Positive and Negative Selective Allosteric Modulators of α5 GABA A Receptors: Effects on Emotionality,Motivation,and Motor Function in the 5xFAD Model of Alzheimer’s Disease

Abstract

Background:

Positive and negative allosteric modulators of α5 GABA_A receptors (PAM and NAM, respectively) are worthy of investigation as putative treatments of Alzheimer’s disease (AD). However, their potential to modify a dynamic range of behaviors in AD models needs to be systematically examined.

Objective:

The study aimed to assess effects of MP-III-022 as PAM and PWZ-029 as NAM on emotional reactivity, motivation, and motor function, as well as on gene expression of GABRA2, GABRA3 and GABRA5 subunit of GABA_A receptors in prefrontal cortex (PFC) and hippocampus (HC) in 5xFAD mice, as an early-onset transgenic AD model.

Methods:

The 6-month-old 5xFAD transgenic and non-transgenic mice of both genders underwent a battery of reflexes and behavioral tests (sensorimotor tests, elevated plus maze, and open field) after 10-day intraperitoneal treatment with MP-III-022, PWZ-029, or solvent. The behavioral battery was followed by qPCR analysis of gene expression.

Results:

MP-III-022 induced a decline in motor function, while PWZ-029 further decreased emotionality of transgenic males, as compared to the transgenic control. No interfering effects on non-cognitive behavior were observed in female mice. In HC, both treatments reversed reciprocal GABRA2 and GABRA3 changes in transgenic females. In PFC, MP-III-022 decreased GABRA5 in both genders, while PWZ-029 increased GABRA2 in male transgenic animals.

Conclusion:

Gender-dependent protracted effects of PAMs and NAMs in AD model, with detrimental impact on motor capabilities of PAM, and attenuation of emotionality elicited by NAM in transgenic males, were revealed. This favors future research of α5 GABA_A receptor modulation in females as more promising.

Keywords

5xFAD Alzheimer’s disease emotionality motor function motivation negative allosteric modulation positive allosteric modulation

INTRODUCTION

Alzheimer’s disease (AD) is one of the major causes of disability in the elderly [1], with an accompanying need for caregivers’ assistance and grim predictions of the future toll worldwide [2]. As neither the causal nor reliably effective symptomatic therapy is available so far, the development of new therapeutics is critical [3]. Besides the decline of memory and other cognitive abilities, the condition is intertwined with a number of additional pathological changes, including compromised emotional reactivity [4], unfavorable changes in motivation [5], and motoric disfunction [6]. Thus, it is vital for any drug discovery program to thoroughly consider the influences of the putative treatment on these non-cognitive domains of performance.

The major inhibitory neurotransmission in the brain is provided by the GABAergic system [7], the role of which in AD is yet to be established in detail [8 –10]. Nonetheless, the GABAergic system has come to be seen as a promising therapeutic target for AD [10], mainly based on the findings that GABA type A receptors (GABA_A Rs) in various brain regions are severely affected in AD [11 –13]. Particularly, consistent with their established role in modulation of learning and memory [14], GABA_A receptors containing the α5 subunit were selected as an intriguing target in this research field [15]. They are predominantly expressed in hippocampal pyramidal cells and layer 5 pyramidal cell dendrites, and through tonic inhibition are capable of altering neural oscillations, which may profoundly affect cognitive behavior [15]. Apparently paradoxically, both positive and negative modulation of these receptors was proposed as a legitimate approach for development of putative AD treatment strategies [16]. However, the majority of research on this topic so far has dealt with learning and memory processes [17], while other aspects of behavior, although being subject to modulation via α5 GABA_A receptors as well [15], were mainly left untested in appropriate AD models. Specifically, in stress models, a potential of α5-selective PAMs to decrease emotionality [18, 19], and of α5-selective NAMs to reverse anhedonia and exert rapid antidepressant-like effects [20], is reported.

In this study, we aimed to investigate the influence of MP-III-022 [21] and PWZ-029 [22], as a positive and a negative selective modulator of α5 GABA_A receptors, respectively, on motor learning and capability, emotionality, and arousal component of motivation in 5xFAD mice. This is a transgenic mouse model based on the transfer of mutations of human genes for amyloid-β protein precursor (AβPP) and presenilin1 (PSE1) [23]. Acknowledging the impossibility to model all the symptoms relevant to AD [24], the advantages of 5xFAD model reflect on the early onset of amyloid pathology, loss of synapses, and confirmation of cognitive impairments [25].

The study was designed so that to assess possible protective or deteriorating influence of protracted modulation of α5 GABA receptors in the vulnerable mature adult period of 6-month-old 5xFAD mice, when first consistent behavioral and biochemical changes reminiscent of AD pathology have been reported [23]. Besides the three selected non-cognitive behavioral domains, the influences of treatments on GABRA2, GABRA3, and GABRA5 mRNA expression in the prefrontal cortex (PFC) and hippocampus (HC) were additionally determined. Namely, PFC is one of the key structures related to motivated behavior and its changes are corelated with apathy in AD [26], while HC is involved in regulation of numerous behavioral outputs, including cognitive flexibility with dynamic and adaptive potential [27]. Moreover, these two structures contribute to neural circuits implicated in anxiety [28], and more generally in emotional processing [29, 30], as well as motor regulation [31, 32].

On the other hand, the selected gene expression receptor repertoire (GABRA2, GABRA3, and GABRA5) plays a role in emotions and/or mood control with potential for therapeutic manipulation [33, 34]. In this vein, possible expression changes in 5xFAD mice, pathologically and/or during the modulation of α5 GABAA receptors, may indicate a range of wanted and unwanted effects of treatments based on such modulation.

METHODS

Subjects

The experiments were performed on transgenic 5xFAD mice that harbor five human mutations of APP and PSEN1: the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations in APP, and the M146L and L286V mutations in PSEN1, as well as their non-transgenic littermates. The colony was kindly provided by the Institute for Biological Research “Siniša Stanković”, University of Belgrade, Belgrade, Serbia, and the animals were born and reared in the vivarium of Faculty of Pharmacy –University of Belgrade, Belgrade, Serbia. 5xFAD mice of both sexes at age of 6 months±2 weeks were used in this study and kept in Plexiglas cages under 12:12 h light–dark cycle (lights on at 06.00 h), with food and water ad libitum. All behavioral experiments were conducted in the light phase (from 07.00 to 17.00 h). The study was conducted within the project approved by the Ethical Council for the Protection of Experimental Animals of the Ministry of Agriculture, Forestry and Water Management of the Republic of Serbia.

Substances

PWZ–029 ((methyl(8–chloro–5,6–dihydro–5–methyl–6–oxo–4H–imidazo[1,5–α ][1,4, 1,4]benzodiazepin–3–yl) methyl ether)) is a negative allosteric modulator with functional preference for α5 GABA_A receptors, and MP–III–022 ((R)–8–ethynyl–6–(2–fluorophenyl)–N,4–dimethyl–4H–benzo[f]imidazo[1,5–a][1,4, 1,4]diazepine–3–carboxamide) is a binding- and efficacy-selective positive allosteric modulator of α5 GABA_A receptors. The substances were synthesized at the Department of Chemistry and Biochemistry, University of Wisconsin—Milwaukee, USA. The control groups received only solvent (SOL; 85%distilled water, 14%propylene glycol, and 1%Tween 80) in which both ligands were dissolved. The treatments were administered intraperitoneally (i.p.), at 5 mg/kg once per day during 10 days before start of behavioral testing. Overall, six treatment groups per gender were tested, with transgenic and non-transgenic 5XFAD mice, respectively, subjected to SOL (TgS and NtgS), MP-III-022 (TgM and NtgM), or PWZ-029 (TgP and NtgP).

Pharmacokinetic study

The C57BL/6 mice were used for determination of brain concentrations of PWZ-029 and MP-III-022. Both substances were administered i.p. in doses of 1 mg/kg/day and 5 mg/kg/day, and samples were collected at four time points: 20 min and 24 h after the single dose, and 20 min after the third dose, for both ligands, while samples from 24 h after the third dose were obtained only for MP-III-022. The experimental details are given in Supplementary Material.

Behavioral battery

The protocol consisted of consecutive tests for reflex responses, motor and locomotor activity, and anxiety–like behavior, in the order as they were listed below.

The righting, pinna, corneal, and clasping reflexes are estimated to assess neurological and sensorimotor deficits. Additionally, rotarod and beam walking test were used to evaluate motor learning and capability, while the basket test only assessed motor capability of animals. The food localization test was used for estimation of olfaction as previously described [35]. For detailed protocols, see the Supplementary Material.

Elevated plus maze (EPM) is a standard test of anxiety–like behaviors in rodents. The apparatus consists of two open (40×8 cm) and two enclosed arms (40×8×30 cm), separated with a central zone (5×5 cm). The subject animal is placed to the central zone and allowed to explore maze for 5 min. Each test is recorded and analyzed in Anymaze tracking software (Stoelting, Wood Dale, IL).

Open field (OF), as a test of exploratory and locomotor activity, was performed in a rectangular Plexiglas arena (40×30 cm). The animal behavior during 15 min, without any previous habituation, was tracked and analyzed in Anymaze software.

Tissue preparation and qPCR

After animals were euthanized by ketamine overdose (100 mg/kg, i.p.), the PFC and HC were removed, deep frozen in liquid nitrogen, and underwent the Trizol protocol for total RNA isolation. After RNA concentration measurement and reverse transcription reaction, the obtained cDNA was subjected to a real time polymerase chain reaction (qPCR) in order to determine GABRA2, GABRA3 and GABRA5 expression levels. Protocol details, utilized primers, and dCt calculation are given in the Supplementary Material.

Statistical analysis

The behavioral data were analyzed via three-way ANOVA (factors: gender, genotype, and treatment) followed by Sidak post hoc testing in IBM SPSS Statistics 25 software. The parameters measured in qPCR experiments were evaluated via two-way ANOVA (factors: genotype and treatment) in different brain structures.

The z-score was defined as previously published by Guilloux et al. [36]. Firstly, z-score for each behavioral parameter within one test for each animal is calculated according to formula: z = (x –μ)/σ, where μ is mean for solvent-treated non-transgenic group, σ is standard deviation for solvent-treated non-transgenic group and×is value obtained in test for selected behavioral parameter. If the parameter was inversely proportional to behavioral domain, the sign minus was added in front of the σ value in calculation formula. Further, if more than one parameter is used from one test, their mean z value is taken as test z-score for further analysis. Otherwise, the z-score for parameter is test z score. Total z-score for each animal for behavioral domain is calculated as sum of test z scores for that animal that contribute to selected domain, divided by number of test z scores for each animal. The total z scores were used for two-way ANOVA followed by Holm–Sidak post hoc, and parameters included in total z-score were: duration and number of slips in beam walking for motor score, open arm time in EPM, and percent of peripheral distance during the first 10 min in OF for emotionality score, and closed arm entries in EPM and total distance during the last 5 min in OF for motivation score. The representative parameters in EPM and OF were selected similarly as previously published [36].

The statistical significances are shown as ^* for 0.01 < p < 0.05, ^**for 0.001 < p < 0.01, ^***for p < 0.001 and ∼ for trend effect 0.05 < p < 0.1 on graphs plotted in GraphPad Prism 9.

RESULTS

Pharmacokinetic dose selection

The pharmacokinetic study determined that the dose of 5 mg/kg would be optimal for protracted treatment with both ligands, PWZ-029 and MP-III-022, to achieve effective selectivity for α5 GABA_A receptors in behavioral experiments. See the Supplementary Material for detailed results.

Reflexes and sensorimotor behavior

The results for clasping reflex in males and females are exemplified in Supplementary Figure 1. In females, higher clasping scores were detected in TgP compared to NtgP group (p = 0.020), and in TgM compared to NtgM group (p = 0.019). Statistical differences shown in the rotarod and basket tests are summarized in the Supplementary Material. No statistical difference between groups was observed for corneal and righting reflexes, as well as for food localization test.

All results from the beam walking test were shown in Fig. 1. The post hoc test revealed statistical difference in time spent on crossing the beam between TgP and TgM; TgP group had lower values in males (p = 0.038). Furthermore, TgM showed increased time for crossing the beam than TgS in males (p = 0.038). Similarly, TgS animals showed prolonged crossing on the beam than NtgS in females (p = 0.005). The TgS group had a trend of increasing number of slips when compared to NtgS in both males and females (p = 0.098 and p = 0.069, respectively). TgS males showed a trend effect on time needed for traversing the beam compared to TgS females, spending less time on beam (trend, p = 0.062). TgP males likewise needed less time to cross the beam compared to TgP females (p = 0.011).

Fig. 1

Motor function. The results obtained in the beam walking test –time needed to traverse the beam (A) and number of slips (B) of the 5XFAD animals of both genders after treatment with PWZ-029 (P), MP-III-022 (M), and solvent (S) are shown. Composite parameter for assessment of motor function (total z-score for motor function) in this model after aforementioned treatment is encircled by rectangular shape (C). Statistical differences between groups: 0.01 < p < 0.05, 0.001 < p < 0.01 were presented as ^*, **, respectively. The trend effects (0.05 < p < 0.1) were labeled as ∼ or p value is written. All error bars represent SEM. The overall effects of gender (g), genotype (gt) or treatment (t), if significant, are summarized in the upper right corner of each graph.

NtgM males tended to have a lower number of slips compared to NtgM females (trend, p = 0.052).

Total z-score for motor capability (z-motor)

The total z-score for motor function in both genders, together with significant overall effects of genotype, treatment, or genotype x treatment interaction, are shown in Fig. 1. MP-III-022 treatment decreased z-motor in transgenic males compared to TgP (p = 0.037). Further, MP-III-022 treatment showed a trend effect on lowering z-motor in transgenic males compared to TgS (trend, p = 0.057). In females, TgS showed lower z-motor compared to NtgS (p = 0.004), and the same stands for TgP compared to NtgP (p < 0.001). The gender trend effect, with males tending to have lower values than females, was observed for two groups, TgM (p = 0.063) and NtgP (p = 0.081).

Open field

Significant overall effects in the open field test in 5xFAD mice are presented in Figs. 2 3. In males, post hoc comparisons showed the percent of peripheral distance in first 10 min (%peripheral distance) was decreased in NtgP as well as NtgM group compared to NtgS group (p = 0.002 and p = 0.001, respectively). However, in females, %peripheral distance was increased in TgM compared to TgS group (p = 0.035). In males, %peripheral distance was decreased in TgS when compared to NtgS (p = 0.044). The TgS and NtgS groups differed for %peripheral distance in males compared to females, with higher values in the former (p = 0.047 and p = 0.004, respectively).

Fig. 2

Emotionality. Results obtained in 5xFAD mice treated with PWZ-029 (P), MP-III-022 (M), or solvent (S) revealed the changes in emotionality in transgenic 5XFAD males and females. Percent of open arm time (A) was obtained in EPM. Percent of peripheral distance in first 10 min (B) was selected as an OF parameter for anxiety in the rectangle arena. The total z-score for measurement of emotional reactivity is pointed out with rectangular shape (C). Statistical differences between groups: 0.01 < p < 0.05, 0.001 < p < 0.01, p < 0.001 were presented as ^*, **, ***, respectively. The trend effects (0.05 < p < 0.1) were labeled as ∼ or p value is written. All error bars represent SEM. The overall effects of gender (g), genotype (gt) or treatment (t), if significant, are summarized in the upper right corner of each graph.

Fig. 3

Motivation. Results related to motivation in 5xFAD mice of both genders treated with PWZ-029 (P), MP-III-022 (M), or solvent (S) are shown. The parameter closed arm entries (A) was utilized to describe locomotor activity of animals in the EPM. Distance in last 5 min (B) was measured in animals habituated to the OF arena. Total z-score for measurement of motivation is encircled by rectangular shape (C). Statistical differences between groups: 0.01 < p < 0.05, 0.001 < p < 0.01, p < 0.001 are presented as ^***, ^***, respectively. The trend effects (0.05 < p < 0.1) was labeled as∼ or p value is written. All error bars represent SEM. The overall effects of gender (g), genotype (gt) or treatment (t), if significant, are summarized in the upper right corner of each graph.

Elevated plus maze

For the results from the EPM, significant overall effects of genotype, treatment, or genotype×treatment interaction were included in Figs. 2 3. In females, NtgM group had an increased number of closed arm entries as compared to NtgS group (p = 0.031). TgP had an increased number of closed arms entries in males compared to females (p = 0.035). The trend effect was shown for closed arm entries between NtgM groups in males and females, where NtgM males had lower values (p = 0.051). In males, TgP group had an increased percent of open arm time when compared to TgS group (p = 0.041). A trend difference between male and female NtgS mice in percent of open arm time was revealed, with males having lower values (trend, p = 0.076).

Total z-score for emotional reactivity (z-emotionality)

The total z-score for emotionality in both genders is showed in Fig. 2. Furthermore, the overall effects of genotype, treatment, or genotype x treatment interaction, if significant, are also included in Fig. 2. PWZ-029 treatment decreased z-emotionality in transgenic males compared to TgS (p = 0.003). TgS had lower z-emotionality than NtgS (p = 0.014) in males. A similar decrease of z-emotionality was also present in NtgP group compared to NtgS (p < 0.001), as well as in NtgM compared to NtgS (p < 0.001) in males. Additionally, PWZ-029 showed a trend effect in TgP male group compared to TgM male group on z-emotionality, with TgP males having decreased values (p = 0.055). The gender effect, with males having lower values than females, was observed for TgM (p < 0.001) and TgP (p < 0.001), as well as for NtgM (p < 0.001) and NtgP (p < 0.001).

Total z-score for motivation (z-motivation)

The total z-score for motivation in both gender is shown in Fig. 3. Furthermore, the overall effects of genotype, treatment, or genotype x treatment interaction, if significant, are also included in this figure.

The MP-III-022 treatment decreased z-motivation in transgenic males, as well as in transgenic females compared to their respective NtgM groups (p = 0.013 and p = 0.023, respectively). The PWZ-022 administration showed a trend of decreasing z-motivation in transgenic males compared to NtgP males (p = 0.074). The MP-III-022 treatment had a trend of increasing z-motivation in non-transgenic females compared to NtgS females (p = 0.054).

The gender effect, with males having lower values than females, was observed for TgM (p = 0.042) and TgP (p < 0.001), and a gender trend was revealed for TgS (p = 0.058).

PCR results

Results from qPCR for GABRA2, GABRA3, and GABRA5 mRNA expression in HC and PFC for males and females are illustrated in Fig. 4.

Fig. 4

The results obtained from qPCR are represented as dCT values that are reversely proportional to GABRA2, GABRA3 or GABRA5 mRNA levels. The expressions of GABRA2, GABRA3, or GABRA5 mRNA were determined in the hippocampus of males (A, B, or C, respectively) and females (G, H, or I, respectively) as well as in the prefrontal cortex of males (D, E, or F, respectively) and females (J, K, or L, respectively). Statistical differences between groups: 0.01 < p < 0.05, 0.001 < p < 0.01, p < 0.001 were presented as ^*, ** and ^***, respectively. The trend effect (0.05 < p < 0.1) was labeled as ∼ or p value is written. All error bars represent SEM. The overall effects of genotype (g) or treatment (t), if significant, are summarized in the upper right corner of each graph.

In male HC, TgS group had higher GABRA5 dCt values as compared to NtgS group (p = 0.032). Post hoc tests pointed that GABRA5 dCt in HC was non-significantly lower in TgP group compared to TgS group (p = 0.069). In female HC, TgS group showed downregulated GABRA2 and GABRA5 (a trend), and upregulated GABRA3 mRNA compared to NtgS (p = 0.011, p = 0.065 and p = 0.016, respectively). The treatment with MP-III-022 and PWZ-029 in transgenic females, compared to TgS, increased expression levels in HC of GABRA2 (p < 0.001 and p < 0.001, respectively), while decreased GABRA3 expression (p = 0.011 and p = 0.001, respectively). Additionally, PWZ-029 induced higher GABRA2 mRNA expression in HC in non-transgenic females in comparison with NtgS (p = 0.011).

In males, GABRA5 dCt values in the PFC were significantly higher in TgM as compared to TgP (p = 0.002) and TgS (p = 0.016). Further, increased GABRA2 levels were found in TgP group compared to TgS in male PFC (p = 0.034). TgM showed higher values for GABRA5 dCt in PFC than TgS in both genders (p = 0.016 for males and p = 0.015 for females). In female PFC, a non-significant trend of higher values of GABRA5 was revealed for NtgM compared to NtgS group (p = 0.055).

DISCUSSION

The development of GABA_A receptor subtype-selective ligands is hoped to result in a new generation of clinically effective drugs, with possible application in various neuropsychopharmacological disorders [37]. GABA_A receptors that contain the α5 subunit are an especially promising target [15, 38]; among α5 GABA_A receptor-selective ligands, the negative modulator PWZ-029 [39] and the positive modulator MP-III-022 [21] were introduced as a successful experimental tool (cf. [37]). The 5xFAD arose as a valuable AD model widely used [23], but the literature resources on α5 GABA_A positive and negative modulator effects in this model are limited [40].

There is seemingly paradoxical evidence suggesting that both types of modulation of α5 GABA_A receptors may be beneficial in models of AD [10]. The underlying hypothesis is that neither positive nor negative modulation of these receptors will have detrimental impact on non-cognitive behavior, which is of importance for further drug development in this research field. Here, we investigated the prolonged effects of positive and negative modulation of α5 GABA_A receptors in aging (6 months old) 5xFAD transgenic and non-transgenic animals of both genders. The major aim was to analyze changes in behavioral domains of motor ability, emotionality, and motivation, as well as in GABRA2, GABRA3, and GABRA5 mRNA expression after 10-day treatment with PWZ-029, MP-III-022, or solvent. In general, the behavioral changes observed were dependent on genetic status, treatment, and gender, but rarely were reciprocal while comparing the influence of the negative versus positive modulation, which would be expected from the pharmacological point of view. PWZ-029 further decreased emotionality assessed in transgenic males, while MP-III-022 induced a decline in motor function in comparison to the transgenic control. To the contrary, no interfering effects on non-cognitive behavior were observed in female mice, making the transgenic females more suitable for future research of cognitive effects of α5 GABA_A receptor selective PAMs and NAMs in 5xFAD model.

The pharmacokinetic study, conducted prior to behavioral battery with the goal to obtain selectivity of modulation at α5 GABA_A receptors, suggested that PWZ-029 and MP-III-022 may exert only negligible activity on α1, α2, or α3 GABA_A receptors when the chosen doses were applied. Namely, both ligands are mild PAMs on GABA_A receptors containing the α1, α2, or α3 subunit, but only at high concentrations that cannot be reached with the selected doses. Furthermore, in order to avoid any acute effects, the behavioral tests were performed after completion of 10-day treatment administration. With such a protocol, the consequences of α5 GABA_A receptor modulation in a preclinical model of AD could be translated to the effects on mature humans exposed to an approximately 1-year-admistation of such ligands (cf. [41]).

As to the behavioral domains focused at in the present study, emotional processing of both positively and negatively valenced stimuli may be relatively preserved in AD [42, 43], although many clinical studies revealed an impaired emotional processing [44, 45]. Such differences may reflect different patterns of underlying neuropathology and disease presentation. On the other hand, the motivated behavior is consistently affected in AD, with apathy as a quantitative reduction of goal-directed activity affecting up to 70%of AD patients. Indeed, it was proposed that the suppressed motivation in AD patients is a pertinent target for possible therapeutic intervention [26]. Finally, the neurological deficits and impaired motor function are the standard part of the overall AD symptomatology and may be encountered during the preclinical phase of the disease development [28 , 46]. In this vein, providing an adequate insight into possible activity at the level of non-cognitive domains of AD represents an important facet of drug discovery in this field.

In preclinical AD, it is accepted that the motor function impairment correlates with cognitive deterioration [47]. In our study, there was an obvious motor decline in control transgenic females, probably due to a faster pathology development in transgenic females compared to males [23]. MP-III-022 treatment precipitated the motor dysfunction in transgenic males when compared to the transgenic control. Since MP-III-022 did not further deteriorate motor capability in transgenic females compared to pathological control group, it could be supposed that positive modulation at α5 GABA_A receptors may have a role in motor dysfunction in disease progression in a gender-dependent manner. On the other hand, PWZ-029 administration did not affect motor function when compared to the respective solvent-treated transgenic or non-transgenic animals. The latter finding is in accordance with the lack of motor effects when another NAM of α5 GABA_A receptors was tested [20].

The established behavioral tests for measurement of emotional responses in rodents such as OF [48] and EPM [49] are based on triggering anxiety as a negative emotion. Humans suffering from AD commonly experience comorbid anxiety, sometimes associated with agitation [50]. In our study, there was a decrease in emotional reactivity in control transgenic males compared to healthy control. While apparently paradoxical, this finding could be interpreted in the context of decreased anxiety that corresponds with disinhibitory tendencies seen in AD patients (cf. [51]). In transgenic males, the emotionality score was further decreased after PWZ-029 application when compared to transgenic controls, while both ligands resulted in decreased emotionality in non-transgenic animals. The PWZ-029 like-effect on emotionality is previously observed after another NAM administration in tau-depositing mice as a model of AD [52]. On the other hand, PAM treatment did not affect emotionality in transgenic males compared to their pathological control, while in transgenic females, MP-III-022 showed an unexpected tendency to increase emotionality compared to transgenic control, at least with respect to the percent of peripheral distance parameter. The latter finding could be interpreted in part by thigmotaxic influences of positive modulation of α5 GABA_A receptors, as deduced from the Morris water maze [53].

In comparison with goal-directed motivation which could be determined by task accomplishment, arousal motivation is measurable by means of locomotion parameters [54]. Factor analysis shed light into different vector orientations of closed arm entries compared to percent of open arm time and entries in EPM, as well as total distance compared to activity in central zone in OF [55]. The arousal component of motivation was not affected in control transgenic animals compared to their healthy control, while both types of modulation contributed to a genotype effect of decreased motivation in transgenic males, with significant differences occurring when compared to analogously treated non-transgenic males. A similar suppressing influence on motivation was observed when transgenic males were compared to analogously treated females, with control transgenic males having only a trend of arousal decline compared to control transgenic females. Such uni-directional PAM and NAM impact on motivation in transgenic males could be a mechanism involved in disease progression. It is noteworthy that MP-III-022 exerted a trend of increasing arousal motivation score, based on the significant effect on closed arm entries in the EPM, in non-transgenic females compared to healthy control, which could be beneficial to mood, and is in line with proposed anti-depressive effects of PAMs [34].

The GABRA5 reduction in the HC is documented in AD patients [13]. Although the literature is inconsistent, it appears that severity of AD symptoms correlates with decrease of α5 GABAA receptors in this region (reviewed in [56]). The GABRA5 expression is a robust parameter for aging in mice since it is well preserved in the HC [57]. The lower GABRA5 level in the HC in transgenic compared to non-transgenic 5xFAD male animals was already published [58]. Our results demonstrated that solvent–treated transgenic animals of both genders had lower GABRA5 levels compared to healthy controls. Further, the protracted treatment with PWZ-029 showed a trend of increasing GABRA5 mRNA in whole HC of transgenic male, but not female mice, which could be of relevance for cognition.

Changes in prefrontal GABRA5 levels in AD patients, in comparison with control, were not revealed [12]. The GABRA5 mRNA expression in the PFC was unaltered between solvent–treated transgenic and non-transgenic animals of both genders. While PWZ-029 was devoid of any action, MP-III-022 reduced GABRA5 in non-transgenic females in comparison with solvent–treated non-transgenic animals. These data differ from the results obtained in C57BL6 female mice chronically treated with an α5-PAM that underwent unpredictable chronic mild stress [18], probably due to substantial differences in these models.

The GABRA2 downregulation in AD brain [12], and more specifically in entorhinal cortex [59], is reported. On the other hand, the GABRA3 expression in AD seems to be higher compared to control [60], albeit with possible dysfunctional α3 GABA_A receptor [61]. Our results demonstrated such divergent GABRA2 and GABRA3 expression pattern only in HC in control transgenic females, implying the region- and gender-dependent effects. The reduced anxiety or disinhibition in control transgenic males could be partly explained by potential GABRA2 and GABRA3 mRNA expression changes in other anxiety-related brain structures that were not part of this study. MP-III-22 and PWZ-029 reversed the changes of GABRA2 and GABRA3 expression in transgenic females to healthy control levels. While the pharmacokinetic validation of dosing regimen ensured that direct ligand effects were mediated by α5 GABA_A receptors, the indirect influence on GABRA2 and GABRA3 expression level may have occurred in female hippocampus. Additionally, PWZ-029 induced higher GABRA2 expression in PFC of transgenic males, as well as in HC of non-transgenic females. As the α2 GABA_A receptors mediate control of anxiety [62], the detected expression changes may be relevant for suppressed anxiety in EPM after PWZ-029 treatment in transgenic males.

In conclusion, depending on behavioral domain and gender, pharmacologically opposite modulation of α5 GABA_A receptors may elicit certain unwanted effects in 5xFAD mice. The clinical implication is that protracted administration of PAM in the prodromal phases of AD may result in adverse consequences on motor function in males, while NAM may have potential to lower the emotional reactivity in males. As untoward dynamic range of behaviors could adversely affect the research on cognition, females may be singled out as a more suitable model for PAM and NAM ligand development in AD. Further studies are needed for the better understanding of α5 GABA_A receptor function in AD, and both types of modulation should be examined in more advanced stages as well as in other behavioral domains in 5xFAD mouse model of AD.

Footnotes

ACKNOWLEDGMENTS

This research was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia through Grant Agreement with University of Belgrade-Faculty of Pharmacy No: 451-03-9/2021-14/200161. We wish to acknowledge the NIH for generous financial support (DA-043204, R01NS076517). We also thank the Milwaukee Institute for Drug Discovery and the University of Wisconsin-Milwaukee’s Shimadzu Laboratory for Advanced and Applied Analytical Chemistry for help with spectroscopy and the National Science Foundation, Division of Chemistry [CHE-1625735].

We acknowledge the expert technical assistance of Dr. Bojan Marković.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

References

Cao

, Tan

, Xu

, Hu

, Cao

, Dong

, Tan

, Yu

(2020) The prevalence of dementia: A systematic review and meta-analysis. J Alzheimers Dis 73, 1157–1166.

Andreakou

, Papadopoulos

, Panagiotakos

, Niakas

(2016) Assessment of health-related quality of life for caregivers of Alzheimer’s disease patients. Int J Alzheimers Dis 2016, 9213968.

Winblad

, Amouyel

, Andrieu

, Ballard

, Brayne

, Brodaty

, Cedazo-Minguez

, Dubois

, Edvardsson

, Feldman

, Fratiglioni

, Frisoni

, Gauthier

, Georges

, Graff

, Iqbal

, Jessen

, Johansson

, Jönsson

, Kivipelto

, Knapp

, Mangialasche

, Melis

, Nordberg

, Rikkert

, Qiu

, Sakmar

, Scheltens

, Schneider

, Sperling

, Tjernberg

, Waldemar

, Wimo

, Zetterberg

(2016) Defeating Alzheimer’s disease and other dementias: A priority for European science and society. Lancet Neurol 15, 455–532.

Sturm

, Yokoyama

, Seeley

, Kramer

, Miller

, Rankin

(2013) Heightened emotional contagion in mild cognitive impairment and Alzheimer’s disease is associated with temporal lobe degeneration. Proc Natl Acad Sci U S A 110, 9944–9949.

Forstmeier

, Maercker

(2015) Motivational processes in mild cognitive impairment and Alzheimer’s disease: Results from the Motivational Reserve in Alzheimer’s (MoReA) study. BMC Psychiatry 15, 293.

Albers

, Gilmore

, Kaye

, Murphy

, Wingfield

, Bennett

, Boxer

, Buchman

, Cruickshanks

, Devanand

, Duffy

, Gall

, Gates

, Granholm

, Hensch

, Holtzer

, Hyman

, Lin

, McKee

, Morris

, Petersen

, Silbert

, Struble

, Trojanowski

, Verghese

, Wilson

, Xu

, Zhang

(2015) At the interface of sensory and motor dysfunctions and Alzheimer’s disease. Alzheimers Dement 11, 70–98.

Farrant

, Nusser

(2005) Variations on an inhibitory theme: Phasic and tonic activation of GABA(A) receptors. Nat Rev Neurosci 6, 215–229.

Solas

, Puerta

, Ramirez

(2015) Treatment options in Alzheimer’s disease: The GABA story. Curr Pharm Des 21, 4960–4971.

, Sun

, Chen

, Xu

, Bu

, Zheng

(2016) Implications of GABAergic neurotransmission in Alzheimer’s Disease. Front Aging Neurosci 8, 31.

10.

Calvo-Flores Guzmán

, Vinnakota

, Govindpani

, Waldvogel

, Faull

RLM

, Kwakowsky

(2018) The GABAergic system as a therapeutic target for Alzheimer’s disease. J Neurochem 146, 649–669.

11.

Kwakowsky

, Calvo-Flores Guzmán

, Pandya

, Turner

, Waldvogel

, Faull

(2018) GABAA receptor subunit expression changes in the human Alzheimer’s disease hippocampus, subiculum, entorhinal cortex and superior temporal gyrus. J Neurochem 145, 374–392.

12.

Limon

, Reyes-Ruiz

, Miledi

(2012) Loss of functional GABA A receptors in the Alzheimer diseased brain. Proc Natl Acad Sci U S A 109, 10071–10076.

13.

Rissman

, Mobley

(2011) Implications for treatment: GABAA receptors in aging, Down syndrome and Alzheimer’s disease. J Neurochem 117, 613–622.

14.

Möhler

, Rudolph

(2017) Disinhibition, an emerging pharmacology of learning and memory. F1000Res 6, F1000FacultyRev-101.

15.

Soh

, Lynch

(2015) Selective modulators of α5-containing GABAA receptors and their therapeutic significance. Curr Drug Targets 16, 735–746.

16.

Jacob

(2019) Neurobiology and therapeutic potential of α5-GABA type A receptors. Front Mol Neurosci 12, 179.

17.

Maramai

, Benchekroun

, Ward

, Atack

(2020) Subtype selective γ-aminobutyric acid type A receptor (GABAAR) modulators acting at the benzodiazepine binding site: An update. J Med Chem 63, 3425–3446.

18.

Piantadosi

, French

, Poe

, Timić

, Marković

, Pabba

, Seney

, Oh

, Orser

, Savić

, Cook

, Sibille

(2016) Sex-dependent anti-stress effect of an α5 subunit containing GABAA receptor positive allosteric modulator. Front Pharmacol 7, 446.

19.

Fee

, Prevot

, Misquitta

, Knutson

, Li

, Mondal

, Cook

, Banasr

, Sibille

(2021) Behavioral deficits induced by somatostatin-positive GABA neuron silencing are rescued by alpha 5 GABA-A receptor potentiation. Int J Neuropsychopharmacol 13, pyab002.

20.

Zanos

, Nelson

, Highland

, Krimmel

, Georgiou

, Gould

, Thompson

(2017) A negative allosteric modulator for α5 subunit- containing GABA receptors exerts a rapid and persistent antidepressant-like action without the side effects of the NMDA receptor antagonist ketamine in mice. eNeuro 4, 1–11.

21.

Stamenić

, Poe

, Rehman

, Santrac

, Divović

, Scholze

, Ernst

, Cook

, Savić

(2016) Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit. Eur J Pharmacol 791, 433–443.

22.

Milić

, Timić

, Joksimović

, Biawat

, Rallapalli

, Divljaković

, Radulović

, Cook

, Savić

(2013) PWZ-029, an inverse agonist selective for α₅ GABAA receptors, improves object recognition, but not water-maze memory in normal and scopolamine-treated rats. Behav Brain Res 241, 206–213.

23.

Oakley

, Cole

, Logan

, Maus

, Shao

, Craft

, Guillozet-Bongaarts

, Ohno

, Disterhoft

, Van Eldik

, Berry

, Vassar

(2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: Potential factors in amyloid plaque formation. J Neurosci 26, 10129–10140.

24.

Veening-Griffioen

, Ferreira

, Meer Van

PJK

, Boon

WPC

, Gispen-de Wied

, Moors

EHM

, Schellekens

(2019) Are some animal models more equal than others? A case study on the translational value of animal models of efficacy for Alzheimer’s disease. Eur J Pharmacol 859, 172524.

25.

Jankowsky

, Zheng

(2017) Practical considerations for choosing a mouse model of Alzheimer’s disease. Mol Neurodegener 12, 89.

26.

van Dyck

, Arnsten

AFT

, Padala

, Brawman-Mintzer

, Lerner

, Porsteinsson

, Scherer

, Levey

, Herrmann

, Jamil

, Mintzer

, Lanctôt

, Rosenberg

(2021) Neurobiologic rationale for treatment of apathy in Alzheimer’s disease with methylphenidate. Am J Geriatr Psychiatry 29, 51–62.

27.

Bast

, Pezze

, McGarrity

(2017) Cognitive deficits caused by prefrontal cortical and hippocampal neural disinhibition. Br J Pharmacol 174, 3211–3225.

28.

Park

, Moghaddam

(2017) Impact of anxiety on prefrontal cortex encoding of cognitive flexibility. J Neurosci 345, 193–202.

29.

Salzman

, Fusi

(2010) Emotion, cognition, and mental state representation in amygdala and prefrontal cortex. Annu Rev Neurosci 33, 173–202.

30.

Philippi

, Botzung

, Noblet

, Rousseau

, Després

, Cretin

, Kremer

, Blanc

, Manning

(2015) Impaired emotional autobiographical memory associated with right amygdalar-hippocampal atrophy in Alzheimer’s disease patients. Front Aging Neurosci 7, 21.

31.

Suzuki

, Miyai

, Ono

, Oda

, Konishi

, Kochiyama

, Kubota

(2004) Prefrontal and premotor cortices are involved in adapting walking and running speed on the treadmill: An optical imaging study. Neuroimage 23, 1020–1026.

32.

Burman

(2019) Hippocampal connectivity with sensorimotor cortex during volitional finger movements: Laterality and relationship to motor learning. PLoS One 14, e0222064.

33.

Rudolph

, Knoflach

(2011) Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 10, 685–97.

34.

Engin

, Benham

, Rudolph

(2018) An emerging circuit pharmacology of GABAA receptors. Trends Pharmacol Sci 39, 710–732.

35.

Luo

, Cannon

, Wekesa

, Lyman

, Vandenbergh

, Anholt

RRH

(2002) Impaired olfactory behavior in mice deficient in the α subunit of Go. Brain Res 941, 62–71.

36.

Guilloux

, Seney

, Edgar

, Sibille

(2011) Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: Relevance to emotionality and sex. J Neurosci Methods 197, 21–31.

37.

Sieghart

, Savić

(2018) International union of basic and clinical pharmacology. CVI: GABAA receptor subtype-and function-selective ligands: Key issues in translation to humans. Pharmacol Rev 70, 836–878.

38.

Clayton

, Poe

, Rallapalli

, Biawat

, Savić

, Rowlett

, Gallos

, Emala

, Kaczorowski

, Stafford

, Arnold

, Cook

(2015) A review of the updated pharmacophore for the alpha 5 GABA(A) benzodiazepine receptor model. Int J Med Chem 2015, 1–54.

39.

Savić

, Clayton

, Furtmüller

, Gavrilović

, Samardzić

, Savić

, Huck

, Sieghart

, Cook

(2008) PWZ-029, a compound with moderate inverse agonist functional selectivity at GABAA receptors containing α5 subunits, improves passive, but not active, avoidance learning in rats. Brain Res 1208, 150–159.

40.

, Guo

, Gearing

, Chen

(2014) Tonic inhibition in dentate gyrus impairs long-term potentiation and memory in an Alzheimer’s [corrected] disease model. Nat Commun 5, 4159.

41.

Rae

, Brown

(2015) The problem of genotype and sex differences in life expectancy in transgenic AD mice. Neurosci Biobehav Rev 57, 238–251.

42.

Guzmán-Vélez

, Feinstein

, Tranel

(2014) Feelings without memory in Alzheimer disease. Cogn Behav Neurol 27, 117–129.

43.

Broster

, Blonder

, Jiang

(2012) Does emotional memory enhancement assist the memory-impaired? Front Aging Neurosci Mar 4, 2.

44.

Hamann

, Monarch

, Goldstein

(2000) Memory enhancement for emotional stimuli is impaired in early Alzheimer’s disease. Neuropsychology 14, 82–92.

45.

Kensinger

, Brierley

, Medford

, Growdon

, Corkin

(2002) Effects of normal aging and Alzheimer’s disease on emotional memory. Emotion 2, 118–134.

46.

Lee

, Choi

, Martin

(2020) Roles of the prefrontal cortex in learning to time the onset of pre-existing motor programs. PLoS One 15, e0241562.

47.

Buchman

, Bennett

(2011) Loss of motor function in preclinical Alzheimer’s disease. Expert Rev Neurother 11, 665–676.

48.

Seibenhener

, Wooten

(2015) Use of the Open Field Maze to measure locomotor and anxiety-like behavior in mice. J Vis Exp (96), e52434.

49.

Bailey

, Crawley

(2009) Anxiety-related behaviors in mice. In: Methods of Behavior Analysis in Neuroscience. 2nd edition, Buccafusco JJ, ed. CRC Press/Taylor & Francis, Boca Raton (FL). Available from: https://www.ncbi.nlm.nih.gov/books/NBK5221/

50.

Kwak

, Yang

, Koo

(2017) Anxiety in dementia. Dement Neurocogn Disord 16, 33–39.

51.

Jawhar

, Trawicka

, Jenneckens

, Bayer

, Wirths

(2012) Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer’s disease. Neurobiol Aging 33, 196.e29–40.

52.

, Ernst

, Treven

, Cerne

, Wakulchik

, Li

, Jones

, Gleason

, Morrow

, Schkeryantz

, Rahman

, Li

, Poe

, Cook

, Witkin

(2018) Negative allosteric modulation of alpha 5-containing GABAA receptors engenders antidepressant-like effects and selectively prevents age-associated hyperactivity in tau-depositing mice. Psychopharmacology (Berl) 235, 1151–1161.

53.

Savić

, Milinković

, Rallapalli

, Clayton

, Joksimović

, Van Linn

, Cook

(2009) The differential role of α1- and α5- containing GABAA receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats. Int J Neuropsychopharmacol 12, 1179–1193.

54.

Ward

(2016) Methods for dissecting motivation and related psychological processes in rodents. Curr Top Behav Neurosci 27, 451–470.

55.

Frohlich

, Morgan

, Ogawa

, Burton

, Pfaff

(2002) Statistical analysis of hormonal influences on arousal measures in ovariectomized female mice. Horm Behav 42, 414–423.

56.

Mohamad

, Has

ATC

(2019) The α5-containing GABAA receptors-a brief summary. J Mol Neurosci 67, 343–351.

57.

Palpagama

, Sagniez

, Kim

, Waldvogel

, Faull

, Kwakowsky

(2019) GABAA receptors are well preserved in the hippocampus of aged mice. eNeuro 6, ENEURO.049618.2019.

58.

Neuner

, Wilmott

, Hoffmann

, Mozhui

, Kaczorowski

(2017) Hippocampal proteomics defines pathways associated with memory decline and resilience in normal aging and Alzheimer’s disease mouse models. Behav Brain Res 322, 288–298.

59.

Grubman

, Chew

, Ouyang

, Sun

, Choo

, McLean

, Simmons

, Buckberry

, Vargas-Landin

, Poppe

, Pflueger

, Lister

, Rackham

OJL

, Petretto

, Polo

(2019) A single-cell atlas of entorhinal cortex from individuals with Alzheimer’s disease reveals cell-type-specific gene expression regulation. Nat Neurosci 22, 2087–2097.

60.

Antonell

, Lladó

, Altirriba

, Botta-Orfila

, Balasa

, Fernández

, Ferrer

, Sánchez-Valle

, Molinuevo

(2013) A preliminary study of the whole-genome expression profile of sporadic and monogenic early-onset Alzheimer’s disease. Neurobiol Aging 34, 1772–1778.

61.

Berggaard

, Witter

, van der Want

JJL

(2019) GABAA receptor subunit α3 in network dynamics in the medial entorhinal cortex. Front Syst Neurosci 13, 10.

62.

Atack

(2010) GABAA receptor α2/α3 subtype-selective modulators as potential nonsedating anxiolytics. Curr Top Behav Neurosci 2, 331–360.