
==== Front
J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02120-3
10.1016/j.jbc.2024.107619
107619
Research Article
Proactive M2 blockade prevents cognitive decline in GRK5-deficient APP transgenic mice via enhancing cholinergic neuronal resilience
Zhang Qiang 1
Singh Prabhakar 1
Peng David W. 1
Peng Evelyn Y. 1
Burns Jeffery M. 234
Swerdlow Russell H. 234
Suo William Z. William.Suo@va.gov
1234∗
1 Laboratory for Alzheimer’s Disease and Aging Research, Kansas City Veterans Affairs Medical Center, Kansas City, Missouri, USA
2 Department of Neurology, University of Kansas Medical College, Kansas City, Kansas, USA
3 Department of Physiology, University of Kansas Medical College, Kansas City, Kansas, USA
4 The University of Kansas Alzheimer’s Disease Center, Kansas City, Kansas, USA
∗ For correspondence: William Z. Suo William.Suo@va.gov
02 8 2024
9 2024
02 8 2024
300 9 1076195 2 2024
9 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Alzheimer's disease (AD) poses an immense challenge in healthcare, lacking effective therapies. This study investigates the potential of anthranilamide derivative (AAD23), a selective M2 receptor antagonist, in proactively preventing cognitive impairments and cholinergic neuronal degeneration in G protein-coupled receptor kinase-5–deficient Swedish APP (GAP) mice. GAP mice manifest cognitive deficits by 7 months and develop senile plaques by 9 months. A 6-month AAD23 treatment was initiated at 5 months and stopped at 11 months before behavioral assessments without the treatment. AAD23-treated mice exhibited preserved cognitive abilities and improved cholinergic axonal health in the nucleus basalis of Meynert akin to wildtype mice. Conversely, vehicle-treated GAP mice displayed memory deficits and pronounced cholinergic axonal swellings in the nucleus basalis of Meynert. Notably, AAD23 treatment did not alter senile plaques and microgliosis. These findings highlight AAD23's efficacy in forestalling AD-related cognitive decline in G protein-coupled receptor kinase-5–deficient subjects, attributing its success to restoring cholinergic neuronal integrity and resilience, enhancing resistance against diverse degenerative insults.

Keywords

Alzheimer's disease
prevention
GRK5 deficiency
M2 receptor
antagonist
cognitive decline
neuronal resilience
amyloid burden
brain inflammation
Abbreviations

AAD23 anthranilamide derivative

Aß β-amyloid

Ach acetylcholine

AD Alzheimer's disease

BF basal forebrain

CAS cholinergic axonal swellings

ChEI cholinesterase inhibitor

GAP G protein-coupled receptor kinase-5–deficient Swedish APP

GRK G protein–coupled receptor kinase

GRK5 G protein-coupled receptor kinase-5

GPCR G protein–coupled receptor

IR immunoreactivity

KO knockout

MCI mild cognitive impairment

NBM nucleus basalis of Meynert

NOI novel object introduction

SP senile plaques

ROI region of interest

WT wildtype

Reviewed by members of the JBC Editorial Board. Edited by Elizabeth J. Coulson
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pmcAlzheimer's disease (AD) remains the predominant cause of dementia, accounting for 60 to 80% of dementia cases (1). It is distinguished by an irreversible decline in cognitive abilities due to neurodegenerative changes. Once established, there are currently no known methods for reversing or curing this disease (2). Consequently, our most effective strategy lies in proactively combating AD by focusing on prevention (3).

The clinical presentation of AD varies widely, reflecting substantial heterogeneity in its pathogenesis and etiology (4, 5). These complexities have led to multiple hypotheses about the origins of AD, some of which even conflict with each other. Nevertheless, a prevailing consensus highlights selective neurodegeneration as the fundamental anatomical basis of the disease (6, 7). Therefore, strategies to prevent AD must revolve around averting this specific neurodegeneration to safeguard cognitive function.

When it comes to a selective neurodegeneration in AD, it is inevitable to notice the cholinergic hypothesis in AD (8), one of the major hypotheses driven the pharmaceutical research of AD in the last half century (9), yet facing controversial evidence and consensus. On one hand, pathological or neurochemical studies from limited individual postmortem brain samples failed to find evidence of basal forebrain (BF) cholinergic neurodegeneration in early AD or mild cognitive impairment (MCI) (10, 11, 12). The suspicion of the cholinergic hypothesis was echoed by the limited clinical efficacy of cholinesterase inhibitors (ChEIs) in disease-modifying effect of AD except for the limited symptom management efficacy (13, 14, 15). On the other hand, studies using high-resolution structural magnetic resonance imaging in hundreds of live patients discovered significant BF atrophy in MCI and early AD (16, 17, 18, 19, 20, 21). Moreover, a clinical trial on 332 prodromal AD patients found that Donepezil reduced the BF atrophy (22). In this regard, Ballinger et al. in the journal of Neuron provided a valuable summary (23) and before reaching any consensus, more investigations in the relevant areas are necessary.

As for a selective impact on the cholinergic system, G protein-coupled receptor kinase-5 (GRK5) deficiency emerges. GRK5 belongs to a small family of protein kinases known as G protein–coupled receptor kinases (GRKs). The primary role of GRKs is to "uncouple" G proteins from their coupled G protein–coupled receptors (GPCRs) up on their activation (24). This GPCR regulatory mechanism for turning off the signaling (25) was novel and highly impactful that earned the Nobel Prize in Chemistry in 2012 for Dr Robert Lefkowitz (26). The regulation of GPCRs by GRKs exhibits both selectivity and redundancy (27). This redundancy allows GRKs to compensate for each other's loss or deficiency without significantly impacting the overall efficiency of timely attenuation of GPCR signaling. Meanwhile, studies in individual GRK knockout (KO) mice have unequivocally shown selective regulation by GRKs in vivo. For example, mice deficient in GRK2, 3, 5, and 6 display selectively impaired desensitization of adrenergic, odorant, muscarinic, and dopaminergic receptors, respectively (28, 29, 30, 31). This is where the selective impact on the cholinergic system comes up with the GRK5 KO or deficiency. Moreover, the selectivity of GRK5 deficiency on the cholinergic system was found to only affect muscarinic receptors, particularly the Gi/o-coupled M2 and M4 receptors, without impacting M1/M3/M5 or nonmuscarinic receptors (27, 28, 32, 33). In fact, the relevance of GRK5 to AD research became evident when rapid loss of functional/membrane GRK5 due to β-amyloid (Aß) exposure was identified (34). Since then, GRK5 deficiency has been observed in the brains of an AD transgenic model and human autopsy samples (25, 34). Pathogenically, GRK5 deficiency alone in aged GRK5KO mice was found to cause amnestic MCI-associated limbic axonopathy (35). When co-existing with overexpression of Swedish mutant Aβ precursor protein in GRK5-deficient APP (GAP) mice, GRK5 deficiency was found to exaggerate Aβ pathology, inflammation, and more importantly the cholinergic selective neurodegeneration (36, 37, 38).

GRK5 deficiency in vivo selectively impairs the "decoupling" of muscarinic type 2 receptors from the associated inhibitory G protein (Gi) (27, 28, 32, 33). This results in prolonged M2 signaling or "presynaptic M2 hyperactivity," leading to prolonged suppression of cAMP/PKA/CREB signaling. The delicate balance between prosurvival and proapoptotic members of the BCL-2 family determines cell fate, including that of neurons (39, 40, 41). The cAMP/PKA/CREB signaling pathway is a prosurvival pathway that enhances neuronal resilience to environmental insults (42, 43). The prolonged suppression of cAMP/PKA/CREB prosurvival signaling consequently led the cholinergic neurons to become selectively susceptible due to a shift from prosurvival to proapoptotic signaling. After all, a failed M2 muscarinic receptor desensitization underlies most, if not all, pathologic impact of GRK5 deficiency (32), which led to our hypothesis to prevent MCI and AD by using M2 antagonist to block the "presynaptic M2 hyperactivity" in the GRK5-deficient subjects (32, 33, 38, 44).

Neurodegeneration can arise from diminished neuronal resilience, heightened neurodegenerative insults, or a combination of both factors (6, 7, 45). Accordingly, prevention strategies can adopt two primary approaches: fortifying neuronal resilience, reducing, or eliminating degenerative insults or employing a combination of both strategies (33, 38). Our prior hypothesis suggests that strengthening neuronal resilience is crucial in elevating the threshold at which neurons undergo apoptosis. This approach may protect against a wide range of degenerative insults, without requiring precise knowledge of the specific insults involved.

The therapeutic strategy of antagonizing M2 hyperactivity specifically targets the condition of GRK5 deficiency. The present study represents the first in vivo test of this hypothesis in GAP mice, utilizing a selective M2 antagonist known as an anthranilamide derivative (AAD23). The results not only validated the hypothesis but also provided preclinical evidence for the first pharmaceutical treatment that may prevent AD.

Results

AAD23 increases brain acetylcholine release and rectifies cholinergic hypofunction caused by GRK5 deficiency

AAD23 dosing in vivo was performed in 3-month-old C57Bl6 wildtype (WT) mice via oral gavage feeding. Acetylcholine (ACh) release measured in brain microdialysate was used as the readout given that is a known effect of M2 antagonists in vivo (46). The experiment revealed that AAD23 began to significantly increase the hippocampal ACh level at a dose as low as 0.5 mg/kg and led to increased ACh release in a dose-dependent manner (Fig. 1A). This response plateaued at doses of 2.5 and 5.0 mg/kg, indicating a maximal effective dosage of AAD23 at 2.5 mg/kg.Figure 1 Effects of AAD23 in GRK5-deficient mice. Changes in the hippocampal ACh release of 3-month-old WT and GRK5KO mice were analyzed (n = 6) to evaluate AAD23’s in vivo effects. A, dose-finding experiment. AAD23 was orally administered via gavage at indicated doses to WT mice. Microdialysates were automatically collected every 20 min using Culex Automated Sampling Systems for ACh measurements. Two baseline samples were taken. The time began to count immediately after the treatment was given (0 min). The dose finding study revealed an Emax of AAD23 at 2.5 mg/kg in vivo. Statistical analysis revealed the dose finding study had an observed power of 1.0 with F = 15.827; p = 0.000; post hoc comparisons of AAD23 doses with untreated (Scheffe) showed p = 0.000 for 1.0, 2.5, and 5.0 mg/kg except for 0.5 mg/kg with a p = 0.896. Post hoc comparisons of each dose at individual time point were also performed (Bonferroni) that revealed significant differences at the earliest time point at 100 min for 0.5 mg/kg, 80 min for 1.0 mg/kg, and 40 min for both 2.5 and 5.0 mg/kg, respectively. ∗p < 0.05, ∗∗p < 0.01 as compared to the untreated at the corresponding time point. B, effect of AAD23 on cholinergic hypofunction in GRK5KO mice. The total ACh release was undistinguishable in any of the groups between −20 and 40 min. After 40 min, however, the ACh levels in the untreated KO mice quickly declined and became significantly lower than those in the WT mice (#p < 0.05 compared to WT). This cholinergic hypofunction was not observed in the AAD23-treated KO mice, and both WT and KO in the presence of AAD23 had significantly higher peaks in the latter phase of the recording up to 100 min. In fact, the AAD23-treated WT and KO mice were undistinguishable, and they both were significantly higher than the untreated KO mice (∗∗p < 0.01). Two-way ANOVA revealed significant genotype (p < 0.05) and AAD23 (p < 0.01) effects as well as significant (p < 0.05) interactions between the genotype and AAD23 treatment. The time point 0 indicates when the treatments (NOI and AAD23) were given. AAD23, anthranilamide derivative; Ach, acetylcholine; KO, knockout; NOI, novel object introduction; WT, wildtype.

Potential impact of AAD23 on cholinergic hypofunction was assessed in GRK5KO mice. As used by others (46, 47), novel object introduction (NOI) was used to trigger the hippocampal ACh release in both WT and KO mice in the absence and presence of 2.5 mg/kg of AAD23 (Fig. 1B). Without AAD23, baseline ACh levels at −20 and 0 min did not differ notably between WT and KO mice. Following the NOI stimulation, initial ACh levels were similar across genotypes. However, in KO mice, ACh levels sharply dropped after 40 min, significantly falling below WT levels at 60 to 100 min (∗p < 0.05 compared to WT). With 2.5 mg/kg AAD23, the overall ACh release did not notably differ before 40 min in any group, but AAD23-treated WT and KO groups showed significantly higher peaks from 40 min until the end of the 100-min recording. This observation aligns with AAD23's effect in boosting ACh release. Notably, while ACh levels in untreated KO mice were much lower than untreated WT mice, this substantial difference vanished in AAD23-treated mice, indicating AAD23 at the given dose effectively corrected the cholinergic hypofunction caused by GRK5 deficiency.

Proactive use of AAD23 prevented cognitive decline in GAP mice

To ensure the 12-month-old GAP mice were indeed cognitive impaired without any other significant issues that could compromise the prevention trial, we behaviorally assessed a group of 12-month-old female GAP mice using a previously established behavioral battery that measures the sensorimotor and anxiogenic tendencies as well as the cognitive changes (48). As shown in Figure 2, while the 12-month-old GAP mice were normal in the sensorimotor and anxiogenic tasks, they were cognitively impaired in Y maze and MWM tasks. The 12-month-old female GAP mice displayed significantly impaired cognition with matured AD-like pathology and were therefore suitable for using as the termination time point for analyzing the AAD23’s efficacies.Figure 2 Cognitive impairments in GAP mice at 12-months of age. 12-month-old female WT (n = 10) and GAP mice (n = 18) were behaviorally assessed using our standard behavioral test battery (Method 2.4) as indicated in the subtitle of each panel (A–H). F–H, univariate ANOVA revealed statistical significances in Y maze and MWM with the F and p values as indicated in panels F–H. The statistical powers in the panels F–H were 0.800, 0.678, and 0.846, respectively. Although the time on the platform (G) showed insufficient power the latency for the same task had a statistical power of 84.6%, which was sufficient to support the conclusion that the 12-month-old female GAP mice were cognitively impaired in both Y maze and MWM tasks while their sensorimotor function and anxiogenic tendencies remained unaffected. GAP, G protein-coupled receptor kinase-5–deficient Swedish APP.

As shown in Figure 3, the vehicle (D8626)-treated GAP mice displayed significant impairments in both Y maze spontaneous alteration test and MWM tasks, whereas the 6-months AAD23 treatment completely prevented the cognitive decline in GAP mice. Moreover, this preventive effect of AAD23 persisted for at least 4 months beyond the onset age of 7-month-old and continued for over an additional month after the treatment withdrawal and before the end of the experiment at 12-month-old age. Therefore, these results suggest that proactive use of AAD23 can prevent cognitive decline in GRK5-deficient subjects.Figure 3 AAD23 prevented the cognitive decline in GAP mice. After 6 months of AAD23 treatment and 12 days of drug withdrawal, the treated (n = 8) and untreated (D8626 n = 22) female GAP mice were subject to our standard behavioral test battery for behavioral assessment that was concluded at 12 months of age. A–H, univariate ANOVA revealed statistical significances in Y maze and MWM with the F and p values as indicated in all the panels. The statistical powers in the panels A–E that revealed a significance (p < 0.05) were 0.956, 0.851, 0.808, 0.858, and 0.619, respectively. While the entry into the goal quadrant (E) had low power, but the platform entries, time, and latency in MWM had all had sufficient power and statistical significance. Therefore, these results suggest that AAD23 completely prevented cognitive impairments in both Y maze spontaneous alteration test and MWM task. AAD23, anthranilamide derivative; GAP, G protein-coupled receptor kinase-5–deficient Swedish APP.

AAD23 mitigated cholinergic axonal swellings but not the brain Aβ deposition nor microgliosis

The pathological analyses in AAD23-treated mice included cholinergic neurodegenerative changes, brain Aβ burden, and inflammation using the same protocols that we established previously. Given no significant loss of total neurons (NeuN+) in GAP mice as we already analyzed before (38), we focused our efforts in this study on stereological analysis of cholinergic (ChAT+)-specific neurons. As shown in Figure 4, we did not find a significant decrease in cholinergic neurons in the nucleus basalis of Meynert (NBM), caudate putamen, and the rest of the nucleus basalis, or in the entire basal forebrains (Fig. 4, E and F). Nevertheless, our stereological evaluations revealed a distinctive cholinergic neurodegenerative change—a notable increase in the number of cholinergic axonal swellings (CAS) within the NBM of 12-month-old GAP mice (Fig. 4, A–D). Importantly, the AAD23 treatment effectively prevented this augmented cholinergic axonal degeneration, as the CAS numbers in the AAD23-treated GAP mice mirrored those in the age-matched WT mice. Therefore, though no significant loss of total neurons was observed, stereological analysis revealed an increased CAS in NBM of untreated GAP mice. Treatment with AAD23 mitigated this neurodegeneration, evidenced by CAS levels comparable to age-matched WT mice.Figure 4 AAD23 prevented BF cholinergic neurodegenerative changes in GAP mice. A–C, representative images of CASs (indicated by arrows) in the NBM from WT, control (D8626), and AAD23-treated GAP mice, respectively. Scale bar = 10 μm. D, stereological quantification of total number of CASs in the NBM of WT, untreated (D8626), and AAD23-treated GAP mice as indicated. Univariate ANOVA revealed statistical significances when the untreated GAP mice were compared to either WT or the AAD23-treated GAP mice with the F and p values indicated in the panel. The observed power was 0.93. E and F, stereological quantification of total number ChAT+ neurons in the NBM and entire BF of WT, untreated, and AAD23-treated GAP mice, respectively. No significance was found in either analysis. AAD23, anthranilamide derivative; BF, basal forebrain; CAS, cholinergic axonal swellings; GAP, G protein-coupled receptor kinase-5–deficient Swedish APP; NBM, nucleus basalis of Meynert;WT, wildtype.

As for the brain Aβ burden and inflammation, we performed immunofluorescent staining with antibodies targeting pan Aβ and CD45 (a microglial marker). The results revealed a notable increase in amyloid plaques in both the hippocampal and cortical regions of 12-month-old GAP mice (Fig. 5). However, upon image analysis of the immunoreactivity (IR), it became evident that the administration of AAD23 had no discernible impact on the extent of either Aβ or CD45+ microgliosis. This result implicates that the efficacy of AAD23 in preventing the cognitive decline in GAP mice was not achieved via mitigating amyloid burdens or inflammation.Figure 5 AAD23 failed to modify the amyloid burdens or inflammation in GAP mice. A–C, representative images of cortical SPs as indicated by anti-Aβ and CD45 IF staining (Red, Aβ; green, CD45; blue, DAPI) in the WT, untreated and AAD23-treated GAP mice, respectively. Scale bar = 50 μm. D and E, quantification of CD45 IR using image analysis in the cortex (parietal) and hippocampus (Hp) of WT, untreated, and AAD23-treated GAP mice as indicated. F and G, quantification of Aβ IR using image analysis in the cortex (Ct) and hippocampus (Hp) of WT, untreated, and AAD23-treated GAP mice as indicated. Univariate ANOVA failed to reveal statistical significance for AAD23 treatment in either Aβ or CD45 as outcome, except for the comparisons between WT and the untreated GAP mice (the F and p values indicated in the panel). Therefore, these results suggest that AAD23 did not significantly impact the brain amyloid burden and inflammation. AAD23, anthranilamide derivative; Aß, β-amyloid; GAP, G protein-coupled receptor kinase-5–deficient Swedish APP; IF, immunofluorescent; IR, immunoreactivity; WT, wildtype.

Discussion

Strengthening neuronal resilience: Multifaceted benefits

The proactive use of the selective M2 antagonist AAD23 in GAP mice showcased a remarkable delay in the onset of cognitive impairments, extending the onset age from 7 to 12 months without affecting Aβ accumulation or microgliosis. Notably, AAD23 did not significantly alter the levels of Aβ or CD45 IR, but it effectively suppressed the progressive increase of CAS in the NBM, preserving cholinergic neurons' integrity. This preservation of neuronal integrity, evident through the reduced CAS numbers, indicates AAD23's potential to halt or slow cholinergic axonal degeneration.

Critically, irrespective of the underlying mechanisms, the key criterion for this prevention trial—functional prevention of cognitive decline—was unequivocally achieved. This demonstrated AAD23's profound in vivo efficacy, providing crucial proof-of-concept for potential clinical use in AD prevention. The gained 5-month period of normal cognition in treated GAP mice (equivalent to ∼15 human years (49)) and the restoration of normal cognitive performance at 12 months imply the potential for further delays if treatment persists. A 15-years or longer delay in AD onset could have a transformative impact on combating AD.

This drug trial aimed to counter the presynaptic M2 hyperactivity triggered by GRK5 deficiency. Mechanistically, AAD23's role in blocking the M2 autoreceptor aimed to halt the backward action of ACh on the cholinergic neuron itself. The brief (seconds) activation of the presynaptic M2 autoreceptor only restricts further ACh release, but its prolonged (hours) activity in the context of GRK5 deficiency hampers cAMP/PKA/CREB prosurvival signaling in the cholinergic neuron (32, 33, 38, 44). This signaling alteration not only hinders axonal transportation (50), leading to more axonal swellings, but also diminishes the cholinergic neuron's resilience against various degenerative insults (39, 40, 41, 42, 43). This molecular mechanism elucidates how GRK5 deficiency makes specific cholinergic neurons vulnerable to degeneration (33, 38, 48). While the blockade of M2R does not alter GRK5 deficiency, it can impede presynaptic M2 hyperactivity, safeguard cholinergic axonal health, and bolster neuronal resilience, thus staving off cognitive decline. The study's findings confirmed the anticipated impact on cholinergic axonal swellings. However, because the chosen timepoint was early to detect notable cholinergic neuronal loss in untreated GAP mice, further investigation is required to understand its effects on cholinergic neuronal loss.

In considering AAD23's potential impact on the β-amyloidogenic pathway, previous studies have highlighted that heightened cholinergic signaling can impede Aβ accumulation (32, 36, 51, 52). While the primary objective of using AAD23 to block M2R was to safeguard the upstream cholinergic neurons, it was expected to enhance ACh release, potentially inhibiting Aβ production and accumulation. Surprisingly, our study did not reveal significant effects on brain Aβ or CD45 IR. Nevertheless, intriguingly, despite the notable accumulation of Aβ and CD45 IR in the AAD23-treated GAP mice, there was no observed cognitive decline, unlike the untreated GAP mice. This observation supports the notion that bolstering neuronal resilience and elevating the neuronal apoptotic threshold might shield neurons from various insults, including Aβ and inflammation. Additionally, our unpublished data indicate that AAD23 treatment in GRK5-deficient mice also prevents cognitive impairments triggered by other insults, like chronic intermittent hypoxia (48), suggesting broader potential applications of AAD23 in various neurodegenerative conditions.

Indeed, despite the similar levels of brain Aβ and microgliosis observed in GAP mice (Fig. 5), AAD23 treatment effectively guarded against cholinergic degeneration and cognitive decline. This observation does not undermine the significance of Aβ and inflammation in the pathogenesis of AD as sources of degenerative insults. Rather, it suggests that there exists a critical time window for Aβ and other insults to accumulate to a level that is toxic enough to kill neurons. Strengthening intrinsic cellular defense mechanisms, as achieved by AAD23, can significantly expand this time window, enhancing neuronal resilience. This study lends support to the concept of "one stone for multiple birds" in therapeutic strategy (33, 38, 48), highlighting the advantage of fortifying internal cellular defenses without necessitating specific therapies against individual insults. This approach is particularly advantageous for diseases like AD with multifaceted origins and diverse etiology. If a singular treatment like AAD23 can delay dementia onset by approximately 15 years (49), combining it with Aβ monoclonal antibodies, like lecanemab (Leqembi) and aducanumab (Aduhelm)—known to modify the disease in later AD phases (53)—could offer a more potent and effective therapeutic avenue.

Targeting presynaptic cholinergic sites: AAD23 versus ChEIs

AAD23, categorized as an M2 antagonist, belongs to the class of cholinergic modifying drugs, extensively studied in AD prompted by the cholinergic hypothesis (8, 54, 55, 56). Notably, ChEIs, the best-known cholinergic modifying drugs for AD, disappointed due to their limited efficacy in managing symptoms without disease modification (13, 14, 15). The distinguishing factor between AAD23 and ChEIs lies in their target mechanisms for addressing AD's underlying pathology. ChEIs focus on inhibiting cholinesterase to preserve ACh levels in the synaptic cleft, primarily compensating for cholinergic hypofunction and ameliorating associated symptoms (13). In contrast, AAD23 targets presynaptic M2 hyperactivity, preventing the oversuppression of cAMP/PKA/CREB signaling in cholinergic neurons (33, 38, 48). By bolstering the intrinsic defenses of these neurons, AAD23 aims to enhance resilience against degenerative insults. Although AAD23 might increase ACh release, it was not the primary goal of its design. Moreover, the cognitive tests in this study were performed after the drug was withdrawn for at least 2 weeks in the absence of AAD23 treatment. Such an experimental design eliminated any cognitive impact due to the acute influence in ACh release by the drug. If any, the cognitive tests were performed at the drug withdrawal period, which in the case of ChEI discontinuation or withdrawal, a worsened symptom could occur (57, 58, 59, 60). Thus, improvements in cognition observed in this study were likely attributed to the preservation of cholinergic neurons rather than functional compensation for cholinergic hypofunction.

Theoretical significance and future perspectives

The study highlights a novel opportunity for preventing neurodegenerative disorders, particularly AD, by focusing on enhancing the intrinsic defense mechanisms of neurons to bolster resilience. This strategy aims to elevate the apoptotic threshold, rendering neurons more resistant to various degenerative triggers like Aβ, hyperphosphorylated tau, free radicals, and inflammatory mediators (33, 38, 48). The significant advantage lies in a singular treatment that could effectively combat multiple insults—a concept termed "one stone for multiple birds."

Additionally, the study urges a reevaluation of previous therapeutic approaches linked to the cholinergic hypothesis in AD. While acknowledging the involvement of cholinergic hypofunction in AD pathogenesis, the study proposes that it might be a functional consequence of a compromised cholinergic neuronal system. Traditional therapies aimed at compensating for this functional aspect, exemplified by ChEIs, might only address symptoms rather than modifying the disease. The study suggests shifting therapeutic focus upstream to the presynaptic level to prevent cholinergic neuronal degeneration, as demonstrated by AAD23.

However, the study's potential was limited due to resource constraints, necessitating more comprehensive investigations with varying doses, durations, and time points. Expanding the study to include other AD transgenic models beyond GAP mice and considering the collection of fresh brain tissue for mechanistic analysis in future studies would be beneficial. Moreover, exploring the duration of AAD23's effects beyond the 12-months age mark and assessing its therapeutic efficacy in early or moderate AD models, either alone or in combination with other anti-Aβ strategies, remains essential for future research.

Overall, this study has yielded compelling proof-of-concept evidence supporting the efficacy of AAD23 in the prevention and substantial delay of cognitive impairments in mice, translating to a considerable extension in human terms, potentially over a decade. Crucially, this delay was achieved, while Aβ burdens and brain inflammation remained unchanged. These findings signify a groundbreaking approach for AD and similar neurodegenerative diseases, highlighting the significance of fortifying neuronal intrinsic defenses against ongoing neurodegenerative insults when specific treatments targeting these insults are yet to be developed.

The study's implications extend beyond the laboratory, offering a novel window for preventive and therapeutic interventions. By focusing on strengthening the neuronal intrinsic defense mechanisms, this strategy allows for the tolerance of existing neurodegenerative insults, potentially extending the symptom-free phase before disease onset. For instance, envisioning a delay from an onset age of 65 to 80 years old (considering an average life expectancy) provides an invaluable functional gain that could significantly benefit the elderly population and society at large.

Such an extended period of preserved cognitive function offers a crucial opportunity for individuals to enjoy a longer, more productive, and independent life before the onset of debilitating cognitive decline. This delayed onset could substantially alleviate the burden on caregivers, healthcare systems, and society as a whole, improving the quality of life for those affected by neurodegenerative disorders.

Conclusion

In conclusion, this study demonstrates that proactive use of AAD23 can significantly delay the cognitive decline and cholinergic degenerative changes in the GRK5-deficent subjects even though such a relief was not achieved via mitigating the brain amyloid burdens.

Experimental procedures

Study design and preparation

The purpose of this study was to determine if proactive use of AAD23 can prevent AD in GRK5-deficient animal models. As an initial optimization of the inclusion/exclusion criteria, GAP double transgenic mice, or GAP mice, were selected over GRK5KO mice because the former displayed more prominent AD pathogenic characteristics (36, 37, 38). Although senile plaques and gliosis began to emerge in individual animals at 9-month-old GAP mice, they only became consistently apparent in all mice around 12-month-old age (36, 37, 38). The onset of cognitive impairments in GAP mice began earlier than the pathology and remarked around approximately 7-month-old (38), assuming they are cognitively impaired at 12-month-old age if it is used as the study termination point for the intended comparisons. Also, as part of the inclusion/exclusion criteria, only female GAP mice were used in this study because of the known gender differences in these models (61, 62).

This study first optimized the dose of AAD23 via acute experiments using hippocampal ACh release as a readout. For acute administration of AAD23, gavage feeding worked fine, but a pilot test of daily gavage feeding for over 2 weeks failed due to increased mortality rates and variations in behavioral tests. This was at least in part because GAP mice have had higher mortality rate and were sensitive to stress (36, 37, 38). This had forced us to use an alternative administration protocol, a mouse dietary incorporation via Teklad. In brief, the proprietary compound CN168, incorporating AAD23 as the active pharmaceutical ingredient with a purity exceeding 98% (custom synthesized by Enamine), was incorporated at 18.75 ppm into a standard mouse diet (D8626) obtained from Teklad. To facilitate differentiation from the regular diet, nontoxic food coloring was added. The stability of these diets at 4 °C for at least 12 months was confirmed. Control mice were fed the D8626 diet. Dosing calculations were based on the average daily consumption of 4 g of the drug-containing diet by an approximate 30 g mouse, leading to an equivalent oral administration of 2.5 mg/kg AAD23 daily.

The 6-months AAD23 treatment began at 5 months of age and stopped at 11 months of age. After withdrawal of AAD23, a 12-days drug washing off period was followed to prevent potential acute drug effects on ACh release and cognition. This period was overlapped with the behavioral room adaptation period, which was then followed by the behavioral assessment. During the 22-days behavioral evaluation period, the mice were entirely off the drug. Consequently, the GAP mice were behaviorally assessed under no influence of the drug until the end of the experiment at 12 months of age, which was then followed by tissue collection for pathology.

Animals

The GRK5KO mice, which involve the targeted deletion of exons 7 and 8 of the grk5 gene (28), were bred with the Swedish APP mice Tg2576 line to yield needed genotypes: WT (GRK5+/+/APPsw−/−), GRK5KO (GRK5−/−/APPsw−/−), and GAP (GRK5+/−/APPsw+/−) mice. To establish a homogeneous background, these mice underwent backcrossing to the C57/BL6 background for over eight generations. Genotyping was conducted using tail DNA isolation and polymerase chain reaction amplification as previously described (37). The animal procedures were strictly in accordance with the guidelines set and approved by the Kansas City Veterans Affairs Medical Center Institutional Animal Care and Use Committee.

Brain cannula implantation and microdialysis for ACh measurement in mice

GRK5KO mouse (3 months old) underwent surgical preparation according to the method described by Cheng (36). A 2-cm midline skin incision was made on top of the skull. The animal was then placed in a stereotaxic frame with a mouse adapter (David Kopf). The implantation of brain cannula into the left ventral hippocampus (AP-2.6, L3.5, V2.5 to bregma) was made by following the previously described procedure (36). Following a recovery period of 1 week, microdialysis was conducted using the Automated BASi Raturn Sampling Caging System within the Culex workstation. ACh measurement was performed via the delivery of artificial cerebrospinal fluid containing 10 nM of Eserine at 1.0 μl/min using a microdialysis pump. Brain microdialysate samples were collected at 20-min intervals for subsequent analysis utilizing an ACh ELISA kit (Abcam).

Behavioral assessments

Behavioral tests were conducted in a dedicated behavioral lab division equipped with the ANY-maze Video Tracking System and Stoelting mouse behavioral battery devices. Mice underwent a 2-week habituation period before undertaking a battery of tests assessing sensorimotor function, anxiogenic tendencies, and mnemonic performance, as described (48). The blinded operators conducted these tasks in a predetermined order: (1) Swimming screening consists of a 15-s rest on a platform followed by 75 s of free swimming and exploration. Free-floating, idling, or involuntarily circling mice were removed from subsequent tests; (2) open field: evaluates activity/exploratory behavior. The total travel time and distance over a 3-min period were recorded; (3) balance beam: evaluates vestibular and general motor balance. Latency to fall from a beam was recorded for three successive trials (60 s); (4) string agility: evaluates agility and grip capacity. Animals were permitted to grasp a suspended string only by their forepaws and then released. Within 60 s, each animal was assessed using a 0 to 5 rating system; (5) elevated plus maze: evaluates level of anxiety. Closed and open arm entries over a 5-min period were recorded; (6) elevated platform: evaluates anxiety levels. Animals were allowed to freely explore the center, middle, and outer zones of an elevated platform for a 5-min period. The time spent and distance traveled in the middle and outer zones were used to estimate the animals’ anxiogenic tendencies. Elevated platform is a novel task that we developed that has better sensitivity for detecting anxiogenic changes than the elevated plus maze (63); (7) Y Maze: evaluates spontaneous alternation behavior and spatial working memory. Mice were allowed 5 min to explore a nontransparent wall Y-maze with three arms. Each arm measured 35 × 5 cm with 10 cm high walls. Mice were placed in the center of the maze facing the center area and allowed to explore for 5 min, with the number and sequence of arm choices being recorded. General activity was measured as the total number of arm entries, while basic mnemonic function was measured as a percent spontaneous alternation (the ratio of arm choices differing from the previous two choices divided by the total number of entries). For example, the sequence of arm entries (2,1,3,2,3,1,3,2) has six alternation opportunities (total entries minus 2) and the percent alternation would be 67%; and (8) MWM: evaluates reference (spatial) learning over 5 days and reference memory in two intermediate and one final probe trial on day 6. Average latency to find the submerged platform was obtained and averaged for each day of acquisition. On the day following acquisition testing, memory retention was evaluated in a single 60-s probe trial in which the submerged platform was removed, and the animal released from the quadrant opposite the former platform-containing quadrant (Quadrant 2; Q2). Percent of time spent in each quadrant and number of annulus crossings were determined from video tracking records.

In addition to the standard battery, the NOI (64, 65) paradigm was employed to induce hippocampal ACh release. Two novel objects were introduced and immobilized in opposite sides of the Automated BASi Raturn Sampling caging system at the beginning of the sampling (0 min) to allow free exploration by the mouse throughout the sampling period.

Tissue processing

Following the behavioral tests, animals were anesthetized and underwent perfusion with cold phosphate-buffered saline followed by 4% paraformaldehyde. The collected brain tissues were postfixed, cryopreserved, and sectioned at a thickness of 25 μm for subsequent analyses (35, 38). To ensure quality, randomly selected brains from each group were outsourced to NeuroScience Associates for multibrain embedding and sectioning services. In brief, the brains were cut at a thickness of 25 μm in a continuous series and collected into 24 series groups with options to select multiple intervals as necessary. The sections were preserved at −20 °C in cryopreservation solution (30% glycerol, 30% ethylene glycol in 0.1 M phosphate-buffered saline) before staining.

Immunohistochemistry

The cryopreserved sections in each group were transferred to 0.1 M phosphate buffer, free floating in 6-well plates, for immunohistochemistry staining with a polyclonal antibody raised against human ChAT (1:1000 dilution, Millipore). The sections were washed in Tris-buffered saline (TBS) and then incubated in 0.3% H2O2 in methanol for 30 min, rinsed three times in TBS, incubated for 30 min in TBS containing 0.25% Triton X-100 and 3% bovine serum, incubated with the primary antibody for 48 h (goat-anti-human ChAT diluted in TBS containing 1% Triton X-100 and 1% bovine serum), washed 3 times, incubated with biotinylated bovine anti-goat secondary antibody (Santa Cruz Biotechnology, 1:500) for 1 h, washed 3 times, incubated with the avidin-biotin–peroxidase complex (ABC Elite kit standard; Vector Laboratories), and finally developed using an enhanced ImmpactDAB substrate solution (ImmpactDAB kit, Vector Laboratories) according to the manufacturer’s recommendations. For double staining with the neuronal marker NeuN, the ChAT-stained sections were further stained with a monoclonal anti-NeuN antibody (Santa Cruz Biotechnology), followed by the ImmPRESS alkaline phosphatase polymer-based reagent and VectorRed substrate kit according to the manufacturer’s instructions (Vector Laboratories). This double-staining method resulted in a two-colored profile: brown for ChAT and red for NeuN. Immunofluorescent staining was performed as described previously (35). For triple staining of Aβ/CD45/DAPI, we used goat pAb-ChAT (1:300)/donkey pAb-Rabbit-IgG-Texas Red (1:600), rat mAb to CD45 (MCA1388, Clone: IBL-3/16, Serotec; 1:25)/donkey pAb-Rat-IgG-Alexa488 (1:800), and DAPI (Invitrogen Corporation). The detailed image quantification methods were previously described (35, 37, 38).

Unbiased stereology

The stereological analysis employed a Leica AF6000 microscope equipped with an x/y/z movement-sensitive stage and controlled by Stereologer software (Stereology Resource Center, Inc). Stereologer contains most commonly used protocols, including the standard fractionator sampling, rare event, and Space Balls protocols that were used for this study. The unbiased stereological quantification was achieved by counting positive cells/structures in a known fraction of the sections that pass through a region of interest (ROI) (66, 67), including the BF and its subregions, as outlined previously (38, 68). Because the largest variation in stereology comes from defining the boundaries of an ROI that has no clear boundary (i.e., the medial septum), we divided the whole basal forebrain into two easily distinguishable areas, the caudate putamen and rest of the nucleus basalis. An additional subregion focus was the NBM. To quantify the numbers of ChAT+ and NeuN+ neurons, a systematic random series of every eighth (200-μm interval) section throughout the entire structure was taken for stereological counts. For each of the selected sections, the ROI was outlined at low magnification (4× objective), and the outlined region was measured at high magnification (63× dry) using a systematic random design with known dissector counting frames. The average section thickness was measured, and 2 μm guard heights at the top and bottom of each section were excluded. Optical counting rules were used to count the cells, and the examiners were blind to the identities of the samples. The counting criteria were ChAT+/NeuN+ (cholinergic) or ChAT-/NeuN+ (noncholinergic) IR in the cell body with a neuronal phenotype. For rare events (i.e., CASs in the NBM), every positive structure inside the frame was counted, rather than using fractionator sampling. Again, NBM was outlined at low magnification, and the CASs were identified at high magnification (63× dry) from the varicosities, based on whether their diameters were larger than 3 μm, as described previously (69). For cholinergic fiber density quantification, either the Aznavour method or the Space Ball dissector method in Stereologer was used, and ROI volume was assessed using Cavalieri’s principle. Statistical analysis involved evaluating sampling scheme reliability and assessing effects related to age, genotype, or treatment.

Statistical analysis

Quantitative data were reported as means ± SD and plotted using box and whiskers to provide information about the data distribution and skewness along with the outliers. The comparisons in this study were limited to AAD23-treated versus untreated groups along with dosing variation, which were analyzed by ANOVA analysis using SPSS 24.0. Post-hoc comparisons of means were performed, and statistical powers were reported as described in each experiment in the relevant figure legends.

Data availability

All data are contained within the manuscript, ad no supplemental data are available.

Conflicts of interest

The authors declare they have no conflicts of interest with the contents of this article.

Author contributions

P. S., W. Z. S., and Q. Z. writing—original draft; P. S., W. Z. S., and Q. Z. project administration; P. S., D. W. P., and Q. Z. methodology; P. S., D. W. P., W. Z. S., and Q. Z. investigation; P. S. and W. Z. S. formal analysis; P. S., D. W. P., E. Y. P., and Q. Z. data curation; J. M. B., R. H. S., and W. Z. S. writing—review & editing; J. M. B., R. H. S., and W. Z. S. resources; W. Z. S. supervision; W. Z. S. funding acquisition; W. Z. S. conceptualization.

Funding and additional information

This work was supported by grants to W. Z. S. from the Alzheimer’s Association (the program of “Novel Pharmacological Strategies to Prevent Alzheimer's Disease” NPSPAD1-11–202149), the Medical Research and Development Service, 10.13039/100000738 Department of Veterans Affairs (Merit Review 1I01 BX001067–01A2 and 1I01BX004739–01A2 ), and resources from the Midwest Veterans’ 10.13039/100011085 Biomedical Research Foundation .
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