Neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD), are characterized by the progressive loss of neurons. PD, in particular, is marked by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the subsequent reduction of dopamine levels in the striatum (Forno, 1996). Oxidative stress has been implicated in the development of PD, leading to dysfunction or death of neuronal cells, contributing to the disease's pathogenesis (Emerit et al., 2004). Environmental exposure to paraquat (PQ) has been linked to an increase risk of developing PD (Chinta et al., 2018). Previous studies have established experimental models using the herbicide paraquat, showing that PQ-induced cell death leads to autophagy disruption and oxidative stress, which are common features of PD (Duarte-Jurado et al., 2023; Ramirez-Moreno et al., 2019).

In brain disorders, such as PD, there is mounting evidence that glial cells play a significant role in neuronal alterations and disease. Pathological network could involve phenotypic alterations of glial cells, which have a neurosupportive and homeostatic role in the healthy brain but adopt reactive states under adverse conditions (Patani et al., 2023).

The use of neuropeptides has been described to have beneficial effects on the dopaminergic neurons of the substantia nigra (Chen et al., 2023). Neurotrophic factors (NTFs) are growth factors that promote neuronal survival and regeneration, as well as glial stimulation, making them potential therapeutic candidates for adult tissue regeneration (Xiao and Le, 2016). Researchers have focused on developing therapies to halt PD progression by delivering NTFs to the brain. For instance, the glial cell line-derived neurotrophic factor (GDNF) has been tested in both animal models (Coll et al., 2020) and clinical trials involving PD patients (Barker et al., 2020; Huttunen et al., 2023; Pakarinen and Lindholm, 2023). Another NTF, the cerebral dopamine neurotrophic factor (CDNF), is an endoplasmic reticulum (ER) luminal protein with trophic activities in various tissues (Jӓntti and Harvey, 2020). Recombinant human CDNF protein, produced in a mammalian cell line, is currently undergoing clinical trials for PD treatment (Huttunen and Saarma, 2019).

Although local delivery of NTFs to the brain has shown promising results in inducing protection of dopaminergic neurons and motor recovery in preclinical models, its success in clinical studies has been limited (Huttunen and Saarma, 2019). This limitation highlights the need to explore novel forms of NTF delivery to the target tissue.

Gene therapy has emerged as a promising alternative for treating neurodegenerative diseases in the brain. Three key areas of focus in gene therapy are vector design, identification of new therapeutic targets, and delivery systems, such as adeno-associated viruses, lentiviruses, and cell-penetrating peptides (CPPs). CPPs, short peptides with fewer than 30 amino acids, efficiently penetrate cell membranes and deliver cargo molecules (Derakhshankhah and Jafari, 2018). CPPs offer advantages like low cytotoxicity and ease of synthesis (Heitz et al., 2009).

In previous studies, a CPP-based delivery system was developed, incorporating the Asn194Lys mutation in the RVG9R peptide (mRVG9R). This mutation is situated at the acetylcholine receptor binding site, enhancing the transfection efficiency of the mRVG9R peptide carrying a transgene (green fluorescent protein reporter gene, GFP) to murine brain cells. This CPP (mRVG9R) can transfect neurons, astrocytes, oligodendrocytes, and microglia in various brain regions, including the cerebral cortex, hippocampus, and striatum. GFP transgene expression persisted for 20 days (Villa et al., 2019; Villa-Cedillo et al., 2017).

The unique characteristics of the mRVG9R peptide make it a promising option for delivering diverse cargo molecules with neurorestorative and neuroprotective effects in neurodegenerative diseases such as PD and AD.

This study's main focus is to evaluate the mRVG9R delivery system as a strategy to analyze the protective and restorative effects of the neurotrophic factor CDNF on neurons and neuroglia through gene therapy in an animal model of Parkinson's disease.