Introduction
Over the last few decades, considerable focus has been on negative
allosteric modulators (NAMs) (previously referred to as inverse
agonists) of the benzodiazepine site of γ -aminobutyric acid
receptors (GABAARs) as a potential therapeutic target
for cognitive impairment in temporal lobe epilepsy (TLE), Huntington’s
disease, Down’s syndrome, schizophrenia and the most common form of
dementia, Alzheimer’s disease (AD), which constitutes one of the most
significant health problems confronting societies with an aging
population.
The ionotropic GABAA R family are heteropentameric
structures consisting of a combination of five subunits (Sieghart et
al., 2002) with the α−subunit being clinically relevant, as it controls
the pharmacological profile of GABAA Rs (McKernan et
al., 1996). Since the understanding that distinct pharmacological
properties of the GABAAR are reliant on the fact that
different brain regions and cell types contain various subunit
compositions, NAMs of the GABAAR at the subunit level
have been widely studied. In particular, GABAARs
containing the α5-subunit have been of interest, given their role
in learning and memory as evidenced by various studies (Caraiscos et
al., 2004; Collinson et al., 2002; Crestani et al., 2002; Dawson et al.,
2006; Ghafari et al., 2017; Yee et al., 2004).
The hippocampus plays a critical role in memory formation and retrieval,
and is significantly affected in AD, which is characterised by
short-term memory deficits as one of the first symptoms of the disease
(Price et al., 2001). The strong evidence to suggest hippocampal
preferential distribution of the α5-containing GABAAR
sub-type (Quirk et al., 1996), together with its diverse pathology in
memory deficit-related disease, and particularly, its preservation in
human brains of AD patients (Howell et al., 2000; Rissman et al., 2007),
has led many researchers to test several α5 subunit-selective compounds
for their potential cognition-enhancing effects (Liu et al., 1996; Quirk
et al., 1996; Savic et al., 2008; Sternfeld et al., 2004).
Originally, Merck, Sharp and Dohme, (MSD) developed the first
GABAAR NAM, known as α5IA, with high efficacy at the
GABAA α5 receptor sub-type without being an anxiogenic
agent (Atack et al., 2006). Following the development of this compound
by MSD, a number of other nootropic drugs (α5 sub-type selective NAMs)
have been developed (e.g. RO4938581; (Ballard et al., 2009)).
Many of these studies reported an impressive pharmacological profile of
this compounds and their potential as cognitive enhancers without
CNS-mediated adverse effects (Ballard et al., 2009; Braudeau et al.,
2011; Chambers et al., 2003; Collinson et al., 2006; Dawson et al.,
2006; Duchon et al., 2019; Eimerbrink et al., 2019; Martinez-Cue et al.,
2014). These studies were initially implemented in rodent models, and
unfortunately, these results were not reproducible in human
subjects/patients to the same extent. Several key molecules consistently
failed clinical trials at different phases including Basmisanil (code,
RO5186582, Roche, 2019), a5IA (Atack, 2010) and MRK-016 (Atack et al.,
2009). Basmisanil was taken through Phase 1 and Phase 2 of clinical
trials for Down’s syndrome and although in the Phase 2, it was shown not
to be efficacious in either adults or adolescents. It appears that
despite a5IA and MRK-016 demonstrating tolerance in young males, some of
these molecules were poorly tolerated in elderly patients with no
cognitive improvement (Atack, 2010), thus reducing the viability of α5
as a therapeutic target. Although these molecules were shown to be
selective for α5 subunit- containing GABAARs, the lack
of efficacy and poor tolerance in human patients could be related to
poor brain penetration of the molecules or an age-related effect.
Whether this failure was due to low drug potency / bioavailability or
due to a general lack of understanding of the synaptic mechanisms
involving α5 receptors during the pathogenesis of the disease is
currently unclear. To address these issues, we synthesised a novel water
soluble α5 GABAAR selective NAM. These receptor
sub-types have been shown to be located in hippocampal extrasynaptic
sites, as well as synaptic sites of postsynaptic pyramidal (Ali et al.,
2008; Glykys et al., 2008; Serwanski et al., 2006). Although it has been
shown that dendrite-targeting interneuron populations elicit α5
GABAAR-mediated inhibition in pyramidal cells (Ali et
al., 2008), it is unclear whether the α5 receptor subtype was expressed
on inhibitory interneurons themselves. This was of particular interest,
as we have shown previously, using theAPPNL-F/NL-F mouse, the first β-amyloid
precursor protein (APP ) knock-in mouse AD model that is thought
to be able to recapitulate the human condition more accurately (see
(Sasaguri et al., 2017), that synaptic excitability is disrupted in
various cortical regions, including the CA1 region (Petrache et al.,
2019), and that this could be related to the alteration of three key
modulatory interneuron populations namely; calretinin- (CR),
cholecystokinin- (CCK), and somatostatin- (SST) expressing interneurons
(Shi et al., 2019). We investigated whether these key modulatory
interneurons located in CA1 stratum radiatum (SR), together with
principal pyramidal cells, expressed the α5 subunit-containing
GABAARs, in the APPNL-F/NL-Fmodel, age-matched to wild-type control mice, and then characterized the
synaptic effects of our newly-developed α5 compound in these 4 sub-types
of neurons.
Methods: