Periodic Reporting for period 3 - FutureTrophicFactors (Elucidating therapeutic effects and mode of action of future trophic factorsin ALS and Parkinson’s disease) Reporting period: 2022-02-01 to 2023-07-31 Summary of the context and overall objectives of the project The prevalence of neurodegenerative diseases such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) is growing rapidly due to an aging population and increased lifeexpectancy. Current treatments for PD and ALS only relieve symptoms and cannot stop the progression of the disease, thus there is an urgent need for new therapies.Neurotrophic factors (NTFs) are secretory proteins that regulate the development, function and survival of neurons. They have been explored as novel drugs for the treatment of PD and ALS but their efficacy in clinicaltrials has been modest.Cerebral dopamine neurotrophic factor (CDNF) is a protein with NTF properties. I have shown that it protects and restores the function of dopamine (DA) neurons in rodent toxin models of PD more effectively than other NTFs making it promising drug candidates for the disease-modifying treatment of PD. CDNF showed neurorestorative effects in non-human primate models of PD. In addition, CDNF was safe and successfully passed phase 1/2clinical trials on PD patients. My group has also previously discovered that CDNF improves motor coordination and protects motoneurons (MNs) in three differentgenetic animal model of ALS (De Lorenzo et al., Bioarchives, under review in Nature Comms)Despite encouraging results with CDNF in animal models and clinical trials of PD and ALS, protein-based treatments have drawbacks, requiring direct delivery to the brain through invasive surgery, sincethey cannot pass through the blood brain barrier (BBB). My recent discovery, however, may overcome this difficulty: novel CDNF variant (C-CDNF) protects DA and motoneurons invitro and in vivo and can pass through the BBB. Importantly, we have found that subcutaneous administration of the novel C-CDNF improves motor function and isneuroprotective in animal models of PD and ALS.This is important for the society, bacause an aging population and increased life expectancy in developed countries are leading to a higher incidence of age-related neurodegenerative diseases such as ALS and PD. Treating these diseases is a growing economic burden for health care systems worldwide. In Europe, the estimated total cost of brain disorders is €798 billion per year in 2010 prices (including direct costs of treatment and care plus indirect cost of lost workdays and lost productivity). ALS and PD are incurable conditions and eventually lead to death. There is thus a huge unmet need for new and more effective treatments that could slow down or even stop disease progression.Neurodegenerative diseases are characterized by the vulnerability of specific neural populations, giving rise to a specific set of symptoms. Nevertheless, pathogenesis of neurodegenerative diseases, such as ALS and PD,shows striking similarities, suggesting, that discoveries in one condition may benefit others.Objectives were:Objective 1) Study and Compare the Mode of Action of C-CDNF and CDNF, 2) study the effect of Systemic Delivery of BBB penetrating C-CDNF in Neurodegenerative Diseases and 3) Developing Human MNs and DA Neurons from Patient-derived iPS cells and Testing the Effects of CDNF and C-CDNF on Survival, ER stress and Reduction of Protein Aggregates in these Cells Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We tested CDNF with C-CDNF in vitro embryonic MN cultures. We were able to show that C-CDNF is as effective as full length CDNF in cell culture. More importantly, we were also able to test C-CDNF and CDNF in vivo in TDP-43 rat model of ALS. CDNF and C-CDNF showed efficacy for improving motor coordination and survival in SOD1-G93A mouse model of ALS. Both CDNF and C-CDNF were also reducing ER stress in vitro and in vivoSpecific objectives were: to test and compare the effect of the CDNF and C-CDNF in vitro and in vivo in SOD1 mice and in TDP-43 rat model of ALS; compare the therapeutic effect of establish the mode of action of CDNF against ER stress and oxidative stress.Significant results: We were able to see good efficacy after single or chronic administration of CDNF in SOD1 mice (Fig. 1). We were also able to show neuroprotection after CDNF infusion in TDP-43 rat model of ALS, in which no other research group has ever reported positive effects with drug candidates. In addition, we were able to see such good effects also after C-CDNF chronic infusion in SOD1 mouse model of ALS and in TDP-43 rat mode of ALS (Fig. 2)We investigated whether CDNF had a neuroprotective effect in vivo in the SOD1-G93A mouse, an ALS in vivo animal model where chronic ER stress response in the corresponding MNs has been previously reported in many studies. To this end, we utilized a single injection of CDNF, which in PD animal models effectively counteracted dopamine neuron degeneration [Lindholm et al., 2007]. As proof-of principle, we first validated that CDNF and 125I-labelled CDNF injected in the brain lateral ventricle efficiently diffuse to different areas of the brain, including cortex, striatum, and substantia nigra, and to the lumbar spinal cord. Moreover, we found that CDNF specifically co-localizes with lumbar MNs. Early symptomatic SOD1-G93A mice and WT littermates of 13 weeks of age received a single i.c.v. injection of 10 μg of human CDNF or phosphate buffered saline (PBS) as vehicle and were examined twice a week to follow changes in symptoms and motor behavior until the final stage of paralysis. At the time of treatment, SOD1-G93A mice displayed measurable tremors in the hind limbs. Upon a single CDNF injection, mutant mice developed gait impairment and paralysis symptoms significantly more slowly than PBS-treated mutant littermates, and survival was increased in both female (7.1%) and male (7.6%) SOD1 mice (Fig. 1A). Along with this, their balance and motor behavior performance were significantly ameliorated by CDNF treatment (Fig. 1B). One week after CDNF or PBS injection, the CDNF group showed an increased ability to run on the smallest 8 mm rod in the multiple static rods experimental paradigm for females and on the 21 and 11 mm rods for males, compared to the PBS group. These gender differences in the experimental tasks are in accordance with previous observations that males develop major symptoms approximately one week earlier than female littermates. In the accelerating rotarod, CDNF-treated mice showed an increased latency to fall compared to vehicle treated-mice. No statistical difference was found in CDNF or vehicle-injected WT littermates over time. In the open field, SOD1-G93A mice exhibited a decreased number of rearings compared to WT mice, which is associated with less strength in the hind limb muscles, and CDNF treatment increased the number of rearings compared to PBS controls at 16 weeks. To correlate the improvement of motor behavior to MN survival, we examined the lumbar spinal cord, which revealed a significantly higher number of MNs present in the CDNF-treated compared to PBS-treated mice (Fig. 1C). Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) We are currently studying and comparing the effects of CDNF and C-CDNF in human motoneurons, after being able to validate the concetrations and culture conditionsWe are also preparing the first manuscript (C-CDNF characterization) that will be submitted to Nature 2022. figures-for-erc-report-october-2021-voutilainen.jpg
