Expression and function of the neurotrophic factors GDF5 and GDNF in the nigrostriatal system during development and in rat models of Parkinson's disease

Growth/differentiation factor 5 (GDF5) and glial cell line-derived neurotrophic factor (GDNF) are neurotrophic factors that promote the survival of midbrain dopaminergic neurons in vitro and in vivo. Both factors have potent neurotrophic and neuroprotective effects in rat models of Parkinson's disease (PD), and may represent promising new therapies for PD. The aim of the present study was to investigate the endogenous expression and function of GDF5 and GDNF in the nigrostriatal dopaminergic system during development and in rat models of PD. Examination of the temporal expression patterns of endogenous GDF5, GDNF, and their respective receptors, in the developing and adult nigrostriatal dopaminergic system suggest that these factors play important roles in promoting the survival and maturation of midbrain dopaminergic neurons during the period of postnatal programmed cell death. The relative levels of GDF5 and GDNF mRNAs in the midbrain and striatum, and their individual temporal expression patterns during development, suggest that their modes of actions are quite distinct in vivo. Furthermore, the sustained expression of GDF5, GDNF, and their receptors into adulthood suggest roles for these factors in the continued support and maintenance of mature nigrostriatal dopaminergic neurons. The present study found that endogenous GDF5, GDNF, and their receptors are differentially expressed in two 6-hydroxydopamine-induced lesion adult rat models of PD. In both terminal and axonal lesion models of PD, GDF5 mRNA levels in the striatum increased at 10 days post-lesion, while GDNF mRNA levels in the nigrostriatal system decreased at 10 and 28 days post-lesion. Thus, despite the fact that exogenous GDF5 and GDNF have similar effects on midbrain dopaminergic neurons in vitro and in vivo, their endogenous responses to a neurotoxic injury are quite distinct. These results highlight the importance of studying the temporal dynamic changes in neurotrophic factor expression during development and in animal models of PD.

The epigenome as a therapeutic target for Parkinson's disease

Parkinson’s disease (PD) is a common, progressive neurodegenerative disease characterised by degeneration of nigrostriatal dopaminergic neurons, aggregation of α-synuclein and motor symptoms. Current dopamine-replacement strategies provide symptomatic relief, however their effectiveness wear off over time and their prolonged use leads to disabling side-effects in PD patients. There is therefore a critical need to develop new drugs and drug targets to protect dopaminergic neurons and their axons from degeneration in PD. Over recent years, there has been robust evidence generated showing that epigenetic dysregulation occurs in PD patients, and that epigenetic modulation is a promising therapeutic approach for PD. This article first discusses the present evidence implicating global, and dopaminergic neuron-specific, alterations in the methylome in PD, and the therapeutic potential of pharmacologically targeting the methylome. It then focuses on another mechanism of epigenetic regulation, histone acetylation, and describes how the histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that mediate this process are attractive therapeutic targets for PD. It discusses the use of activators and/or inhibitors of HDACs and HATs in models of PD, and how these approaches for the selective modulation of histone acetylation elicit neuroprotective effects. Finally, it outlines the potential of employing small molecule epigenetic modulators as neuroprotective therapies for PD, and the future research that will be required to determine and realise this therapeutic potential.

The role of the dopaminergic neurotrophin growth/differentiation factor-5 in the developing rat ventral midbrain

Growth differentiation factor-5 (GDF-5) is a member of the transforming growth factor-β superfamily, a family of proteins that play diverse roles in many aspects of cell growth, proliferation and differentiation. GDF-5 has also been shown to be a trophic factor for embryonic midbrain dopaminergic neurons in vitro (Krieglstein et al. 1995) and after transplantation to adult rats in vivo (Sullivan et al. 1998). GDF-5 has also been shown to have neuroprotective and neurorestorative effects on adult dopaminergic neurons in the substantia nigra in animal models of Parkinson’s disease (Sullivan et al. 1997, 1999; Hurley et al. 2004). This experimental evidence has lead to GDF-5 being proposed as a neurotrophic factor with potential for use in the treatment of Parkinson’s disease. However, it is not know if GDF-5 is expressed in the brain and whether it plays a role in dopaminergic neuron development. The experiments presented here aim to address these questions. To that end this thesis is divided into five separate studies each addressing a particular question associated with GDF-5 and its expression patterns and roles during the development of the rat midbrain. Expression of the GDF-5 in the developing rat ventral mesencephalon (VM) was found to begin at E12 and peak on E14, the day that dopaminergic neurons undergo terminal differentiation. In the adult rat, GDF-5 was found to be restricted to heart and brain, being expressed in many areas of the brain, including striatum and midbrain. This indicated a role for GDF-5 in the development and maintenance of dopaminergic neurons. The appropriate receptors for GDF-5 (BMPR-II and BMPR-Ib) were found to be expressed at high levels in the rat VM at E14 and BMPR-II expression was demonstrated on dopaminergic neurons in the E13 mouse VM. GDF-5 resulted in a three-fold increase in the numbers of dopaminergic neurons in cultures of E14 rat VM, without affecting the numbers of neurones or total cells. GDF-5 was found to increase the proportion of neurons that were dopaminergic. The numbers of Nurr1-positive cells were not affected by GDF-5 treatment, but GDF-5 did increase the numbers of Nurr1- positive cells that expressed tyrosine hydroxylase (TH). Taken together this data indicated that GDF-5 increases the conversion of Nurr1-positive, TH-negative cells to Nurr1-positive, TH-positive cells. In GDF-5 treated cultures, total neurite length, neurite arborisation and somal area of dopaminergic were all significantly increased compared to control cultures. Thus this study showed that GDF-5 increased the numbers and morphological differentiation of VM dopaminergic neurones in vitro. In order to examine if GDF-5 could induce a dopaminergic phenotype in neural progenitor cells, neurosphere cultures prepared from embryonic rat VM were established. The effect of the gestational age of the donor VM on the proportion of cell types generated from neurospheres from E12, E13 and E14 VM was examined. Dopaminergic neurons could only be generated from neurospheres which were prepared from E12 VM. Thus in subsequent studies the effect of GDF-5 on dopaminergic induction was examined in progentior cell cultures prepared from the E12 rat VM. In primary cultures of E12 rat VM, GDF-5 increased the numbers of TH-positive cells without affecting the proliferation or survival of these cells. In cultures of expanded neural progenitor cells from the E12 rat VM, GDF-5 increased the expression of Nurr1 and TH, an action that was dependent on signalling through the BMPR-Ib receptor. Taken together, these experiments provide evidence that GDF-5 is expressed in the developing rat VM, is involved in both the induction of a dopaminergic phenotype in cells of the VM and in the subsequent morphological development of these dopaminergic neurons

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

The role of mitogen activated protein kinase phosphatase-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons: relevance to Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterised by the loss of midbrain dopaminergic neurons from the substantia nigra pars compacta(SNpc), which results in motor, cognitive and psychiatric symptoms. Evidence supports a role for the mitogen-activated protein kinase p38 in the demise of dopaminergic neurons, while mitogen-activated protein kinase phosphatase-1 (MKP-1), which negatively regulates p38 activity, has not yet been investigated in this context. Inflammation may also be associated with the neuropathology of PD due to evidence of increased levels of proinflammatory cytokines such as interleukin-1β (IL-1β) within the SNpc. Because of the specific loss of dopaminergic neurons in a discreet region of the brain, PD is considered a suitable candidate for cell replacement therapy but challenges remain to optimise dopaminergic cell survival and morphological development. The present thesis examined the role of MKP-1 in neurotoxic and inflammatory-induced changes in the development of midbrain dopaminergic neurons. We show that MKP-1 is expressed in dopaminergic neurons cultured from embryonic day (E) 14 rat ventral mesencephalon (VM). Inhibition of dopaminergic neurite growth induced by treatment of rat VM neurons with the dopaminergic neurotoxin 6- hydroxydopamine (6-OHDA) is mediated by p38, and is concomitant with a significant and selective decrease in MKP-1 expression in these neurons. Dopaminergic neurons transfected to overexpress MKP-1 displayed a more complex morphology and contributed to neuroprotection against the effects of 6-OHDA. Therefore, MKP-1 expression can promote the growth and elaboration of dopaminergic neuronal processes and can help protect them from the neurotoxic effects of 6-OHDA. Neural precursor cells (NPCs) have emerged as promising alternative candidates to fetal VM for cell replacement strategies in PD. Here we show that phosphorylated (and thus activated) p38 and MKP-1 are expressed at basal levels in untreated E14 rat VM NPCs (nestin, DCX, GFAP and DAT-positive cells) following proliferation as well as in their differentiated progeny (DCX, DAT, GFAP and βIII-tubulin) in vitro. Challenge with 6-OHDA or IL-1β changed the expression of endogenous phospho-p38 and MKP-1 in these cells in a time-dependent manner, and so the dynamic balance in expression may mediate the detrimental effects of neurotoxicity and inflammation in proliferating and differentiating NPCs. We demonstrate that there was an up-regulation in MKP-1 mRNA expression in adult rat midbrain tissue 4 days post lesion in two rat models of PD; the 6-OHDA medial forebrain bundle (MFB) model and the four-site 6-OHDA striatal lesion model. This was concomitant with a decrease in tyrosine hydroxylase (TH) mRNA expression at 4 and 10 days post-lesion in the MFB model and 10 and 28 days post-lesion in the striatal lesion model. There was no change in mRNA expression of the pro-apoptotic gene, bax and the anti-apoptotic gene, bcl-2 in the midbrain and striatum. These data suggest that the early and transient upregulation of MKP-1 mRNA in the midbrain at 4 days post-6-OHDA administration may be indicative of an attempt by dopaminergic neurons in the midbrain to protect against the neurotoxic effects of 6-OHDA at later time points. Collectively, these findings show that MKP-1 is expressed by developing and adult dopaminergic neurons in the midbrain, and can promote their morphological development. MKP-1 also exerts neuroprotective effects against dopaminergic neurotoxins in vitro, and its expression in dopaminergic neurons can be modulated by inflammatory and neurotoxic insults both in vitro and in vivo. Thus, these data contribute to the information needed to develop therapeutic strategies for protecting midbrain dopaminergic neurons in the context of PD.