Browsing by Subject "GDNF"

Literature review part: The enteric nervous system (ENS) often called “the second brain” is considered its own autonomic division that can independently regulate gut function. The ENS is derived from enteric neural crest-derived cells (ENCCs), which colonize the gut during development. Development of the ENS is a complex process, and many signalling pathways are required for a properly functioning ENS, especially GDNF/Gfrα1/RET signalling controlling survival, proliferation, migration, and differentiation of ENCCs. Hirschsprung’s disease (HSCR) is the most common congenital disease affecting gut motility. The prevalence of HSCR is 1:5000, and it is characterized by a complete lack of enteric neurons (aganglionosis) in the distal colon. Due to impaired intestinal motility, infants may have constipation, emesis, abdominal pain or distention, and, in some cases, diarrhea. The most life-threatening symptom is HSCR-associated enterocolitis (HAEC), which occurs in 30-50% of patients. Routine treatment for HSCR is a surgical operation called “pull through” in which the aganglionic segment is removed, and the remaining ganglionic segment is joined to the anus. However, the risk of developing HAEC after successful surgery still exists. Histopathological analysis has revealed that HAEC is accompanied by various changes in the gut epithelium, especially in mucin-producing goblet cells. These changes include hyperplasia of the goblet cells, altered mucin profile, retention of mucin, damaged and disorganized epithelium structure, inflammation, and bacterial adherence to the epithelium. However, a lack of suitable postnatal HSCR mouse models has partially hindered the progress of pinpointing the exact order of these events. A RET mutation found in half of the patients is overwhelmingly the biggest risk factor for HSCR. RET is a receptor on the cell membrane that mediates the effects in GDNF/Gfrα1/RET signaling pathway. of Knock-out mice of Gdnf, Gfra1 and Ret all have intestinal aganglionosis, resembling HSCR. However, to date, no mouse models of HSCR affecting GDNF/Gfrα1/RET signalling exist because pups are born without kidneys and die soon after birth. Experimental part: The GFRa1 hypomorphic mouse line (Gfra1hypo/hypo) created by Dr. Jaan-Olle Andressoo is the first successful model that survives past birth while manipulating GDNF-Gfrα1-RET signalling and phenocopying HSCR. These mice have 70-80% reduction in the expression of Gfrα1 in the developing gut and kidneys, which is sufficient to cause aganglionosis in the distal colon, yet not enough to impair kidney development.These mice are sacrificed between P7-P25 because of welfare problems yet giving a time window for analysis of the development of HAEC. Histological analyses revealed that Gfra1hypo/hypo mice had goblet cell hyperplasia and a shift away from acidic mucin production in the distal colon. Goblet cell hyperplasia was first observed at P10, but the shift in mucin profile already appeared at P5. It is not known what causes goblet cells to change their mucin production, but it seems to be the earliest histopathological change in HAEC preceding goblet cell hyperplasia. qPCR-analysis revealed that Muc2, the main secreted mucin that protects epithelium from invading pathogens, was upregulated at both P5 and P10. mRNA levels of Tnfa were also upregulated at P10. The aforementioned changes were not observed in the duodenum where the ENS had developed normally despite the reduction in Gfra1 expression. This indicates that the changes observed in the colon are likely due to the lack of ENS innervation, rather than a direct effect from GDNF-GFRa1-RET signalling itself. Finally, serum analysis indicated that systemic inflammation did not occur from P10-P16, although one Gfra1hypo/hypo animal had high levels of IL6 and TNFa at P14-16. This indicates that inflammation is not an early stage event and it is preceded by goblet cells related changes. In conclusion, changes in goblet cells seems to be earliest histopathological findings preceeding HAEC.

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.

Parkinson's disease is a progressive neurodegenerative disease. The incidence of the disease is 1.5-2 per cent after age 60. Typical symptoms are tremor, rigidity and bradykinesia. At the late stage of the disease patients have psychic disorders, for example dementia, anxiety and depression. Motor impairment is caused by degenerative loss of dopamine cells in nigrostriatal tract. Current treatment of the disease relieves the symptoms but it cannot stop or slow the progress of the disease. Neurotrophic factors and gene therapy have been trialled to improve the treatment of Parkinson's disease and the results have been encouraging. Neurotrophic factors are proteins that regulate actions of neurons. It has been discovered that they are neuroprotective and neurorestorative. The results with glial cell line-derived neurotrophic factor (GDNF) have been encouraging in in vivo studies of Parkinson's disease. There has been variability in success of clinical trials though. GDNF degrades quickly in vivo but overexpression of GDNF in cells can be produced with viral vector adeno-associated virus. Two different forms of GDNF, pre-α-pro-GDNF (α-GDNF) and pre-β-pro-GDNF (β-GDNF), are produced as precursors and they are activated proteolytically. Based on in vitro studies, some differences in secretion of precursors have been discovered. α-GDNF is secreted constitutively and secretion of β-GDNF is dependent on physiological stimulation. Previous in vitro studies have focused on α-GDNF, but β-GDNF might be a better solution for treating Parkinson's disease based on physiological regulation system. Cerebral dopamine neurotrophic factor (CDNF) is recently discovered and less studied than GDNF. It has been discovered that CDNF also has neuroprotective and neurorestorative effects in animal models of Parkinson's disease. The aim of the first part of this study was to discover the neurorestorative effect of single injection of CDNF injected above substantia nigra for rats that received injection of 6-hydroxydopamine (6-OHDA) into medial forebrain bundle. One week later rats received PBS, GDNF or CDNF injection. The degree of the lesion was estimated with apomorphine (0.1 mg/kg s.c.) or d-amphetamine sulphate (2.5 mg/kg) induced rotation test. The rats were perfused nine weeks post-lesion and their brains were sliced. Tyrosine hydroxylase (TH) positive dopamine cells were stained by immunohistochemistry. The amount of TH positive cells in substantia nigra was counted and optical density of TH positive fibres in striatum was measured. The aim of the second part of the study was to research the neuroprotective effect of two different precursors of GDNF, dsAAV1-pre-α-pro-GDNF and dsAAV1-pre-β-pro-GDNF, given with viral vectors. The dopamine cells in nigrostriatal tract were destroyed with a 6-OHDA injection into striatum and viral vectors were injected two weeks later. Rats in control group received injection of dsAAV1-GFP. The degree of the lesion was evaluated with d-amphetamine sulphate (2.5 mg/kg) induced rotation tests and cylinder test. The rats were perfused eight weeks post-lesion and their brains were processed for immunohistochemistry. The results of the study were interesting and supporting previous studies. The success of the neurotrophic factor treatment is dependent on a successful injection of protein or viral vector, and the dose is dependent on the size of the lesion. Neurotrophic factors and gene therapy needs to be studied more before wide clinical usage.