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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.

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.

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf(-/-)), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf(-/-) mouse are much greater than in those of Cdnf(+/+) mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf(-/-) mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf(-/-) male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf(-/-) mouse brain is altered. The deficiencies of Cdnf(-/-) mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.

The Cerebral Dopamine Neurotrophic Factor (CDNF) is a neurotrophic factor that has a protective effect in cell and animal models of several neurodegenerative diseases. The molecular mechanism of the protective effect of CDNF is unclear. Many neurodegenerative diseases have been related to a proteostasis dysregulation in the endoplasmic reticulum (ER). A failure of proteostasis produces ER stress, triggering the unfolded protein response (UPR) and, in the long-term, induces cell death. An adaptive UPR solves ER stress by attenuating protein synthesis, inducing chaperones expression, and degradation of misfolded proteins. Since CDNF is an ER resident protein, we investigated whether the role of CDNF is to regulate ER proteostasis. To this end, we determined the effect of CDNF in thapsigargin-induced ER stress in HEK293-T cells and cultured hippocampal neurons. Our results show that CDNF improved the viability of HEK293-T cells exposed to thapsigargin. CDNF increased levels of protective proteins of the early UPR, such as BiP, ATF4, ATF6, and XBP-1 in both HEK293-T cells and neurons. Conversely, expression of CDNF attenuated ER stress-induced apoptotic proteins, CHOP and cleaved caspase-3 in HEK293-T cells and neurons. A mutant CDNF lacking the ER retention sequence failed to protect against ER stress. In conclusion, CDNF regulates proteostasis in the ER by inducing the adaptive UPR response and inhibiting apoptotic pathways triggered by ER stress. We propose that neuroprotection induced by CDNF is mediated by regulating ER proteostasis.

Previous research advocates that exercise is a non-pharmacological therapy for Parkinson's disease (PD). However, few studies have investigated the effects of exercise on central nervous system structures other than the nigrostriatal pathway by using PD animal models. This study investigated the effects of exercise on tyrosine hydroxylase (TH)- and cerebral dopamine neurotrophic factor (CDNF)-containing spinal-cord neurons. Male Swiss mice were divided into 4 groups: sedentary control (SEDCONT), exercise control (EXERCONT), sedentary Parkinson (SEDPD), and exercise Parkinson (EXERPD). The PD groups were submitted to a surgical procedure for stereotaxic bilateral injection of 6-hydroxydopamine into the striatum. TH- and CDNF-containing spinal-cord neurons were evaluated in all groups, using immunohistochemistry and western-blotting. TH content in the ventral horn differed notably between the SEDPD and EXERPD groups. CDNF content was highest in the EXERPD group. SEDPD and EXERPD groups differed the most, as shown by immunohistochemistry and western-blotting. The EXERPD group showed the most intense labeling in immunohistochemistry compared to the SEDCONT and EXERCONT groups. Therefore, we showed here that exercise increased the content of both TH and CDNF in the spinal-cord neurons of a bilateral PD mouse model. We may assume that the spinal cord is affected in a PD model, and therefore this central nervous system region deserves more attention from researchers dealing with PD.

Cerebral dopamine neurotrophic factor (CDNF) has been studied in animal models of Parkinson’s disease, where it was shown to repair and protect dopamine neurons. Alongside its neurotrophic activity, it can also localize in the endoplasmic reticulum (ER) acting as an ER stress response (ERSR) protein to maintain ER homeostasis. Since ER stress plays a major role in the development and progression of cardiovascular diseases, we investigated the role of CDNF in cardiomyocytes during ER stress. Initially, the expression of CDNF was tested by treating H9c2 cells with various concentrations of tunicamycin (TM) and performing reverse-transcriptase polymerase chain reaction and Western blotting. To evaluate the overexpression of CDNF in cardiomyocytes, H9c2 cells were transfected with pcDNA-CDNF and analyzed by Western blotting and indirect immunofluorescence microscopy. The effects of CDNF on cardiomyocytes during ER stress were analyzed with CCK-8 method and TUNEL staining using cells transfected with pcDNA-CDNF or pcDNA3.1. The percentage of TUNEL-positive cells was quantified as the apoptotic level. Our results showed that CDNF protein expression can be induced by activation of ER stress in cultured cardiomyocytes. Moreover, overexpression of CDNF improved cell viability and protected cardiomyocytes from apoptosis induced by ER stress. The findings presented here contribute toward identifying the physiological functions of CDNF in cardiovascular diseases.

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.

Cerebral dopamine neurotrophic factor (CDNF) protects dopaminergic neurons against toxic damage in the rodent brain and is in clinical trials to treat Parkinson's disease patients. Yet the underlying mechanism is poorly understood. To examine its significance for neural circuits and behavior, we examined the development of neurotransmitter systems from larval to male adult mutant zebrafish lacking cdnf. Although a lack of cdnf did not affect overall brain dopamine levels, dopaminergic neuronal clusters showed significant abnormalities. The number of histamine neurons that surround the dopaminergic neurons was significantly reduced. Expression of tyrosine hydroxylase 2 in the brain was elevated in cdnf mutants throughout their lifespan. There were abnormally few GABA neurons in the hypothalamus in the mutant larvae, and expression of glutamate decarboxylase was reduced throughout the brain. cdnf mutant adults showed a range of behavioral phenotypes, including increased sensitivity to pentylenetetrazole-induced seizures. Shoaling behavior of mutant adults was abnormal, and they did not display social attraction to conspecifics. CDNF plays a profound role in shaping the neurotransmitter circuit structure, seizure susceptibility, and complex behaviors in zebrafish. These findings are informative for dissecting the diverse functions of this poorly understood factor in human conditions related to Parkinson's disease and complex behaviors. SIGNIFICANCE STATEMENT A zebrafish lacking cdnf grows normally and shows no overt morphologic phenotype throughout the life span. Remarkably, impaired social cohesion and increased seizure susceptibility were found in adult cdnf KO fish conceivably associated with significant changes of dopaminergic, GABAergic, and histaminergic systems in selective brain areas. These findings suggest that cdnf has broad effects on regulating neurogenesis and maturation of transmitter-specific neuronal types during development and throughout adulthood, rather than ones restricted to the dopaminergic systems.

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.

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.

Parkinson's disease (PD) is characterized by progressive loss of dopamine cells. It is suggested that exercise could be employed as a non-pharmacological approach for reducing the risk of PD incidence. The purpose of this study was to compare the effects of 4-week Mild-intensity (MIEx) and progressive exercise (PEx) protocols on rotational behavior, GFAP, DA, TH, MANF, CDNF and NGF levels in striatum of parkinsonian rats induced by 6-hydroxydopamine.42 Wistar male rats were divided into 6 groups including, healthy and PD controls, MIEx, PEx, healthy MIEx, and healthy PEx. MIEx protocol was performed as follows: 5 days a week, 2 sessions a day of 15 min at a speed of 15 m/min. PEx protocol encompassed a training regimen of 5 days a week initiating by 20 min in the first day reaching 50 min on the fifth day and 60 min in the next 3 weeks. PD was induced after training protocol by injection of 6-OHDA into the striatum of rats. For confirming PD, apomorphine rotational test was employed.The MIEx protocol did not have any positive impacts on the variables except for CDNF (P 0.0001). Levels of DA (P 0.0001) and TH (P = 0.0004) increased significantly after performing PEx protocol. Moreover, PEx protocol considerably reduced rotational behavior of rats (P = 0.0244).The findings of this research confirm positive effects of PEx in protecting against PD. This progressive training protocol has explicitly shown a neuroprotective effect against PD-inducing nervous toxin through increasing neurotrophins.

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.

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.

Background The activation of microglia plays a crucial role in neuroinflammation. Previous studies have shown that cerebral dopamine neurotrophic factor (CDNF) has a protective effect on neuroinflammation, but the mechanisms involved have not been fully studied. AKT is a serine-threonine protein kinase widely expressed in mammals through which the downstream pathway FoxO1/mTOR is closely related to cell inflammation, apoptosis, metabolism, etc. Therefore, we examined whether CDNF regulates neuroinflammation through this pathway. Methods After pretreatment with CDNF and LPS, microglial cells were detected by laser confocal microscopy, coimmunoprecipitation and immunofluorescence to observe whether CDNF was colocalized with AKT. The expression of AKT and its downstream FoxO1/mTOR were determined by Western blot. The effect of CDNF on inflammatory cytokines was detected by ELISA, and the mRNA levels of AKT and FoxO1/mTOR were detected by qRT-PCR. Results Laser confocal and coimmunoprecipitation experiments significantly reveal the occurrence of interactions between AKT and CDNF in microglia. Western blot results show that CDNF incubation suppressed the activation of AKT/FoxO1/mTOR signaling. Moreover, CDNF clearly decreased the expression of inflammatory cytokines. In qRT-PCR, the expression of mRNA in AKT and its downstream FoxO1/mTOR gradually decreased due to CDNF intervention. Conclusions CDNF combined with AKT and regulated the downstream pathway FoxO1/mTOR in microglia, eventually suppressing the secretion of inflammatory factors. Therefore, CDNF might play a protective role in the neuroinflammation of microglia via AKT/FoxO1/mTOR signaling.

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.

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.

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.

Cerebral dopamine neurotrophic factor (CDNF) protects the nigrostriatal dopaminergic (DA) neurons in rodent models of Parkinson’s disease and restores DA circuitry when delivered after these neurons have begun to degenerate. These DA neurons have been suggested to transport striatal CDNF retrogradely to the substantia nigra (SN). However, in cultured cells the binding and internalization of extracellular CDNF has not been reported. The first aim of this study was to examine the cellular localization and pharmacokinetic properties of recombinant human CDNF (rhCDNF) protein after its infusion into rat brain parenchyma. Second, we aimed to study whether the transport of rhCDNF from the striatum to the SN results from its retrograde transport via DA neurons or from its anterograde transport via striatal GABAergic projection neurons. We show that after intrastriatal infusion, rhCDNF diffuses rapidly and broadly, and is cleared with a half-life of 5.5 h. Confocal microscopy analysis of brain sections at 2 and 6 h after infusion of rhCDNF revealed its widespread unspecific internalization by cortical and striatal neurons, exhibiting different patterns of subcellular rhCDNF distribution. Electron microscopy analysis showed that rhCDNF is present inside the endosomes and multivesicular bodies. In addition, we present data that after intrastriatal infusion the rhCDNF found in the SN is almost exclusively localized to the DA neurons, thus showing that it is retrogradely transported.

The potential value of cerebral dopamine neurotrophic factor (CDNF) in treating Parkinson's disease (PD) remains controversial. To evaluate the therapeutic effects of CDNF-expressing bone marrow derived mesenchymal stem cell (CDNF-MSCs) injections in a rat model of Parkinson's disease, we chose three different routes of CDNF-MSC administration, including intra-striatal, intra-ventricular, and intravenous pathways. Parkinsonism was induced by intra-striatal unilateral injection of 6-OHDA and then rats were subsequently randomized into three groups for either intra-striatal, intra-ventricular or intravenous injection for CDNF-MSC grafting. Therapeutic effects were evaluated by observing dopaminergic (DA) neurons both in the substantia nigra compacta (SNc) and within the striatum and by monitoring apomorphine-induced rotational behavior (circling). Data show that one intra-venous administration of CDNF-MSCs was ineffective for treating Parkinson's disease-like neurodegeneration. Conversely, intra-striatal grafts can reduce loss of DA neurons both in the SNc and striatum with improvement of Parkinson's-related behaviors, compared to intra-ventricular injections. Thus, intra-striatal grafts composed of CDNF-MSCs may provide a strategy for therapeutic delivery to treat PD.

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.

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.

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.

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.

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.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the progressive loss of midbrain dopaminergic neurons, which causes motor impairments. Current treatments involve dopamine replacement to address the disease symptoms rather than its cause. Factors that promote the survival of dopaminergic neurons have been proposed as novel therapies for PD. Several dopaminergic neurotrophic factors (NTFs) have been examined for their ability to protect and/or restore degenerating dopaminergic neurons, both in animal models and in clinical trials. These include glial cell line-derived neurotrophic factor, neurturin, cerebral dopamine neurotrophic factor and growth/differentiation factor 5. Delivery of these NTFs via injection or infusion to the brain raises several practical problems. A new delivery approach for NTFs involves the use of recombinant viral vectors to enable long-term expression of these factors in brain cells. Vectors used include those based on adenoviruses, adeno-associated viruses and lentiviruses. Here we review progress to date on the potential of each of these four NTFs as novel therapeutic strategies for PD, as well as the challenges that have arisen, from pre-clinical analysis to clinical trials. We conclude by discussing recently-developed approaches to optimise the delivery of NTF-carrying viral vectors to the brain.

We describe the discovery of a novel, 20 kDa, secreted human protein named mesencephalic astrocyte-derived neurotrophic factor, or MANF. The homologous, native molecule was initially derived from a rat mesencephalic type-1 astrocyte cell line and recombinant MANF subcloned from a cDNA encoding human arginine-rich protein. MANF selectively protects nigral dopaminergic neurons, versus GABAergic or serotonergic neurons. The discovery of MANF marks a more systematic approach in the search for astrocyte-derived, secreted proteins that selectively protect specific neuronal phenotypes. Compared to glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF), MANF was more selective in the protection of dopaminergic neurons at lower (0.05–0.25 ng/mL) and middle (0.5–2.5 ng/mL) concentrations: MANF>GDNF>BDNF. GDNF was more selective at higher concentrations (25–50 ng/ml): GDNF>MANF>BDNF. Two domains in MANF of 39-AA and 109-AA respectively, and eight cysteines are conserved from C. elegans to man. MANF is encoded by a 4.3 Kb gene with 4 exons, and is located on the short arm of human chromosome 3. The secondary structure is dominated by α-helices (47%) and random coils (37%). Studies to determine the localization of MANF in the brains of rat, monkey, and man, as well as the receptor, signaling pathways, and biologically active peptide mimetics are in progress. The selective, neuroprotective effect of MANF for dopaminergic neurons suggests that it may be indicated for the treatment of Parkinson’s disease., DA - 20030609IS - 0895-8696LA - engPT - Journal ArticleRN - 0 (Brain-Derived Neurotrophic Factor)RN - 0 (DNA, Complementary)RN - 0 (MANF protein, human)RN - 0 (Nerve Growth Factors)RN - 0 (Nerve Tissue Proteins)RN - 0 (Neuroprotective Agents)RN - 0 (glial cell-line derived neurotrophic factor)RN - 51-61-6 (Dopamine)SB - IM

Parkinson’s disease (PD) is a progressive and age-associated neurodegenerative disorder. Patients at different stages of the disease course have distinguished features, mainly in the number of dopaminergic neurons. Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered neurotrophic factor, being deemed as a hopeful candidate for PD treatment. Here, we evaluated the efficacy of CDNF in protecting dopaminergic function using the 6-OHDA-induced PD rat model suffering from different levels of neuronal loss and the recombinant adeno-associated virus 8 (AAV8) as a carrier for the CDNF gene. The results showed that AAV8-CDNF administration significantly improved the motor function and increased the tyrosine hydroxylase (TH) levels in PD rats with mild lesions (2 weeks post lesion), but it had limited therapeutic effects in rats with severe lesions (5 weeks post lesion). To better improve the recovery of motor function in severely lesioned PD rats, we employed a strategy using the CDNF gene along with the aromatic amino acid decarboxylase (AADC) gene. This combination therapeutic strategy indeed showed an enhanced benefit in restoring the motor function of severely lesioned PD rats by providing the neuroprotective effect of CDNF and dopamine enhancing effect of AADC as expected. This study may provide a basis for future clinical application of CDNF in PD patients at different stages and offer a new alternative strategy of joint use of CDNF and AADC for advanced PD patients in clinical trials.

Background The anti-inflammatory effect of the cerebral dopamine neurotrophic factor (CDNF) was shown recently in primary glial cell cultures, yet such effect remains unknown both in vivo and in 6-hydroxydopamine (6-OHDA) models of Parkinson’s disease (PD). We addressed this issue by performing an intranigral transfection of the human CDNF (hCDNF) gene in the critical period of inflammation after a single intrastriatal 6-OHDA injection in the rat. Methods At day 15 after lesion, the plasmids p3xNBRE-hCDNF or p3xNBRE-EGFP, coding for enhanced green florescent protein (EGFP), were transfected into the rat substantia nigra (SN) using neurotensin (NTS)-polyplex. At day 15 post-transfection, we measured nitrite and lipoperoxide levels in the SN. We used ELISA to quantify the levels of TNF-α, IL-1β, IL-6, endogenous rat CDNF (rCDNF) and hCDNF. We also used qRT-PCR to measure rCDNF and hCDNF transcripts, and immunofluorescence assays to evaluate iNOS, CDNF and glial cells (microglia, astrocytes and Neuron/Glial type 2 (NG2) cells). Intact SNs were additional controls. Results In the SN, 6-OHDA triggered nitrosative stress, increased inflammatory cytokines levels, and activated the multipotent progenitor NG2 cells, which convert into astrocytes to produce rCDNF. In comparison with the hemiparkinsonian rats that were transfected with the EGFP gene or without transfection, 6-OHDA treatment and p3xNBRE-hCDNF transfection increased the conversion of NG2 cells into astrocytes resulting in 4-fold increase in the rCDNF protein levels. The overexpressed CDNF reduced nitrosative stress, glial markers and IL-6 levels in the SN, but not TNF-α and IL-1β levels. Conclusion Our results show the anti-inflammatory effect of CDNF in a 6-OHDA rat of Parkinson’s disease. Our results also suggest the possible participation of TNF-α, IL-1β and IL-6 in rCDNF production by astrocytes, supporting their anti-inflammatory role. Electronic supplementary material The online version of this article (doi:10.1186/s12974-014-0209-0) contains supplementary material, which is available to authorized users.

Traumatic spinal cord injury (SCI) causes neuron death and axonal damage resulting in functional motor and sensory loss, showing limited regeneration because of adverse microenvironment such as neuroinflammation and glial scarring. Currently, there is no effective therapy to treat SCI in clinical practice. Bone marrow-derived mesenchymal stem cells (BMSCs) are candidates for cell therapies but its effect is limited by neuroinflammation and adverse microenvironment in the injured spinal cord. In this study, we developed transgenic BMSCs overexpressing cerebral dopamine neurotrophic factor (CDNF), a secretory neurotrophic factor that showed potent effects on neuron protection, anti-inflammation, and sciatic nerve regeneration in previous studies. Our results showed that the transplantation of CDNF-BMSCs suppressed neuroinflammation and decreased the production of proinflammatory cytokines after SCI, resulting in the promotion of locomotor function and nerve regeneration of the injured spinal cord. This study presents a novel promising strategy for the treatment of spinal cord injury.

Microglia are important resident immune cells in the central nervous system (CNS) and involved in the neuroinflammation caused by CNS disorders, including brain trauma, ischemia, stroke, infections, inflammation, and neurodegenerative diseases. Our study explores the hypothesis that conserved dopamine neurotrophic factor (CDNF), a secretory neurotrophic factor, may provide a novel therapy for associated with neuroinflammation related to the microglia. We observed that CDNF was upregulated in rat primary microglia treated with 1 μg/mL lipopolysaccharide, an inflammatory inducer, for 24 h. Thus, we hypothesize that CDNF may play a role, mediator or inhibitor, in regulating the inflammation in microglial cells induced by LPS. Finally, our data showed that CDNF significantly attenuated the production of proinflammatory cytokines (PGE2 and IL-1β) and remarkably alleviated the cytotoxicity (percentage of lactate dehydrogenase released) in the LPS-induced microglia by suppressing the phosphorylation of JNK, but not the P38 or ERK pathways. These results demonstrate the anti-inflammatory property of CDNF by inhibition of JNK signaling in LPS-induced microglia, suggesting that CDNF may be a potential novel agent for the treatment of neuroinflammation in the CNS disorders.

Astrocyte inflammation plays important roles both in physiological and pathological processes in the central nervous system (CNS). Ischemic injury in the CNS causes damage to astrocytes and the release of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β, and interleukin-6. This current study investigates whether mesencephalic astrocyte-derived neurotrophic factor (MANF) inhibits oxygen–glucose deprivation (OGD)-induced cell damage and inflammatory cytokine secretion by suppressing endoplasmic reticulum stress in rat primary astrocytes. We found that MANF alleviated OGD-induced astrocyte damage and rescued the cell viability, and the upregulation of GRP78 (endoplasmic reticulum (ER) stress marker) and NF-κB p65 (one of the central mediators of proinflammatory pathways) induced by OGD were significantly reduced by preincubation of MANF. In addition, the increases of secretion and mRNA expression levels of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in astrocytes induced by OGD were significantly suppressed by MANF. These findings demonstrate that MANF shows the potential to alleviate cell damage and inflammation in rat primary astrocytes by suppressing ER stress, indicating that MANF plays an important role in astrocyte inflammation and functioning and may suggest a promising strategy for neuroprotection in the CNS.

Astrocyte damage and the disorders of cytokine secretion induced by endoplasmic reticulum stress (ERS) are crucial pathological processes in ischemic injury of the central nervous system (CNS), (e.g., ischemic reperfusion injury of the brain and spinal cord). ERS stimulates damage to astrocytes and the release of pro-inflammatory cytokines, which deteriorates CNS injury. This current study investigates whether the overexpression of conserved dopamine neurotrophic factor (CDNF) alleviates ER stress-induced cell damage and inflammatory cytokine secretion. We found that primary astrocytes showed both a successful transduction and a significant overexpression of CDNF protein following lentivirus application. Our results show that the percentage of LDH released as a result of ER stress was significantly lower in astrocytes with an overexpression of CDNF than in the control groups without CDNF overexpression, indicating that CDNF alleviates ER stress-induced astrocyte damage. The secretion and mRNA expression levels of pro-inflammatory cytokines were increased by tunicamycin, and this stimulation was significantly suppressed by an overexpression of CDNF, demonstrating that CDNF plays an important role in astrocyte inflammation and functioning by resisting ER stress. These findings suggest that primary astrocytes can be efficiently transduced with CDNF lentiviral vectors and that the overexpression of CDNF in astrocytes shows the potential to alleviate cell damage and proinflammatory cytokine secretion, which may represent a promising strategy for neuroprotection in the CNS.

Vascular endothelial growth factor B (VEGF-B) has recently been shown to be a promising novel neuroprotective agent for several neurodegenerative conditions. In the current study we extended previous work on neuroprotective potential for Parkinson’s disease (PD) by testing an expanded dose range of VEGF-B (1 μg and 10 μg) and directly comparing both neuroprotective and neurorestorative effects of VEGF-B in progressive unilateral 6-hydroxydopamine (6-OHDA) PD models to a single dose of glial cell line-derived neurotrophic factor (GDNF, 10 μg), that has been established by several groups as a standard in both preclinical PD models. In the amphetamine-induced rotational tests the treatment with 1 and 10 μg VEGF-B resulted in significantly improved motor function of 6-OHDA-lesioned rats compared to vehicle-treated 6-OHDA-lesioned rats in the neuroprotection paradigm. Both doses of VEGF-B caused an increase in tyrosine hydroxylase (TH)-positive cell and fiber count in the substantia nigra (SN) and striatum in the neuroprotective experiment. The effect size was comparable to the effects seen with GDNF. In the neurorestoration paradigm, VEGF-B injection had no significant effect in either the behavioral or the immunohistochemical analyses, whereas GDNF injection significantly improved the amphetamine-induced rotational behavior and reduced TH-positive neuronal cell loss in the SN. We also present a strong positive correlation (p = 1.9 e-50) of the expression of VEGF-B with nuclear-encoded mitochondrial genes involved in fatty acid metabolism in rat midbrain, pointing to the mitochondria as a site of action of VEGF-B. GDNF showed a positive correlation with nuclear-encoded mitochondrial genes that was not nearly as strong (p = 0.018). VEGF-B counteracted rotenone-induced reduction of (a) fatty acid transport protein 1 and 4 levels and (b) both Akt protein and phosphorylation levels in SH-SY5Y cells. We further verified VEGF-B expression in the human SN pars compacta of healthy controls and Parkinson’s disease patients, in neuronal cells that show co-expression with neuromelanin. These results have demonstrated that VEGF-B has potential as a neuroprotective agent for PD therapy and should be further investigated.

PARKINSON 's disease results from the progressive degeneration of dopamine neurons that innervate the striatum1,2. In rodents, glial-cell-line-derived neurotrophic factor (GDNF) stimulates an increase in midbrain dopamine levels, protects dopamine neurons from some neurotoxins, and maintains injured dopamine neurons3–9. Here we extend the rodent studies to an animal closer to the human in brain organization and function, by evaluating the effects of GDNF injected intracerebrally into rhesus monkeys that have had the symptomatology and pathophysiological features of Parkinson's disease induced by the neurotoxin 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP)10–14. The recipients of GDNF displayed significant improvements in three of the cardinal symptoms of parkinsonism: bradykinesia, rigidity and postural instability. GDNF administered every four weeks maintained functional recovery. On the lesioned side of GDNF-treated animals, dopamine levels in the midbrain and globus pallidus were twice s high, and nigral dopamine neurons were, on average, 20% larger, with an increased fibre density. The results indicate that GDNF may be of benefit in the treatment of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF), a novel member of the TGF-β superfamily, has been shown to promote the survival and morphological differentiation of fetal dopamine neurons in culture and increase dopamine levels and metabolism in adult rats. Since several other trophic factors are able to rescue specific populations of mature CNS neurons following injury, the present study was designed to investigate a possible neuroprotective role by GDNF for midbrain dopamine neurons in rats exposed to the neurotoxin 6-hydroxydopamine (6-OHDA). Prior to surgery, young adult male Fisher 344 rats were divided into the following groups (n = 7–8/group): (1) intranigral saline + intranigral 6-OHDA; (2) intranigral GDNF + intranigral 6-OHDA; (3) intranigral saline + intrastriatal 6-OHDA; and (4) intranigral GDNF + intrastriatal 6-OHDA. The saline treated groups received a single 2 μl intranigral injection of phosphate buffered saline (PBS) while the GDNF treated rats received 10 μ g 2 μ l GDNF in PBS. Twenty-four hours later, the animals received a unilateral 4 μg/μl 6-OHDA infusion either into the substantia nigra or striatum. The rats were sacrificed two weeks postsurgery and the brains processed for tyrosine hydroxylase (TH) immunocytochemistry. Representative TH immunoreactive (TH-IR) sections were also counterstained with hematoxylin and eosin to determine the total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area. In the nigral lesion groups, there was significantly less loss of TH-IR neurons in the substantia nigra pars compacta of GDNF (47% survival) vs. PBS (9% survival) treated animals. The same was true in the ventral tegmental area, where there was a 90% survival of TH-IR neurons in the GDNF treated animals as compared to a 68% survival in PBS treated animals. In the striatal lesion groups, there was significant sparing of TH-IR neurons in the substantia nigra pars compacta of the GDNF (40% survival) compared to the PBS (16% survival) treated animals. However, in the ventral tegmental area, the protection seen in the GDNF treated animals (69% survival) was not statistically significant when compared to the PBS treated rats (48% survival). In sections counter stained with hematoxylin and eosin, the percentage of neurons surviving in GDNF treated hosts was higher suggesting that the 6-OHDA toxicity may reduce TH expression in some dopamine neurons without inducing cell death. Therefore, in both lesion models, our results demonstrate a substantial neuroprotective effect in rats pretreated with GDNF when compared to the vehicle treated groups.

GLIAL-CELL-LINE-DERIVED neurotrophic factor (GDNF), a recently cloned new member of the transforming growth factor-β superfamily, promotes survival of cultured fetal mesencephalic dopamine neurons1 and is expressed in the developing striatum2,3. There have, however, been no reports about effects of GDNF in situ. We have used the dopaminergic neurotoxin l–methyl–4–phenyl–1,2,3,6–tetrahydropyridine (MPTP), which produces parkinsonian symptoms in man, to determine whether GDNF might exert protective or regenerative effects in vivo in the adult nigrostriatal dopamine system in C57/B1 mice. GDNF injected over the substan-tia nigra or in striatum before MPTP potently protects the dopamine system, as shown by numbers of mesencephalic dopamine nerve cell bodies, dopamine nerve terminal densities and dopamine levels. When GDNF is given after MPTP, dopamine levels and fibre densities are significantly restored. In both cases, motor behaviour is increased above normal levels. We conclude that intracerebral GDNF administration exerts both protective and reparative effects on the nigrostriatal dopamine system, which may have implications for the development of new treatment strategies for Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) belongs to a newly discovered family of evolutionarily conserved neurotrophic factors. We demonstrate for the first time a therapeutic effect of CDNF in a unilateral 6-hydroxydopamine (6-OHDA) lesion model of Parkinson's disease in marmoset monkeys. Furthermore, we tested the impact of high chronic doses of human recombinant CDNF on unlesioned monkeys and analyzed the amino acid sequence of marmoset CDNF. The severity of 6-OHDA lesions and treatment effects were monitored in vivo using 123I-FP-CIT (DaTSCAN) SPECT. Quantitative analysis of 123I-FP-CIT SPECT showed a significant increase of dopamine transporter binding activity in lesioned animals treated with CDNF. Glial cell line-derived neurotrophic factor (GDNF), a well-characterized and potent neurotrophic factor for dopamine neurons, served as a control in a parallel comparison with CDNF. By contrast with CDNF, only single animals responded to the treatment with GDNF, but no statistical difference was observed in the GDNF group. However, increased numbers of tyrosine hydroxylase immunoreactive neurons, observed within the lesioned caudate nucleus of GDNF-treated animals, indicate a strong bioactive potential of GDNF. peerReviewed

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) stress-responsive protein with neuroprotective effects in animal models of neurodegeneration, but the underlying mechanism is not understood. We constructed a set of lentiviral vectors that contain or lack the highly conserved final four amino acids of MANF (“RTDL”), which resemble the canonical ER retention signal (“KDEL”), to study MANF regulation in neuroblastoma cells and rat primary cortical neurons. The RTDL sequence was required for both ER retention and secretory response to ER stress. Overexpression of KDEL receptor paralogs (KDELRs) differentially reduced MANF secretion but had no effect on MANF lacking RTDL. MANF binding to the plasma membrane also required the RTDL sequence and was inhibited with a peptide known to interact with KDELRs, suggesting MANF binds KDELRs at the surface. We detected surface localization of FLAG-tagged KDELRs, with levels increasing following ER stress. Our study provides new insight into the regulation of MANF trafficking and has implications for other secreted proteins containing a KDEL-like retention signal.