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Beyond Plaques: How Amyloid Precursor Protein Affects GABAergic Transmission 

In the quest to unravel the mysteries of Alzheimer’s disease (AD), scientists have long focused on the role of amyloid beta (Aβ) plaques and mutations in the amyloid precursor protein (APP). However, recent research has shed light on another aspect of APP’s function that may play a crucial role in the early stages of the disease. A team of researchers in Belgium investigated the effects of overexpressing wild-type human APP on GABAergic transmission, a key process in the brain’s inhibitory signaling. 

Previous findings suggested that APP overexpression can cause network hyperexcitability and epileptiform spikes in mice, but the exact mechanisms were not fully understood. The new study builds upon these findings by exploring the role of APP in gamma-aminobutyric acid (GABA)ergic transmission. Specifically, the researchers investigated the expression and function of GABA type A receptors (GABAARs) and GABA type B receptors (GABABRs), which mediate the inhibitory effects of GABA. Using a mouse model that overexpresses wild-type human APP, the researchers gained insights into how APP influences neuronal function and highlighted potential implications for neurodegenerative diseases such as Alzheimer’s disease (AD) and Down Syndrome (DS). 

The Importance of Amyloid in Neurodegenerative Disease

APP is a highly conserved single-pass transmembrane protein. The scientific community is still trying to pin down a precise physiological role for APP, but we do know that its metabolism yields a precursor of Aβ and that it plays a central role in the initiation of AD. Aβ peptides arise from the sequential cleavage of APP by β-site APP cleaving enzyme (BACE1) and presenilin-containing γ-secretase peptides, at which point they can aggregate into insoluble amyloid fibrils and form amyloid plaques that contribute to neurodegeneration, a hallmark of AD pathophysiology.

Although AD research has traditionally focused on the role of Aβ plaques in disease progression, there’s mounting evidence to suggest synaptic dysfunction occurs early in AD and can do so independently of amyloid plaque formation. You can see this most clearly in the largely unsuccessful clinical trials targeting Aβ formation or amyloid deposits. And while Aβ plaque treatments with monoclonal antibody drugs like aducanumab have shown promise, their efficacy is still up for debate.

But APP’s involvement goes beyond AD: it’s also implicated in DS, a neurodegenerative disorder characterized by the triplication of chromosome 21. People with DS face a significantly enhanced risk of developing AD, alongside seizure activity and epilepsy. The overlapping neuropathology between AD and DS suggests there are at least some shared pathogenic mechanisms, potentially tied to APP – which play a major role in regulating the balance between excitatory and inhibitory neurotransmission. Something worth noting: in mouse models, it’s the overexpression of APP rather than the resulting overproduction of Aβ that causes EEG abnormalities such as network hyperexcitability and epileptiform spikes. Clearly, APP levels of expression warrant more investigation. 

APP, GABA, and Neurotransmission

If we take a look at excitatory neurotransmission, APP modulates the trafficking and surface expression of the N-methyl-D-aspartate receptor (NMDAR), a key player in synaptic plasticity, learning, and memory. Notably, NMDAR activation decreases the surface expression of APP, promoting amyloidogenesis. In mice overexpressing wild-type APP, as well as those expressing APP with a Swedish double mutation akin to early-onset Alzheimer’s disease (EOAD) in humans, the expression and function of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in neurons is reduced.

The other side of this is inhibitory neurotransmission, where the main player is GABA, operating through GABAARs and GABABRs. GABAAR-mediated responses depend on the intracellular chloride concentration, which is regulated by chloride cation transporters. The balance between the Na+-K+-2Cl cotransporter NKCC1 and the K+-Cl cotransporter KCC2 influences the Cl gradient across the neuronal membrane, which in turn affects the GABAAR-mediated inhibitory responses. Any changes to the expression or function of these transporters can disrupt GABAergic inhibition. Notably, the expression levels of NKCC1 and KCC2 change during neuronal maturation, leading to a shift from excitatory to inhibitory GABAergic signaling, also known as the GABA shift.

Previous work from the Belgian team demonstrated that overexpressing APP in cortical cell cultures disrupts the GABA shift by downregulating KCC2 expression while leaving NKCC1 levels unaltered, resulting in a diminished inhibitory GABA response. Interestingly, APP interacts directly with KCC2, thereby maintaining its stability and function at the cell surface. 

Furthermore, APP is highly expressed in GABAergic interneurons and is part of the GABABR complex. The binding of APP to GABABRs promotes their axonal trafficking, surface expression, and presynaptic inhibition; an interaction that also protects APP from amyloidogenic cleavage by BACE1. Additionally, soluble APP, a product of both amyloidogenic and non-amyloidogenic cleavage pathways, has been shown to bind to presynaptic GABABRs, regulating neurotransmitter release.

Taking a Deeper Look at APP and GABAergic Signaling

With all of this in mind, the Belgian researchers set out to explore APP in GABAergic signaling, independent of Aβ plaques and APP mutations. They made extensive use of a mouse model that overexpressed wild-type human APP (hAPPwt; line I5) under the PDGF promoter, which moderately increased APP levels with minimal Aβ peptide and amyloid plaque formation.

Using this mouse model, the researchers planned to unravel the intricate mechanisms that underlie how APP influences GABAergic transmission, shedding light on its role in neurological disorders and ultimately providing valuable insights into the underlying pathophysiology. These findings could pave the way for the development of novel therapeutic approaches targeting APP-related synaptic dysfunction, offering new avenues for the treatment of disorders involving aberrant GABAergic signaling.

APP Overexpression Affects Cognition and Behavior

Starting with more macro-level effects, the researchers found that the overexpression of APP in mice led to neuronal loss and electrophysiological abnormalities. They saw these effects even in the absence of amyloid plaques and at low levels of Aβ. To complement these data, they conducted behavioral tests to assess the cognitive and emotional functions of hAPPwt mice. The mice showed cognitive impairment, as seen by lowered performance when it came to spatial working memory and spatial reference memory tasks. Additionally, hAPPwt mice showed signs of anxiety, including reduced exploratory behavior in new environments and increased aversion to illuminated areas.

To try to understand the mechanisms behind the observed cognitive impairment and neuronal alterations, the researchers performed in vivo and ex vivo experiments to assess neuronal excitability and synaptic plasticity. Intracranial electroencephalogram (EEG) recordings revealed abnormal spikes and hyperexcitation in hAPPwt mice, which were rescued by treatment with a presynaptic GABABR agonist. Ex vivo field potential recordings showed an increase in short-term facilitation and enhanced long-term potentiation (LTP) in hAPPwt mice, suggesting altered synaptic transmission.

GABA Levels and APP Overexpression

One of the key findings of the study was that the overexpression of APP resulted in neuronal overexcitation, as seen in the abnormal EEG patterns and increased LTP. However, the researchers failed to observe any significant changes in the components of GABAergic (inhibitory) or glutamatergic (excitatory) receptor systems or in GABA production ability. The researchers investigated hippocampus GABA protein levels via western blot using antibodies that included Anti-GABA(A) α1 Receptor (extracellular) Antibody (#AGA-001), Anti-GABA(A) α3 Receptor (extracellular) Antibody (#AGA-003), and Anti-GABA(A) α5 Receptor Antibody (#AGA-025) from Alomone Labs (Figure 1).

What the study did reveal, however, was a decrease in the levels of GABA in the hippocampus of hAPPwt mice (but no change to the amount of glutamate). Further investigation suggested that this decrease in GABA levels could be due to the soluble APP fragments acting on presynaptic GABABRs, reducing GABA release. Administering a GABABR antagonist successfully reduced excitability, indicating that the observed increased excitability was due to decreased GABA content.

Changes to Protein Levels in Response to Overexpressing APP

Figure 1. Protein levels of the main GABAergic and glutamatergic receptors in 6-month-old male hAPPwt mice. (a) Quantification and representative western blots of GABAergic receptor protein levels, including GABABR and GABAAR α1, α2, α3, and α5 subunits, measured in hippocampal tissue lysates of hAPPwt (n = 4) and control (n = 4) animals. (b) Quantification and representative western blots of glutamatergic receptor subunits of NMDA receptor (GluN2A, GluN2B) and AMPA receptor (GluA1, GluA2) measured in hippocampal tissue lysates of hAPPwt (n = 4) and control (n = 4) animals. The protein amount was normalized to β-tubulin and expressed as a percentage (Values are means ± SEM; *P ≤ 0.05, two-tailed unpaired t-test with Welch’s correction). Kreis, A, et al. Sci Rep 11, 17600 (2021).

Structural Changes in Response to APP Overexpression

Looking beyond changes to neurotransmission and the effects on behavior, the study also investigated structural changes in the hippocampus. Nissl staining of brain sections showed how the hAPPwt mice had a thinner hippocampus accompanied by neuronal death (confirmed by positive caspase-3 staining), specifically in the CA1 area (Figure 2).

Importantly, the study emphasized that these effects were observed in the absence of significant amyloid plaque formation and at low levels of Aβ.

APP Overexpression Induces Structural Changes in the Brain

Figure 2. Morphological changes in the CA1 hippocampal region in male hAPPwt and age-matched control animals at 3–24 months of age. (a) Comparison of Nissl stained sagittal brain sections of the hippocampal CA1 area between hAPPwt and age-matched control littermates, starting at 3 months and progressing to 12 months of age. Arrows mark the presence of dark Nissl-stained neurons in hAPPwt mice at 12 months of age, which is a sign of neuronal death. Neuronal degeneration and death were confirmed by caspase-3 staining in 12-month-old hAPPwt animals. (b–d) Quantification of surviving neurons in the CA1 area of 3, 6, and 12-month-old hAPPwt animals and age-matched littermates. Neurons with round and palely stained nuclei were considered viable, while shrunken and dark-stained cells were considered dead. Data are expressed as the number of surviving neurons per analyzed CA1  section (300 μm × 300 μm) in each age group for hAPPwt (n = 8) and control (n = 8) mice. (e) Nissl-stained sagittal brain sections of the hippocampal CA1 area in 24-month-old hAPPwt mice reveal a lateral progression of neuronal degeneration, which is evident by the presence of dark Nissl-stained neurons. Sections were taken later at 0.06 mm, 1.08 mm, and 1.56 mm; values are means ± SEM; ***P ≤ 0.001, ****P ≤ 0.0001, two-tailed unpaired t-test). Image and legend from Kreis, A, et al. Sci Rep 11, 17600 (2021).

APP Effects in the Absence of Amyloid Plaques

This intriguing study set out to explore how APP, in the absence of mutations or amyloid plaque formation, modulates excitatory and inhibitory neurotransmission. With the aid of a mouse model that overexpresses moderate levels of wild-type human APP, the researchers were able to discover a decrease in pyramidal cells in the hippocampal region, accompanied by caspase-positive cells indicating cell death. These findings suggest that both GABAergic and glutamatergic neurons may be affected. The overexpression of hAPPwt also led to impaired adult neurogenesis and abnormal synchronous spiking activity detected in EEG recordings, even at low levels of Aβ and without amyloid plaque formation.

The results seem to indicate that hAPPwt and its non-amyloid metabolites contribute to the observed abnormalities and hyperexcitability, which supports previous observations in patients with APP mutations or APP overexpression. The research also reveals increased levels of APP intracellular domains (AICDs) and phosphorylated tau in hAPPwt mice, which could contribute to neurodegeneration and increased excitability.

This study highlights the presence of APP at synaptic sites, suggesting its role in affecting neurotransmission. Specifically, it found altered expression of GABA receptor subunits in hAPPwt mice, including an increase in the α2 subunit associated with unconditioned anxiety. The balance of GABAergic signaling is regulated by cation-chloride transporters, and while previous studies demonstrated that APP overexpression affects KCC2 expression, this study surprisingly showed stable levels of KCC2 and NKCC1 in hAPPwt mice.

And if we turn our attention to GABA, the research has teased out the interaction between APP and the inhibitory GABABRs. The formation of an APP-GABABR1a complex protected APP from cleavage and reduced Aβ production. It also detected increased production of soluble APP fragments (sAPPα and sAPPβ) in hAPPwt mice, which bound GABABR1a and decreased neurotransmitter release. 

Overall, this fascinating study highlights the role of APP in GABAergic transmission, independent of amyloid plaques or mutations, and emphasizes the potential of enhancing GABAergic inhibition to prevent early seizures and neuronal overexcitation. This contributes to our understanding of synaptic dysfunction in neurological disorders and may offer new insights for developing therapeutic approaches targeting APP-related abnormalities in excitatory and inhibitory signaling.

Related Reagents

Primary Antibodies:

GABA(A) Receptors:

Glutamate Receptors:

Cation-Coupled Cl Transporters:

Explorer Kit: