Dysregulated Neurotransmitters and CB1 Receptor Dysfunction and Their Roles in Agitation Associated with Alzheimer’s Disease

Abstract

Alzheimer’s disease (AD) is characterized by the progressive loss of cognitive function and is frequently accompanied by neuropsychiatric symptoms (NPS). Pathologically, AD is defined by two hallmark features: the extracellular accumulation of β-amyloid and the intracellular hyperphosphorylation of the tau protein. In addition to these primary changes, several other abnormalities are associated with the disease, including neuroinflammation, synaptic loss, oxidative stress, neurotransmitter imbalance, and genetic and epigenetic alterations. NPS in AD encompass a range of symptoms, such as anxiety, apathy, agitation, depression, and psychosis. These symptoms are thought to arise partly from the damage caused by the pathological hallmarks of AD, which impair various neurotransmitter systems. Altered levels of several neurotransmitters, including gamma-aminobutyric acid (GABA), serotonin (5-HT), dopamine (DA), and the cholinergic and noradrenergic systems, have been implicated in the development of agitation. Additionally, reduced endocannabinoid system (ECS) functionality, particularly cannabinoid receptor 1 (CB1R), has been linked to neurobehavioral alterations. Preclinical studies suggest that a decrease in CB1R levels is associated with aggressive behavior, and CB1R agonists have demonstrated beneficial effects in alleviating agitation and related symptoms. Given these findings, the current review focuses on the therapeutic potential of targeting neurotransmitter systems and CB1R dysfunction to manage agitation in AD.

1. Introduction

Alzheimer’s disease (AD) is a common neurodegenerative disease characterized by the progressive loss of cognitive functions and the presence of neuropsychiatric symptoms (NPS). AD is the sixth leading cause of death in the US and ranks as the fifth leading cause of death worldwide [1]. At the histological level, two well-recognized hallmarks are the extracellular accumulation of amyloid-β (Aβ) plaques and intracellular tau neurofibrillary tangles. In 90–97% of cases, AD patients experience NPS during the illness, which burdens caregivers, increases institutionalization, and accelerates cognitive decline [1,2]. NPS comprise several behavioral disturbances, including apathy, depression, sleep disorders, hallucinations, delusions, psychosis, agitation, and aggression [3]. In an earlier study assessing the prevalence of NPS in a sample of 216 participants, irritability/aggression (76.2%) was the most frequent symptom, followed by apathy (72.3%) and depressive symptoms (68.0%) [4]. Approximately 90% of AD subjects have one or more comorbid conditions [4]. Studies have shown that the prevalence of agitation in AD ranges from 30% to 50%, compared to 30% in Lewy body dementia and 40% in frontotemporal and vascular dementia [5,6]. Agitation is characterized by aggressive behaviors, both verbal and physical, such as yelling, cursing, hitting, and kicking. It also includes a range of non-aggressive behaviors typically marked by excessive motor activity, such as wandering, pacing, repetitive mannerisms, and general restlessness. Other manifestations may include hoarding, eating inappropriate substances, or making unusual noises, among others. Agitation damages social relationships and hinders daily living activities [7]. Furthermore, the impaired function of the endocannabinoid system (ECS), especially the cannabinoid type 1 receptor (CB1R), has been associated with neurobehavioral changes in AD [8,9,10,11]. Several studies have indicated that dysregulation of the serotonergic (5-HT), dopaminergic (DA), noradrenergic (NE), cholinergic, and GABAergic pathways is associated with the pathophysiology of agitation in AD [12,13]. Research has revealed that NPS are more strongly correlated with the presence of tau pathology than with β-amyloid pathology [14,15]. Functional imaging studies suggest an imbalance in metabolic brain activity or reduced glucose metabolism in the prefrontal cortex (PFC) and subcortical regions, which are implicated in mediating agitation behavior in AD [16,17,18]. Other findings also suggest that neuroinflammation in the medial temporal region and surrounding areas is linked to the emergence of agitation in AD patients [19]. This review focuses on the roles of GABAergic, 5-HT, DA, NE, and cholinergic systems and their interactions with CB1R in aggression and agitation in AD. CB1R regulates GABA [20], 5-HT release [21,22], and adrenergic receptor function [23], which are known to be linked to behavioral changes in AD [12,13,24].

Neuroinflammation is more likely to be a response to Aβ and tau deposition, rather than a cause of these abnormal protein aggregations [25], and is also involved in the imbalance of neurotransmitters [26,27], as well as synaptic loss [28] (Figure 1). Accumulated amyloid-β and tau are known to impair the balance of chemical neurotransmitters in the brain [27]. Reports have indicated that NPS in dementia progress through three stages: (1) irritability, depression, and changes in nighttime behavior; (2) appetite changes, agitation, and apathy; and (3) motor impairments, hallucinations, and disinhibition [29,30,31]. A large study of 10,000 individuals found that 39.3% of patients with AD had no agitation, 24.9% had mild symptoms, and 35.9% experienced moderate-to-severe agitation requiring treatment [32].

2. Gamma-Aminobutyric Acid (GABA)

GABA is produced by the decarboxylation of glutamate, catalyzed by the enzyme glutamic acid decarboxylase (GAD), and then metabolized by GABA transaminase [33]. In the brain, GABA transporters facilitate the uptake and recycling of GABA in synaptic vesicles [34]. GABA interacts with ionotropic GABA_A and metabotropic GABA_B receptors, both of which are found in presynaptic and postsynaptic terminals [33]. GABA can regulate the release of serotonin [35], acetylcholine, and dopamine in the brain [36].

2.1. Studies in Animal Models of AD

Previous in vitro and in vivo studies in animal models have linked pathological markers of AD with altered GABA signaling. An in vitro study demonstrated that Aβ neurotoxicity alters GABAergic neuron activity and impairs inhibitory postsynaptic potentials by downregulating postsynaptic GABA_A receptors in the rat somatosensory cortex [37]. In line with these findings, studies on animal models of AD have reported the loss of hippocampal GABAergic cells as early as six months of age, with early Aβ deposition observed in TgCRND8 mice [38] and APP/PS1 mice [39]. Furthermore, APP/PS1 mice exhibited a significant reduction in GABA_B receptors in the hippocampus and dentate gyrus as early as six months, with this reduction increasing with age [40]. Similarly, tau is believed to contribute to the reduction in GABA transmission in hippocampal regions [41].

2.2. Clinical Studies of AD

Evidence pointing out the role of GABA and related factors in AD is supported by several postmortem studies, which have reported reduced GABA levels in the cortical and limbic regions [33,42]. Beyond GABA levels, reductions in GAD, GABA receptors, and GAD-65 have been detected in the hippocampal and frontotemporal regions of AD patients [33,43]. Supporting these findings, a recent meta-analysis of 12 studies (including 182 AD patients and 176 controls) showed a 26% reduction in cerebrospinal fluid (CSF) GABA levels in the AD group compared to the control [44].

2.3. Effects on Behavior

Dysregulated GABA neurotransmission has been implicated in various neuropsychiatric symptoms, including aggression, anxiety, and psychosis [17,18,19,20,21,22,23,24]. Previous findings suggest that increased responsiveness of the amygdala to emotional stimuli, linked with inadequate prefrontal regulation, may increase the likelihood of aggressive behavior [45]. GABAergic activity at GABA_A receptors can reduce subcortical reactivity, and, therefore, a reduction in GABAergic activity may lead to increased aggression, as demonstrated in mouse models [46]. Researchers using the BALB/cJ mouse model observed heightened aggression and anxiety compared to BALB/cByJ controls [47,48]. The BALB/cJ and BALB/cByJ mouse strains are widely used to model GABAergic dysregulation and its behavioral effects. BALB/cJ mice, characterized by heightened aggression and anxiety, exhibit these traits due to significant GABAergic disruption. In contrast, BALB/cByJ mice display more moderate behavioral responses, reflecting relatively stable GABAergic function [47]. In BALB/cJ animal models, microarray analysis revealed altered GABA signaling in the anterior cingulate cortex (ACC), characterized by a 40% reduction in GABA levels and a 20-fold increase in the GABA-degrading enzyme [48].

2.4. GABA-Enhancing Drugs’ Effects on Agitation/Aggression

Despite the evidence from both preclinical and clinical studies that a deficit in GABA could contribute to aggressive behavior, some reports have shown that valproic acid, a drug that enhances GABAergic system activity, has limited efficacy in treating agitation in AD. Reduced GABA levels in the brains of Alzheimer’s patients suggest that impairments in this neurotransmitter’s function disrupt normal neurological processes and may contribute to treatment-associated adverse events [49]. A review of off-label clinical studies on gabapentin and pregabalin for treating agitation in AD also showed mixed results. Further reports indicate that randomized, placebo-controlled studies support their efficacy in treating agitation [50]. In a small placebo-controlled trial, carbamazepine showed modest benefits in treating agitation and aggression in AD patients [51]. However, an eight-week, multicenter, randomized, double-blind, placebo-controlled trial on oxcarbazepine revealed no significant improvement in agitation or aggression in patients with dementia [52]. A trend toward reduced scores on the Brief Agitation Rating Scale (BARS; p = 0.07) was observed in the oxcarbazepine group [52]. An 8-week trial with topiramate and risperidone in 48 outpatients from Iran showed that both treatments reduced symptoms, with no significant differences in the Neuropsychiatric Inventory (NPI) or the Cohen-Mansfield Agitation Inventory (CMAI) between the two groups [53]. Benzodiazepines (BDZs), another class of drugs, are CNS depressants that amplify the effects of the neurotransmitter GABA by binding to GABA_A receptors [54]. BDZs act as positive allosteric modulators of GABA-A receptors, enhancing GABAergic transmission. Commonly used to treat anxiety and insomnia, BDZs may also aid in AD by enhancing GABA activity, preventing NMDA-induced neurotoxicity, and inhibiting Aβ aggregation [55,56]. Studies suggest that BDZs with short half-life (e.g., oxazepam) may be effective in treating agitation and aggression in both healthy elderly individuals and AD patients. Despite guidelines recommending their short-term use, BDZs are often taken chronically for years, increasing the risk of falls, dependence, and withdrawal syndromes [57]. Tiagabine, an anticonvulsant medication, is a widely recognized GABA transporter inhibitor used in clinical practice. It functions by blocking the reuptake of GABA, enhancing GABAergic activity in the brain. In one study, the effects of chronic tiagabine administration for 5 weeks were evaluated in individuals with a history of substance abuse and antisocial aggressive behaviors. Participants were randomly assigned to either the placebo or tiagabine group with escalating doses (4 mg, 8 mg, 12 mg). The results showed that aggression decreased over time in the tiagabine group, while there was no significant change in the placebo group [58]. However, this beneficial effect has not been tested in patients with dementia. GABAergic enhancement strategies require further evaluation to prove their efficacy in treating agitation in AD. A CB1R agonist at low doses enhances GABA release in the brain [20,59] and may be beneficial in treating agitation in AD. Multiple studies have reported that synthetic and medical cannabis oil containing CB1R partial agonists can help alleviate agitation in AD patients [8,9,60,61].

3. Serotonin

Serotonin is a neurotransmitter synthesized from the amino acid tryptophan and is essential for mood regulation, appetite control, and sleep. It is primarily synthesized in the raphe nuclei of the brainstem, with projections reaching the cerebral cortex, thalamus, hypothalamus, basal ganglia, brainstem, and spinal cord. These pathways influence mood, sensory processing, motor control, and autonomic functions [62]. Serotonin projections from the median raphe nucleus target midline forebrain regions, such as the hippocampus and the amygdala, where they regulate anxiety, fear, and memory. They also extend to the spinal cord and autonomic nervous system, influencing pain, motor control, blood pressure, heart rate, and digestion [31,62]. The serotonergic system is believed to influence cognition primarily through its activity in the hippocampus and prefrontal cortex. In the hippocampus, 5-HT contributes to spatial navigation, decision-making, and social interactions [62,63]. In the prefrontal cortex, 5-HT is critical for working memory, attention, decision-making, and reversal learning in humans and animals [62,64]. Serotonin’s functional specificity arises from the wide distribution and distinct signaling of its 14 receptor subtypes across different brain regions [62]. Impairments in serotonin signaling are due to reduced serotonin neuron density, fewer terminals, and altered 5-HT_1A receptors. These changes contribute to cognitive decline and behavioral symptoms, including those associated with dementia [65].

3.1. Clinical Studies in AD

AD is associated with significant deficits in the serotonergic system, including the loss of 5-HT neurons in the brainstem’s raphe nuclei and reduced levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in key brain regions, such as the prefrontal cortex, amygdala, hippocampus, and temporal cortex [62]. This dysregulation is linked to increased aggression and agitation, possibly due to disrupted prefrontal cortex and amygdala function. Studies show that lower 5-HT and 5-HIAA levels correlate with increased agitation. Treatment with selective serotonin reuptake inhibitors (SSRIs) has been found to reduce agitation. Additionally, the decreased expression of specific serotonin receptors, particularly 5-HT_1A and 5-HT₆, is associated with agitation in AD [66]. In AD patients, reductions in both 5-HT and 5-HIAA have been observed, particularly in Brodmann area 10 (BA10), where these deficits are associated with increased hyperactivity [62]. Studies also indicate that serotonin assists prefrontal inhibition, and, therefore, insufficient serotonergic activity can enhance aggression [45].

3.2. Effects on Behavior

Serotonin plays a pivotal role in regulating aggression in both animals and humans, a relationship that has also been explored in patients with AD [67,68]. The association between serotonin and aggression is well established, with research highlighting this connection in AD, where aggression is a common symptom [67,68]. Genetic evidence identified the 5-HTT variable number of tandem repeats (5-HTTVNTR) allele 10 as a factor contributing to increased aggression [69]. Variations in serotonin levels, influenced by genetic factors such as the 5-HTTVNTR allele, may lead to heightened aggression in patients with AD. Research also suggests that differences in serotonin signaling, such as those associated with the 5-HTTVNTR polymorphism, play a role in aggressive behavior in both humans and animals, including AD patients [69]. These findings emphasize the impact of 5-HT regulation on behavior, with genetic variations such as the 5-HTTVNTR allele influencing aggressive responses.

3.3. Effects of Serotonergic Receptor-Enhancing Drugs on Agitation/Aggression

Recent studies have explored the potential of serotonergic-receptor-enhancing drugs, particularly 5-HT_1A agonists, in managing agitation/aggression symptoms, revealing their potential effectiveness in reducing them. A preliminary open-label study on tandospirone—a 5-HTR_1A partial agonist—for the treatment of the behavioral and psychological symptoms of dementia demonstrated its efficacy in alleviating agitation, aggression, and irritation in AD patients [70]. In contrast, meta-analyses have indicated that SSRIs show limited effectiveness in managing agitation in AD, especially when compared to antipsychotics [71]. Furthermore, a study of 18,750 dementia registrants in Sweden associated antidepressant use, especially SSRIs and mirtazapine, with accelerated cognitive decline, particularly in severe cases. Escitalopram was associated with a faster cognitive decline than sertraline, while citalopram was linked to slower progression. Higher SSRI doses correlated with a greater decline, severe dementia, mortality, and fractures. However, it remains unclear whether these effects were due to the antidepressants themselves or the underlying conditions [72]. In contrast to these drugs, the CB1R agonist WIN55,212-2 [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl methanone mesylate) enhances serotonin levels at low doses and elicits antidepressant effects in animals [21,73]. The activation of 5-HT_1A receptors may contribute to the regulation of agitation behaviors. However, more randomized placebo-controlled studies are needed to evaluate the efficacy of 5-HT_1A agonists in treating agitation in AD.

4. Norepinephrine

Norepinephrine (NE), also known as noradrenaline, is a key neurotransmitter in the brain that regulates arousal, attention, cognitive function, and stress responses [74]. It is synthesized from L-tyrosine, which is converted to L-3,4-dihydroxyphenylalanine (L-DOPA) by tyrosine hydroxylase, the rate-limiting enzyme in its synthesis. L-DOPA is then transformed into dopamine (DA) by dopa decarboxylase and stored in vesicles. In noradrenergic neurons, dopamine β-hydroxylase (DBH) converts dopamine into norepinephrine, while dopamine-producing neurons lack DBH, preventing NE formation [75]. Norepinephrine interacts with three primary G-protein-coupled receptor families: alpha-1 (α1), alpha-2 (α2), and beta (β) receptors. These receptors differ in their binding affinity for norepinephrine and can produce either excitatory or inhibitory effects based on their specific signaling mechanisms [74]. The locus coeruleus (LC) is the primary source of NE in the brain, with its neurons projecting to key regions, such as the PFC and amygdala [66,76].

4.1. Clinical Studies in AD

Several postmortem studies have consistently highlighted the involvement of the noradrenergic system in AD, showing notably reduced NE levels in various brain regions. Most studies report significant decreases in NE levels across areas such as the frontal medial gyrus, temporal superior gyrus, cingulate gyrus, hippocampus, amygdala, thalamus, hypothalamus, caudate, putamen, and LC. However, other studies have not replicated these findings, possibly due to confounding factors such as postmortem delay [75,77,78]. In AD, the LC is among the earliest brain regions affected, exhibiting tau pathology and NFTs in the initial stages, followed by progressive neuron loss as the disease advances [66,79]. In AD, despite the degeneration of locus coeruleus (LC) neurons, CSF levels of norepinephrine (NE) and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) remain stable or elevated, likely due to compensatory upregulation of NE synthesis [66,80,81]. This is further supported by increased tyrosine hydroxylase expression in LC neurons [81]. Additionally, elevated α1-adrenoceptor expression in the hippocampus and PFC suggests an adaptive mechanism to counteract NE depletion [82]. Lastly, AD is associated with enhanced dendritic and axonal sprouting from the remaining LC neurons, indicating an adaptive effort to sustain noradrenergic signaling [83]. Overall, these changes in NE pathways result in a hyperactive state of α1-adrenoceptor function.

4.2. Effects on Behavior

In AD, agitation is linked to compensatory changes in NE signaling following LC degeneration [84,85]. Greater LC neuron loss and early NFT accumulation are associated with increased aggression [85]. CSF NE levels have been shown to correlate with behavioral symptoms, although the results are inconsistent. Agitation is also tied to altered NE receptor function [86]. A small study on postmortem samples from AD patients indicated that increased α1-adrenoceptor expression is associated with aggressive behavior, particularly following long-term treatment with neuroleptic drugs [87]. Furthermore, studies using yohimbine and clonidine, a selective α2-adrenergic antagonist and an α2-adrenergic agonist, respectively, suggest that heightened noradrenergic system responsiveness may contribute to agitation and aggression in AD [88,89]. A study on α1-adrenoceptor binding in the PFC revealed a positive association between receptor density, receptor affinity, and aggression in patients with AD [87]. The activation of α1-adrenoceptors in the PFC is associated with impaired cognitive function [90]. In contrast, in the amygdala, α1-adrenoceptor activation and the stimulation of LC terminals enhance activity, resulting in increased fear and anxiety-like behaviors in rodents [91,92]. Antagonizing these receptors produces the opposite effect, reducing such behaviors [93,94].

4.3. Drugs Antagonizing Alpha-Adrenergic Receptors and Their Effects on Agitation/Aggression

Evidence supporting alpha-adrenergic receptor antagonists as potential candidates for reducing agitation in AD is consistent with the effect of prazosin, a centrally acting α1-adrenoceptor antagonist that can effectively cross the blood–brain barrier. Prazosin has been used for hypertension and benign prostatic hyperplasia [95]. A small double-blind placebo-controlled study with prazosin involving 22 nursing home and community-dwelling participants with probable or possible AD showed significant improvements in agitation and aggression symptoms [93]. The recently FDA-approved drug brexpiprazole, indicated for treating agitation in AD, acts as an antagonist of noradrenergic α1B and α2C receptors and serotonergic 5-HT_2A receptors. It also functions as a partial agonist of 5-HT_1A and dopaminergic D2 receptors, all with sub-nanomolar affinity [94,96]. Earlier studies have shown that CB1R agonism leads to the functional desensitization of postsynaptic α₂-adrenergic receptors (α₂-ARs) on layer V/VI pyramidal cells in the medial PFC. Furthermore, anatomical evidence indicates multiple interaction sites between CB1Rs and α₂-ARs within the mPFC, including presynaptic terminals, somata, and dendrites [23]. Several studies on synthetic and medical cannabis oil containing CB1R partial agonists have reported improvements in agitation among AD patients [8,9,60,61]. These findings suggest that α1-adrenoceptor antagonists and CB1R agonists provide beneficial effects against agitation in AD. However, the mode of action of the CB1R agonist may involve suppressing the noradrenergic system in addition to its primary action at CB1R.

5. Dopamine

Dopamine synthesis occurs entirely within presynaptic neurons, starting with phenylalanine, which is converted stepwise into tyrosine, L-DOPA, and, finally, dopamine. The rate-limiting step in this pathway is catalyzed by tyrosine hydroxylase, which converts tyrosine to L-DOPA. Another key enzyme, L-amino acid decarboxylase (AADC), then removes a carboxyl group from L-DOPA to generate dopamine. DA binds to five distinct receptors grouped into two categories based on their signaling pathways: D1-like receptors (D1 and D5) and D2-like receptors (D2, D3, and D4). The concentration of DA in the synapse is regulated by the dopamine transporter (DAT), which is located on the presynaptic membranes of dopaminergic neurons. DAT is responsible for the reuptake of DA, terminating its action in the synaptic cleft [97].

5.1. Clinical Studies of AD

Evidence suggests that the relative preservation of dopaminergic function—despite diminished cholinergic or serotonergic activity—may contribute to psychotic symptoms, aggression, and impulsivity in AD [98,99]. Studies have shown a negative correlation between choline acetyltransferase (ChAT)–DA and ChAT–D1 ratios in the temporal cortex and aggressive behavior [100], although DA levels alone have not been directly linked to agitation [101,102]. Further support comes from findings of preserved homovanillic acid (HVA) levels in the CSF of aggressive patients, along with a lack of significant associations between CSF DA metabolites (HVA and DOPAC) and agitation [98,103]. However, one study linked the cerebellar DOPAC–DA ratio to physical agitation [102]. Genetic studies on dopamine receptor polymorphisms in agitated AD patients have yielded mixed or inconsistent results, with some associations found for DRD1 and DAT VNTR polymorphisms, but with no clear links for DRD2, DRD3, or DRD4 [98,104]. While dopaminergic function appears relatively preserved in agitated AD patients, how it interacts with other neurotransmitter systems to influence behavior remains unclear.

5.2. Drugs Acting on the Dopaminergic System and Their Effects on Agitation/Aggression

Numerous trials have studied the effectiveness of the antipsychotic medications risperidone, aripiprazole, and olanzapine in treating severe agitation, aggression, and psychosis (e.g., delusions and hallucinations) in AD patients. While these antipsychotics have shown some benefits, the effect sizes were modest, and placebo responses were common. Therefore, a thorough evaluation of the potential benefits and risks is crucial before starting treatment [105]. The mechanism of action of brexpiprazole, which has recently been approved for treating agitation in AD, is partly attributed to its role as a partial agonist of D2 receptors, among other neurotransmitter interactions [94,96].

5.2.1. Acetylcholine

Acetylcholine (ACh) is a major neurotransmitter in the brain. It is released by cholinergic neurons and is essential for the signal transmissions involved in memory and learning [106]. It binds to nicotinic (nAChR) and muscarinic (mAChR) receptors. nAChRs are ligand-gated cation channels found in key brain regions, such as the hippocampus and substantia nigra [107]. The most common subtypes in the CNS are α7 and α4β2. They function presynaptically to enhance glutamate release and postsynaptically on GABAergic neurons. mAChRs signal via G-proteins and include five subtypes (M1–M5) [108,109]. M1 receptors are the most abundant, making up 35–60% of muscarinic receptors in the brain [108].

5.2.2. Clinical Studies in AD

An earlier study measuring regional cerebral metabolic activity in AD patients using [18F] fluorodeoxyglucose PET scans indicates a relationship between hypocholinergic function and agitation behavior in AD [110]. Pharmacologic studies suggest that cholinergic deficits in AD contribute to agitation. Anticholinergic agents increase hostility [111], while cholinergic treatments (e.g., cholinesterase inhibitors and xanomeline) help reduce agitation, highlighting the role of cholinergic dysfunction in AD-related agitation [13,112]. Postmortem studies suggest a link between cholinergic deficits and the behavioral and psychological symptoms of dementia (BPSD) in AD, and others indicate that cholinergic dysfunction is correlated with aggression and depression [100]. Additionally, in other postmortem brain studies, reduced cholinergic function was associated with aggressive behavior in AD patients [113]. A recent meta-analysis comprising 48 trials with 22,845 patients revealed that acetylcholine esterase inhibitor therapy, which increases the levels of acetylcholine, negatively impacted psychological health, appetite disorders, insomnia, or depression compared to those who received a placebo [114]. Further studies are warranted to understand the role of acetylcholine on agitation behavior.

6. Endocannabinoid System

The ECS includes G-protein-coupled CB1R and CB2R, endocannabinoids (eCBs) such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), and the enzymes that regulate their synthesis and breakdown [115,116]. CB1Rs are abundantly expressed in the brain, especially in regions involved in cognition, memory, and movement. In contrast, CB2Rs are predominantly found in immune cells [117]. CB1R plays a key role in modulating the release of neurotransmitters such as 5-HT, glutamate, DA, NE, and GABA [22].

6.1. CB Receptor’s Role in AD and Animal Models of AD

A study examining postmortem brain samples from individuals with advanced AD revealed an overall decline in CB1R densities in certain areas of the hippocampus and deeper layers of the frontal cortex [118]. In contrast, as AD progresses and neuroinflammation increases, CB2R is upregulated, particularly in response to heightened microglial and astrocyte activity [119]. Other studies on postmortem hippocampal brain tissue have revealed changes in the molecular composition of 2-AG, indicating the presence of changes in the endocannabinoid signaling network near senile plaques. These findings suggest alterations in circulating eCBs [120]. In the AβPPswe/PS1ΔE9 AD mouse model, cognitive deficits are observed, along with reduced eCB levels and enhanced CB receptor coupling in the striatum [121]. The activation of CB1R has been shown to exert neuroprotective effects, including a reduction in tau phosphorylation, in PC12 neuron studies [122]. Another animal study also demonstrated that hippocampal CB1R deletion increased inflammation, reduced cell proliferation, and impaired social memory in adult mice [123]. In other animal AD models, low-dose THC treatment (between 0.2 and 0.02 mg/kg) enhanced spatial learning in aged APP/PS1 mice by lowering oligomeric Aβ, phospho-tau, and total tau expression levels while reducing GSK-3β activity, without inducing psychotropic effects [124]. This supports the neuroprotective effect of CB1R agonism in AD models.

6.2. Role of CBRs in Neuroinflammation Regulation

CB1R and CB2R play crucial roles in regulating neuroinflammation in the brain. CB1R is a G-protein-coupled receptor predominantly found in neurons, with minimal expression in microglia and astrocytes [125]. On the other hand, glial cells primarily exhibit low levels of CBR2 [126]. During neuroinflammation, CB1R and CB2R interact through soluble factors, with CB1R playing a key role in reducing microglial activation and limiting the release of pro-inflammatory factors. CB1R expression is low in resting astrocytes and microglia, but increases upon activation during neuroinflammation [123]. Endocannabinoids such as 2-arachidonoylglycerol (2-AG) and anandamide, along with synthetic cannabinoids, activate CB1R and modulate microglial activity. This activation facilitates the transition of glial cells from a pro-inflammatory (M1) state to an anti-inflammatory (M2) state, helping to reduce neuroinflammation [116,123,127]. Blocking CB1R can accelerate the onset of inflammation [128], whereas activating CB1R with an agonist can reduce inflammation [129]. These findings highlight the potential therapeutic role of CB1R modulation in neuroinflammatory conditions.

Another key mechanism through which CB1R regulates AD-related neuroinflammation is by targeting the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) inflammasome. Chronic neuroinflammation is a key factor in the progression of AD. One of the main drivers of AD-related neuroinflammation is the NLRP3 inflammasome, a protein complex that plays a role in immune system activation [130]. Research suggests that NLRP3 activation not only contributes to AD, but also influences aggressive behavior in animal models [131]. For example, studies using the resident–intruder paradigm show that NLRP3-driven neuroinflammation increases aggression in mice. Notably, tetrahydrocannabinol (THC)—a partial agonist of cannabinoid receptors—has been found to inhibit NLRP3 inflammasome activation in in vitro models [132]. This suggests that targeting inflammasome activity with THC could help to reduce inflammation-related behavioral changes, including aggression [131]. Supporting this idea, preclinical studies have shown that low doses of THC (0.2–2 mg/kg body weight) reduce aggression in a dose-dependent manner in mice, rats, and squirrel monkeys, as observed in resident–intruder tests [133].

Animal studies suggest a link between CB2R and AD pathology. Research shows that CB2R-deficient mice exhibit tau neuropathology, hippocampus-dependent memory impairment, and mitochondrial dysfunction, supporting CB2R’s potential role in AD progression [134]. CB2R activation (via agonists such as JWH-015, JWH-133, and MDA7) promotes Aβ clearance, reduces neuroinflammation, and improves cognitive function in AD mouse models [135,136,137]. Chronic JWH-133 administration in Tg APP 2576 mice reduced TNF-α levels, microglial activation, and Aβ-plaque loads, leading to improved cognitive performance [137]. CB2R has been shown to play an important role in regulating NLRP3 functions in various in vitro models, where CB2R agonists inhibit activated NLP3 function [138,139]. THC is also a partial agonist of CB2R [140], which may exert synergic effects with CB1R in regulating the NLRP3 function and behavior [131]. Notably, the frequency and severity of NPS increase as cognitive impairment worsens [141,142]. Activation of CB2R helps in reducing the pathological markers of AD and may reduce the impact on the neurotransmitter imbalances altered by tau and beta-amyloid [26,27]. Finally, it may act to mitigate NPS.

6.3. Effects on Behavior

The ECS regulates behavior through various receptor pathways. CB1R loss has been linked to increased aggression in CB1R knockout mice, an effect that is reversible with acute CB1R agonist administration [10]. Reduced hippocampal and frontal cortex CB1R levels have also been observed in AD patients, potentially contributing to behavioral disturbances [118]. Animal studies indicate that low doses of THC (0.2–2 mg/kg body weight) exert a dose-dependent anti-aggressive effect in various animal species (mice, rats, and squirrel monkeys) [133]. Additionally, the ECS plays a key role in the regulation of neurotransmitter release and receptor functions at GABAergic, serotonergic, and adrenergic synapses. Deficiencies in GABAergic and serotonergic signaling are associated with agitation in AD [12]. Research suggests that CB1R activation enhances [³H]-GABA release in rats [20], indicating that CB1R agonists may help to restore neurotransmitter balance and alleviate behavioral symptoms in AD. Previous studies have shown that CB1R activation induces the functional desensitization of postsynaptic α₂-adrenergic receptors (α₂-ARs) on layer V/VI pyramidal neurons in the mPFC [23] and inhibits 5-HT reuptake in vitro [21].

Open-label clinical trials have suggested that dronabinol—synthetic THC—improves appetite, body weight, and nocturnal motor activity while reducing agitation in AD patients [143,144]. In patients with severe dementia, dronabinol treatment led to significant reductions in all domains of the Pittsburgh Agitation Scale, along with improvements in Clinical Global Impression scores, sleep duration, and meal consumption. Although adverse events were reported, none led to treatment discontinuation [8]. In a randomized placebo-controlled trial, nabilone—a THC analog and partial CB1R agonist—showed efficacy in treating agitation, with potential benefits for overall NPS, caregiver burden, and nutritional status [60]. Similarly, an open-label add-on study found that low doses of medical cannabis oil with THC significantly decreased Neuropsychiatric Inventory and Clinical Global Impression severity scale scores in AD patients [61].

These findings support the potential of CB1 receptor activation in treating NPS, including agitation. At present, several placebo-controlled randomized studies are underway to evaluate the efficacy of THC in treating behavioral symptoms in AD (NCT02792257 and NCT05543681). While the randomized controlled trial (RCT) on dronabinol (NCT02792257) has been completed, the results have not been released. These trials will provide insights into the efficacy of CB1R partial agonists in treating agitation and NPS in AD.

7. Discussion

Agitation in AD is a complex behavioral syndrome that progresses alongside the disease. The exact pathophysiology of agitation in AD is not fully understood. Still, preclinical and clinical studies suggest that altered pathways involving neuroinflammation, neurotransmitter imbalances, synaptic loss, and ECS dysfunction could contribute to the onset of agitation and aggression (Figure 1). Existing evidence indicates that multiple neurotransmitter systems and additional receptor pathways are likely to be involved in triggering agitation behavior in AD, with earlier clinical evidence demonstrating that inflammation associated with more severe tau pathology is more closely linked to NPS than beta-amyloid pathology [14,15]. Functional imaging studies have suggested that reduced metabolic activity between the PFC and subcortical regions plays a role in mediating agitation behaviors in AD [16,17,18]. Additionally, research has shown that neuroinflammation in the medial temporal region and adjacent areas is linked to the onset of agitation in AD patients [19]. Neuroinflammation likely arises as a response to Aβ and tau deposition rather than being the primary cause of these abnormal protein aggregates [25], while also contributing to neurotransmitter imbalances [26].

In postmortem brain samples from AD patients, CB1 receptor activity was higher in the early stages of AD, particularly in the hippocampus and frontal cortex, but decreased at later stages [118]. This pattern suggests the initial hyperactivity of the endocannabinoid system in regions without classical histopathological markers, potentially compensating for early synaptic impairments. However, as the disease progresses, CB1R function declines, suggesting that early CB1 stimulation may have therapeutic potential. The loss of CB1R is associated with aggressive behavior in animals, and additional studies indicate that selective CB1R loss in hippocampal regions results in increased neuroinflammation, cognitive decline, and decreased cell proliferation [123]. CB1R regulates inflammation, and the neurotransmitter systems of 5-HT, DA, GABA, and adrenergic receptors play a role in stabilizing neurotransmitter imbalances and mitigating neuroinflammation. This neuroinflammation appears to be a key factor in behavioral symptoms in AD. The activation of CB1R by partial agonists (THC, dronabinol, or nabilone) could offer therapeutic benefits not only for agitation, but also for other NPS.

Memory deficits caused by high doses of cannabinoids may result from excessive, nonselective CB1 receptor occupancy, which interferes with the precise spatiotemporal regulation of endocannabinoid-mediated synaptic plasticity, as previously suggested by Carlson et al. [145]. In the general population, where CB1R receptors are preserved, higher doses of THC have been linked to cognitive impairments. However, the scenario may differ in the AD patient population due to CB1R loss, which suggests that controlled CB1R activation may have beneficial therapeutic effects. For instance, chronic low-dose THC treatment stabilizes dendritic spines and cognitive decline in 18-month-old mice, but not in younger mice. This effect is dependent on CB1R receptors in the glutamatergic cells of the forebrain [146,147]. In addition, a study showed that the CB1R agonist WIN55,212-2 has a biphasic dose-dependent effect on hippocampal acetylcholine (ACh) release. A low dose (0.5 mg/kg) stimulates ACh release, while a high dose (5 mg/kg) inhibits it. CB1 receptors mediate these effects, but involve different brain regions: the hippocampus for inhibition and the septum for stimulation. Dopamine D1 and D2 receptors further modulate these responses. These findings suggest that endocannabinoids regulate neuronal activity in a dose-dependent manner through distinct neural pathways [148]. Thus, CB1R activation by its agonists may have differential cognitive effects depending on age, with lower doses showing potential therapeutic benefits for treating agitation and beyond in AD patients.

8. Conclusions

In summary, activation of CB1R by its partial agonists may provide early-onset therapeutic effects on agitation and other NPS. It plays a key role in managing neuroinflammation, neurotransmitter levels, and synaptic plasticity at low doses, offering beneficial effects against agitation and potentially improving cognitive function [149,150]. Additionally, selective therapeutic agents that modulate alpha-adrenergic receptor activation and CB1R activation may be potential candidates for treating agitation in AD. More randomized placebo-controlled studies are warranted to understand their potential efficacy in treating agitation and other NPS.