ABSTRACT
Background and Purpose: Oligomeric Aβ1-42(oAβ42) exhibits agonist-like action at human α7- and
α7β2-nicotinic receptors (collectively, α7*-nAChR).
Aβ1-42 and an N-terminal Aβ peptide fragment (N-Aβ
fragment: Aβ1-15/16) have been shown to modulate
presynaptic Ca2+ and enhance hippocampus-based
synaptic plasticity via α7* nAChR. Both the N-Aβ fragment and its
essential core sequence, the N-Aβcore hexapeptide
(Aβ10-15), protect against Aβ-associated synapto- and
neurotoxicity, also involving nAChR. Here, we investigated how
oAβ42, the N-Aβ fragment and N-Aβcore regulate the
functional activity of α7*-nAChRs.
Experimental approach : Single-channel patch clamp recordings
measured the impact of ACh, oAβ42, the N-Aβ fragment and
the N-Aβcore on the function of concatenated, human α7- and
α7β2-containing nAChR expressed in nAChR-null SH-EP1 cells. Molecular
dynamics simulations identified potential sites of interaction between
the N-Aβ fragment and the orthosteric α7*-nAChR binding interfaces.
Key Results : Relative to the effects of ACh alone,
oAβ42 preferentially enhanced α7β2-nAChR open
probability and open-dwell times. Co-application with the N-Aβcore
neutralized these effects. Further, we demonstrate that the N-Aβ
fragment alone, or in combination with ACh or oAβ42,
resulted in selective enhancement of α7-nAChR single-channel open
probability and open-dwell times (compared to ACh or
oAβ42).
Conclusions and Implications : Our findings show the functional
diversity of Aβ peptides in regulating α7*-nAChR function, with
implications for a wide range of nAChR-mediated functions in AD.
Single-channel recordings of the differential effects of
oAβ42, N-Aβ fragment and/or N-Aβcore on α7*-nAChR
isoform function revealed the complexities of their interactions with
α7*-nAChR, with new insights into the neuroprotective actions of these
N-Aβ-derived peptides.
INTRODUCTION
Alzheimer’s disease (AD) is a neurodegenerative disorder that robs
individuals of their language, reasoning, and memory. An estimated 6.7
million Americans > 65 years of age and 20-30
million individuals worldwide are thought to suffer from AD. AD is
typically confirmed via in vivo imaging or postmortem assessment
of distinct neuropathological biomarkers: amyloid plaques primarily
composed of Aβ1-42), neurofibrillary tangles (filaments
of hyperphosphorylated tau), and neurodegeneration in select areas of
the brain (Jack et al., 2018).
Evidence now suggests that accumulation of amyloid plaques and
neurofibrillary tangles contribute marginally to the development of
cognitive decline in AD patients (Castellani et al., 2007; Marchesi,
2012). Instead, there is increased appreciation of the role(s) played by
the elevation of soluble, oligomeric forms of Aβ1-42(oAβ42) in AD etiopathogenesis during a long prodromal
phase, up to 15 years prior to diagnosis (Cline, Bicca, Viola, & Klein,
2018; Inayathullah & Teplow, 2011), particularly in regard to the
development and progression of synapse dysfunction and loss.
Specifically, oAβ42 is released at the synapse in
response to neuronal activity (Cirrito et al., 2005) and physiological
levels of the neuropeptide increase presynaptic Ca2+and positively modulate hippocampus-based synaptic plasticity and
contextual fear conditioning (Lawrence et al., 2014; Puzzo et al.,
2008), indicating that Aβ normally functions as a synaptic regulator.
Among the classes of molecular targets for Aβ and derivatives in their
specific forms are nicotinic acetylcholine receptors (nAChR), including
those that contain α7 and perhaps other subunits (α7*-nAChR; *indicates
the possible incorporation of other nAChR subunits) (Lukas et al., 1999;
Whiteaker & George, 2023). Whereas α7*-nAChRs primarily exist as
homopentamers (α7-nAChR) in the majority of brain regions, a minority
also contain β2 subunits (α7β2-nAChR). These heteromeric α7β2-nAChR form
functional receptors with several features distinct from those of
homomeric α7-nAChR (Khiroug et al., 2002; Moretti et al., 2014; Murray
et al., 2012). α7β2-nAChR are enriched in specific populations of
cholinergic and non-cholinergic neurons of the basal forebrain (Azam,
Winzer-Serhan, & Leslie, 2003; George et al., 2021; Thinschmidt,
Frazier, King, Meyer, & Papke, 2005; Whiteaker & George, 2023).
α7β2-nAChRs are particularly sensitive to functional modulation by
various Aβ forms (Liu, Huang, Shen, Steffensen, & Wu, 2012; Liu et al.,
2009). At the synapse, oAβ42 at physiological
concentrations (pM-nM) has been shown to activate presynaptic α7*-nAChRs
(Lawrence et al., 2014), including α7β2-nAChR (Mehta et al., 2009), via
direct agonistic interaction with the ligand binding domain (Tong et
al., 2011), this signaling pathway largely accounting for the Aβ-induced
regulation of presynaptic Ca2+.
Amyloidogenic Aβ peptides are produced from the amyloid precursor
protein (APP) via variable cleavage by β- and γ-secretases, yielding the
more abundant Aβ1-40 and the more toxic
Aβ1-42, the latter being the predominant isoform in
plaques. Aβ1-42 harbors a hydrophilic, N-terminal and a
hydrophobic C-terminal domain derived from APP extracellular and
transmembrane domains, respectively (Vassar et al., 1999). Through an
alternative, non-amyloidogenic α-secretase-mediated pathway, a 15-16
amino acid (1-DAEFRHDSGYEVHHQK-16) N-terminal fragment of
Aβ1-42 (N-Aβ fragment) is generated and is present in
significant amounts in human cerebrospinal fluid (Portelius et al.,
2011; Portelius et al., 2007), largely as the 15-mer as the lysine in
position 16 is rapidly removed by carboxypeptidase. In rodent models,
like oAβ42, the N-Aβ fragment has been shown to regulate
presynaptic Ca2+ and enhance synaptic plasticity and
contextual fear conditioning at pM to nM concentrations (Lawrence et
al., 2014; Portelius et al., 2011), thus also functioning as a
neuromodulator. In addition, the N-Aβ fragment was found to safeguard
against elevated oAβ42-induced neuronal toxicity,
synaptic dysregulation, compromised contextual fear memory and
gliotoxicity (Forest, Taketa, Arora, Todorovic, & Nichols, 2021),
involving nAChR (Lawrence et al., 2014). Through a combined
structure-function and mutational analysis, it was demonstrated that the
concentration-dependent neuromodulatory and protective actions of the
N-Aβ fragment against Aβ toxicities are localized in a unique core
hexapeptide sequence (10-YEVHHQ-15; N-Aβcore; (Forest et al., 2021).
Neuronal and network-level excitotoxicity is a feature of AD and has
been reported in humans and in numerous mouse models of AD pathology
(Minkeviciene et al., 2009; Palop et al., 2007; Palop & Mucke, 2009;
Vossel et al., 2013). Recently, we have shown that oAβ42exposure i) acutely activates both human α7- and α7β2-nAChR; ii)
preferentially enhances α7β2-nAChR open-dwell times; and iii) when
chronically exposed, induces hyperexcitability of basal forebrain
cholinergic neurons (BFCNs) within the medial septum and horizontal
diagonal band (George et al., 2021). These findings demonstrate specific
cellular and intrinsic-level mechanisms through which
oAβ42 enhances BFCN excitability through the activation
of α7β2-nAChR.
Here, we tested the hypothesis that smaller N-terminal domain fragments
of Aβ may mimic (or block) actions of oAβ42 on α7*-nAChR
function, perhaps without altering effects of the natural
neurotransmitter, acetylcholine (ACh). We defined the effects of the
human N-Aβ fragment and N-Aβcore on the single-channel properties of
human α7- and α7β2-nAChR heterologously expressed in human, native
nAChR-null SH-EP1 cells.
MATERIALS AND METHODS
Chemicals
Standard reagents were purchased from Sigma-Aldrich (St. Louis, MO,
USA). Fresh stock drug solutions were made daily and diluted as
required.
Amyloid peptides
Solutions of soluble, oligomeric amyloid (oAβ42; Echelon
Biosciences, Salt Lake City, UT) were prepared from aqueous stock
solutions as previously described and shown to exist predominantly in
the oligomeric state (George et al., 2021; Stine, Jungbauer, Yu, &
LaDu, 2011). The N-Aβ fragment (1-DAEFRHDSGYEVHHQ-15), the N-Aβcore
(10-YEVHHQ-15), and the inactive N-Aβcore hexapeptide (N-InAβcore;
10-SEVAAQ-15; (Forest et al., 2021) used as a control were prepared from
aqueous stock solutions of peptides synthesized and isolated at
>98% purity (American Peptide; Anaspec). Concentrations of
the peptides used were based on previous studies of their effects on
synaptic plasticity and neuronal intrinsic excitability (George et al.,
2021; Lawrence et al., 2014).
Constructs for human concatenated α7- and α7β2-nAChR
Fully pentameric α7- and α7β2-nAChR concatemers were constructed as
previously described (George et al., 2021). Human α7- and α7β2-nAChR
cDNA constructs were subcloned into a high-expression mammalian vectors
(pCEP4 and pCDNA 3.1+; respectively) that harbored the Kozak consensus
sequence and native α7- and α7β2-nAChR signal peptide sequences (Figure
1). All α7*-nAChR constructs were engineered to express the fluorescent
protein mCherry, facilitating the identification of α7*-nAChR
successfully transfected SH-EP1 cells. Genes that encoded nAChR subunits
were arranged in the order 5′- α7-α7-α7-α7-α7-3′ for homomeric α7-nAChR
and 5′-α7-β2-α7-β2-α7-3′ for heteromeric α7β2-nAChR. Sequences of all
subunits, together with their associated partial linkers, were confirmed
by DNA sequencing (Thermo Fisher Scientific, Waltham, MA, USA), and the
assembly of each translated pentamer was verified by restriction digest.
For α7-nAChR, orthosteric acetylcholine (ACh) binding sites are formed
at all α7/α7 subunit interfaces and involve the principal (+) face
containing subunit loop C of one subunit and the adjacent subunit’s
complementary (-) face containing loop D. As previously demonstrated for
α4β2-containing nAChR (Lucero et al., 2016), α7/β2 subunit pairs of the
α7β2-nAChR construct may form ACh binding pockets between the principal
(+) face of the α7 subunit and the complementary (-) face of the β2
subunit. Whether there is ligand interaction at β2(+)/α7(-) interfaces
is not clear. To enhance α7*-nAChR cell-surface expression, SH-EP1 cells
were co-transfected with the human nAChR chaperone protein NACHO (Gu et
al., 2016). Human NACHO was engineered in the mammalian expression
vector pIRES (Addgene, Watertown, MA) to facilitate expression of
ZsGreen1 fluorophore and to identify successfully-transfected cells.
Fluorescence microscopy imaging was used both to assess efficacy of
transfections and to identify cells exhibiting both mCherry and ZsGreen1
fluorescence as optimal candidates for patch clamp recording (Figure 1).
Single-channel electrophysiology of α7- and α7β2-nAChRs transiently
expressed in SH-EP1 cells
SH-EP1 cells were passaged and maintained as described previously (Peng
et al., 1999). Briefly, wild-type SH-EP1 (native nAChR-null) were
transfected using Qiagen’s Effectene transfection kit (Qiagen, Hilden,
Germany). Single-channel α7- and α7β2-nAChR–mediated currents were
recorded (Axopatch 200B amplifier; Molecular Devices) from successfully
co-transfected SH-EP1 cells at 22oC under a
cell-attached configuration as previously described (George et al.,
2021). Patches were clamped at a transmembrane potential of −100 mV.
Patch pipettes were fabricated from thick-walled borosilicate glass
(1.12 mm; WPI, Sarasota, FL, USA), and tips were micro-forged to a final
resistance of 13 mΩ. Concentrations used for oAβ42,
N-Aβcore or N-Aβ fragment adhered to previously published methodologies
(Forest et al., 2018; George et al., 2021; Lawrence et al., 2014). To
elicit single-channel events, pipettes were filled with artificial
cerebral spinal fluid (ACSF + 1.5 µM atropine) solution that contained:
(i) ACh alone (300 µM), (ii) oAβ42 alone (100 nM), (iii)
N-Aβcore alone (1 µM), (iv) N-Aβ fragment alone (1µM), (v) ACh (300 µM)
+ N-Aβcore (1 µM), (vi) ACh (300 µM) + N-Aβ fragment (1 µM), (vii)
oAβ42 (100 nM) + N-Aβcore (1 µM), or 8)
oAβ42 (100 nM) + N-Aβ fragment (1 µM). The ACh
concentration corresponded to the EC50 value for each
construct as determined by two-electrode voltage clamp (TEVC) (Moretti
et al., 2014) and was used to ensure collection of sufficient open
channel events for analysis without producing significant overlap of
unitary events or open channel blockade of receptors.
For quality control, patches with seal qualities <10 GΩ were
immediately discarded. Single-channel recordings were filtered at 5 kHz
and sampled at 50 kHz by using pClamp 10.4 (Molecular Devices). Under
these conditions, no single-channel events were observed in recordings
from untransfected SH-EP1 cells, from cells using ligand-free patch
pipettes, or from cells transfected with α7*-nAChR cDNA constructs using
patch pipettes containing the N-InAβcore control.
All single-channel recordings were analyzed using QuB software
[(v1.4.0.132; http://www.qub.buffalo.edu; (Qin, Auerbach, & Sachs,
2000b)], including for preprocessing and determining closed-dwell
times (Supplementary Figure S1), open- and closed-dwell times within
bursts, single-channel amplitudes, open probability, and burst duration.
Recorded traces were baseline corrected and single-channel events were
idealized according to a half-amplitude, threshold-crossing criterion.
Single-channel amplitudes, as well as forward and backward gating rates,
were derived from the idealized trace by fitting the raw data to simple
closed–open (C↔O) kinetic models. At the single-channel level, α7- and
α7β2-nAChR sojourns occur as brief, isolated openings with intermittent
bursts/clusters of channel opening events with longer-lived closed
intervals interspersed between bursts of channel activity, which
reflected times when all potentially activatable channels in the patch
are closed or desensitized (Grosman & Auerbach, 2001). Additional
closed states were added to the C↔O model until the log-likelihood score
failed to improve by >10 U (Qin, Auerbach, & Sachs,
2000a). The opening and closing rate constants were estimated by using
maximum interval likelihood, which optimizes rate constants of a
user-defined kinetic model according to the interval durations detected
by the half-amplitude, threshold-crossing criterion. Components with
time constants less than the selected dead time (0.2 ms, representing
twice the value of the filter rise time) were thereby eliminated from
consideration during fitting.
As previously described (George et al., 2021), we defined the main
amplitude state for α7- and α7β2-nAChR constructs used in this study by
using the α7*-specific PAM PNU-120596 to isolate the main conductance
state for both α7- and α7β2-nAChR. Stability plots of single-channel
amplitudes were generated to determine the quality of each
single-channel recording over time and between patches and to calculate
the mean amplitude of single events within bursts for each patch.
Amplitude stability plots were generated from single-channel events
elicited in the presence of ACh & PNU-120596 or oAβ42& PNU-120596 for α7-containing (linked and concatenated) nAChR and the
mean single-channel amplitude was defined for both α7- and
α7β2-containing nAChR. Only the single-channel events that matched this
pre-defined amplitude state were selected for further analyses.
Single-channel bursts corresponding to these precise amplitudes were
segregated from isolated openings, and only bursts were used for
single-channel analysis (highlighted in Figure S1). Bursts of
single-channel activity were defined as series of openings separated by
closures shorter than the minimum inter-burst closed duration [or
Tcrit; (Grosman & Auerbach, 2000)] and from other
such episodes by prolonged channel desensitization Chakrapani, Bailey,
and Auerbach (2004). For all groups tested, Tcrit was
calculated by using QuB software. Bursts that contained overlapping
currents, which indicate two simultaneously active channels, were rare,
and were discarded from the analysis. The advantages of using burst
analysis has been described (Ciuraszkiewicz et al., 2013), as it
increases the probability that adjacent openings arise from the same
receptor.
Molecular Dynamic Simulations
The α7-nAChR homopentamer model was built based on the cryo-electron
microscopy (cryo-EM) structure from the Protein Data Bank (PDBID:7KOO).
We removed the transmembrane and cytoplasmic domains and kept the
extracellular domain only. As there is no pentameric structure of the
α7β2-nAChR stoichiometry, we extracted β2 subunits from the cryo-EM
structure of the α4β2-nAChR stoichiometry (PDBID:6CNK) and replaced two
α7 subunits in the α7 homopentamer to generate the model of the
α7β2-nAChR heteropentamer. We used Auto Dock Vina (Eberhardt,
Santos-Martins, Tillack, & Forli, 2021) to dock the N-Aβ fragment onto
one of five α7+/α7- interfaces in the α7-nAChR homopentamer (Figure 6,
top panels) and one of two α7+/β2- interfaces in α7β2-nAChR
heteropentamer (Figure 6, bottom panels). The search space is defined to
be a cube with a side length of 50 Å surrounding the midpoint from loop
C of the (+) subunit interface to loops D and E of the (-) subunit
interface. For each of the α7- and α7β2-nAChR stoichiometries, we
selected the top 10 (out of 100) binding poses predicted by Auto Dock
Vina and performed molecular dynamics simulations. The modeled
nAChR/N-Aβ fragment complex structures were solvated in the TIP3P water
model (Jorgensen, Chandrasekhar, Buckner, & Madura, 1986) and
neutralized with K+ and Cl- at 0.15
M. The van der Waals interactions were smoothly switched off over 10–12
Å using a force-based switching function and the long-range
electrostatic interactions were calculated using the particle-mesh Ewald
method (York, Wlodawer, Pedersen, & Darden, 1994). Each system was
first equilibrated in the canonical ensemble (NVT) for 125 ps. The
production run was performed in the isothermal–isobaric ensemble (NPT)
at 310.15K and 1 bar with hydrogen mass repartitioning and 4 femtosecond
time steps . The production run was checked every 50 ns for 400 ns and
it was terminated if the N-Aβ fragment completely moved out of the
binding site. All simulations were performed with OpenMM package and
CHARMM36(m) force field . The simulation systems and input files were
generated by CHARMM-GUI Solution Builder, and visualization was done
using VMD (Humphrey, Dalke, & Schulten, 1996).
Data analysis for single-channel recordings
Single-channel amplitudes, closed-dwell times, and open-dwell times were
determined from individual patches, and tau (τ) values for each
exponential fit were averaged across multiple patches. Averaged
single-channel amplitudes, closed-dwell times, open probabilities
(Popen), and open-dwell times for α7- or α7β2-nAChR
(elicited with N-Aβcore or N-Aβ fragment) were compared to results
obtained in the presence or absence of ACh or oAβ42. For
each group, single-channel open-dwell durations were pooled from
multiple patches and histograms were generated by using Qub software.
All open-dwell time duration histograms were best fit with two
exponentials. Histograms of closed-dwell time durations within bursts
(Supplementary Figure S2) were best fit with a single exponential. Each
individual exponential and their respective time constants (τ) for burst
duration were calculated using Qub software. Time constants and
proportions of each exponential (i.e., short and long burst durations)
were compared between α7- and α7β2-nAChR.
Data are presented as means ± SEM overlaid with individual data points,
except when error estimates are calculated for gaussian or exponential
distributions, in which case single-channel open and closed times are
represented as histograms with the best-fit value ± SEM. Data points for
each figure represent individual patches pooled from multiple cells
transfected on different experimental days. For all tests, an α level of
<0.05 was considered significant. Single-channel data were
analyzed using a two-tailed Student’s t test when comparing the effects
of N-Aβcore or N-Aβ fragment on each α7*-nAChR or by one-way or two-way
ANOVA and Dunnet’s or Tukey’s multiple comparison test when comparing
the means across experimental conditions for each nAChR construct
(GraphPad Prism; San Diego, CA).
RESULTS