CDNF and MANF are members of a recently discovered and evolutionarily conserved neurotrophic factor family implicated in supporting the survival and protection of midbrain dopaminergic neurons in the nigrostriatal pathway, which degenerate in Parkinson’s Disease (PD). Increasing evidence demonstrated that MANF overexpression resulted in significant protection and repair of TH+ cells and DA neurons in the substantia nigra (SN). In addition, continuous infusion of CDNF demonstrated greater protection of TH-positive neurons in the SNc and fibers in striatum than GDNF in the 6-OHDA neurotoxin model. Current literature suggests that CDNF and MANF are involved in regulating ER stress and are upregulated in vitro and in vivo during the unfolded protein response (UPR). Thus, this study sought to investigate whether selective knockdown (K/D) of MANF and CDNF causes pathophysiological conditions that lead to the behavioural manifestation of PD in preclinical models. Male Sprague-Dawley rats underwent stereotaxic surgery, whereby 2 μL at 0.5 μL/minute of MANF, CDNF, MANF and CDNF combined, or a scrambled negative control (N=44) of rat lentiviral-mediated shRNA formulations were infused into the SN in reference to bregma: Anterior/Posterior=-5.3 mm, Medial/Lateral=±2.3 mm, Dorsal/Ventral=-7.8 mm. Rats were tested on a battery of behavioural tests for the assessment of PD phenotypes, such as impairments in balance, gait and motor coordination. MANF K/D rats demonstrated PD phenotypes in the rearing duration, beam traversal, rotarod and cylinder test (P <0.05). These results were largely mirrored in the combined MANF and CDNF K/D group, however, CDNF K/D rats failed to demonstrate consistent motor deficits (P >0.05). Additionally, CDNF mRNA expression from the platelets of PD patients revealed no significant differences compared to healthy controls (P >0.05). In conclusion, the etiology of PD remains to be elucidated, and this is the first study to demonstrate that MANF K/D rats recapitulate key motor features of parkinsonism. / Thesis / Master of Science (MSc) / CDNF and MANF are members of a recently discovered and evolutionarily conserved neurotrophic factor family implicated in supporting the survival and protection of midbrain dopaminergic neurons in the nigrostriatal pathway, which degenerate in Parkinson’s Disease (PD). Increasing evidence demonstrated that MANF overexpression resulted in significant protection and repair of DA neurons in the substantia nigra (SN). Current literature suggests that CDNF and MANF are involved in regulating ER stress and are upregulated in cells and in rodents during the unfolded protein response (UPR). Thus, this study sought to investigate whether selective knockdown (K/D) of MANF and CDNF causes the underlying changes in the brain that lead to the behavioural manifestation of PD in preclinical models. 2 μL at 0.5 μL/minute of MANF, CDNF, MANF and CDNF combined, or a scrambled negative control (N=44) of rat lentiviral-mediated shRNA formulations were infused into the SN. Rats were tested on a battery of behavioural tests for the assessment of PD phenotypes, such as impairments in balance, gait and motor coordination. MANF K/D rats demonstrated PD phenotypes in the rearing duration, beam traversal, rotarod and cylinder test (P <0.05). These results were largely mirrored in the combined MANF and CDNF K/D group, however, CDNF K/D rats failed to demonstrate consistent motor deficits (P >0.05). Additionally, CDNF mRNA expression from the platelets of PD patients revealed no significant differences compared to healthy controls (P >0.05). In conclusion, the etiology of PD remains to be elucidated, and this is the first study to demonstrate that MANF K/D rats recapitulate key motor features of parkinsonism.

no abstract provided / Thesis / Master of Science (MSc)

Mitochondrial dysfunction has been identified as a key factor in the progression of Parkinson's disease. Mitochondrial dysfunction has been shown to induce stress pathways, leading to neuronal dysfunction and cell death. Our lab has previously identified that, in neurons, reconfiguring the mitochondria using supercomplex assembly factors is protective against excitotoxic stress. For this thesis, we sought to characterize the stress pathways and synaptic impairment in an in vitro mitochondrial dysfunction model. Then, to determine if we can rescue the deficits shown, we manipulated mitochondrial integrity using the inner mitochondrial membrane targeted isoform of MCL1, which has previously been shown to regulate cristae structure and mitochondrial supercomplex assembly. We demonstrate that the integrated stress response is activated upon mitochondrial dysfunction. Next, we show mitochondrial dysfunction leads to a downregulation of synaptic genes involved in neurotransmission. Finally, our results show that both the antiapoptotic outer mitochondrial membrane-targeted isoform, and MCL1-Matrix are able to prevent cell death in response to mitochondrial dysfunction; however, MCL1-Matrix confers greater reduction in ISR activation and reactive oxygen species production. These data suggest that manipulating mitochondrial integrity, using MCL1-Matrix, confers a broad protective effect against neuronal stressors and may be used as a novel approach to preventing Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative Lewy body disorder characterized by severe motor deficits, followed by cognitive dysfunction with progression of the disease. Environmental exposure has been suggested as a possible contributor to the development of PD and this view is linked to the discovery of the nigrostriatal neurotoxin MPTP. MPTP can induce dopamine specific degeneration within the basal ganglia often resulting in motor deficits similar to PD. MPTP used in the C57BL/6 mouse is a widely used animal model of PD. The pyrethroid permethrin (PM), and the organophosphate chlorpyrifos (CPF), can produce changes in dopaminergic nigrostriatal neurons, the primary target of PD and MPTP-induced neurotoxicity. Such insecticide induced changes in the basal ganglia could exacerbate the onset or severity of PD. Chronic exposure to the metal manganese (Mn) can damage the globus pallidus (GP) of the BG, and produce motor deficits similar to PD. Since the GP is part of the BG circuitry essential for motor control, and is synaptically integrated with the nigrostriatal pathway, Mn may exacerbate MPTP-induced neurotoxicity. Because the BG is disynaptically linked to the mesocortical pathway, a dopaminergic pathway that is important for cognition, Mn induced damage in the BG could indirectly affect the mesocortical pathway as well. This study investigated the pesticides, permethrin and chlorpyrifos, and the heavy metal, manganese as possible environmental compounds that could exacerbate PD in the MPTP treated C57BL/6 mouse. The first part of this dissertation used immunohistochemistry to examine insecticide induced effets on MPTP-induced neurotoxicity in the dorsolateral striatum of the C57BL/6 mouse, the principal target of the nigrostriatal pathway. Tyrosine hydroxylase (TH) was used as a marker for loss of dopaminergic neuropil and glial fibrillary acidic protein (GFAP) was used as a marker of glial activation in the striatum. Three experiments assessed effects of 1) PM (200 mg/kg), 2) CPF (50 mg/kg) & 3) PM + CPF, on MPTP (30 mg/kg) neurotoxicity. Immunohistochemistry revealed a decrease in TH staining and an increase in GFAP staining with MPTP (30 mg/kg). A main effect increase in GFAP was observed for PM (200 mg/kg), but not for CPF (50 mg/kg) or PM+CPF. Insecticides, alone or combined, did not alter MPTP-induced toxicity. . However, the absence of the PM-induced increase in GFAP staining following combined insecticide treatment suggests a neuroprotective effect. The next set of experiments in this dissertation looked at the effect of Mn on MPTP-induced neurotoxicity in the nigrostriatal and mesocortical dopaminergic pathways of the C57BL/6 mouse. Inductively Coupled Plasma atomic emission spectrometry revealed striatal Mn levels were significantly increased with multiple dose 100, 50, and 25 mg/kg MnCl2. Administration of Mn (MnCl2 s.c., Days 1, 4, & 7) in the MPTP (20 mg/kg i.p., Day 8) treated C57BL/6 mouse revealed Mn and MPTP interactions for locomotor activity, grip strength, and repeated measures of learning. Mn attenuated the effect of MPTP on striatal DOPAC, and facilitated the effect of MPTP on cortical DA and DOPAC. Mn also attenuated the MPTP induced decrease in cortical DAT. While these data support the notion that insecticides can produce tissue damage in the nigrostriatal pathway, in this case, these insecticide induced changes were not found to be strong enough to facilitate PD-like tissue damage. While Mn did not always facilitate MPTP neurotoxicity in the mesocortical and nigrostriatal dopaminergic pathways, these results demonstrate Mn and MPTP can interact in a complex way to alter dopaminergic function as well as motor and cognitive behavior. Differences in brain uptake mechanisms and metabolism of Mn and MPTP, could explain why combined administration of Mn and MPTP differentially affect dopaminergic activity in the nigrostriatal and mesocortical pathways. / Ph. D.

The basal ganglia provide a major neural system through which the cortex affects behavior. Most notable among these effects are those related to the voluntary control of movement as seen in neurodegenerative disorders like Parkinson’s disease (PD). Well known tests of visual perception in PD “explicitly” measure object recognition (a high-level visual process) but “implicitly” rely on intact mid-level visual processes like grouping and figure-ground segmentation to structure the image. Hence, exploring the importance of the basal ganglia in perceptual organization (PO) abilities by examining the specific impairments incurred with the damage of such a vital structure is imperative. Therefore, this study attempted to investigate PD performance in tasks in computerized classic gestalt perception experiments with the aim of identifying any mid-level visuo-perceptual deficits. Differences were observed in the grouping by proximity dot counting task but not in other tasks that involved figure-ground segregation, part detection in embedded contexts or shape discrimination. / M.S. / Damage to the basal ganglia, a group of structures in the subcortical part of the brain (below the cerebral cortex), has long been associated primarily with Parkinson’s Disease (PD), a neurological disorder that manifests with symptoms like muscle rigidity and tremors. While several key visual and perceptual problems have also been connected to this area, very few studies have tried to describe the mechanisms by which PD functionally alters their ability to perceive the visual world. Hence, this study attempted to investigate PD performance in computerized classic perception experiments with the aim of exploring mechanisms that organize incoming visual information to structure the image called perceptual organization (PO). Differences were observed in tasks that tested their ability to group “dots” when they are varied by proximity to each other but not in other tasks that involved their ability to segregate figures from the ground, detect parts of shapes in embedded contexts or discriminate between shapes.

1-Methyl-4-(1-methylpyrrol-2-yl)-1 ,2,3,6-tetrahydropyridine (TMMP) is a neurotoxic analog of the parkinsonian inducing agent MPTP. TMMP and its putative metabolites 1-methyl-4-(1-methylpyrrol-2-yl)-2,3-dihydropyridinium (MMDP+) and 1-methyl-4-(1-methylpyrrol-2-yl)pyridinium (MMP+) were synthesized and fully characterized. Substrate/inactivation properties of TMMP and its analog N-propargyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine with MAO-B were investigated. Kinetic data was obtained, including Km and Vmax for TMMP as an MAO-B substrate, and KI and kinact values for N-propargyl-4-(1-methylpyrrol-2-yl)-1,2,3,6-tetrahydropyridine. The metabolic studies of TMMP and MMDP+ were conducted with an HPLC diode array assay. Both in-vivo and in-vitro metabolic studies showed that TMMP is oxidized to its dihydropyridinium species (MMDP+) in a reaction catalyzed by MAO-B. MMDP+ undergoes autoxidation to form the pyridinium species (MMP+), the mechanism of this conversion is not clear. In-vitro studies show that MAO-B is not responsible for this conversion and the oxidation of MMDP+ to MMP+ is likely to be enzyme catalyzed. Toxicity investigations include dopamine depletion studies of TMMP and MMDP+, mitochondrial respiration and microdialysis studies of MMDP+ and MMP+. The above studies show that TMMP is an MPTP-type neurotoxin. / Master of Science

Parkinson's disease (PD) is a debilitating motor disorder that primarily afflicts older individuals (> 50yrs). Although its cause is unknown, many factors are thought to contribute to the disease. There is growing epidemiological evidence supporting a link between pesticide exposure and PD. The present immunohistochemical study was undertaken to characterize the role of insecticide exposure in the etiology of idiopathic PD. The insecticides selected for study were the pyrethroid permethrin (PE) and the organophosphate chlorpyrifos (CP), both of which possess properties that could damage or disrupt the nigrostriatal pathway, which is the principal neurodegenerative target in PD. The present study examined possible alteration of the amount of dopamine re-uptake transporter protein (DAT), within the striatum of the C57BL/6 mouse, using DAT antibodies, following low (0.8, 1.5 & 3.0 mg/kg) and high (200 mg/kg) doses of PE, respectively. Possible nigrostriatal terminal degeneration was examined using antibodies to tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with 50 mg/kg of CP alone or in combination with the high dose of PE. For both the high dose of PE alone and for the combined PE/CP treatment, glial fibrillary acidic protein (GFAP) antibodies were used to examine the possibility of non-degenerative tissue injury. Groups of matched treated/vehicle-control mice received three IP injections of the insecticide/dose of interest over a 2-week period. Counts of immunoreactive (IR) neuropil in the dorsolateral striatum were made from four pre-selected fields per striatal tissue section. Counts were compared between matched sections, processed on the same slide, from a treated mouse and its vehicle control. A mean difference score, across slides, for each treated/vehicle control pair was determined. All low dose PE groups showed a trend of decreased DAT IR neuropil, but only the 3.0mg/kg group showed a statistically significant reduction (p<.0078). The 200 mg/kg PE group showed a trend toward reduced TH IR neuropil that was not statistically significant, but a significant increase in GFAP IR (p = .048) was observed. No significant change in TH IR neuropil was observed for CP (50mg/kg) alone. A significant increase was observed for GFAP IR neuropil for the PE/CP (200/50 mg/kg) combination dose (p = .033). The combined insecticide treatment failed, however, to produce a significant change in TH IR within the striatum, compared to vehicle controls. These data suggest that the significant increases in GFAP IR neuropil, in the striatum, reflect some form of tissue insult, following exposure to a high dose of PE, or PE/CP in combination, that is insufficient to induce degeneration of dopaminergic terminals within the temporal interval investigated. Although such damage may be sufficient to account for previously reported decreases in maximal dopamine uptake observed with high doses of these compounds, the DAT IR data appear to indicate that this damage is unlikely to be a change in the amount of DAT in these high dose conditions. The decreases in striatal DAT IR neuropil observed for low doses of PE suggest an alteration in the normal integrity of the nigrostriatal pathway and in the route by which environmental toxins may enter dopaminergic neurons. / Master of Science

No description available.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

In this study we aimed to investigate the effects of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) and rotenone neurotoxins on dopaminergic (DAergic) neuronal survival using ventral mesencephalic (VM) organotypic cell culture derived from postnatal rat pups (P4-5) and immunocytochemistry for tyrosine hydroxylase (TH) as a marker of DAergic cells. In addition, we examined the neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) on TH-ir cells exposed to MPTP and rotenone as a possible treatment for PD. The TH-ir cells in co-cultures with striatum (ST) as a target grew better then when VM was cultured alone and that TH-ir cells in co-cultures could be maintained without using conditioned and trophic media. We treated 7 day and 14 day co-cultures at different times with varying MPTP and rotenone concentrations and found 14 day old cultures were more vulnerable than 7 day old co-cultures to the effects of either neurotoxin with TH-ir cell numbers significantly lower in 14 day cultures compared to 7 day cultures. Both neurotoxins induced a dose-dependent TH-ir cell reduction in the co-cultures. In addition we compared the toxicity of MPTP and its active metabolite 1-methyl-4- phenylpyridinium (MPP+) as the neurotoxic effects of MPTP on DAergic cells depends on its conversion to MPP+ by astrocytes. We found no significant difference in TH-ir cell reduction in co-cultures treated with MPTP and MPP+. Rotenone was more toxic than MPTP with less TH-ir cell survival in the weeks post treatment. GDNF exposure produced increased cell size and significant increases in TH-ir cell branching in cocultures in a dose-dependent manner. Post treatment of GDNF against MPTP and rotenone provided significant neuroprotection as TH-ir cell survival was at the lower neurotoxin doses and not at the higher doses. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1346931 / Thesis (Ph.D.) - University of Adelaide, School of Medical Sciences, 2008