A Thesis Submitted to the School of Graduate Studies in Partial Fulfilment of the Requirements for the Degree Doctor of Philosophy in Neuroscience / Bipolar disorder (BD) is increasingly being recognized as a neuroprogressive illness characterized by progressively worsening cognitive function and structural brain changes. Although lithium remains the gold standard treatment for BD, its ability to halt neuroprogression is a crucial, yet not fully understood aspect of its therapeutic benefit. Accumulating evidence suggests that lithium may be neuroprotective through alleviating ER stress, a feature highly implicated in the pathogenesis of BD. Over the last 20 years, Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF), an ER resident protein, has gained significant attention due to its ability to alleviate ER stress, reduce inflammation, and enhance cellular survival. In this thesis, we investigated the relationship between lithium and MANF using in vitro cellular models and in vivo rodent models. We establish that lithium upregulates MANF expression via the AP-1 signalling pathway. Moreover, we show that MANF plays a crucial functional role in lithium’s neuroprotective effects, as inhibiting the AP-1 pathway negated lithium’s protection against ER stress-induced neurotoxicity and reduced MANF expression. Furthermore, we demonstrate that MANF deficiency increases striatal cell susceptibility to amphetamine-induced neurotoxicity, highlighting its protective function in preclinical models of mania. In vivo experiments further showed that overexpressing MANF in amphetamine-treated rats reduced hyperlocomotion, prevented the upregulation of ER stress markers, and prevented amphetamine-associated death. Collectively, these findings suggest that MANF may be a critical mediator of lithium’s ability to halt neuroprogression in BD and advance our understanding of BD pathophysiology. Importantly, we present MANF as a promising therapeutic candidate for future therapeutic interventions aimed at halting neuroprogression in BD and preventing fatality due to amphetamine overdose. / Dissertation / Doctor of Philosophy (PhD) / Bipolar disorder (BD) is a psychiatric condition characterized by extreme episodic mood swings of highs (mania) and severe lows (depression). Over time, BD patients can experience changes in the brain, worsening symptoms, and cognitive decline – a process understood as neuroprogression. Lithium, a common treatment for BD, has been shown to slow down this detrimental progression, although exactly how it is accomplished is not fully understood. This thesis focused on a protein called MANF, which has been shown to protect and keep brain cells healthy by reducing stress within cells. We found that lithium increases MANF levels in brain cells, and this increase is crucial for lithium’s neuroprotective effects. Furthermore, we show that MANF is protective against the toxic drug amphetamine in brain cells and stops overdose- mediated death from occurring. By understanding how MANF works, we propose a new treatment intended to prevent the progression of BD and potential treatment for amphetamine overdose.

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.

Disorders of the central nervous system (CNS) continuously pose problems for current therapeutics. In part, this is due to the uncertainty of underlying pathophysiological changes that give rise to specific disorders. Parkinson’s disease (PD) specifically is a neurodegenerative CNS disorder with unknown origins of dopaminergic degeneration in the substantia nigra. Current therapies are reactive in nature and no existing neuroprotective therapies are available. Two hypotheses have been proposed to contribute to dopaminergic degeneration in PD: endoplasmic reticulum (ER) stress and oxidative stress. This thesis investigates molecular targets involved in each of these responses (mesencephalic astrocyte-derived neurotrophic factor (MANF) and cyclin-dependent 5 (CDK5)/p25 respectively) to support a multi-hit hypothesis in PD neural degeneration. Using behavioural and biochemical analysis, a reduction in MANF was found to participate in the ER stress hypothesis and CDK5/p25 hyperactivation is a viable neuroprotective target related to the oxidative stress hypothesis. Both pathways are evidenced in PD pathology and this thesis proposes specific targets for both pathways in the development of necessary neuroprotective therapies. Subsequently, included in this thesis is a chapter about the unmet pharmacological alleviation of negative and cognitive symptom domains in another CNS disorder of unknown pathophysiology: schizophrenia (SZ). These untreated symptoms are thought to be caused by irregularities in the signalling of multiple neurotransmitter systems. This chapter investigates the role of synapsin II, a protein involved in regulating signalling of multiple neurotransmitters, in manifesting negative and cognitive SZ symptoms and analyzes brain glucose metabolism. Reduced synapsin II levels were consistently implicated in the underlying physiology, and therefore synapsin II is proposed as a potential pharmacological target for these unmedicated symptomologies. Overall this thesis uses interrelated studies to propose novel molecular targets to address unmet therapeutic needs based on evidence of their involvement in the pathophysiology of PD and SZ. / Thesis / Doctor of Philosophy (PhD) / Brain diseases like Parkinson’s disease (PD) and Schizophrenia (SZ) are difficult to treat because their cause has not been discovered. PD shows degeneration of cells in the brain but the cause for degeneration is unknown. This makes developing treatments to protect cells from dying difficult. Two pathways are suggested to cause cell death in PD. This thesis proposes that both pathways are responsible for degeneration through a combined effort. Here, both pathways are shown to lead to cell death resembling PD and specific molecules are suggested as targets for developing protective treatments. Like PD, SZ has symptoms that cannot be treated because the cause is unclear. A protein was investigated for producing SZ-like symptoms and found to have potential for treatment design. This thesis aims to understand molecular changes in the brain leading to PD, with a look at SZ and how they can be used for better treatment design.

Nothing is perfect, and this includes the ability to maintain homeostasis within the cell with age. Factors such as aging, chemicals, and gene dysfunction disrupt cellular homeostasis, leading to increased stress and compromising the ability of animals to maintain a healthy lifespan. Dysregulated homeostasis can be detrimental on an organismal level, impacting locomotion, and on a cellular level causing proteins to misfold and become aggregates, which are toxic to cells. Toxic protein aggregation and loss of locomotory function are key hallmarks of several age-related diseases. My Ph.D. work examined the collapse of homeostasis on electrotaxis, the age-associated increase in proteotoxicity, the decline in longevity, and neuronal and muscle health. On a behavioural level I demonstrated that loss of various components of the MT-UPR, ER-UPR, and HSR modulated the speed of animals. Additionally, I found that activation of stress responses due to chemicals and exercise reduced and increased the speed of animals respectively. On a cellular level I elucidated potential mechanisms by which Mesencephalic Astrocyte Derived Neurotrophic Factor (MANF) affects the stress response to maintain homeostasis and prevent protein aggregation. I observed the novel localization and role of MANF in lysosomes to potentially act as a critical regulator of the stress response to maintain proteostasis, neuronal health and longevity, thereby bringing balance to the cell. Furthermore, the broad tissue expression of MANF revealed its localization to muscles. This supports the ability of MANF to act as more than a neurotrophic factor as it was found to be required for muscular health in animals in an age-dependent manner. Overall, my Ph.D. research has provided new insights into the stress response and behaviour and the precise role of MANF in mediating stress response signaling to promote organismal and cellular fitness. / Dissertation / Doctor of Science (PhD) / Cellular perturbations or stress disrupt homeostasis, activating multiple stress responses. Activation of the stress response can determine the fate of an organism and is crucial to its health. Although the stress response pathways are generally understood, little is known about how the stress responses preserve animal behaviour or how they are regulated to promote organismal survival. My work has provided a basis for how stress responses affect behaviour positively and negatively in animals. I found that the stress response required mesencephalic astrocyte derived neurotrophic factor (MANF) to promote organismal survival. My thesis determined that MANF acts as more than a neurotrophic factor. MANF was found to not only be essential in neuronal health but also longevity and muscle health. Overall, this thesis demonstrated the impact of stress response on behaviour and the potential mechanism by which MANF is cytoprotective in whole organisms.