Browsing by Subject "CDNF"

Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.

Multiple sclerosis (MS) is a demyelinating autoimmune disease in which peripheral immune cells infiltrate the CNS and damage the insulating myelin sheaths surrounding neurons, creating demyelinated lesions in the spinal cord and the brain. MS is an incurable, life-long disease which causes a range of symptoms resulting from CNS degeneration. Current treatments mostly focus on preventing autoimmune attacks and the formation of lesions, but do not reduce the damage caused by the attacks, or impact the gradual degeneration of the axons of MS patients. This study aimed to establish the potential of MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) as treatments for MS. MANF and CDNF are endoplasmic reticulum (ER) located proteins with unique structure and mode of action. UPR is a cellular stress response that, when triggered by inflammation in MS, can cause the apoptosis of myelinating oligodendrocytes and neurodegeneration. MANF and CDNF are also capable of modulating immune responses and improving regenerative processes in damaged tissues. The capability of these two molecules to protect CNS tissue was tested on mice induced with experimental autoimmune encephalomyelitis (EAE), a disease model for MS. Intravenous injections of MANF or CDNF in two doses were performed every 2nd day for 28 days after disease induction. Behavioral testing (rotarod and open field tests) indicated that both proteins improved motor function before the onset of paralysis. Daily clinical scoring showed a brief therapeutic window after the onset of paralysis, during which MANF and CDNF were able to halt disease progression. Flow cytometry analysis of mice spleens and brains showed no effect on immune cell populations at the end of the 28-day testing period. Immunohistological staining at the end of the experiment showed no differences in levels of neuroinflammation between treatment groups and control mice but showed that treatment with MANF and CDNF clearly reduced the formation of demyelinated lesions over the duration of the disease. These findings suggest the improved motor performances and protection from paralysis provided by treatment by MANF and CDNF may be due to their ability to protect CNS tissue from UPR caused by autoimmune demyelinating attacks. Further research is required to elucidate the mechanics behind this neuroprotective ability, and lead to more effective use of MANF and CDNF.

Parkinson's disease is a neurodegenerative disorder where dopaminergic neurons in substantia nigra are gradually destroyed. Less than 10% of Parkinson's disease cases are genetic. For example mutations in α-synuclein, LRRK2-, parkin-, PINK1- and DJ-1 are known to cause Parkinson's disease. There is still no curative treatment for Parkinson's disease. Alpha-synuclein is linked to Parkinson's disease through Lewy bodies. Three point mutations causing Parkinson's disease have been found in a gene coding α-synuclein. Alpha-synuclein has been expressed in Drosophila melanogaster, C. elegans and mouse. Main function of LRRK2-protein is thought to be kinase activity. Mutations in LRRK2-gene are the most common known cause of Parkinson's disease. LRRK has been expressed in Drosophila melanogaster, C. elegans and mouse. LRRK2 knock-out Drosophila melanogaster and mouse have also been studied. Parkin is a neuroprotective protein and its deficiency results in a loss of neurons in substantia nigra. Mutations in Parkin cause 50% of recessive Parkinson's disease. Parkin knock-out Drosophila melanogaster and mouse and Drosophila melanogaster and mouse expressing human Parkin are Parkin animal models. PINK1 is a mitochondrial protein coded by nucleus. DJ-1 is thought to have a part in mitochondria maintenance and protection. Both PINK1- and DJ-1 knock-out Drosophila melanogaster and mouse have been studied. None of the genetic animal models of Parkinson's disease is identical to symptoms and pathology of human Parkinson's disease. The purpose of the experimental part of this thesis was to examine non-drug induced behavioural test and Cerebral dopamine neurotrophic factor (CDNF) in 6-OHDA lesioned rats. CDNF protects and restores dopaminergic neurons. The non-drug induced behavioural tests included in this study were stepping test, cylinder test and staircase test. An old and widely used drug induced test for Parkinson's disease, amphetamine-induced rotation test, has problems that have led to a seek for replacing and complementary test methods. In amphetamine-induced rotation test dopamine agonist is given to a unilaterally 6-OHDA lesioned animal. The agonist causes rotational behaviour that can be measured with designed equipment. The stepping test measures forelimb akinesia in rats. In the experimental setting the rat is moved sideways when it is held only one front paw on a table and adjusting steps are counted. In the cylinder test front paw preference is measured. In the experimental setting the rat is placed in a transparent cylinder and the front paw preference is counted on rears and on ground contacts after a rear. The staircase test measures front paw coordination and function. In the experimental setting the number of sucrose pellets picked up from a double staircase is counted. There were no significant differences between lesioned groups in stepping test, cylinder test or in staircase test. It is possible that the 6-OHDA lesion used in the experiment was not extensive enough. Different non-drug induced behavioural tests supplement each other and they should be combined for the best result. Combining different behavioural tests enables more reliable results and versatile information than the amphetamine-induced rotation test alone.

Parkinson's disease is a progressive degenerative brain disease that causes degeneration of dopaminergic neurons in the substantia nigra. Characteristic motor symptoms in Parkinson's disease are caused by dopamine deficiency in striatum. Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the dopamine biosynthesis. Because of this TH has a significant role in the function of the dopaminergic system. TH activity is regulated by several mechanisms. The most important regulatory mechanism is phosphorylation of TH protein by spesific protein kinases. Alterations in the function of TH have been associated with Parkinson's disease. The most prominent findings are decreased TH protein and TH mRNA content in the nigrostriatal dopaminergic neurons. A possible pathogenic role of TH in Parkinson's disease has also been suggested. In addition TH might be a potential therapeutic protein for gene therapy. One possible approach is viral vector-mediated gene transfer of TH gene directly into the brain. Simultaneous gene transfer of TH gene and neurotrophic factor gene could both enhance dopamine synthesis and prevent remaining dopaminergic neurons from dying. None of the current treatments of Parkinson's disease can halt or retard dopaminergic neuron degeneration. Novel treatments are being developed and amongst other strategies neurotrophic factors have proven promising candidates for the treatment of Parkinson's disease. Member of CDNF/MANF family of neurotrophic factors, cerebral dopamine neurotrophic factor (CDNF), is currently being studied. Previous studies have demonstrated the neuroprotective and neurorestorative effects of CDNF but more research is needed for optimal administration technique and dose. The aim of this work was to study the neuroprotective effect of AAV vector-mediated delivery of CDNF (AAV-CDNF) in a rat model of Parkinson's disease. Rats' brains were unilaterally lesioned with intrastriatal injection of 6-OHDA two weeks after viral vector injections and amphetamine-induced rotational behavior was monitored for ten weeks. The CDNF protein expression after intrastriatal AAV vector-mediated gene transfer was analyzed with immunohistochemical staining of brain sections. We confirmed that CDNF protein is expressed in rat brain after intrastriatal injection of AAV-CDNF. AAV-CDNF treatment also reduced the amphetamineinduced ipsilateral rotations nearly as much as AAV-GDNF treatment. AAV-CDNF treatment also had an effect on the amount of remaining TH-immunoreactive cells in the substantia nigra pars compacta and the optical density of striatal TH-immunoreactive fibers but these results did not reach statistical significance. The immunohistochemical measures did not correlate completely with the behavioral data and further studies are needed to confirm the results obtained here. The results of this research support the conclusion that AAV-CDNF treatment has a neuroprotective effect on nigrostriatal dopaminergic neurons.