Small Rho GTPases as pharmacological targets in age-related cognitive decline: experimental evaluation of two distinct therapeutic approaches

Introduction Brain ageing is characterized by structural and functional changes in key brain areas and neuronal circuits, which together contribute to cognitive decline. It is well established that age is a major risk factor for neurodegenerative diseases and dementia. While considerable attention has been devoted to the study of pathological brain ageing, the relative lack of research focusing on the characteristics of healthy brain aging is noteworthy. Rho GTPases are key regulators of synaptic plasticity, positioning this pathway as a promising therapeutic target for age-related brain dysfunction. In this thesis, we investigated the effects of two distinct pharmacological strategies targeting Rho GTPase signaling: the synthetic molecule fasudil, and the protein toxin cytotoxic necrotizing factor 1 (CNF1). Fasudil is a Rho-associated kinase (ROCK) inhibitor that acts as a vasodilator, is clinically used for cerebrovascular conditions. Recently have been explored for drug repurposing in neurodegenerative disorders; however, its neuroprotective mechanisms, as well as the effects of chronic treatment, remain poorly understood. CNF1 is a modulator of Rho GTPases activity, which has been studied for its role in improving synaptic plasticity in neurodegenerative models. As a model of brain ageing, we used socially isolated aged mice since isolation induces neural and synaptic changes that mimic hallmarks of brain ageing, accelerating cognitive decline in the elderly. This is particularly relevant since a substantial proportion of the elderly population in Western countries is exposed to social isolation. Aims Aim one: Investigate the effect of chronic fasudil administration in young healthy mice to evaluate the long-term effects on cognitive brain function and potential off-target mechanisms. Aim two: Investigate the effects of CNF1-mediated Rho GTPase modulation in age-related brain functional and cognitive impairments in a mouse model of socially isolated aged mice. Methods For Aim one, CD1 one-month old-male mice received daily oral administration of fasudil 100mg/kg/d for 12 weeks or were left untreated as controls (n=12). For Aim-two, CD1 male mice of twelve-months old were socially isolated till the end of experiments. At 18 months of aged old mice received either CNF1 intracerebroventricularly (1.0 × 10⁻⁴ M) or vehicle. A group of mice were left undisturbed as age-matched controls (n=12). After treatments mice underwent to a battery of behaviorual tests assessing brain function including neuromuscular strength (grip strength), motor coordination (rotarod), exploratory behavior and anxiety-like behavior (open field test), recognition memory (novel object recognition), and fear memory (fear conditioning). After behavioral testing, a subset of animals from each group underwent magnetic resonance brain analysis including diffusion tensor imaging (DTI) to study structural alteration and magnetic resonance spectroscopy (MRS) to study metabolites in key brain areas including hippocampus (HIP) and prefrontal cortex (PFC). Extracts from this brain areas were analyzed to assess Rho GTPases activity. Finally synaptic markers expression such as GABA‑A receptor, and postsynaptic density protein 95 (PSD‑95) were evaluated. For Aim one, protein comparative analyses and in silico docking assays were performed to assess the possible binding affinity of fasudil for the GABA‑A receptor. Results Aim one Computational analysis revealed a strong binding affinity of fasudil for the GABA-A receptor. In vivo chronic drug treatment impaired contextual fear-related memory. These behavioural changes were accompanied by a reduction in the γ2 subunit of the GABA-A receptor in both the HIP and the PFC. In the PFC a decrease in PSD-95 expression and an increase in Rac1 activity were also detected. The molecular and behavioural effects of fasudil closely resemble those induced by chronic benzodiazepine prolonged treatment. Magnetic resonance analyses revealed elevated glutamate levels in the PFC, associated with a general increase in total choline and a mild deterioration of white matter integrity in the corpus callosum, alterations that have been described as characteristic of benzodiazepine withdrawal. Aim two Aged male mice subjected to prolonged social isolation developed an anxiety-like behaviour. That behavioral change was accompanied by structural alterations detected via DTI in both the HIP and PFC, but not in the amygdala. Consistent with structural alterations, we observed region-specific metabolic changes in both HIP and PFC, affecting metabolites related to membrane turnover, energy metabolism, and particularly neurotransmission reflecting the imbalance between inhibitory and excitatory systems observed in early stages of dementia. CNF1 restored anxiety-like behavior, paralleled by rebalancing of brain metabolites and neurotransmitter levels in both HIP and PFC. This effect was accompanied by a reduction in gephyrin and GABAAα5 subunit expression and increased Rac1 activity in the PFC Conclusions Our data indicate that fasudil modulates GABA-A-γ2 receptor expression and may act as an allosteric modulator. Chronic treatment appears to exert a negative impact on the PFC in mice, inducing behavioural and molecular effects similar to those observed following prolonged benzodiazepine exposure. These findings highlight potential adverse effects associated with long-term fasudil administration and suggest that similar precautions to those applied to benzodiazepines should be considered if applied for brain ageing related disorders. Conversely, CNF1 effectively restored the behavioural and metabolic alterations observed in aged and socially isolated mice, promoting the re-establishment of excitatory–inhibitory balance in the cognitive areas of these models. These findings highlight a central role for Rho GTPase signaling in the regulation of brain metabolic and neurotransmission homeostasis during ageing processes and suggest that targeting this pathway may delay or prevent the progression toward overt cognitive impairment.