase I–II clinical study in moderately advanced PD patients. Here, we review the history of growth factor-based clinical trials in PD, and discuss how CDNF differs from the previously tested growth factors.

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are proteins that have received increasing attention in the last decades. Although they are called neurotrophic factors they are drastically different from neurotrophic factors in their expression and physiological actions.They are located in the lumen of the endoplasmic reticulum (ER) and their basal secretion from neurons is very low. However their secretion is stimulated upon ER calcium depletion by chemical probes such as thapsigargin, a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor. Exogenous MANF and CDNF possess therapeutic properties in several neurological dkodse models, including Parkinson disease and stroke. Endogenoes MANF expression has been shown to be neuroprotective, as well as administration of either CDNF or MANF into the extracellular space. In this review, we focus on their therapeutic effects, regulation of expression and secretion, comparison of their mechanisms of action, and their application to the brain parenchyma as recombinant proteins.

Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease, and early stage AD is characterized by synaptic dysfunction generally ascribed to soluble oligomers of amyloid-beta (Aβ). Neurotrophic factors are promising for AD treatment and are integrally involved in neuronal growth, survival and maintenance. Cerebral dopamine neurotrophic factor (CDNF) was recently discovered to have beneficial effects on long-term memory. The present study explored the synaptoprotective effects of CDNF in Aβ-treated primary hippocampal cells. Immunofluorescent analysis of synaptophysin and postsynaptic density protein 95 (PSD95) puncta densities in the group of pretreatment with CDNF before Aβ exposure revealed significant improvements compared to Aβ group. In addition, pretreatment with CDNF reduced the expression levels of endoplasmic reticulum (ER) stress-related proteins, including Bip (also known as GRP78), phosphorylation of eukaryotic translation initiation factor 2 subunit α (peIF2α), phosphorylation of c-Jun N-terminal kinase (pJNK), and cleaved caspase 3, which are increased by Aβ treatment at early stage. Our results revealed protective effects of CDNF on Aβ-induced synaptotoxicity and ER stress, implying that CDNF may protect against Aβ-induced synaptotoxicity through suppression of ER stress. CDNF could be a potential drug candidate for early AD treatment.

Cerebral dopamine neurotrophic factor (CDNF) has been considered as potent candidates for the therapy of Parkinson's disease (PD) for which can promote the survival of midbrain dopaminergic neurons. In addition to secret out from cells like other classical neurotrophic factors (NTFs), CDNF can locate in the endoplasmatic reticulum (ER), where they can function as ER stress response protein to regulate ER stress. In our previous studies, we have found two helices, α1 and α7, which can regulate the intracellular trafficking and secretion of CDNF. α1 distruction can significantly retain CDNF protein in the ER, but α7 distruction induce most CDNF protein secreting out the cells. Then α1 and α7 regulate protein trafficking and secretion in opposite side. However, the exact secretion level of CDNF affected by α1 or α7 have not been sensitively quantified. In this study, we used nanoluciferase to quantify the secretion level of CDNF protein so that we could evaluate the impact of α1 and α7 on CDNF secretion or function.

Regeneration is an important process in multicellular organisms, responsible for homeostatic renewal and repair of different organs after injury. Immune cell activation is observed at early stages of the regenerative response and its regulation is essential for regenerative success. Thus, immune regulators play central roles in optimizing regenerative responses. Neurotrophic factors (NTFs) are secreted molecules, defined by their ability to support neuronal cell types. However, emerging evidence suggests that they can also play important functions in the regulation of immune cell activation and tissue repair. Here we discuss the literature supporting a role of NTFs in the regulation of inflammation and regeneration. We will focus, in particular, in the emerging roles of mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) in the regulation of immune cell function and in the central role that immune modulation plays in their biological activity in vivo. Finally, we will discuss the potential use of these factors to optimize regenerative success in vivo, both within and beyond the nervous system.

Regulation of adult neurogenesis plays an important role in therapeutic strategies for various neurodegenerative diseases. Recent studies have suggested that the enhancement of adult neurogenesis can be helpful in the treatment of Parkinson's disease (PD). In this study, we investigated whether Korean red ginseng (KRG) can enhance neurogenesis in the subventricular zone (SVZ) of a PD mouse model. To accomplish this, male 8-week-old C57BL/6 mice were injected with vehicle or 20 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) four times at 2 h intervals. After the final injection, they were administered water or 100 mg/kg of KRG extract and injected intraperitoneally with 50 mg/kg of 5'-bromo-2'-deoxyuridine-monophosphate (BrdU) once a day for 14 consecutive days. After the last pole test, dopaminergic neuronal survival in the striatum and the substantia nigra (SN), cell proliferation in the SVZ and mRNA expression of neurotrophic factors and dopamine receptors in the striatum were evaluated. KRG administration suppressed dopaminergic neuronal death induced by MPTP in the striatum as well as the SN, augmented the number of BrdU- and BrdU/doublecortin (Dcx)-positive cells in the SVZ and enhanced the expression of proliferation cell nuclear antigen, brain derived neurotrophic factor (BDNF), glial cell derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), ciliary neurotrophic factor (CNTF), dopamine receptor D3 (DRD3) and D5 mRNAs. These results suggest that KRG administration augments neurogenesis in the SVZ of the PD mouse model.

Neurotrophic factors (NTFs) are important for the development, function, and survival of neurons in the mammalian system. Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are two recently identified members of a novel family of NTFs in vertebrates that function to protect dopaminergic neurons. Although these genes are conserved across eukaryotes, their mechanism of neuroprotection is not fully understood. Sequence searches for MANF/CDNF homologs in invertebrates have identified a single orthologue that is most related to MANF. Here we report the in vivo characterization of the MANF gene, manf-1, in the nematode Caenorhabditis elegans. We found that manf-1 mutants have an accelerated, age-dependent decline in the survival of dopaminergic neurons. The animals also show increased endoplasmic reticulum (ER) stress, as revealed by reporter gene expression analysis of hsp-4, an ER chaperone BiP/GRP78 homolog, suggesting that a failure to regulate the ER unfolded protein response (ER-UPR) may be a contributing factor to dopaminergic neurodegeneration. Expression studies of manf-1 revealed that the gene is broadly expressed in a pattern that matches closely with hsp-4. Consistent with the requirements of MANF-1 in the ER-UPR, we found that α-Synuclein protein aggregation in body wall muscles, a major constituent of Lewy bodies, was significantly enhanced in manf-1 mutant animals. Overall, our work demonstrates the important role of MANF-1 in dopaminergic neuronal survival and the maintenance of ER homeostasis in C. elegans.

Neurotrophic factors (NTFs) hold potential as disease-modifying therapies for neurodegenerative disorders like Parkinson’s disease. Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF), and mesencephalic astrocyte-derived neurotrophic factor (MANF) have shown neuroprotective and restorative effects on nigral dopaminergic neurons in various animal models of Parkinson’s disease. To date, however, their effects on brain neurochemistry have not been compared using in vivo microdialysis. We measured extracellular concentration of dopamine and activity of dopamine neurochemistry-regulating enzymes in the nigrostriatal system of rat brain. NTFs were unilaterally injected into the striatum of intact Wistar rats. Brain microdialysis experiments were performed 1 and 3 weeks later in freely-moving animals. One week after the treatment, we observed enhanced stimulus-evoked release of dopamine in the striatum of MANF-treated rats, but not in rats treated with GDNF or CDNF. MANF also increased dopamine turnover. Although GDNF did not affect the extracellular level of dopamine, we found significantly elevated tyrosine hydroxylase (TH) and catechol-O-methyltransferase (COMT) activity and decreased monoamine oxidase A (MAO-A) activity in striatal tissue samples 1 week after GDNF injection. The results show that GDNF, CDNF, and MANF have divergent effects on dopaminergic neurotransmission, as well as on dopamine synthetizing and metabolizing enzymes. Although the cellular mechanisms remain to be clarified, knowing the biological effects of exogenously administrated NTFs in intact brain is an important step towards developing novel neurotrophic treatments for degenerative brain diseases. Electronic supplementary material The online version of this article (10.1007/s12035-018-0872-8) contains supplementary material, which is available to authorized users.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) protein that can be secreted and protects dopamine neurons and cardiomyocytes from ER stress and apoptosis. The mechanism of action of extracellular MANF has long been elusive. From a genetic screen for mutants with abnormal ER stress response, we identified the gene Y54G2A.23 as the evolutionarily conserved C. elegans MANF orthologue. We find that MANF binds to the lipid sulfatide, also known as 3-O-sulfogalactosylceramide present in serum and outer-cell membrane leaflets, directly in isolated forms and in reconstituted lipid micelles. Sulfatide binding promotes cellular MANF uptake and cytoprotection from hypoxia-induced cell death. Heightened ER stress responses of MANF-null C. elegans mutants and mammalian cells are alleviated by human MANF in a sulfatide-dependent manner. Our results demonstrate conserved roles of MANF in sulfatide binding and ER stress response, supporting sulfatide as a long-sought lipid mediator of MANF’s cytoprotection., MANF is a secreted ER stress-inducible protein that protects neurons, pancreatic β cells and cardiomyocytes from cell death under oxidative stress, hypoxic or ischemic conditions. Here the authors show that MANF confers cytoprotection through direct binding to sulfatide followed by cellular uptake in both C. elegans and mammalian cells.

Parkinson's disease (PD) is a progressive neurodegenerative disorder where dopamine (DA) neurons in the substantia nigra degenerate and die. Since no cure for PD exists, there is a need for disease-modifying drugs. Glial cell line-derived neurotrophic factor (GDNF) and related neurturin (NRTN) can protect and repair DA neurons in neurotoxin animal models of PD. However, GDNF was unable to rescue DA neurons in an α-synuclein model of PD, and both factors have shown modest effects in phase two clinical trials. Neurotrophic factors (NTFs), cerebral DA NTF (CDNF) and mesencephalic astrocyte-derived NTF (MANF) form a novel family of evolutionarily conserved, endoplasmic reticulum (ER) located and secreted NTFs. CDNF and MANF have a unique structure and an unparalleled dual mode of action that differs from other known NTFs. Both protect cells from ER stress, and regulate the unfolded protein response via interacting with chaperons, and CDNF dissolves intracellular α-synuclein aggregates. By binding to putative plasma membrane receptors, they promote the survival of DA neurons similarly to conventional NTFs. In animal models of PD, CDNF protects and repairs DA neurons, regulates ER stress, and improves motor function more efficiently than other NTFs.

Cerebral dopamine neurotrophic factor (CDNF) protects and repairs dopamine neurons in animal models of Parkinson's disease, which motivated us to investigate its therapeutic effect in an animal model of Alzheimer's disease (AD). We employed an established APP/PS1 mouse model of AD and gave intrahippocampal injections of CDNF protein or CDNF transgene in an AAV2 viral vector to 1-year-old animals. We performed a behavioral test battery 2 weeks after the injections and collected tissue samples after the 3-week test period. Intrahippocampal CDNF-therapy improved long-term memory in both APP/PS1 mice and wild-type controls, but did not affect spontaneous exploration, object neophobia or early stages of spatial learning. The memory improvement was not associated with decreased brain amyloid load or enhanced hippocampal neurogenesis. Intracranial CDNF treatment has beneficial effects on long-term memory and is well tolerated. The CDNF molecular mechanisms of action on memory await further studies.

UNLABELLED Mice were exposed to 1 month of space flight on the Russian biosatellite BION-M1 to determine its effect on the expression of genes involved in the maintenance of the mouse brain dopamine system. The current article focuses on the genes encoding glial cell line-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF). Space flight reduced expression of the GDNF gene in the striatum and hypothalamus but increased it in the frontal cortex and raphe nuclei area. At the same time, actual space flight reduced expression of the gene encoding CDNF in the substantia nigra but increased it in the raphe nuclei area. To separate the effects of space flight from environmental stress contribution, we analyzed expression of the investigated genes in mice housed for 1 month on Earth in the same shuttle cabins that were used for space flight and in mice of the vivarium control group. Shuttle cabin housing failed to alter the expression of the GDNF and CDNF genes in the brain structures investigated. Thus, actual long-term space flight produced dysregulation in genetic control of GDNF and CDNF genes. These changes may be related to downregulation of the dopamine system after space flight, which we have shown earlier. © 2015 Wiley Periodicals, Inc. SIGNIFICANCE Our results provide the first evidence of microgravity effects on expression of the GDNF and CDNF neurotrophic factor genes. A considerable decrease in mRNA level of GDNF and CDNF in the nigrostriatal dopamine system was found. Because both GDNF and CDNF play a significant role in maintenance and survival of brain dopaminergic neurons, we can assume that this dysregulation in genetic control of GDNF and CDNF genes in substantia nigra could be among the reasons for the deleterious effects of space flight on the dopamine system.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF) are a novel evolutionary conserved neurotrophic factor (NTF) family. There are two distinct domains in MANF and CDNF 3-dimentional structure, N-terminal saposin-like domain and C-terminal SAP-domain, which suggest their unique mode of action. Although identified for their neurotrophic activity, recent studies have shown MANF and CDNF can protect cells during endoplasmic reticulum (ER) stress. This review summarizes the unique structure and related potential protective role for cells during ER stress of MANF and CDNF.

Cerebral Dopamine Neurotrophic Factor (CDNF) and Mesencephalic Astrocyte-derived Neurotrophic factor (MANF) are members of a recently discovered family of neurotrophic factors (NTFs). Here, we used intranigral or intrastriatal lentiviral vector-mediated expression to evaluate their efficacy at protecting dopaminergic function in the 6-OHDA model of Parkinson's disease (PD). In contrast to the well-studied Glial-Derived Neurotrophic Factor (GDNF), no beneficial effects were demonstrated by striatal overexpression of either protein. Interestingly, nigral overexpression of CDNF decreased amphetamine-induced rotations and increased tyroxine hydroxylase (TH) striatal fiber density but had no effect on numbers of TH(+) cells in the SN. Nigral MANF overexpression had no effect on amphetamine-induced rotations or TH striatal fiber density but resulted in a significant preservation of TH(+) cells. Combined nigral overexpression of both factors led to a robust reduction in amphetamine-induced rotations, greater increase in striatal TH-fiber density and significant protection of TH(+) cells in the SN. We conclude that nigral CDNF and MANF delivery is more efficacious than striatal delivery. This is also the first study to demonstrate that combined NTF can have synergistic effects that result in enhanced neuroprotection, suggesting that multiple NTF delivery may be more efficacious for the treatment of PD than the single NTF approaches attempted so far.Molecular Therapy (2014); doi:10.1038/mt.2014.206.

In Drosophila melanogaster, mesencephalic astrocyte-derived neurotrophic factor(DmMANF) is an evolutionarily conserved ortholog of mammalian MANF and cerebral dopamine neurotrophic factor (CDNF), which have been shown to promote the survival of dopaminergic neurons in the brain. We observed especially high levels of DmMANF in the visual system of Drosophil a, particularly in the first optic neuropil (lamina). In the lamina, DmMANF was found in glial cells (surface and epithelial glia), photoreceptors and interneurons. Interestingly, silencing of DmMANF in all neurons or specifically in photoreceptors or L2 interneurons had no impact on the structure of the visual system. However, downregulation of DmMANF in glial cells induced degeneration of the lamina. Remarkably, this degeneration in the form of holes and/or tightly packed membranes was observed only in the lamina epithelial glial cells. Those membranes seem to originate from the endoplasmic reticulum, which forms autophagosome membranes. Moreover, capitate projections, the epithelial glia invaginations into photoreceptor terminals that are involved in recycling of the photoreceptor neurotransmitter histamine, were less numerous after DmMANF silencing either in neurons or glial cells. The distribution of the alpha subunit of Na+/K+-ATPase protein in the lamina cell membranes was also changed. At the behavioral level, silencing of DmMANF either in neurons or glial cells affected the daily activity/sleep pattern, and flies showed less activity during the day but higher activity during the night than did controls. In the case of silencing in glia, the lifespan of flies was also shortened. The obtained results showed that DmMANF regulates many functions in the brain, particularly those dependent on glial cells.

Visual Abstract, Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) resident protein with neuroprotective effects. Previous studies have shown that MANF expression is altered in the developing rodent cortex in a spatiotemporal manner. However, the role of MANF in mammalian neurogenesis is not known. The aim of this study was to determine the role of MANF in neural stem cell (NSC) proliferation, differentiation, and cerebral cortex development. We found that MANF is highly expressed in neural lineage cells, including NSCs in the developing brain. We discovered that MANF-deficient NSCs in culture are viable and show no defect in proliferation. However, MANF-deficient cells have deficits in neurite extension upon neuronal differentiation. In vivo, MANF removal leads to slower neuronal migration and impaired neurite outgrowth. In vitro, mechanistic studies indicate that impaired neurite growth is preceded by reduced de novo protein synthesis and constitutively activated unfolded protein response (UPR) pathways. This study is the first to demonstrate that MANF is a novel and critical regulator of neurite growth and neuronal migration in mammalian cortical development.

Neurotrophins play a major role in adult neuronal survival, maintenance and regeneration. Alterations in their levels have been implicated in various neurodegenerative disorders, including Parkinson’s disease (PD). It has been reported that, the pathophysiology of PD progress is essentially depends on various striatal signaling cascade, which consists handful of neurotrophic factors namely, cerebral dopamine neurotrophic factor (CDNF), glial derived neurotrophic factor (GDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), brain derived neurotrophic factor (BDNF), nerve growth factor (NGF). Although, the exact pathophysiology of PD is remained elusive however, the loss of dopaminergic neurons and dopamine deficiency has considered as a major consequence for the movement disability as seen in PD. It has been proposed that loss of dopaminergic neurons in PD may be conducted by inadequate neurotrophic activity which leads to neuronal apoptosis. In addition, stimulation of neurotrophic factors in the striatal brain region has been reported to be beneficial in experimental models of PD. In the current review we have detailed out the neurotrophic factors and their role in pathogenesis of Parkinson’s disease.

Summary: All forms of diabetes mellitus (DM) are characterized by the loss of functional pancreatic β cell mass, leading to insufficient insulin secretion. Thus, identification of novel approaches to protect and restore β cells is essential for the development of DM therapies. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-inducible protein, but its physiological role in mammals has remained obscure. We generated MANF-deficient mice that strikingly develop severe diabetes due to progressive postnatal reduction of β cell mass, caused by decreased proliferation and increased apoptosis. Additionally, we show that lack of MANF in vivo in mouse leads to chronic unfolded protein response (UPR) activation in pancreatic islets. Importantly, MANF protein enhanced β cell proliferation in vitro and overexpression of MANF in the pancreas of diabetic mice enhanced β cell regeneration. We demonstrate that MANF specifically promotes β cell proliferation and survival, thereby constituting a therapeutic candidate for β cell protection and regeneration. : Potent strategies for diabetes mellitus (DM) prevention and treatment are urgently needed. MANF is an endoplasmic reticulum stress-inducible protein, but its physiological role in mammals is currently unknown. Lindahl et al. now find that MANF-deficient mice develop diabetes. Importantly, MANF protein enhances β cell proliferation in vitro and pancreatic MANF overexpression in diabetic mice enhances β cell regeneration. Because MANF is expressed in human β cells, it might have therapeutic potential for diabetes treatment.

Parkinson’s disease (PD) is a neurodegenerative disorder associated with a progressive loss of dopaminergic (DAergic) neurons of the substantia nigra (SN) and the accumulation of intracellular inclusions containing α-synuclein. Current therapies do not stop the progression of the disease, and the efficacy of these treatments wanes over time. Neurotrophic factors (NTFs) are naturally occurring proteins promoting the survival and differentiation of neurons and the maintenance of neuronal contacts. CDNF (cerebral dopamine NTF) and GDNF (glial cell line-derived NTF) are able to protect DAergic neurons against toxin-induced degeneration in experimental models of PD. Here, we report an additive neurorestorative effect of coadministration of CDNF and GDNF in the unilateral 6-hydroxydopamine (6-OHDA) lesion model of PD in rats. NTFs were given into the striatum four weeks after unilateral intrastriatal injection of 6-OHDA (20 µg). Amphetamine-induced (2.5 mg/kg, i.p.) rotational behavior was measured every two weeks. Number of tyrosine hydroxylase (TH)-positive cells from SN pars compacta (SNpc) and density of TH-positive fibers in the striatum were analyzed at 12 weeks after lesion. CDNF and GDNF alone restored the DAergic function, and one specific dose combination had an additive effect: CDNF (2.5µg) and GDNF (1µg) coadministration led to a stronger trophic effect relative to either of the single treatments alone. The additive effect may indicate different mechanism of action for the NTFs. Indeed, both NTFs activated the survival promoting PI3 kinase (PI3K)-Akt signaling pathway, but only CDNF decreased the expression level of tested endoplasmatic reticulum (ER) stress markers ATF6, glucose-regulated protein 78 (GRP78), and phosphorylation of eukaryotic initiation factor 2α subunit (eIF2α).

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) promote the survival of midbrain dopaminergic neurons which degenerate in Parkinson's disease (PD). However, CDNF and MANF are structurally and functionally clearly distinct from the classical, target-derived neurotrophic factors (NTFs) that are solely secreted proteins. In cells, CDNF and MANF localize in the endoplasmic reticulum (ER) and evidence suggests that MANF, and possibly CDNF, is important for the maintenance of ER homeostasis. MANF expression is particularly high in secretory tissues with extensive protein production and thus a high ER protein folding load. Deletion of MANF in mice results in a diabetic phenotype and the activation of unfolded protein response (UPR) in the pancreatic islets. However, information about the intracellular and extracellular mechanisms of MANF and CDNF action is still limited. Here we will discuss the structural motifs and physiological functions of CDNF and MANF as well as their therapeutic potential for the treatment of neurodegenerative diseases and diabetes. Currently available knockout models of MANF and CDNF in mice, zebrafish and fruit fly will increase information about the biology of these interesting proteins.

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔCMANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔCMANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNFRTDL and CDNFSARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNFKTEL and CDNFHPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANFQTEL and MANFYPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANFKTEL and MANFHPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.

Neuroinflammation is recognized as a major factor in Parkinson's disease (PD) pathogenesis and increasing evidence propose that microglia is the main source of inflammation contributing to the dopaminergic degeneration observed in PD. Several studies suggest that astrocytes could act as physiological regulators preventing excessive microglia responses. However, little is known regarding how astrocytes modulate microglial activation. In the present study, using Zymosan A-stimulated midbrain microglia cultures, we showed that astrocytes secrete factors capable of modulating microglial activation, namely its phagocytic activity and the production of reactive oxygen species since both parameters were highly diminished in cells incubated with astrocytes conditioned media (ACM). Glial cell line-derived neurotrophic factor (GDNF), cerebral dopamine neurotrophic factor (CDNF) and brain-derived neurotrophic factor (BDNF), known to have a neuroprotective role in the nigrostriatal system, are among the candidates to be astrocyte-secreted molecules involved in the modulation of microglial activation. The effect of ACM on Zymosan A-induced microglial activation was abolished when the GDNF present in the ACM was abrogated using a specific antibody, but not when ACM was neutralized with anti-CDNF, anti-BDNF or with a heat-inactivated GDNF antibody. In addition, media conditioned by astrocytes silenced for GDNF were not able to prevent microglial activation, whereas supplementation of non-conditioned media with GDNF prevented the activation of microglia evoked by Zymosan A. Taken together, these results indicate that astrocyte-derived GDNF plays a major contribution to the control of midbrain microglial activation, suggesting that GDNF can protect from neurodegeneration through the inhibition of neuroinflammation.

Neurotrophic factors support the survival of dopaminergic neurons. The cerebral dopamine neurotrophic factor (CDNF) is a novel neurotrophic factor with strong trophic activity on dopaminergic neurons comparable to that of glial cell line-derived neurotrophic factor (GDNF). To investigate whether rare or common variants in CDNF are associated with Parkinson disease (PD), we performed mutation analysis of CDNF and a genetic association study between CDNF polymorphisms and PD. We screened 110 early-onset Parkinson disease (EOPD) patients for CDNF mutations. Allelic and genotype frequencies of 3 CDNF single nucleotide polymorphisms (SNPs) (rs1901650, rs7094179, and rs11259365) were compared in 215 PD patients and age- and sex-matched controls. We failed to identify any mutations in CDNF among the EOPD patient sample population. We observed a trend towards increased risk for PD in patients carrying the C allele of SNP rs7094179 (odds ratio (OR) = 1.27, 95% confidence interval (CI) 0.96–1.67). Patients carrying the C allele were susceptible to PD in both dominant (CC + CA vs. AA; OR = 7.20, 95% CI 0.88–59.1) and recessive (CA + AA vs. CC; OR = 0.64, 95% CI 0.41–0.99) models. Genotype and allele frequencies of SNPs rs1901650 and rs11259365 did not differ between PD patients and controls. Our study suggests that the C allele of an intronic CDNF SNP (rs7094179) might be an allele for susceptibility to PD. Further studies with larger sample size are required to confirm our results.

Cerebral dopamine neurotrophic factor (CDNF) is a novel evolutionary conserved protein which can protect and restore the function of dopaminergic neurons in the rat model of Parkinson's disease, suggesting that CDNF might be beneficial for the treatment of Parkinson's disease. CDNF is widely expressed in neurons in several brain regions including cerebral cortex, hippocampus, substantia nigra, striatum and cerebellum. Human CDNF is glycosylated and secreted from transiently transfected cells; however, the mechanism underlying CDNF secretion is currently unclear. In this study, we found that CDNF could be secreted primarily via the regulated secretion pathway in PC12 cells. The glycosylation of CDNF is not required for its secretion. Moreover, we identified two key subdomains in CDNF which are important for its intracellular localization and secretion. Disrupting helix-1 of CDNF significantly reduces its constitutive and regulated secretion and the helix-1 mutant is retained in the endoplasmic reticulum. Although helix-7 mutation only decreases CDNF regulated secretion and has no effect on its constitutive secretion, which is further supported by the reduction in co-localization of helix-7 mutant with secretory granules. In all, these findings will advance our understanding of the molecular mechanism of CDNF trafficking and secretion.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secreted protein which reduces endoplasmic reticulum (ER) stress and has neurotrophic effects on dopaminergic neurons. Intracortical delivery of recombinant MANF protein protects tissue from ischemic brain injury in vivo. In this study, we examined the protective effect of adeno-associated virus serotype 7 encoding MANF in a rodent model of stroke. An AAV vector containing human MANF cDNA (AAV-MANF) was constructed and verified for expression of MANF protein. AAV-MANF or an AAV control vector was administered into three sites in the cerebral cortex of adult rats. One week after the vector injections, the right middle cerebral artery (MCA) was ligated for 60 min. Behavioral monitoring was conducted using body asymmetry analysis, neurological testing, and locomotor activity. Standard immunohistochemical and western blotting procedures were conducted to study MANF expression. Our data showed that AAV-induced MANF expression is redistributed in neurons and glia in cerebral cortex after ischemia. Pretreatment with AAV-MANF reduced the volume of cerebral infarction and facilitated behavioral recovery in stroke rats. In conclusion, our data suggest that intracortical delivery of AAV-MANF increases MANF protein production and reduces ischemic brain injury. Ischemia also caused redistribution of AAV-mediated MANF protein suggesting an injury-induced release.

Cerebral dopamine neurotrophic factor (CDNF), previously known as the conserved dopamine neurotrophic factor, belongs to the evolutionarily conserved CDNF/mesencephalic astrocyte-derived neurotrophic factor MANF family of neurotrophic factors that demonstrate neurotrophic activities in dopaminergic neurons. The function of CDNF during brain ischemia is still not known. MANF is identified as an endoplasmic reticulum (ER) stress protein, however, the role of CDNF in ER stress remains to be fully elucidated. Here, we test the neuroprotective effect of CDNF on middle cerebral artery occlusion (MCAO) rats and neurons and astrocytes treated with oxygen&ndash, glucose depletion (OGD). We also investigate the expression of CDNF in cerebral ischemia and in primary neurons treated with ER stress-inducing agents. Our results show that CDNF can significantly reduce infarct volume, reduce apoptotic cells and improve motor function in MCAO rats, while CDNF can increase the cell viability of neurons and astrocytes treated by OGD. The expression of CDNF was upregulated in the peri-infarct tissue at 2 h of ischemia/24 h reperfusion. ER stress inducer can induce CDNF expression in primary cultured neurons. Our data indicate that CDNF has neuroprotective effects on cerebral ischemia and the OGD cell model and the protective mechanism of CDNF may occur through ER stress pathways.

Degeneration of the locus coeruleus noradrenergic system is thought to play a key role in the pathogenesis of Parkinson's disease (PD), whereas pharmacological approaches to increase noradrenaline bioavailability may provide neuroprotection. Noradrenaline inhibits microglial activation and suppresses pro-inflammatory mediator production (e.g., tumor necrosis factor-α, interleukin-1β & inducible nitric oxide synthase activity), thus limiting the cytotoxicity of midbrain dopaminergic neurons in response to an inflammatory stimulus. Neighbouring astrocyte populations promote a neurotrophic environment in response to β2-adrenoceptor (β2-AR) stimulation via the production of growth factors (e.g., brain derived neurotrophic factor, cerebral dopamine neurotrophic factor & glial cell derived neurotrophic factor which have shown promising neuroprotective and neuro-restorative effects in the nigrostriatal dopaminergic system. More recent findings have demonstrated a role for the β2-AR in down-regulating expression levels of the human α-synuclein gene SNCA and relative α-synuclein protein abundance. Given that α-synuclein is a major protein constituent of Lewy body pathology, a hallmark neuropathological feature in Parkinson's disease, these findings could open up new avenues for pharmacological intervention strategies aimed at alleviating the burden of α-synucleinopathies in the Parkinsonian brain. In essence, the literature reviewed herein supports our hypothesis of a tripartite neuroprotective role for noradrenaline in combating PD-related neuropathology and motor dysfunction via (1) inhibiting nigral microglial activation & pro-inflammatory mediator production, (2) promoting the synthesis of neurotrophic factors from midbrain astrocytes and (3) downregulating α-synuclein gene expression and protein abundance in a β2-AR-dependent manner. Thus, taken together, either pharmacologically enhancing extra-synaptic noradrenaline bioavailability or targeting glial β2-ARs directly makes itself as a promising treatment option aimed at slowing/halting PD progression.

Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known as Arginine-rich, Mutated in Early Stage of Tumors (ARMET), is a secreted protein which reduces endoplasmic reticulum (ER) stress. Previous studies have shown that MANF mRNA expression and protein levels are increased in the cerebral cortex after brain ischemia, a condition which induces ER stress. The function of MANF during brain ischemia is still not known. The purpose of this study was to examine the protective effect of MANF after ischemic brain injury. Recombinant human MANF was administrated locally to the cerebral cortex before a 60-min middle cerebral artery occlusion (MCAo) in adult rats. Triphenyltetrazolium chloride (TTC) staining indicated that pretreatment with MANF significantly reduced the volume of infarction at two days after MCAo. MANF also attenuated TUNEL labeling, a marker of cell necrosis/apoptosis, in the ischemic cortex. Animals receiving MANF pretreatment demonstrated a decrease in body asymmetry and neurological score as well as an increase in locomotor activity after MCAo. Taken together, these data suggest that MANF has neuroprotective effects against cerebral ischemia, possibly through the inhibition of cell necrosis/apoptosis in cerebral cortex.

We have solved the structures of mammalian mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF). CDNF protects and repairs midbrain dopaminergic neurons in vivo; MANF supports their survival in culture and is also cytoprotective against endoplasmic reticulum (ER) stress. Neither protein structure resembles any known growth factor but the N-terminal domain is a saposin-like lipid-binding domain. MANF and CDNF may thus bind lipids or membranes. Consistent with this, there are two patches of conserved lysines and arginines. The natively unfolded MANF C-terminus contains a CKGC disulphide bridge, such as reductases and disulphide isomerases, consistent with a role in ER stress response. The structure thus explains why MANF and CDNF are bifunctional; neurotrophic activity may reside in the N-terminal domain and ER stress response in the C-terminal domain. Finally, we identified three changes, (MANF)I10-->K(CDNF), (MANF)E79-->M(CDNF) and (MANF)K88-->L(CDNF), that may account for the biological differences between the proteins.

In Parkinson's disease, brain dopamine neurons degenerate most prominently in the substantia nigra. Neurotrophic factors promote survival, differentiation and maintenance of neurons in developing and adult vertebrate nervous system. The most potent neurotrophic factor for dopamine neurons described so far is the glial-cell-line-derived neurotrophic factor (GDNF). Here we have identified a conserved dopamine neurotrophic factor (CDNF) as a trophic factor for dopamine neurons. CDNF, together with its previously described vertebrate and invertebrate homologue the mesencephalic-astrocyte-derived neurotrophic factor, is a secreted protein with eight conserved cysteine residues, predicting a unique protein fold and defining a new, evolutionarily conserved protein family. CDNF (Armetl1) is expressed in several tissues of mouse and human, including the mouse embryonic and postnatal brain. In vivo, CDNF prevented the 6-hydroxydopamine (6-OHDA)-induced degeneration of dopaminergic neurons in a rat experimental model of Parkinson's disease. A single injection of CDNF before 6-OHDA delivery into the striatum significantly reduced amphetamine-induced ipsilateral turning behaviour and almost completely rescued dopaminergic tyrosine-hydroxylase-positive cells in the substantia nigra. When administered four weeks after 6-OHDA, intrastriatal injection of CDNF was able to restore the dopaminergic function and prevent the degeneration of dopaminergic neurons in substantia nigra. Thus, CDNF was at least as efficient as GDNF in both experimental settings. Our results suggest that CDNF might be beneficial for the treatment of Parkinson's disease.

Parkinson's disease (PD is a progressive neurological disorder characterized by the degeneration and death of midbrain dopamine and non-dopamine neurons in the brain leading to motor dysfunctions and other symptoms, which seriously influence the quality of life of PD patients. The drug L-dopa can alleviate the motor symptoms in PD, but so far there are no rational therapies targeting the underlying neurodegenerative processes. Despite intensive research, the molecular mechanisms causing neuronal loss are not fully understood which has hampered the development of new drugs and disease-modifying therapies. Neurotrophic factors are by virtue of their survival promoting activities attract candidates to counteract and possibly halt cell degeneration in PD. In particular, studies employing glial cell line-derived neurotrophic factor (GDNF) and its family member neurturin (NRTN), as well as the recently described cerebral dopamine neurotrophic factor (CDNF) and the mesencephalic astrocyte-derived neurotrophic factor (MANF) have shown positive results in protecting and repairing dopaminergic neurons in various models of PD. Other substances with trophic actions in dopaminergic neurons include neuropeptides and small compounds that target different pathways impaired in PD, such as increased cell stress, protein handling defects, dysfunctional mitochondria and neuroinflammation. In this review, we will highlight the recent developments in this field with a focus on trophic factors and substances having the potential to beneficially influence the viability and functions of dopaminergic neurons as shown in preclinical or in animal models of PD.

International audience; Cerebral dopamine neurotrophic factor (CDNF) is a promising therapeutic agent for Parkinson disease. As such, there has been great interest in studying its mode of action, which remains unknown. The three-dimensional crystal structure of the N terminus (residues 9-107) of CDNF has been determined, but there have been no published structural studies on the full-length protein due to proteolysis of its C-terminal domain, which is considered intrinsically disordered. An improved purification protocol enabled us to obtain active full-length CDNF and to determine its three-dimensional structure in solution. CDNF contains two well folded domains (residues 10-100 and 111-157) that are linked by a loop of intermediate flexibility. We identified two surface patches on the N-terminal domain that were characterized by increased conformational dynamics that should allow them to embrace active sites. One of these patches is formed by residues Ser-33, Leu-34, Ala-66, Lys-68, Ile-69, Leu-70, Ser-71, and Glu-72. The other includes a flexibly disordered N-terminal tail (residues 1-9), followed by the N-terminal portion of α-helix 1 (residues Cys-11, Glu-12, Val-13, Lys-15, and Glu-16) and residue Glu-88. The surface of the C-terminal domain contains two conserved active sites, which have previously been identified in mesencephalic astrocyte-derived neurotrophic factor, a CDNF paralog, which corresponds to its intracellular mode of action. We also showed that CDNF was able to protect dopaminergic neurons against injury caused by α-synuclein oligomers. This advises its use against physiological damages caused by α-synuclein oligomers, as observed in Parkinson disease and several other neurodegenerative diseases.

Neurotrophic factors (NTFs) hold great potential as therapeutic agents in the treatment of neurodegenerative conditions, including Parkinson's disease (PD), in which the progressive loss of dopamine neurons in the substantia nigra pars compacta causes severe motor symptoms. There is extensive evidence that in preclinical animal models of PD NTFs are both neuroprotective and neurorestorative. In particular, glial cell line-derived neurotrophic factor (GDNF), neurturin (NRTN), cerebral dopamine neurotrophic factor, and mesencephalic astrocyte-derived neurotrophic factor have shown great potential to restore dopamine neurocircuitry. Although some previous clinical trials have demonstrated limited efficacy of GDNF and NRTN, there are several concerns raised with these studies. Moreover, open-label studies with GDNF as well as a study with NRTN showed clinical improvement, particularly in patients with early-stage PD. Indeed, as previous clinical trials with NTFs were associated with several technical problems, there is a great need for further investigations. In this review we discuss the emerging and existing possibilities to use NTFs as neurorestorative agents and the ways to improve their efficacy, and compare gene therapy and recombinant protein therapy approaches for restoring the dopamine circuitry in PD.

Microbubbles (MBs) are small (1-5 μm diameter), perfluorcarbon gas filled lipid microspheres used for contrast enhancement during ultrasound imaging. MBs (Definity®, others) are approved for contrast echocardiography and widely used (6 M patient injections) as ultrasound contrast agents with established pharmacokinetics and clearance data. Recent research has shifted to the therapeutic potential of MBs and the NRC and Artenga Inc. developed a novel strategy (Patent to be filed) to load biologics onto MBs with high, consistent loading using a novel clinically scalable methodology. Investigators at Sunnybrook Research Institute have pioneered MR guided focused ultrasound (MRgFUS) plus MB techniques to deliver a wide range of therapeutic agents through the blood-brain-barrier (BBB) and developed a state of the art MRgFUS system for animal models. MRgFUS can precisely target the substantia nigra (SN) and striatum (ST) for simultaneous targeting of multiple brain regions. Neurotrophic factors (NTFs) are being developed in the hope of halting or reversing the progression of neuronal loss in chronic neurodegenerative diseases, however, their clinical utility is limited by drug delivery issues. Recently the University of Helsinki discovered a novel NTF, cerebral dopamine neurotrophic factor (CDNF), with a unique structure and mode of action. CDNF has demonstrated improved efficacy compared to GDNF in various preclinical models of PD. Artenga, Inc., Sunnybrook Research Institute, the National Research Council of Canada (NRC), and the University of Helsinki plan is to use MR guided focused ultrasound (MRgFUS) and neurotrophic drug-loaded microbubbles (NTF-MBs) for targeted, noninvasive, blood brain barrier (BBB) drug delivery of CDNF.