Alternative Targets To Fight Alzheimer's Disease: Focus On Astrocytes Part 4

Recent studies identified the Janus kinase 2-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway as a key pathway for the induction and maintenance ofastrocyte reactivity. 

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Using adenoviral delivery techniques, authors either downregulated orupregulated the JAK2-STAT3 pathway specifically in hippocampal astrocytes. They foundthat the JAK2-STAT3 pathway is necessary and sufficient to trigger astrocyte reactivity inthe hippocampus of transgenic APP mice, controlling also for gene expression of a varietyof genes, of which many involve the inflammatory process. 

The downregulation of thispathway reduced also A deposits and improved mice's spatial learning but not memoryretrieval. On the other hand, the upregulation of the JAK2-STAT3 pathway resulted inopposite and deleterious results [207]. 

Astrocytes are involved in both the production and clearance of ROS, concurring withthe oxidative stress found in AD, whose reduction has been tested as a potential therapeutictarget. Interestingly, mobile phone radiofrequency electromagnetic fields (EMF) have beenshown to reduce both A and H2O2-induced ROS production in human and rat primaryastrocytes, as well as the co-localization between the cytosolic (p47-phox) and membrane (gp91-phox) subunits of NADPH oxidase, indicating the suppression of its activity [212]. 

Other antioxidant anthocyanin compounds have recently been investigated [277]. Amongthem, pelargonidin, which acts as an estrogen receptor agonist, has been tested in ratsthat received an intrahippocampal injection of A (25–35). 

Pelargonidin treatment resultedin improved Morris water maze test performance. Higher hippocampal catalase andacetylcholinesterase activities have been detected, accompanied by lower GFAP proteinexpression, but no change in inducible nitric oxide synthase (iNOS), compared to controlanimals [213].Recently, the compound monascin has been found to activate the expression of several antioxidative genes such as SOD-1, SOD-2, SOD-3, and HSP16.2 and reduce A toxicity in C. elegans strain [214], suggesting its antioxidant potential. 

In addition, resveratrol [278], tocotrienol [279], epicatechins [280], H-1,2-dithiole-3-thione [281], curcumin, andepigallocatechin-3-gallate [282] have shown in vitro and in vivo anti/oxidant properties inseveral models of A -mediated toxicity and AD.As fundamental regulators of brain homeostasis, astrocytes also regulate the intracellular Ca2+ concentration through an intermediate conductance calcium-activated potassiumchannel, KCa3.1. 

This channel is actively involved in the phenotypic change of astrocytesduring astrogliosis observed in AD. By using KCa3.1 knockout mice, memory deficits,neuronal loss, glial activation, tau phosphorylation, and insulin signaling deficits wereameliorated compared with control animals, making this channel an interesting pharmacological target in AD [215]. 

During the neuroinflammatory process, ATP and ADPare released around plaques, leading to the activation of the metabotropic P2Y1 purinoreceptors (P2Y1Rs) expressed by astrocytes, which increases the rate of spontaneouscalcium events [283]. 

Chronic intracerebroventricular infusion of P2Y1R inhibitors resultedin structural and functional restoration of astrocytes and the preservation of memorydeficits [216].Since AD patients show increased levels of the Gs-coupled adenosine receptor A2A inastrocytes, Orr et al. studied in vivo the ablation of astrocytic A2A receptors demonstrating that it enhances long-term memory [284]. 

The adenosine tone on the astrocytic A2Areceptors has also been modulated through a new BBB-permeable equilibrative nucleosidetransporter (ENT) inhibitor, J4, tested in APP/PS1 mice. 

In particular, J4 inhibited therecycling of adenosine from the extracellular space performed by ENTs, resulting in theprevention of the decline in spatial memory, a common feature in AD patients [217]. Additionally, istradefylline, a selective antagonist of A2A receptors, enhanced the performancein behavioral tests in transgenic APP mice [218].

3.7. Modulation of Astrocytes According to Their Morphofunctional State: The Caseof Palmitoylethanolamide

All these rearrangements could lead to either a gain or loss of one or more functions [126].Thus, pathological changes in astrocytes should not just refer to hypertrophy. Indeed,also morphological atrophy could contribute to AD early synaptic failures and cognitivedeficits [126,285]. For these reasons, molecules able to modulate astrocyte morphologyand functions according to their reactive or atrophic status could be potentially valuabletherapeutics. 

To the best of our knowledge, the only molecule that has so far shown someindications to exert such effects is palmitoylethanolamide (PEA).PEA is a naturally occurring amide of ethanolamide and palmitic acid, firstly isolatedfrom soy lecithin, egg yolk, and peanut meal. It acts as a lipid messenger that mimicsseveral endocannabinoid-driven actions, even though it does not bind to cannabinoidreceptors [286]. 

We and other groups have shown that PEA exerts anti-inflammatoryand neuroprotective properties in several preclinical models of A -induced toxicity andAD [287]. PEA in vitro attenuates A -induced astrocyte expression of GFAP and S100Band the release of pro-inflammatory molecules [273,288]. In a surgical model of A neurotoxicity PEA treatment reduced astrocyte hypertrophy and markers of inflammation, including iNOS, cyclooxygenase (COX)-2, IL-1 , and TNF- [289]. 

PEA also demonstratedthe ability to protect A -induced neuronal reduced viability and loss in vitro, ex vivo,and in vivo [208,209,286,289,290]. These results have been confirmed also in primaryastrocytes derived from the prefrontal cortex of 3xTg-AD mice, in which PEA promotedneuronal viability [210]. All these reports concurred to demonstrate that PEA exertedthese effects through the PPAR by the use of selective antagonists, corroborated byexperiments in models where the receptor was genetically ablated [291–293]. 

However,studies showed that PEA effects could involve also the orphan G-protein coupled receptor55 [294], and the transient receptor potential vanilloid type 1 channel [295]. 

Moreover, PEAcan exert an indirect activation of cannabinoid receptors, via the so-called entourageeffect [296], working as a false substrate for fatty acid amide hydrolase, an enzyme involvedin the metabolism of the endocannabinoid anandamide (AEA) [297]. Indeed, due to thereduction of its catabolism, AEA levels rise. Thus, in turn, AEA could bind to cannabinoidreceptors. 

One additional peculiar feature of PEA is its ability to act as an autacoid localinjury antagonist, thus dampening mast cells that are now considered critical effectorsduring AD progression [298]. 

In this way, PEA contributes to protecting neurons fromexcitotoxicity [297]. Interestingly, the modulation of the cross-talk between mast cellsand glial cells is emerging as a valuable approach to treating several neuroinflammatorybrain pathologies, including AD [299]. Some articles present an extensive review of PEAbiological functions in the CNS [296,297,300]. 

Different formulations of PEA have been synthesized to improve its bioavailabilityand efficacy, including the ultramicronized (um-PEA) and PEA-oxazoline forms as well asthe combination of PEA with luteolin (Lut), an antioxidant compound, ultramicronized together (co-ultra PEA/Lut). Pretreatment with um-PEA of rat hippocampal slices challengedacutely with A 42 significantly reduced iNOS and GFAP expression [301]. 

It also restoredcell viability of glioma and neuroblastoma impaired by lipopolysaccharide and interferon-gamma treatment, reducing protein expression of both iNOS and COX-2 [211]. Um-PEAdemonstrated oral bioavailability and its chronic administration reduced neuroinflammatory markers and showed neuroprotective effects in 3xTg-AD mice [210,219,302,303].

Whencomparing the hippocampi of 6-month-old with 12-month-old 3xTg-AD mice, the youngeranimals did not show astrocyte hypertrophy (measured as an increase in GFAP immunoreactivity) but exhibited an ongoing intense neuroinflammatory process with high levelsof iNOS, TNF- , chemokines, and interleukins, whereas older mice showed significantastrocyte atrophy without elevation in neuroinflammatory markers. 

Chronic subcutaneouspretreatment with um-PEA for 3 months prevented the establishment of the phlogisticprocess in the hippocampi of 6-month-old 3xTg-AD mice, compared to vehicle-treated ones.Um-PEA also prevented the altered performance in cognitive tasks and reduced A formation and phosphorylation of tau protein in the hippocampus [219]. 

Astrocyte hypertrophywas detected in the cortices of vehicle-treated 6-month-old mice, and um-PEA chronictreatment decreased both GFAP mRNA and protein expression [210]. Interestingly, 3xTgAD mice that received um-PEA subcutaneous administration for 3 months, before beingtested at 12 months of age, showed restored astrocyte GFAP immunoreactivity to the levelof non-Tg controls, also improving their outcome in behavioral assessment of short-termmemory [219]. 

Collectively these reports show that um-PEA acted to prevent either astrocyte hypertrophy or atrophy. This indicates that PEA behaved as a modulator ofastrocyte morphology and cell reactivity state. This is by the currentview seeing astrocyte reactivity as an evolving and reversible process caused by extrinsictriggers [126,304]. 

Another formulation that combines the aforementioned PEA effects with the antioxidant actions of Lut has been tested in preclinical AD models. Co-ultra PEA/Lut showedanti-inflammatory and antiapoptotic effects in A 42-challenged rat hippocampal slicesand neuroblastoma cells [301]. 

In vivo, co-ultra PEA/Lut administration for two weeks inrats that received a single intrahippocampal infusion of A 42 prevented the A -inducedastrocyte hypertrophy, as well as the upregulation in gene expression of pro-inflammatory cytokines and enzymes found in rats treated with vehicle. Moreover, co-ultra PEA/Lutprevented the A -mediated decrease in gene expression of both glial-derived and brain-derived neurotrophins [35]. 

Despite having these promising features, no studies haveyet elucidated the synergic mechanisms of actions of the association of PEA with Lut.Regardless, since co-ultra PEA/Lut administration started the same day of the surgicalinfusion, to model the very first phase of A 42 accumulation as in the prodromal stage ofAD, the above-reported study mimicked a potential therapeutic intervention at the earlieststage of the disease. The results support the thesis that targeting astrocytes at the beginningof the pathology could have a positive impact. 

Other very recent studies endorse this view.Reports from Dr. Escartin's group modulated the activation of astrocytes in 9-month-old3xTg-AD mice. The downregulation of the JAK2-STAT3 pathway fully restored mice earlysynaptic and long-term potentiation alterations [207], improved short-term memory, andreduced anxiety behavior [176], thus supporting the hypothesis that targeting astrocytes atthe very early stages of AD could be beneficial. 

The potential translational value of ultramicronized or co-micronized PEA as a preventive therapeutic strategy in AD is corroborated by its safety and tolerability, as it isalready in the human and veterinary market as food for special medical purposes and complementary feed, respectively. 

Some single or few-cases human studies have been carriedout showing favorable results in improving MCI and frontotemporal dementia [305,306],in recovering from stroke [307], and in managing neuropathic pain associated with neuroinflammation [308].

4. Conclusions

Despite the spasmodic basic and medical research and the existence of approvedtherapies, there is a huge unmet clinical need for effective therapies for AD, especiallytreatments that are intended to address the biological basis of the pathology to favorablymodify its long-term course. Currently approved drugs do not target the underlyingpathology of AD since they only provide modest beneficial effects to a small subset ofpatients. 

Our previous study indeed showed the ability of PEA to normalize the astrocyte alterations observed in an experimental model of AD, the 3xTg-AD mice, endowed with face,construct, and predictive validities, bringing them back to a homeostatic condition. Thatand other possibilities of new therapeutic approaches represent an important springboardfor the development of therapies for a still incurable disease, such as AD.

Author Contributions: Conceptualization, C.S.; resources, C.S. and L.S.; writing-original draftpreparation, R.F., M.V., and C.S.; figures and tables preparation, G.M., R.F., and M.V.; writing-reviewand editing, M.V., L.S., and C.S. All authors have read and agreed to the published version ofthe manuscript.

Conflicts of Interest: Marta Valenza discloses a previous work-term contract (2019–2020) withEpitech Group S.p.A., which was already closed at the time of preparation of the present reviewarticle. The other authors declare no conflict of interest.

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