Merja H. Voutilainen

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.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Is Neurorestorative in Rat Model of Parkinson's Disease

Neurotrophic factors are promising candidates for the treatment of Parkinsons 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 125I-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 125I-MANF was transported to the frontal cortex, whereas 125I-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.

Chronic infusion of CDNF prevents 6-OHDA-induced deficits in a rat model of Parkinson's disease.

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) constitute a novel, evolutionarily conserved family of neurotrophic factors (NTF) expressed in vertebrates and invertebrates. The effects of two-week infusions of CDNF, MANF and glial cell line-derived neurotrophic factor (GDNF) were studied in a rat 6-hydroxydopamine (6-OHDA) hemiparkinsonian model. Degeneration of nigrostriatal dopamine nerve tract after toxin injection was assessed by measuring amphetamine-induced rotational behavior, and at the end of the experiment by quantifying tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta (SNpc) and TH-positive fibers in the striatum. The diffusion of the NTFs into the brain tissue following chronic infusion was also studied. Finally, we examined the transportation of intrastriatally injected (125)I-CDNF within the brain. The amphetamine-induced rotational behavior was gradually normalized in rats treated with CDNF for two weeks following the intrastriatal 6-OHDA injection. CDNF was also able to inhibit 6-OHDA-induced loss of TH-immunoreactive cells of the SNpc and TH-positive fibers in the striatum. MANF and GDNF had no statistically significant effect in any of the above measures. The volume of distribution for MANF in the striatum was significantly larger than that of GDNF after 3-day infusions. Both (125)I-CDNF and (125)I-GDNF were retrogradely transported from the striatum to the SN. No behavioral signs of toxicity were observed during treatment with the three NTFs. These results imply that CDNF may have potential as a neuroprotective or even neurorestorative therapy of PD.

CDNF Protects the Nigrostriatal Dopamine System and Promotes Recovery after MPTP Treatment in Mice

Cerebral dopamine neurotrophic factor (CDNF) is a recently discovered protein, which belongs to the evolutionarily conserved CDNF/MANF family of neurotrophic factors. The degeneration of dopamine neurons following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment is well characterized, and efficacy in this model is considered a standard criterion for development of parkinsonian therapies. MPTP is a neurotoxin, which produces parkinsonian symptoms in humans and in C57/Bl6 mice. To date, there are no reports about the effects of CDNF on dopamine neuron survival or function in the MPTP rodent model, a critical gap. Therefore, we studied whether CDNF has neuroprotective and neurorestorative properties for the nigrostriatal dopamine system after MPTP injections in C57/Bl6 mice. We found that bilateral striatal CDNF injections, given 20 h before MPTP, improved horizontal and vertical motor behavior. CDNF pretreatment increased tyrosine hydroxylase (TH) immunoreactivity in the striatum and in the substantia nigra pars reticulata (SNpr), as well as the number of TH-positive cells in substantia nigra pars compacta (SNpc). Posttreatment with CDNF, given 1 week after MPTP injections, increased horizontal and vertical motor behavior of mice, as well as dopamine fiber densities in the striatum and the number of TH-positive cells in SNpc. CDNF did not alter any of the analyzed dopaminergic biomarkers or locomotor behavior in MPTP-untreated animals. We conclude that striatal CDNF administration is both neuroprotective and neurorestorative for the TH-positive cells in the nigrostriatal dopamine system in the MPTP model, which supports the development of CDNF-based treatment for Parkinsons disease.

Gene therapy with AAV2-CDNF provides functional benefits in a rat model of Parkinson's disease

Cerebral dopamine neurotrophic factor (CDNF) protein has been shown to protect the nigrostriatal dopaminergic pathway when given as intrastriatal infusions in rat and mouse models of Parkinsons disease (PD). In this study, we assessed the neuroprotective effect of CDNF delivered with a recombinant adeno‐associated viral (AAV) serotype 2 vector in a rat 6‐hydroxydopamine (6‐OHDA) model of PD. AAV2 vectors encoding CDNF, glial cell line–derived neurotrophic factor (GDNF), or green fluorescent protein were injected into the rat striatum. Protein expression analysis showed that our AAV2 vector efficiently delivered the neurotrophic factor genes into the brain and gave rise to a long‐lasting expression of the proteins. Two weeks after AAV2 vector injection, 6‐OHDA was injected into the rat striatum, creating a progressive degeneration of the nigrostriatal dopaminergic system. Treatment with AAV2‐CDNF resulted in a marked decrease in amphetamine‐induced ipsilateral rotations while it provided only partial protection of tyrosine hydroxylase (TH)‐immunoreactive cells in the rat substantia nigra pars compacta and TH‐reactive fibers in the striatum. Results from this study provide additional evidence that CDNF can be considered a potential treatment of Parkinsons disease.

Therapeutic potential of the endoplasmic reticulum located and secreted CDNF/MANF family of neurotrophic factors in Parkinson's disease

Parkinsons 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.

Transient transfection of human CDNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat substantia nigra

BackgroundThe 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.MethodsAt 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.ResultsIn 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.ConclusionOur 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.

Development and plasticity of meningeal lymphatic vessels

The recent discovery of meningeal lymphatic vessels (LVs) has raised interest in their possible involvement in neuropathological processes, yet little is known about their development or maintenance. We show here that meningeal LVs develop postnatally, appearing first around the foramina in the basal parts of the skull and spinal canal, sprouting along the blood vessels and cranial and spinal nerves to various parts of the meninges surrounding the central nervous system (CNS). VEGF-C, expressed mainly in vascular smooth muscle cells, and VEGFR3 in lymphatic endothelial cells were essential for their development, whereas VEGF-D deletion had no effect. Surprisingly, in adult mice, the LVs showed regression after VEGF-C or VEGFR3 deletion, administration of the tyrosine kinase inhibitor sunitinib, or expression of VEGF-C/D trap, which also compromised the lymphatic drainage function. Conversely, an excess of VEGF-C induced meningeal lymphangiogenesis. The plasticity and regenerative potential of meningeal LVs should allow manipulation of cerebrospinal fluid drainage and neuropathological processes in the CNS.

Characterization of a new low-dose 6-hydroxydopamine model of Parkinson's disease in rat.

Intrastriatal administration of 6‐hydroxydopamine (6‐OHDA) induces partial degeneration of the nigrostriatal pathway, mimicking the pathology of Parkinsons disease (PD). Setting up the partial lesion model can be challenging because a number of experimental settings can be altered. This study compares seven experimental settings in a single study on d‐amphetamine‐induced rotations, tyrosine hydroxylase (TH)‐positive neurites in the striatum, dopamine transporter (DAT)‐positive neurites in the striatum, and TH‐positive cells in the substantia nigra pars compacta (SNpc) in rats. Moreover, we validate a new algorithm for estimating the number of TH‐positive cells. We show that the behavior and immunoreactivity vary greatly depending on the injection settings, and we categorize the lesions as progressive, stable, or regressive based on d‐amphetamine‐induced rotations. The rotation behavior correlated with the degree of the lesion, analyzed by immunohistochemistry; the largest lesions were in the progressive group, and the smallest lesions were in the regressive group. We establish a new low‐dose partial 6‐OHDA lesion model in which a total of 6u2009μg was distributed evenly to three sites in the striatum at a 10° angle. The administration of low‐dose 6‐OHDA produced stable and reliable rotation behavior and induced partial loss of striatal TH‐positive and DAT‐positive neurites and TH‐positive cells in the SNpc. This model is highly suitable for neurorestoration studies in the search for new therapies for PD, and the new algorithm increases the efficacy for estimating the number of dopamine neurons. This study can be extremely useful for laboratories setting up the partial 6‐OHDA model.

Differentiation and molecular heterogeneity of inhibitory and excitatory neurons associated with midbrain dopaminergic nuclei.

Local inhibitory GABAergic and excitatory glutamatergic neurons are important for midbrain dopaminergic and hindbrain serotonergic pathways controlling motivation, mood, and voluntary movements. Such neurons reside both within the dopaminergic nuclei, and in adjacent brain structures, including the rostromedial and laterodorsal tegmental nuclei. Compared with the monoaminergic neurons, the development, heterogeneity, and molecular characteristics of these regulatory neurons are poorly understood. We show here that different GABAergic and glutamatergic subgroups associated with the monoaminergic nuclei express specific transcription factors. These neurons share common origins in the ventrolateral rhombomere 1, where the postmitotic selector genes Tal1, Gata2 and Gata3 control the balance between the generation of inhibitory and excitatory neurons. In the absence of Tal1, or both Gata2 and Gata3, the GABAergic precursors adopt glutamatergic fates and populate the glutamatergic nuclei in excessive numbers. Together, our results uncover developmental regulatory mechanisms, molecular characteristics, and heterogeneity of central regulators of monoaminergic circuits. Summary: Tal1 and Gata transcription factors select between inhibitory GABAergic and excitatory glutamatergic fates of neurons regulating the dopaminergic system.