JM-20 treatment prevents neuronal damage and memory impairment induced by aluminum chloride in rats

Highlights

• JM-20 improved different types of memory in a model of cognitive decline and neuronal damage in prefrontal cortex and hippocampus of adult rats.

• Mitoprotective and antiapoptotic mechanisms are suggested as central players mediating JM-20 effects.

• JM-20 showed therapeutic potential as a promising candidate for the treatment of memory impairment and Alzheimer-like pathologies.

Abstract

The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl₃) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl₃ -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration.

Introduction

The number of patients with dementia has increased with numbers at 46.8 million towards the end of the 2018, a figure set to increase to 152 million by 2050 (Patterson, 2018). Alzheimer's disease (AD) is the most common form of dementia, accounting for approximately 60 % of all types, which will prove to be a great financial burden to society in the future (Patterson, 2018). AD is a complex and multifactorial disorder, for which aging is the main identified risk factor, whereas Ca²⁺ dyshomeostasis, oxidative stress, energy deficits, and mitochondrial dysfunction are all emerging as potential biochemical factors (Dorszewska et al., 2016; Onyango et al., 2016; Prince et al., 2015). Currently, symptomatic treatment for the cognitive deficits targets either cholinergic (with acetylcholinesterase inhibitors) or glutamatergic transmission (through N-methyl-d-aspartate (NMDA) receptor antagonists, e.g., memantine). However, treatments with a single symptomatic agent have shown insufficient efficacy (Birks and Harvey, 2006; Raina et al., 2008; Tan et al., 2014). Therefore, it is imperative to find new drugs that could delay the onset, slow the progression or improve the cognitive symptoms, to save lives and reduce the economic and social burden of the disease. It has been extraordinarily difficult to develop new therapies. Attempts to develop disease-modifying therapies have had a 100 % failure rate (Cummings et al., 2018) and no treatments have been approved since 2003 (Cummings et al., 2014). Every trial, however, presents opportunities to learn and improve the drug development process (Cummings, 2018; Cummings et al., 2018). In this context, small molecules with multitarget effects provide a promissory strategy on smart drug design focused to target key components in the neurodegenerative pathway (Nunez-Figueredo et al., 2014b).

JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-4,11-dihydro-1H-pyrido[2,3-b][1,5] benzodiazepine), is a novel hybrid molecule with a 1,5 benzodiazepine fraction covalently linked to a dihydropyridine ring, forming an innovative entity with proven multisite neuroprotective activity (Nunez-Figueredo et al., 2014a, 2015; Nunez-Figueredo et al., 2014b, c; Ramirez-Sanchez et al., 2015), that includes anti-excitotoxic and anti-Ca²⁺ effects (Nunez-Figueredo et al., 2014a), the reduction of vesicular glutamate uptake (Nunez-Figueredo et al., 2015), antioxidant and mitoprotective effects. Notably, this molecule reduced mortality, neurological and motor impairment in experimental models of ischemic stroke and Parkinson’s disease in rats (Fonseca-Fonseca et al., 2021, 2019) without amnesic or toxic effects (Nunez-Figueredo et al., 2014a, 2015; Nunez-Figueredo et al., 2014b, c; Ramirez-Sanchez et al., 2015). In addition, other pharmacological effects were detected as direct or indirect action of JM-20 in the complex scenery of the pathological models of memory impairment, like the inhibition of acetylcholinesterase enzyme (Wong-Guerra et al., 2019; Da Silva et al., 2020). Oral administration of JM-20 prevented memory impairment, in episodic memory and acquisition-consolidation processes against acute scopolamine treatment (Wong-Guerra et al., 2019). Previous results also suggest the anti-apoptotic effect of JM-20 (Ramirez-Sanchez et al., 2015; 2018) and further pieces of evidence are presented in this work that support this notion. Drawing from all these positive antecedents, we hypothesized that this molecule could revert and prevent neurotoxic chronic damage that involves more complex long-term mechanisms.

Aluminum is a neurotoxic metal that contributes to the progression of several neurodegenerative diseases including AD by combining several mechanisms. Numerous investigations have shown that oral administration of aluminum to rodents recapitulates several pathologic mechanisms involved in AD including cognitive decline. The neurodegenerative effects involve mitochondrial dysfunction, oxidative stress, neuroinflammation and apoptosis (Drago et al., 2008; Sethi et al., 2008; Mesole et al., 2020). Apoptosis is a type of programmed cell death mediated by diverse molecules such as p21, p38, mitogen-activated protein kinase (MAPK), p53, caspases 2, 3, 8 and 9, BCL-XS and Bax apoptosis-inducing factors, Par-4 (prostate apoptosis response 4), while Bcl-XL and Bcl-2B (cell lymphoma/leukemia-2 protein) inhibit apoptosis by different mechanisms. Apoptosis is essential for normal homeostasis and elimination of dysfunctional cells, then the balance between the antagonistic effects of the proapoptotic and antiapoptotic members has been involved in the maintenance of the cell integrity. Particularly, mitochondria are cytoplasmic organelles that regulate both metabolic and intrinsic apoptotic signaling pathways; mitochondrial failure is associated with early events in the pathogenesis of aging-related neurodegenerative disorders (Leuner et al., 2007; Obulesu and Lakshmi, 2014). On the other side, neuroinflammation and TNF-α release, activate extrinsic apoptotic signaling pathways leading to neuronal cell loss and cognitive impairment (Elmore, 2007; Cantarella et al., 2015; Burgaletto et al., 2020).

Thus, the present study was designed to explore the impact of JM-20 on aluminum-induced memory impairment in rats. We hypothesized that treatment with JM-20 is capable of rescuing mitochondria from aluminum-induced neurotoxicity, preventing apoptotic signals that lead to neuronal death and thus preventing the impairment of multiple types of memory in this model.