Loss of cholinergic receptor muscarinic 1 impairs cortical mitochondrial structure and function: implications in Alzheimer’s disease

Abstract

Introduction: Cholinergic Receptor Muscarinic 1 (CHRM1) is a G protein-coupled acetylcholine (ACh) receptor predominantly expressed in the cerebral cortex. In a retrospective postmortem brain tissues-based study, we demonstrated that severely (≥50% decrease) reduced CHRM1 proteins in the temporal cortex of Alzheimer’s patients significantly correlated with poor patient outcomes. The G protein-mediated CHRM1 signal transduction cannot sufficiently explain the mechanistic link between cortical CHRM1 loss and the appearance of hallmark Alzheimer’s pathophysiologies, particularly mitochondrial structural and functional abnormalities. Therefore, the objective of this study was to analyze the molecular, ultrastructural, and functional properties of cortical mitochondria using CHRM1 knockout (Chrm1-/-) and wild-type mice to identify mitochondrial abnormalities.Methods: Isolated and enriched cortical mitochondrial fractions derived from wild-type and Chrm1-/- mice were assessed for respiratory deficits (oxygen consumption) following the addition of different substrates. The supramolecular assembly of mitochondrial oxidative phosphorylation (OXPHOS)-associated protein complexes (complex I-V) and cortical mitochondrial ultrastructure were investigated by blue native polyacrylamide gel electrophoresis and transmission electron microscopy (TEM), respectively. A cocktail of antibodies, specific to Ndufb8, Sdhb, Uqcrc2, Mtco1, and Atp5a proteins representing different subunits of complexes I-V, respectively was used to characterize different OXPHOS-associated protein complexes.Results: Loss of Chrm1 led to a significant reduction in cortical mitochondrial respiration (oxygen consumption) concomitantly associated with reduced oligomerization of ATP synthase (complex V) and supramolecular assembly of complexes I-IV (Respirasome). Overexpression of Chrm1 in transformed cells (lacking native Chrm1) significantly increased complex V oligomerization and respirasome assembly leading to enhanced respiration. TEM analysis revealed that Chrm1 loss led to mitochondrial ultrastructural defects and alteration in the tinctorial properties of cortical neurons causing a significant increase in the abundance of dark cortical neurons (Chrm1-/- 85% versus wild-type 2%).Discussion: Our findings indicate a hitherto unknown effect of Chrm1 deletion in cortical neurons affecting mitochondrial function by altering multiple interdependent factors including ATP synthase oligomerization, respirasome assembly, and mitochondrial ultrastructure. The appearance of dark neurons in Chrm1-/- cortices implies potentially enhanced glutamatergic signaling in pyramidal neurons under Chrm1 loss condition. The findings provide novel mechanistic insights into Chrm1 loss with the appearance of mitochondrial pathophysiological deficits in Alzheimer’s disease.

Hippocampal versus cortical deletion of cholinergic receptor muscarinic 1 in mice differentially affects post-translational modifications and supramolecular assembly of respiratory chain-associated proteins, mitochondrial ultrastructure, and respiration: implications in Alzheimer’s disease

“…The reduced Atp5a oligomerization in Chrm1 −/− cortical mitochondria corresponded with abnormal cristae structure in the cortical neuropil synaptic bouton mitochondria, supporting a role for Chrm1 in regulating cristae shape by controlling ATP synthase oligomerization ( Sabbir et al, 2023 ). Our study established a cause-and-effect relationship between Chrm1 signaling loss and mitochondrial structural-functional deficits in cortical neurons ( Sabbir et al, 2023 ). In light of these findings, the objective of this study is to analyze molecular, structural, and physiological characteristics of hippocampal mitochondria in Chrm1 −/− and wild-type mice to understand the molecular basis of any brain region-specific effect on mitochondrial phenotypes and to extrapolate those findings to our retrospective human brain region-specific observations to determine why hippocampal CHRM1 loss was not associated with poor survival of Alzheimer’s patient ( Sabbir et al, 2022 ).…”

“…A Chrm1 promoter-trapped enhanced green fluorescence protein (eGFP) reporter mouse-based study under the Gene Expression Nervous System Atlas (GENSAT) project revealed that Chrm1 is expressed in mouse hippocampal pyramidal neurons ( Tomishima et al, 2007 ; Schmidt et al, 2013 ) ( Figures 1A, B ). Therefore, to study the effect of Chrm1 loss in hippocampal mitochondrial structure and function, we harvested EHMFs using a differential centrifugation technique that was extensively validated in our previous studies ( Sabbir, 2019 ; Sabbir et al, 2021 ; Sabbir et al, 2023 ) for effectiveness in isolation and enrichment of ECMFs from mouse brain ( Sabbir et al, 2023 ). TEM images of the EHMF revealed the presence of free isolated mitochondria ( Supplementary Figures S1A, B , blue arrows) as well as pre- (electron-dense and potential neurotransmitter containing vesicles, Supplementary Figure S1B , green arrows) and postsynaptic (relatively less electron-dense with few vesicular structures, Supplementary Figure S1B , orange arrows) dendritic membrane-enclosed mitochondria ( Supplementary Figure S1B , pink and yellow arrows, respectively).…”

“…Enriched hippocampal mitochondrial fractions (EHMFs) were prepared from freshly harvested adult male mice brains by a method previously described by Sabbir et al (2020) , Sabbir et al (2021) and Sabbir et al (2023) . The hippocampus was first diced on ice and added to a mitochondrial isolation buffer (MIB) containing 70 mM sucrose, 210 mM mannitol, 5 mM HEPES pH 7.2, and 1 mM EGTA.…”

“…Therefore, to understand the role of CHRM1 to sustain normal mitochondrial function, mitochondrial pathophysiologies arising from the loss of CHRM1 may inform the mechanisms leading to the progression of AD. Recently, we characterized the molecular biology of cortical mitochondria in Chrm1 deleted (Chrm1 −/− ) transgenic mice ( Sabbir et al, 2023 ). Our findings indicate that Chrm1 loss severely reduced cortical mitochondrial respiration, associated with a significantly decreased supramolecular assembly of OXPHOS-associated protein complexes, specifically the Atp5a subunit of ATP synthase.…”

“…Additionally, an abnormal cortical mitochondrial ultrastructure was revealed. These are important regulators of mitochondrial function ( Sabbir et al, 2023 ). It has been suggested that long rows of oligomerized ATP synthase dimers facilitate the convexity of the mitochondrial inner membrane at the apex of the cristae shaping its structure and appearance ( Nesci and Pagliarani, 2019 ).…”