Neurotrophic factors are proteins involved in the maturation, differentiation and survival of neurons. Due to their neuroprotective properties, they have been regarded as potent candidates for the treatment of neurodegenerative diseases. Recently, a novel family of neurotrophic factors was discovered comprising mesencephalic astrocyte-derived neurotrophic factor (MANF) and cerebral dopamine neurotrophic factor (CDNF). These factors have been shown to protect against the degeneration of nigrostriatal dopaminergic neurons in mammalian models of Parkinson's disease, however their neuroprotective mechanisms of action are not yet understood. Although distinct in vertebrates, MANF and CDNF constitute a single homolog in invertebrates. In the present study, we have characterized the in vivo expression and function of the C. elegans homolog manf-1. We have shown that manf-1 is not essential for neuronal development, however when knocked down, mutants exhibit enhanced age-related dopaminergic neuronal degeneration accompanied by an increase in the endogenous ER stress response. Loss of manf-1 function also results in enhanced alpha-synuclein expression and aggregation, a pathological hallmark of Parkinson’s disease. / 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 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.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder primarily affecting the aging population over the age of sixty. Characterized by the significant degeneration of dopaminergic (DAergic) neurons of the substantia nigra causing severe motor dysfunction. Although the exact pathogenesis of this disease is still unknown endoplasmic reticulum stress and mitochondrial dysfunction are believed to play a role. PD is diagnosed after severe DAergic neuron degeneration, and yet is still often misdiagnosed. There is a need for a definitive diagnostic test for the early detection of PD. Current therapies only relieve symptoms and do not stop disease progression. Neurotrophic factors (NTF) are naturally occurring proteins that promote the survival, differentiation and maintenance of neurons and present a promising candidate for the treatment of PD. Cerebral dopamine neurotrophic factor (CDNF) is a novel NTF that protects and rescues DAergic neurons. The present study investigated the role of DAergic activity and CDNF mRNA expression in C. elegans, as well as understanding how does PD affect the endogenous levels of CDNF protein and mRNA expression. We demonstrated that of the various dopamine (DA) synthesis and transport mutants tested, the impaired synthesis of DA from levodopa is linked to the up regulation of CDNF. Also, following unilateral 6-hydroxydopamine (6-OHDA) lesioning protein and mRNA expression of CDNF was not affected implicating ER stress as inducing a possible compensatory up regulation of CDNF, thus returning levels to normal. CDNF mRNA expression was determined to decline with age and possibly increase ones vulnerability to developing a neurodegenerative disorder. An increase mRNA expression of CDNF in the PD patient population was found to be specific to platelets. Stroke patients showed an increase in CDNF expression in whole blood. In conclusion, these findings highlight the importance of the relationship between CDNF and ER stress and warrants further investigation. / Thesis / Master of Science (MSc)