Titolo della tesi: Neuroprotective Effects of Exogenous Manipulations in the Tg2576 mice, a Murine Model of Alzheimer's Disease: Novel Approaches for the Prevention and Treatment of Neurodegenerative Diseases.
Alzheimer's disease (AD), the most common cause of dementia (A. Kumar et al., 2024), is a progressive neurodegenerative disorder affecting molecular, cellular, and cognitive functions (Knopman et al., 2021). In addition to cognitive decline, AD is often accompanied by neuropsychiatric symptoms (NPSs), such as apathy, depression, agitation, and sleep disturbances (Kales et al., 2015; Theleritis et al., 2014), which worsen quality of life (Kales et al., 2014).
The pathophysiology primarily entails the accumulation of tau protein in the brain, particularly in the medial temporal lobe and neocortical structures, resulting in intracellular neurofibrillary tangles, as well as the aggregation of amyloid-β (Aβ) peptide, leading to the extracellular amyloid plaques formation (Knopman et al., 2021). Aβ and tau accumulation in the brain are crucial agents implicated in the neurodegeneration, the cognitive decline, and the NPSs typical of AD patients (Dang et al., 2023; Gonzalez-Ortiz et al., 2024; Ossenkoppele et al., 2022; Villemagne et al., 2013).
Neuroinflammation refers to an inflammatory response within the central nervous system (CNS) triggered by factors such as infection, trauma, or toxins. This response involves the release of pro-inflammatory cytokines (e.g., interleukin (IL)-1β, IL-6, TNF), chemokines, and signaling molecules (e.g., nitric oxide (NO), reactive oxygen species (ROS)) by CNS immune cells, primarily microglia and astrocytes (DiSabato et al., 2016). In AD, microglia show diverse activation patterns and are found near amyloid plaques, suggesting a link between microglial activity and amyloid pathology (Gao et al., 2023; Miao et al., 2023). The amyloid cascade-inflammation hypothesis proposes that microglial activation contributes to tau pathology (J. P. Hayes et al., 2012; Kitazawa et al., 2004; McGeer & McGeer, 2013). Pro-inflammatory mediators disrupt synaptic function, induce neuronal death, and inhibit neurogenesis (Lyman et al., 2014). For example, IL-1β promotes synaptic loss by increasing N-methyl D-aspartate (NMDA) receptor activation (Mishra et al., 2012). Furthermore, tumor necrosis factor (TNF) triggers neuronal apoptosis by activating TNF receptor 1 (TNFR1) and recruiting caspase 8 when the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway is impaired (Tummers & Green, 2017). Various Aβ aggregates can induce glial activation and the release of pro-inflammatory cytokines, anti-inflammatory cytokines, chemokines, NO, and ROS, all contributing to neuronal dysfunction and death (S. Kumar et al., 2023; Tummers & Green, 2017). These processes activate molecular pathways that alter microglial phenotypes (Alawieyah Syed Mortadza et al., 2018; Husemann et al., 2001; S. Liu et al., 2012; Venegas & Heneka, 2017). Cytokines such as IL-1β and TNF, produced by microglia, may enhance β-secretase activity, promoting Aβ production through the NF-κB pathway (C.-H. Chen et al., 2012; Hawcroft et al., 2003). Aβ also activates NF-κB in astrocytes, increasing complement C3 release, which disrupts neurons and activates microglia (Lian et al., 2015, 2016). Additionally, activated microglia can induce neurotoxic astrocytes via IL-1α and TNF, forming a self-amplifying inflammatory feedback loop (Liddelow et al., 2017). Dysregulated neuron-glia interactions further exacerbate this inflammatory response in AD.
Currently, there is no definitive cure for AD, and pharmacological treatments mainly target symptom relief. Various antibodies have been tested in preclinical studies with transgenic AD-like mice (Corsetti et al., 2020; Latina, Giacovazzo, Cordella, et al., 2021; Wilcock et al., 2004), sporadic AD models (Latina, Giacovazzo, Cordella, et al., 2021), and immunotherapy trials (Yadollahikhales & Rojas, 2023). Recently, aducanumab and lecanemab were approved by the U.S. Food and Drug Administration (FDA). While there are safety concerns and mixed clinical results (Iwatsubo, 2023; Yadollahikhales & Rojas, 2023), these third-generation anti-amyloid therapies show high amyloid clearance rates and represent a major advancement in AD treatment. They offer a better risk-to-benefit ratio compared to existing drugs, though clinical trials remain ongoing.
Developing strategies to improve symptoms, slow disease progression, or prevent the conversion from MCI to AD is crucial. This has driven research toward non-pharmacological approaches that complement therapies. Unhealthy lifestyles are risk factors for neurodegenerative diseases like AD (Calder et al., 2017; Madore et al., 2020). Nutrition, in particular, has gained attention, with studies showing that dietary changes and supplements can positively impact emotional and cognitive functions by modulating brain activity in aging and neurodegeneration (Fekete et al., 2022; Kalache et al., 2019; X. Li et al., 2023; Scales et al., 2018).
Palmitoylethanolamide (PEA) is an endogenous fatty acid amide and lipid modulator associated with the endocannabinoid system and found in foods (D’Agostino et al., 2012). It is synthesized in the brain by neurons, microglia, and astrocytes in response to stress, contributing to homeostasis. PEA has notable anti-inflammatory, neuroprotective, and analgesic effects, primarily mediated by PPAR activation. In vitro studies demonstrate that PEA reduces amyloid-beta (Aβ1-42)-induced neuroinflammation and neurodegeneration (Scuderi et al., 2011, 2012; Scuderi & Steardo, 2013). In vivo, PEA administration in rats with Aβ1-42-induced neuroinflammation exhibits significant anti-inflammatory and neuroprotective effects through peroxisome proliferator-activated receptor (PPAR)-α activation (Scuderi et al., 2014).
The Tg2576 mouse is a widely used AD model that overexpresses a mutant form of amyloid precursor protein (APP) with the Swedish mutation, leading to increased Aβ levels and plaque formation (Hsiao et al., 1996). These mice show cognitive deficits starting at around 5 months, with Aβ accumulation observed by 8-9 months and amyloid plaques by 12 months (Jacobsen et al., 2006; Taglialatela et al., 2009). Neuroinflammation markers, such as glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (Iba1), increase significantly by 9-12 months, alongside elevated IL-1β levels (Evans et al., 2020; J.-H. Lee et al., 2022). Oxidative stress is also noted at 12 months, with heightened levels of 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 in the hippocampus and cortex (P. Jin et al., 2013; B. Zhang et al., 2005).
In the first study, we explored the therapeutic potential of chronic PEA administration in the Tg2576 mouse model of AD. Our results indicate that chronic PEA administration via subcutaneous pellets offers several neuroprotective benefits, including reducing nitrosative stress, alleviating neuroinflammation, modulating microglial activity, preserving synaptic function, and restoring cognitive performance. These findings underscore the promising therapeutic action of chronic PEA treatment for AD patients, warranting further investigation into its mechanisms of action and suggesting the potential for developing nutraceutical-based therapies leveraging PEA's anti-inflammatory and neuroprotective properties.
Non-invasive brain stimulation (NIBS) is emerging as a promising non-pharmacological intervention for preventing the progression from MCI to AD and reducing symptoms in early-stage patients. NIBS seeks to restore brain frequency patterns to align more closely with those of healthy individuals. In both MCI and early AD patients, as well as AD animal models, there are reports of excitation/inhibition balance disruption, neuronal hyperexcitability and altered brain oscillations, including decreases in alpha, beta, and gamma frequency bands and increases in the theta band (Bhattacharya et al., 2011; Busche et al., 2008; Koenig et al., 2005; Noebels, 2011; Palop et al., 2007).
Gamma oscillations are crucial for coordinating neuronal activity involved in complex cognitive functions and are disrupted in MCI and AD, contributing to cognitive deficits (Koenig et al., 2005; Missonnier et al., 2010; J. Y. Park et al., 2012). Both patient groups show reduced gamma activity, which is also observed in AD mouse models (Guillon et al., 2017; Iaccarino et al., 2016). These oscillations rely on fast-spiking parvalbumin interneurons (PV-INs) and are essential for higher-order cognitive functions (Bartos et al., 2007; Cardin et al., 2009).
PV-INs are crucial for network synchrony and play an essential role in learning and memory. Their removal in the hippocampal Cornu Ammonis (CA)1 region impairs spatial working memory (Murray et al., 2011). Additionally, disruptions to excitatory inputs onto PV-INs negatively impact learning and memory, along with associated network changes (Y.-J. Chen et al., 2010; Fuchs et al., 2007; He et al., 2021).
Further research is needed to investigate the relationship between PV-INs and GABAergic activity in AD and how restoring PV-INs function could mitigate AD progression. Notably, optogenetic stimulation of 40 Hz gamma activity in PV-INs of 5XFAD mice enhanced neuroprotection, preserved brain structure, reduced Aβ, and improved cognitive performance (Iaccarino et al., 2016).
Studies on 40 Hz flicker stimulation revealed it increases cytokines that promote microglial phagocytosis and activates the NF-κB and MAPK pathways, enhancing cytokine expression within an hour (Garza et al., 2020). Additionally, this stimulation alters microglial morphology and increases cytokine production in healthy mice, suggesting that brain rhythms influence function through NF-κB signaling (Prichard et al., 2023). Furthermore, multisensory gamma stimulation at 40 Hz enhances Aβ clearance by promoting cerebrospinal fluid flow and increasing aquaporin-4 polarization in 5XFAD mice (Murdock et al., 2024).
These findings indicate that restoring gamma oscillations through 40 Hz brain stimulation may effectively improve AD pathology by reducing inflammation, delaying neurodegeneration, and enhancing cognition in transgenic mice. Consequently, noninvasive 40 Hz stimulation has emerged as a promising therapeutic approach for addressing gamma oscillopathy and alleviating cognitive deficits associated with AD (Chan et al., 2021; Manippa et al., 2022; McDermott et al., 2018; Thomson, 2018).
In the second study, we explored the therapeutic effects of hippocampal optogenetic 40 Hz gamma stimulation in Tg2576 mice. At 6 months, these mice exhibited specific working memory deficits, indicated by reduced spontaneous alternations in the Y-maze Spontaneous Alternation Test (Y-maze), while recognition memory remained intact. The decreased immobility observed in the tail suspension test (TST) alongside increased locomotor activity in the Y-maze suggests hyperactivity as a notable characteristic at this stage. Although no significant differences in dendritic spine density were found at 12 months, these results underscore the necessity for further investigation, and we plan to increase the sample size for future analyses.