Unfolded protein response markers Grp78 and eIF2alpha are upregulated with increasing alpha-synuclein levels in Lewy body disease.

AIMS: Endoplasmic reticulum stress followed by the unfolded protein response is one of the cellular mechanisms contributing to the progression of α-synuclein pathology in Parkinson's disease and other Lewy body diseases. We aimed to investigate the activation of endoplasmic reticulum stress and its correlation with α-synuclein pathology in human post-mortem brain tissue. METHODS: We analysed brain tissue from 45 subjects-14 symptomatic patients with Lewy body disease, 19 subjects with incidental Lewy body disease, and 12 healthy controls. The analysed brain regions included the medulla, pons, midbrain, striatum, amygdala and entorhinal, temporal, frontal and occipital cortex. We analysed activation of endoplasmic reticulum stress via levels of the unfolded protein response-related proteins (Grp78, eIF2α) and endoplasmic reticulum stress-regulating neurotrophic factors (MANF, CDNF). RESULTS: We showed that regional levels of two endoplasmic reticulum-localised neurotrophic factors, MANF and CDNF, did not change in response to accumulating α-synuclein pathology. The concentration of MANF negatively correlated with age in specific regions. eIF2α was upregulated in the striatum of Lewy body disease patients and correlated with increased α-synuclein levels. We found the upregulation of chaperone Grp78 in the amygdala and nigral dopaminergic neurons of Lewy body disease patients. Grp78 levels in the amygdala strongly correlated with soluble α-synuclein levels. CONCLUSIONS: Our data suggest a strong but regionally specific change in Grp78 and eIF2α levels, which positively correlates with soluble α-synuclein levels. Additionally, MANF levels decreased in dopaminergic neurons in the substantia nigra. Our research suggests that endoplasmic reticulum stress activation is not associated with Lewy pathology but rather with soluble α-synuclein concentration and disease progression.

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons in the spinal cord, brainstem and motor cortex, leading to paralysis and eventually to death within 3-5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the endoplasmic reticulum-resident protein cerebral dopamine neurotrophic factor in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and aetiology: (i) the conditional choline acetyltransferase-tTA/TRE-hTDP43-M337V rat model previously described; (ii) the widely used SOD1-G93A mouse model; and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the endoplasmic reticulum stress response in motor neurons, we used three main methods: (i) primary cultures of motor neurons derived from embryonic Day 13 embryos; (ii) immunohistochemical analyses of spinal cord sections with choline acetyltransferase as spinal motor neuron marker; and (iii) quantitative polymerase chain reaction analyses of lumbar motor neurons isolated via laser microdissection. We show that intracerebroventricular administration of cerebral dopamine neurotrophic factor significantly halts the progression of the disease and improves motor behaviour in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. Cerebral dopamine neurotrophic factor rescues motor neurons in vitro and in vivo from endoplasmic reticulum stress-associated cell death and its beneficial effect is independent of genetic disease aetiology. Notably, cerebral dopamine neurotrophic factor regulates the unfolded protein response initiated by transducers IRE1α, PERK and ATF6, thereby enhancing motor neuron survival. Thus, cerebral dopamine neurotrophic factor holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

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

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

CDNF and MANF regulate ER stress in a tissue-specific manner.

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) display cytoprotective effects in animal models of neurodegenerative diseases. These endoplasmic reticulum (ER)-resident proteins belong to the same protein family and function as ER stress regulators. The relationship between CDNF and MANF function, as well as their capability for functional compensation, is unknown. We aimed to investigate these questions by generating mice lacking both CDNF and MANF. Results showed that CDNF-deficient Manf-/- mice presented the same phenotypes of growth defect and diabetes as Manf-/- mice. In the muscle, CDNF deficiency resulted in increased activation of unfolded protein response (UPR), which was aggravated when MANF was ablated. In the brain, the combined loss of CDNF and MANF did not exacerbate UPR activation caused by the loss of MANF alone. Consequently, CDNF and MANF deficiency in the brain did not cause degeneration of dopamine neurons. In conclusion, CDNF and MANF present functional redundancy in the muscle, but not in the other tissues examined here. Thus, they regulate the UPR in a tissue-specific manner.

The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-stress-regulated protein exhibiting cytoprotective properties through a poorly understood mechanism in various in vitro and in vivo models of neuronal and non-neuronal damage. Although initially characterized as a secreted neurotrophic factor for midbrain dopamine neurons, MANF has recently gained more interest for its intracellular role in regulating the ER homeostasis, including serving as a cofactor of the chaperone glucose-regulated protein 78 (GRP78). We aimed for a better understanding of the neuroprotective mechanisms of MANF. Here we show for the first time that MANF promotes the survival of ER-stressed neurons in vitro as a general unfolded protein response (UPR) regulator, affecting several UPR pathways simultaneously. Interestingly, MANF does not affect naïve neurons. We hypothesize that MANF regulates UPR signaling toward a mode more compatible with neuronal survival. Screening of MANF interacting proteins from two mammalian cell lines revealed a conserved interactome of 15 proteins including several ER chaperones such as GRP78, GRP170, protein disulfide isomerase family A member 1, and protein disulfide isomerase family A member 6. Further characterization confirmed previously published finding that MANF is a cofactor of GRP78 interacting with its nucleotide binding domain. Using microscale thermophoresis and nuclear magnetic resonance spectroscopy, we discovered that MANF is an ATP binding protein and that ATP blocks the MANF-GRP78 interaction. Interestingly, functional analysis of the antiapoptotic properties of MANF mutants in cultured neurons revealed divergent roles of MANF as a GRP78 cofactor and as an antiapoptotic regulator of UPR. We conclude that the co-factor type interaction with GRP78 is dispensable for the survival-promoting activity of MANF in neurons.

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

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

MANF protects human pancreatic beta cells against stress-induced cell death.

AIMS/HYPOTHESIS: There is a great need to identify factors that could protect pancreatic beta cells against apoptosis or stimulate their replication and thus prevent or reverse the development of diabetes. One potential candidate is mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein. Manf knockout mice used as a model of diabetes develop the condition because of increased apoptosis and reduced proliferation of beta cells, apparently related to ER stress. Given this novel association between MANF and beta cell death, we studied the potential of MANF to protect human beta cells against experimentally induced ER stress. METHODS: Primary human islets were challenged with proinflammatory cytokines, with or without MANF. Cell viability was analysed and global transcriptomic analysis performed. Results were further validated using the human beta cell line EndoC-ßH1. RESULTS: There was increased expression and secretion of MANF in human beta cells in response to cytokines. Addition of recombinant human MANF reduced cytokine-induced cell death by 38% in human islets (p < 0.05). MANF knockdown in EndoC-ßH1 cells led to increased ER stress after cytokine challenge. Mechanistic studies showed that the protective effect of MANF was associated with repression of the NF-κB signalling pathway and amelioration of ER stress. MANF also increased the proliferation of primary human beta cells twofold when TGF-ß signalling was inhibited (p < 0.01). CONCLUSIONS/INTERPRETATION: Our studies show that exogenous MANF protein can provide protection to human beta cells against death induced by inflammatory stress. The antiapoptotic and mitogenic properties of MANF make it a potential therapeutic agent for beta cell protection.

Unconventional neurotrophic factors CDNF and MANF: Structure, physiological functions and therapeutic potential.

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.

In vivo screening reveals interactions between Drosophila Manf and genes involved in the mitochondria and the ubiquinone synthesis pathway.

BACKGROUND: Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) and Cerebral Dopamine Neurotrophic Factor (CDNF) form an evolutionarily conserved family of neurotrophic factors. Orthologues for MANF/CDNF are the only neurotrophic factors as yet identified in invertebrates with conserved amino acid sequence. Previous studies indicate that mammalian MANF and CDNF support and protect brain dopaminergic system in non-cell-autonomous manner. However, MANF has also been shown to function intracellularly in the endoplasmic reticulum. To date, the knowledge on the interacting partners of MANF/CDNF and signaling pathways they activate is rudimentary. Here, we have employed the Drosophila genetics to screen for potential interaction partners of Drosophila Manf (DmManf) in vivo. RESULTS: We first show that DmManf plays a role in the development of Drosophila wing. We exploited this function by using Drosophila UAS-RNAi lines and discovered novel genetic interactions of DmManf with genes known to function in the mitochondria. We also found evidence of an interaction between DmManf and the Drosophila homologue encoding Ku70, the closest structural homologue of SAP domain of mammalian MANF. CONCLUSIONS: In addition to the previously known functions of MANF/CDNF protein family, DmManf also interacts with mitochondria-related genes. Our data supports the functional importance of these evolutionarily significant proteins and provides new insights for the future studies.

Brain structural deficits and working memory fMRI dysfunction in young adults who were diagnosed with ADHD in adolescence.

When adolescents with ADHD enter adulthood, some no longer meet disorder diagnostic criteria but it is unknown if biological and cognitive abnorma lities persist. We tested the hypothesis that people diagnosed with ADHD during adolescence present residual brain abnormalities both in brain structure and in working memory brain function. 83 young adults (aged 20-24 years) from the Northern Finland 1986 Birth Cohort were classified as diagnosed with ADHD in adolescence (adolescence ADHD, n = 49) or a control group (n = 34). Only one patient had received medication for ADHD. T1-weighted brain scans were acquired and processed in a voxel-based analysis using permutation-based statistics. A sub-sample of both groups (ADHD, n = 21; controls n = 23) also performed a Sternberg working memory task whilst acquiring fMRI data. Areas of structural difference were used as a region of interest to evaluate the implications that structural abnormalities found in the ADHD group might have on working memory function. There was lower grey matter volume bilaterally in adolescence ADHD participants in the caudate (p < 0.05 FWE corrected across the whole brain) at age 20-24. Working memory was poorer in adolescence ADHD participants, with associated failure to show normal load-dependent caudate activation. Young adults diagnosed with ADHD in adolescence have structural and functional deficits in the caudate associated with abnormal working memory function. These findings are not secondary to stimulant treatment, and emphasise the importance of taking a wider perspective on ADHD outcomes than simply whether or not a particular patient meets diagnostic criteria at any given point in time.

DTI abnormalities in adults with past history of attention deficit hyperactivity disorder: a tract-based spatial statistics study.

BACKGROUND: Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique enabling visualization and measurement of white matter tracts. Attention deficit hyperactivity disorder (ADHD) has been studied with DTI earlier with variable results, yet there is little research on remitted ADHD. PURPOSE: To compare the brain white matter between ADHD drug naïve subjects whose ADHD symptoms have mostly subsided and healthy controls. MATERIAL AND METHODS: Tract-based spatial statistics (TBSS) was used to compare 30 subjects with adolescent ADHD with control subjects at the age of 22-23 years. The study population was derived from a population-based Northern Finland Birth Cohort 1986. Fractional anisotropy (FA), mean diffusivity (MD), and measures of diffusion direction (λ1-3) were calculated. Permutation testing was used to test for differences in mean values of FA, MD, and λ1-3 between the groups. The results were corrected for multiple comparisons across the whole white matter skeleton. RESULTS: The ADHD group showed increased FA related to decreased radial diffusivity in the left forceps minor (P < 0.05). In the vicinity along the same tract, axial diffusion was significantly decreased without any significant effect on FA. No between-group difference in MD was observed. Regressor analysis revealed no gender-, IQ- or GAF-related changes. After removal of left handed subjects the statistical significance was only barely lost. CONCLUSION: In a setting with remitted ADHD, the results may represent a compensatory mechanism in the left forceps minor.

MANF protein expression is upregulated in immune cells in the ischemic human brain and systemic recombinant MANF delivery in rat ischemic stroke model demonstrates anti-inflammatory effects.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has cytoprotective effects on various injuries, including cerebral ischemia, and it can promote recovery even when delivered intracranially several days after ischemic stroke. In the uninjured rodent brain, MANF protein is expressed almost exclusively in neurons, but post-ischemic MANF expression has not been characterized. We aimed to investigate how endogenous cerebral MANF protein expression evolves in infarcted human brains and rodent ischemic stroke models. During infarct progression, the cerebral MANF expression pattern both in human and rat brains shifted drastically from neurons to expression in inflammatory cells. Intense MANF immunoreactivity took place in phagocytic microglia/macrophages in the ischemic territory, peaking at two weeks post-stroke in human and one-week post-stroke in rat ischemic cortex. Using double immunofluorescence and mice lacking MANF gene and protein from neuronal stem cells, neurons, astrocytes, and oligodendrocytes, we verified that MANF expression was induced in microglia/macrophage cells in the ischemic hemisphere. Embarking on the drastic expression transition towards inflammatory cells and the impact of blood-borne inflammation in stroke, we hypothesized that exogenously delivered MANF protein can modulate tissue recovery processes. In an attempt to enhance recovery, we designed a set of proof-of-concept studies using systemic delivery of recombinant MANF in a rat model of cortical ischemic stroke. Intranasal recombinant MANF treatment decreased infarct volume and reduced the severity of neurological deficits. Intravenous recombinant MANF treatment decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokine IL-10 in the infarcted cortex one-day post-stroke. In conclusion, MANF protein expression is induced in activated microglia/macrophage cells in infarcted human and rodent brains, and this could implicate MANF's involvement in the regulation of post-stroke inflammation in patients and experimental animals. Moreover, systemic delivery of recombinant MANF shows promising immunomodulatory effects and therapeutic potential in experimental ischemic stroke.

Novel neurotrophic factor CDNF protects and rescues midbrain dopamine neurons in vivo.

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.

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

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

Aging disrupts MANF-mediated immune modulation during skeletal muscle regeneration.

Age-related decline in skeletal muscle regenerative capacity is multifactorial, yet the contribution of immune dysfunction to regenerative failure is unknown. Macrophages are essential for effective debris clearance and muscle stem cell activity during muscle regeneration, but the regulatory mechanisms governing macrophage function during muscle repair are largely unexplored. Here, we uncover a new mechanism of immune modulation operating during skeletal muscle regeneration that is disrupted in aged animals and relies on the regulation of macrophage function. The immune modulator mesencephalic astrocyte-derived neurotrophic factor (MANF) is induced following muscle injury in young mice but not in aged animals, and its expression is essential for regenerative success. Regenerative impairments in aged muscle are associated with defects in the repair-associated myeloid response similar to those found in MANF-deficient models and could be improved through MANF delivery. We propose that restoring MANF levels is a viable strategy to improve myeloid response and regenerative capacity in aged muscle.

MANF regulates neuronal survival and UPR through its ER-located receptor IRE1α.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in neurons and pancreatic ß cells in vitro and in models of neurodegeneration and diabetes in vivo. However, the exact mode of MANF action has remained elusive. Here, we show that MANF directly interacts with the ER transmembrane unfolded protein response (UPR) sensor IRE1α, and we identify the binding interface between MANF and IRE1α. The expression of wild-type MANF, but not its IRE1α binding-deficient mutant, attenuates UPR signaling by decreasing IRE1α oligomerization; phosphorylation; splicing of Xbp1, Atf6, and Txnip levels; and protecting neurons from ER stress-induced death. MANF-IRE1α interaction and not MANF-BiP interaction is crucial for MANF pro-survival activity in neurons in vitro and is required to protect dopamine neurons in an animal model of Parkinson's disease. Our data show IRE1α as an intracellular receptor for MANF and regulator of neuronal survival.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) has a unique mechanism to rescue apoptotic neurons.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) protects neurons and repairs the Parkinson disease-like symptoms in a rat 6-hydroxydopamine model. We show a three-dimensional solution structure of human MANF that differs drastically from other neurotrophic factors. Remarkably, the C-terminal domain of MANF (C-MANF) is homologous to the SAP domain of Ku70, a well known inhibitor of proapoptotic Bax (Bcl-2-associated X protein). Cellular studies confirm that MANF and C-MANF protect neurons intracellularly as efficiently as Ku70.

Mesencephalic astrocyte-derived neurotrophic factor is neurorestorative in rat model of Parkinson's disease.

Neurotrophic factors are promising candidates for the treatment of Parkinson's disease (PD). Mesencephalic astrocyte-derived neurotrophic factor (MANF) belongs to a novel evolutionarily conserved family of neurotrophic factors. We examined whether MANF has neuroprotective and neurorestorative effect in an experimental model of PD in rats. We also studied the distribution and transportation of intrastriatally injected MANF in the brain and compared it with glial cell line-derived neurotrophic factor (GDNF). Unilateral lesion of nigrostriatal dopaminergic system was induced by intrastriatal injection of 6-hydroxydopamine (6-OHDA). Amphetamine-induced turning behavior was monitored up to 12 weeks after the unilateral lesion. The local diffusion at the injection site and transportation profiles of intrastriatally injected MANF and GDNF were studied by immunohistochemical detection of the unlabeled growth factors as well as by autoradiographic and gamma counting detection of (125)I-labeled trophic factors. Intrastriatally injected MANF protected nigrostriatal dopaminergic nerves from 6-OHDA-induced degeneration as evaluated by counting tyrosine hydroxylase (TH)-positive cell bodies in the substantia nigra (SN) and TH-positive fibers in the striatum. More importantly, MANF also restored the function of the nigrostriatal dopaminergic system when administered either 6 h before or 4 weeks after 6-OHDA administration in the striatum. MANF was distributed throughout the striatum more readily than GDNF. The mechanism of MANF action differs from that of GDNF because intrastriatally injected (125)I-MANF was transported to the frontal cortex, whereas (125)I-GDNF was transported to the SN. Our results suggest that MANF is readily distributed throughout the striatum and has significant therapeutic potential for the treatment of PD.

Regulation of brain-derived neurotrophic factor (BDNF) and cerebral dopamine neurotrophic factor (CDNF) by anti-parkinsonian drug therapy in vivo.

Available treatment for Parkinson's disease (PD) is mainly symptomatic instead of halting or reversing degenerative processes affecting the disease. Research on the molecular pathogenesis of PD has suggested reduced trophic support as a possible cause or mediator of neurodegeneration. In animal models of the disease, neurotrophic factors prevent neurodegeneration and induce behavioral recovery. Some anti-Parkinsonian drugs show neuroprotective activity, but it is not known whether the drug-induced neuroprotection is mediated by neurotrophic factors. In this study, we have investigated the influence of two neuroprotective anti-Parkinsonian drugs, the monoamine oxidase B inhibitor selegiline and the adenosine A(2A) antagonist SCH 58261, on the levels of brain-derived neurotrophic factor (BDNF) and cerebral dopamine neurotrophic factor (CDNF) in the mouse brain. Protein levels of BDNF and CDNF were quantified by western blot after 2 weeks of treatment with either of the drugs or placebo. CDNF levels were not significantly influenced by selegiline or SCH 58261 in any brain area studied. Selegiline treatment significantly increased BDNF levels in the anterior cingulate cortex (1.55 +/- 0.22, P < 0.05, Student's t-test). In the striatum, selegiline increased BDNF content by 32%, but this change did not reach statistical significance (1.32 +/- 0.15, P < 0.13, Student's t-test). Our data suggest that neurotrophic factors, particularly BDNF may play a role in the neuroprotective effects of selegiline, but do not support the hypothesis that anti-Parkinsonian drugs would work by increasing the levels of CDNF in brain.

Cerebral dopamine neurotrophic factor is essential for enteric neuronal development, maintenance, and regulation of gastrointestinal transit.

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 of 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.