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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that mostly strikes the elderly. However, the exact molecular and cellular pathogenesis of AD, especially the dynamic changes of neurons during disease progression, remains poorly understood. Here we used single-nucleus RNA sequencing (snRNA-seq) to access the transcriptional changes of hippocampal neurons in APP23 mouse model of AD. We performed snRNA-seq using a modified Smart-seq2 technique on 3,280 neuronal nuclei from the hippocampus of young and aged APP23 and control mice and identified four distinct subpopulations. Comparative transcriptional analysis showed multiple changes in different subtypes of hippocampal neurons of APP23 mice in comparison to control mice, as well as the transcriptional changes in these neurons during disease progression. Our findings revealed multiple neuronal subtype-specific transcriptional changes that may lead to targets for future studies of AD.

The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory β1-subunits. Although the short cytosolic N-terminal domain of the β1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the β1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β1-subunit N terminus.

Objective To propose a new hypothesis that GABAergic dysfunction in excitatory and inhibitory (E/I) imbalance drives the pathogenesis of Alzheimer's disease (AD). Background Synaptic dysfunction and E/I imbalance emerge decades before the appearance of cognitive decline in AD patients, which contribute to neurodegeneration. Initially, E/I imbalance was thought to occur first, due to dysfunction of the glutamatergic and cholinergic systems. However, new evidence has demonstrated that the GABAergic system, the counterpart of E/I balance and the major inhibitory neurotransmitter system in the central nervous system, is altered enormously and that this contributes to E/I imbalance and further AD pathogenesis. New hypothesis Alterations to the GABAergic system, induced by multiple AD pathogenic or risk factors, contribute to E/I imbalance and AD pathogenesis. Major challenges for the hypothesis This GABAergic hypothesis accounts for many critical questions and common challenges confronting a new hypothesis of AD pathogenesis. More specifically, it explains why amyloid beta (Aβ), β-secretase (BACE1), apolipoprotein E4 gene (APOE e4), hyperactive glia cells, contributes to AD pathogenesis and why age and sex are the risk factors of AD. GABAergic dysfunction promotes the spread of Aβ pathology throughout the AD brain and associated cognitive impairments, and the induction of dysfunction induced by these varied risk factors shares this common neurobiology leading to E/I imbalance. In turn, some of these factors exacerbate GABAergic dysfunction and E/I imbalance. Moreover, the GABAergic system modulates various brain functions and thus, the GABAergic hypothesis accounts for nonamnestic manifestations. Furthermore, corrections of E/I balance through manipulation of GABAergic functions have shown positive outcomes in preclinical and clinical studies, suggesting the potential of the GABAergic system as a therapeutic target in AD. Linkage to other major theories Dysfunction of the GABAergic system is induced by multiple critical signaling pathways, which include the existing major theories of AD pathogenesis, such as the Aβ and neuroinflammation hypotheses. In a new perspective, this GABAergic hypothesis accounts for the E/I imbalance and related excitotoxicity, which contribute to cognitive decline and AD pathogenesis. Therefore, the GABAergic system could be a key target to restore, at least partially, the E/I balance and cognitive function in AD patients.

Background: The role of post-translational modification of regulatory β-subunits in the control of large conductance potassium (BK) channels is largely unknown. Results: β4-subunit palmitoylation controls surface trafficking of BK channel α-subunit splice variants. Conclusion: Palmitoylation of β4 masks an α-subunit trafficking motif to control surface delivery. Significance: Palmitoylation of regulatory subunits provides a dynamic mechanism to control surface trafficking of specific BK channel variants., Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels.

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium-and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by similar to 55% in the C53:54:56 A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.