CAMKK2 regulates mitochondrial function by controlling succinate dehydrogenase expression, post-translational modification, megacomplex assembly, and activity in a cell-type-specific manner.

BACKGROUND: The calcium (Ca2+)/calmodulin (CAM)-activated kinase kinase 2 (CAMKK2)-signaling regulates several physiological processes, for example, glucose metabolism and energy homeostasis, underlying the pathogenesis of metabolic diseases. CAMKK2 exerts its biological function through several downstream kinases, therefore, it is expected that depending on the cell-type-specific kinome profile, the metabolic effects of CAMKK2 and its underlying mechanism may differ. Identification of the cell-type-specific differences in CAMKK2-mediated glucose metabolism will lead to unravelling the organ/tissue-specific role of CAMKK2 in energy metabolism. Therefore, the objective of this study was to understand the cell-type-specific regulation of glucose metabolism, specifically, respiration under CAMKK2 deleted conditions in transformed human embryonic kidney-derived HEK293 and hepatoma-derived HepG2 cells. METHODS: Cellular respiration was measured in terms of oxygen consumption rate (OCR). OCR and succinate dehydrogenase (SDH) enzyme activity were measured following the addition of substrates. In addition, transcription and proteomic and analyses of the electron transport system (ETS)-associated proteins, including mitochondrial SDH protein complex (complex-II: CII) subunits, specifically SDH subunit B (SDHB), were performed using standard molecular biology techniques. The metabolic effect of the altered SDHB protein content in the mitochondria was further evaluated by cell-type-specific knockdown or overexpression of SDHB. RESULTS: CAMKK2 deletion suppressed cellular respiration in both cell types, shifting metabolic phenotype to aerobic glycolysis causing the Warburg effect. However, isolated mitochondria exhibited a cell-type-specific enhancement or dampening of the respiratory kinetics under CAMKK2 deletion conditions. This was mediated in part by the cell-type-specific effect of CAMKK2 loss-of-function on transcription, translation, post-translational modification (PTM), and megacomplex assembly of nuclear-encoded mitochondrial SDH enzyme complex subunits, specifically SDHB. The cell-type-specific increase or decrease in SDHs protein levels, specifically SDHB, under CAMKK2 deletion condition resulted in an increased or decreased enzymatic activity and CII-mediated respiration. This metabolic phenotype was reversed by cell-type-specific knockdown or overexpression of SDHB in respective CAMKK2 deleted cell types. CAMKK2 loss-of-function also affected the overall assembly of mitochondrial supercomplex involving ETS-associated proteins in a cell-type-specific manner, which correlated with differences in mitochondrial bioenergetics. CONCLUSION: This study provided novel insight into CAMKK2-mediated cell-type-specific differential regulation of mitochondrial function, facilitated by the differential expression, PTMs, and assembly of SDHs into megacomplex structures. Video Abstract.

CAMKK2-CAMK4 signaling regulates transferrin trafficking, turnover, and iron homeostasis.

BACKGROUND: Circulatory iron is a hazardous biometal. Therefore, iron is transported in a redox-safe state by a serum glycoprotein - transferrin (TF). Different organs acquire iron from the systemic circulation through a tightly regulated mechanism at the blood-tissue interface which involves receptor-mediated internalization of TF. Thus, abnormal TF trafficking may lead to iron dyshomeostasis associated with several diseases including neurodegeneration. Iron -induced toxicity can cause neuronal damage to iron-sensitive brain regions. Recently, it was discovered that CAMKK2, a calcium (Ca2+)/calmodulin-activated kinase, controls receptor-mediated TF trafficking in mouse tissues, specifically in the brain. The biological function of CAMKK2 is mediated through multiple downstream effectors. Both CAMKK2 and one of its downstream kinase, CAMK4, exhibit overlapping expression in mouse brain. The role of CAMK4 in vesicular transport has been reported and loss of CAMKK2 or CAMK4 leads to cognitive defects in mouse. Therefore, it was hypothesized that CAMKK2-CAMK4 signaling regulates receptor-mediated TF trafficking and iron homeostasis which may be responsible for the neuronal malfunction observed in CAMKK2- or CAMK4-deficient mice. METHODS: CAMK4-/- mouse was used to study tissue-specific turnover of TF, TF-receptor (TFRC) and iron. CRISPR/Cas9-based CAMKK2 and/or CAMK4 deleted human embryonic kidney-derived HEK293 cell clones were used to study the molecular defects in receptor-mediated TF trafficking. Further, a "zero functional G protein" condition in HEK293 cell was exploited to study CAMKK2-CAMK4 signaling-mediated regulation of intracellular Ca2+ homeostasis which was linked to calcium signaling during TF trafficking. RESULTS: Loss of CAMK4 leads to abnormal post-translational modifications (PTMs) and turnover of TF in mouse cerebellum and liver which was associated with iron dyshomeostasis in these tissues. The HEK293 cell-based study revealed that the absence of CAMKK2-CAMK4 signaling altered intracellular Ca2+ homeostasis and lead to abnormal calcium signaling during TF trafficking. Also, CAMKK2-CAMK4 signaling deficiency affected the molecular interaction of TF and TF-receptor-associated protein complexes which indicated a potential failure in the recruitment of interacting proteins due to differential PTMs in TF. CONCLUSION: Overall, this study established a novel mechanistic link between intracellular Ca2+ level, receptor-mediated TF trafficking, and iron homeostasis, all regulated by CAMKK2-CAMK4 signaling. Video Abstract.

Cholinergic Receptor Muscarinic 1 Co-Localized with Mitochondria in Cultured Dorsal Root Ganglion Neurons, and Its Deletion Disrupted Mitochondrial Ultrastructure in Peripheral Neurons: Implications in Alzheimer's Disease.

Background: Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) has been linked to the pathogenesis of Alzheimer's disease (AD). Our recent study found significantly lower CHRM1 protein levels in AD patient cortices, linked to reduced survival. Furthermore, using knockout mice (Chrm1-/-) we demonstrated that deletion of Chrm1 alters cortical mitochondrial structure and function, directly establishing a connection between its loss and mitochondrial dysfunction in the context of AD. While CHRM1's role in the brain has been extensively investigated, its impact on peripheral neurons in AD remains a crucial area of research, especially considering reported declines in peripheral nerve conduction among AD patients. Objective: The objective was to characterize Chrm1 localization and mitochondrial deficits in Chrm1-/- dorsal root ganglion (DRG) neurons. Methods: Recombinant proteins tagged with Green or Red Fluorescent Protein (GFP/RFP) were transiently expressed to investigate the localization of Chrm1 and mitochondria, as well as mitochondrial movement in the neurites of cultured primary mouse DRG neurons, using confocal time-lapse live cell imaging. Transmission electron microscopy was performed to examine the ultrastructure of mitochondria in both wild-type and Chrm1-/- DRGs. Results: Fluorescence imaging revealed colocalization and comigration of N-terminal GFP-tagged Chrm1 and mitochondrial localization signal peptide-tagged RFP-labelled mitochondria in the DRGs neurons. A spectrum of mitochondrial structural abnormalities, including disruption and loss of cristae was observed in 87% neurons in Chrm1-/- DRGs. Conclusions: This study suggests that Chrm1 may be localized in the neuronal mitochondria and loss of Chrm1 in peripheral neurons causes sever mitochondrial structural aberrations resembling AD pathology.

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

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.

Introduction: In a previous retrospective study using postmortem human brain tissues, we demonstrated that loss of Cholinergic Receptor Muscarinic 1 (CHRM1) in the temporal cortex of a subset of Alzheimer's patients was associated with poor survival, whereas similar loss in the hippocampus showed no such association. Mitochondrial dysfunction underlies Alzheimer's pathogenesis. Therefore, to investigate the mechanistic basis of our findings, we evaluated cortical mitochondrial phenotypes in Chrm1 knockout (Chrm1-/-) mice. Cortical Chrm1 loss resulted in reduced respiration, reduced supramolecular assembly of respiratory protein complexes, and caused mitochondrial ultrastructural abnormalities. These mouse-based findings mechanistically linked cortical CHRM1 loss with poor survival of Alzheimer's patients. However, evaluation of the effect of Chrm1 loss on mouse hippocampal mitochondrial characteristics is necessary to fully understand our retrospective human tissue-based observations. This is the objective of this study. Methods: Enriched hippocampal and cortical mitochondrial fractions (EHMFs/ECMFs, respectively) derived from wild-type and Chrm1-/- mice were used to measure respiration by quantifying real-time oxygen consumption, supramolecular assembly of oxidative phosphorylation (OXPHOS)-associated proteins by blue native polyacrylamide gel electrophoresis, post-translational modifications (PTMs) by isoelectric focusing (IEF), and mitochondrial ultrastructure by electron microscopy. Results: In contrast to our previous observations in Chrm1-/- ECMFs, EHMFs of Chrm1-/- mice significantly increased respiration with a concomitant increase in the supramolecular assembly of OXPHOS-associated proteins, specifically Atp5a and Uqcrc2, with no mitochondrial ultrastructural alterations. IEF of ECMFs and EHMFs from Chrm1-/- mice showed a decrease and an increase, respectively in a negatively charged (pH∼3) fraction of Atp5a relative to the wild-type mice, with a corresponding decrease or increase in the supramolecular assembly of Atp5a and respiration indicating a tissue-specific signaling effect. Discussion: Our findings indicate that loss of Chrm1 in the cortex causes structural, and physiological alterations to mitochondria that compromise neuronal function, whereas Chrm1 loss in the hippocampus may benefit neuronal function by enhancing mitochondrial function. This brain region-specific differential effect of Chrm1 deletion on mitochondrial function supports our human brain region-based findings and Chrm1-/- mouse behavioral phenotypes. Furthermore, our study indicates that Chrm1-mediated brain region-specific differential PTMs of Atp5a may alter complex-V supramolecular assembly which in turn regulates mitochondrial structure-function.

Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) Protein in the Hippocampus and Temporal Cortex of a Subset of Individuals with Alzheimer's Disease, Parkinson's Disease, or Frontotemporal Dementia: Implications for Patient Survival.

BACKGROUND: Dysfunction of cholinergic neurotransmission is a hallmark of Alzheimer's disease (AD); forming the basis for using acetylcholine (ACh) esterase (AChE) inhibitors to mitigate symptoms of ACh deficiency in AD. The Cholinergic Receptor Muscarinic 1 (CHRM1) is highly expressed in brain regions impaired by AD. Previous analyses of postmortem AD brains revealed unaltered CHRM1 mRNA expression compared to normal brains. However, the CHRM1 protein level in AD and other forms of dementia has not been extensively studied. Reduced expression of CHRM1 in AD patients may explain the limited clinical efficacy of AChE inhibitors. OBJECTIVE: To quantify CHRM1 protein in the postmortem hippocampus and temporal cortex of AD, Parkinson's disease (PD), and frontotemporal dementia (FTD) patients. METHODS: Western blotting was performed on postmortem hippocampus (N = 19/73/7/9: unaffected/AD/FTD/PD) and temporal cortex (N = 9/74/27: unaffected/AD/PD) using a validated anti-CHRM1 antibody. RESULTS: Quantification based on immunoblotting using a validated anti-CHRM1 antibody revealed a significant loss of CHRM1 protein level (<50%) in the hippocampi (78% AD, 66% PD, and 85% FTD) and temporal cortices (56% AD and 42% PD) of dementia patients. Loss of CHRM1 in the temporal cortex was significantly associated with early death (<65-75 years) for both AD and PD patients. CONCLUSION: Severe reduction of CHRM1 in a subset of AD and PD patients can explain the reported low efficacy of AChE inhibitors as a mitigating treatment for dementia patients. Based on this study, it can be suggested that future research should prioritize therapeutic restoration of CHRM1 protein levels in cholinergic neurons.

Antisense overlapping long non-coding RNA regulates coding arachidonate 12-lipoxygenase gene by translational interference.

The arachidonate 12-lipoxygenase (ALOX12) enzyme catalyzes polyunsaturated fatty acids and facilitates generation of bioactive lipid mediators associated with various biological processes and disease pathologies. The human genome assembly revealed that the ALOX12 gene overlaps an antisense non-coding gene designated as ALOX12-antisense 1 (ALOX12-AS1). This arrangement indicates that the uncharacterized ALOX12-AS1 long non-coding RNA (lncRNA) may bind to the sense coding ALOX12 mRNA to form an antisense-sense duplex providing the basis of a novel ALOX12 regulatory mechanism. Therefore, this study was designed to determine whether the interaction of ALOX12-AS1 with ALOX12 mRNA functions as an anti-sense/sense duplex-mediated regulatory mechanism controlling the cellular content of ALOX12. Our findings indicate that two major isoforms of ALOX12-AS1 lncRNA are ubiquitously expressed in a variety of primary adult human tissues and different transformed cell types. RNA-FISH revealed cell-type-specific cytosolic as well as nuclear and nucleolar localization of the lncRNA. Interestingly, phorbol ester-induced nucleo-cytoplasmic translocation of the lncRNA in monocytic THP-1 cells resulted in a reduction of ALOX12 protein without a concomitant change in its mRNA level. This indicated ALOX12-AS1 operates via an antisense-sense duplex-mediated translational downregulation mechanism. This deduction was validated by demonstrating sense/antisense duplex formation and an association of the duplex with ribosomal proteins in HEK293 cells. Overall, this study revealed a hitherto unknown mechanism of antisense lncRNA-mediated translational downregulation of ALOX12 that adds to the existing regulatory mechanisms for the modulation of potent bioactive lipid mediators that contribute to both health and disease.

Loss of ß-Arrestins or six Gα proteins in HEK293 cells caused Warburg effect and prevented progesterone-induced rapid proteasomal degradation of progesterone receptor membrane component 1.

Hormonal dysregulation plays a significant role in the metabolic switching during malignant transformation. Progesterone Receptor Membrane Component 1 (PGRMC1) is a single-pass transmembrane receptor activated by the binding of progesterone (P4), a sex hormone. In a previous study, P4 treatment caused rapid (within 30 min) induction of aerobic glycolysis in transformed HEK293 cells, a hallmark malignant phenotype known as the Warburg effect. This metabolic reprogramming was associated with the proteasomal degradation of a 70 kilodalton (kDa) PGRMC1. PGRMC1 interacts with a variety of proteins, including G protein-coupled receptors (GPCRs) and P4-PGRMC1 signaling modulates cyclic adenosine monophosphate (cAMP) production. Therefore, we hypothesized that the P4-induced Warburg effect and proteasomal degradation of PGRMC1 involve G proteins and ß-Arrestins (ARRBs). In the present study, we investigated P4-induced aerobic glycolysis, proteasomal degradation of p70 PGRMC1, as well as abundance and subcellular translocation of PGRMC1 along with two key glycolytic enzymes Hexokinase 1 (HK1) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) in six Gα subunit (Gsix) proteins or ARRB1/2-deficient HEK293 cells. Loss of ARRB1/2 or Gsix proteins inhibited P4-induced p70 PGRMC1 degradation but failed to prevent the P4-induced Warburg effect. Also, deficiency of ARRB1/2 or Gsix proteins differentially affected the basal as well as P4-induced abundance and subcellular translocation of PGRMC1, HK1, and GAPDH proteins. Overall, the findings indicate that P4-PGRMC1-mediated metabolic reprogramming in HEK293 cells depends on ß-Arrestins and Gα proteins suggesting the involvement of an underlying GPCR signal transduction pathway.

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: clinical and prognostic significance.

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin alpha RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion/promoter methylation/expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion/methylation) of hMLH1, ITGA9, and RBSP3 were high (45-55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT-PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5-aza-2'-deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less-active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low/intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco-addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck.

Hypomorphic CAMKK2 in EA.hy926 endothelial cells causes abnormal transferrin trafficking, iron homeostasis and glucose metabolism.

We recently reported that loss of calcium/calmodulin-dependent protein kinase kinase-2 (CAMKK2), a serine/threonine kinase activated by intracellular calcium, in mice leads to tissue-specific aberrant turnover of transferrin (TF), a receptor-mediated iron-transporter that supplies iron to tissues. Iron dyshomeostasis is associated with the pathogenesis of several diseases, making TF transport relevant to health. In this study, we used hemizygous CAMKK2 hypomorphic human endothelial cell (EA.hy926) clones to demonstrate that cells with reduced CAMKK2 exhibit increased TF uptake and transcytosis, and decreased intracellular trafficking to subcellular organelles compared to wild-type. The abnormal TF trafficking in CAMKK2 hypomorphic cells correlated with a reduction in intracellular iron content and defective glucose metabolism including glycolysis and mitochondrial respiration. CAMKK2 deficiency also caused reduction in GAPDH and VDAC1 protein level which correlated to defective bioenergetics function. These findings have identified a novel mechanistic link between abnormal calcium signaling, iron dyshomeostasis and metabolic dysfunction involving CAMKK2.

Muscarinic Toxin 7 Signals Via Ca2+/Calmodulin-Dependent Protein Kinase Kinase ß to Augment Mitochondrial Function and Prevent Neurodegeneration.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M1R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca2+/calmodulin-dependent protein kinase kinase ß (CaMKKß) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKß to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M1R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 µM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 µM) or short hairpin RNA to CaMKKß. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 µM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M1R-modulated, CaMKKß-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Progesterone induced Warburg effect in HEK293 cells is associated with post-translational modifications and proteasomal degradation of progesterone receptor membrane component 1.

Progesterone (P4) is a major steroid hormone that has important effects on metabolism. The progesterone receptor membrane component 1 (PGRMC1) is a non-canonical P4 binding protein. The biological functions affected by PGRMC1 include cholesterol/steroid biosynthesis and metabolism, iron homeostasis and heme trafficking, autophagy, regulation of cell cycle and proliferation, cell migration and invasion. PGRMC1 has been an attractive target for therapeutic intervention in cancer and neurodegenerative disorders due to its biological role in promoting cell survival. P4 has been used in a number of clinical applications and is considered neuroprotective. The involvement of PGRMC1 in P4-mediated regulation of cellular glucose metabolism is not well studied. PGRMC1 is a 21 kDa protein but complex post-translational modifications (PTMs) lead to the existence of several high molecular mass proteins whose molecular function, intracellular distribution, and physiological relevancies are not fully known. Therefore, in this study, P4-PGRMC1-mediated cellular glucose metabolism and PTMs of PGRMC1 were studied using wild-type and CRISPR/Cas9 mediated PGRMC1 knockout (KO) human embryonic kidney-derived (HEK293) cell lines. A 70 kDa (p70) and 100 kDa (p100) PGRMC1 proteins were identified that are predominantly associated with endoplasmic reticulum/mitochondria and nuclear fractions in the cells, respectively. Phosphorylation, acetylation, ubiquitination, and sumoylation of native PGRMC1 under serum starvation were identified which provided an explanation for the higher molecular masses. This study indicates that P4-PGRMC1 signaling caused a rapid increase in glycolysis in the presence of oxygen (aerobic glycolysis) and a corresponding decrease in cellular respiration, known as the Warburg effect. Further, it was demonstrated that the P4-induced increase in glycolysis is associated with rapid proteasomal degradation of the p70 and reduction of the nuclear p100 protein level. P4 treatment also caused significant alteration in the dynamics of PGRMC1 PTMs and its association with potential interacting proteins. Overall, this study provides a hitherto unknown aspect of P4-PGRMC1 mediated signaling that changes basic cellular metabolism in HEK293 cells.

Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells.

Bone marrow-derived allogeneic (donor derived) mesenchymal stem cells (MSCs) are immunoprivileged and are considered to be prominent candidates for regenerative therapy for numerous degenerative diseases. Even though the outcome of initial allogeneic MSCs based clinical trials was encouraging, the overall enthusiasm, of late, has dimmed down. This is due to failure of long-term survival of transplanted cells in the recipient. In fact, recent analyses of allogeneic MSC-based studies demonstrated that cells after transplantation turned immunogenic and were subsequently rejected by host immune system. The current study reveals a novel mechanism of immune switch in MSCs. We demonstrate that hypoxia, a common denominator of ischemic tissues, induces an immune shift in MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by downregulation or the absence of major histocompatibility complex class II (MHC-II) molecules. We found that 26S proteasome-mediated intracellular degradation of MHC-II helps maintain the absence of MHC-II expression on cell surface in normoxic MSCs and preserves their immunoprivilege. The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC-II and, as a result, the MSCs became immunogenic. Furthermore, we found that hypoxia-induced decrease in the levels of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs preserved the immunoprivilege of MSCs. Therefore, hypoxia-induced inactivation of 26S proteasome assembly instigates loss of immunoprivilege of allogeneic mesenchymal stem cells. Maintaining 26S proteasome activity in mesenchymal stem cells preserves their immunoprivilege.

Loss of Ca2+/Calmodulin Dependent Protein Kinase Kinase 2 Leads to Aberrant Transferrin Phosphorylation and Trafficking: A Potential Biomarker for Alzheimer's Disease.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine kinase that is activated following an increase in the intracellular Ca2+ concentration and activates multiple signaling cascades that control physiologically important neuronal processes. CaMKK2 has been implicated in schizophrenia, bipolar disease, neurodegeneration, and cancer. Using isoelectric focusing (IEF) and mass spectrometry-based proteomic analysis, it was found that knockdown (KD) of CaMKK2 in cultured adult primary dorsal root ganglion (DRG) neurons resulted in the reduction of transferrin (TF) phosphorylation at multiple functionally relevant residues which corresponded to loss of an acidic fraction (pH~3-4) of TF. In vitro studies using CRISPR/Cas9 based CaMKK2 knockout (KO) HEK293 and HepG2 cells lines validated previous findings and revealed that loss of CaMKK2 interfered with TF trafficking and turnover. TF is an iron transporter glycoprotein. Abnormal accumulation of iron and/or deregulated Ca2+ homeostasis leads to neurodegeneration in Alzheimer's disease (AD). Therefore, it was hypothesized that aberrant CaMKK2 in AD may lead to aberrant phosphorylated transferrin (P-TF: pH~3-4 fraction) which may serve as a hallmark biomarker for AD. A significant reduction of P-TF in the brain and serum of CaMKK2 KO mice and a triple-transgenic mouse model of AD (3xTg-AD) supported this hypothesis. In addition, analysis of early (< 65 years) and late-stage (>65 years) postmortem human AD cerebrospinal fluid (CSF) and serum samples revealed that aberrant P-TF (pH~3-4 fraction) profile was associated with both early and late-stage AD compared to age-matched controls. This indicates P-TF (pH~3-4 fraction) profile may be useful as a minimally invasive biomarker for AD. In addition, this study provides a link between aberrant CaMKK2 with TF trafficking and turnover which provides a novel insight into the neurodegeneration process.

Muscarinic receptor antagonists activate ERK-CREB signaling to augment neurite outgrowth of adult sensory neurons.

A major cellular effector activated by G protein coupled receptors is extracellular signal-regulated kinase (ERK). The ERK signaling cascade regulates a variety of cellular processes including growth and proliferation. Both G protein and ß-arrestin-mediated signaling lead to ERK activation by phosphorylation through different kinases. Recently, we have shown muscarinic acetylcholine type 1 receptor (M1R) antagonists, muscarinic toxin 7 (MT7) and pirenzepine, elevated neurite outgrowth and protected from small and large fiber neuropathy in adult sensory neurons in various animal models. Thus, we tested the novel hypothesis that muscarinic antagonists could drive neurite outgrowth through altered M1R-ERK signaling. We have used two dimensional isoelectric focusing/SDS-PAGE combined with analysis using multiple phospho-epitope specific antibodies to study ERK1/2 phosphorylation and activation of its downstream nuclear effector cyclic response element binding protein (CREB). Activated CREB is known to exhibit neuroprotective and growth promoting effects. One hour of treatment with MT7 and pirenzepine activated ERK through M1R and induced a significant increase in levels of pCREB(S133) in cultured sensory neurons. Further, pharmacological blockade or siRNA based knockdown of ERK abolished the MT7 and pirenzepine mediated neuritogenic effect. In addition, we have shown drug-induced alterations of charged protein fractions that may possess additional post-translationally modified forms of ERK and CREB. For the first time we show that long-term treatment, e.g. 1 h, with muscarinic antagonists selective or specific for M1R can activate a biased ß-arrestin dependent ERK-CREB signal cascade. Our study gives novel insight into muscarinic antagonist-mediated modulation of M1R-ERK-CREB signaling which could be exploited for therapy in neuropathic diseases.

High glucose concentration suppresses a SIRT2 regulated pathway that enhances neurite outgrowth in cultured adult sensory neurons.

In peripheral nerve under hyperglycemic conditions high flux of d-glucose through the polyol pathway drives an aberrant redox state contributing to neurodegeneration in diabetic sensorimotor polyneuropathy (DSPN). Sirtuins, including SIRT2, detect the redox state via the NAD+/NADH ratio to regulate mitochondrial function via, in part, AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). In adult dorsal root ganglia (DRG) sensory neurons mitochondrial dysfunction has been proposed as an etiological factor in dying-back neuropathy in diabetes. We tested the hypothesis that a high concentration of d-glucose depleted SIRT2 expression via enhancement of polyol pathway activity. We posited that this would lead to impaired mitochondrial function and suppression of neurite outgrowth in cultured sensory neurons. The use of dominant negative mutants or neurons from SIRT2 knockout (KO) mice to block SIRT2 signaling revealed that neurons derived from control or type 1 diabetic rodents required SIRT2 for optimal neurite outgrowth. Over-expression of WT-SIRT2 elevated neurite outgrowth in normal and diabetic cultures. SIRT2 protein isoforms 2.1 and 2.2 were reduced by 20-30% in DRG of type 1 diabetic mice (p < .05). After 72 h exposure to high d-glucose (25 mM vs 5 mM) cultured sensory neurons showed a significant 2-fold (p < .05) decrease in SIRT2 expression, P-AMPK, levels of respiratory Complexes II/III and respiratory capacity. DRG neurons expressed aldose reductase and the aforementioned deficits were prevented by treatment with aldose reductase inhibitors (lidorestat or sorbinil) or sorbitol dehydrogenase inhibitor (SDI-158). In cultures derived from type 1 diabetic rats treatment with SDI-158 elevated expression of SIRT2, P-AMPK/PGC-1α and neurite outgrowth (p < .05). SIRT2 KO neurons exhibited deficits in the LKB-1/AMPK/PGC-1α pathway and mitochondrial function. In cultured neurons the SIRT2 pathway enhances axonal outgrowth and this signaling axis encompassing activation of AMPK/PGC-1α is impaired in DSPN, in part, due to enhanced polyol pathway activity caused by hyperglycemia.

Insulin prevents aberrant mitochondrial phenotype in sensory neurons of type 1 diabetic rats.

Diabetic neuropathy affects approximately 50% of diabetic patients. Down-regulation of mitochondrial gene expression and function has been reported in both human tissues and in dorsal root ganglia (DRG) from animal models of type 1 and type 2 diabetes. We hypothesized that loss of direct insulin signaling in diabetes contributes to loss of mitochondrial function in DRG neurons and to development of neuropathy. Sensory neurons obtained from age-matched adult control or streptozotocin (STZ)-induced type 1 diabetic rats were cultured with or without insulin before determining mitochondrial respiration and expression of mitochondrial respiratory chain and insulin signaling-linked proteins. For in vivo studies age-matched control rats and diabetic rats with or without trace insulin supplementation were maintained for 5months before DRG were analyzed for respiratory chain gene expression and cytochrome c oxidase activity. Insulin (10nM) significantly (P<0.05) increased phosphorylation of Akt and P70S6K by 4-fold and neurite outgrowth by 2-fold in DRG cultures derived from adult control rats. Insulin also augmented the levels of selective mitochondrial respiratory chain proteins and mitochondrial bioenergetics parameters in DRG cultures from control and diabetic rats, with spare respiratory capacity increased by up to 3-fold (P<0.05). Insulin-treated diabetic animals exhibited improved thermal sensitivity in the hind paw and had increased dermal nerve density compared to untreated diabetic rats, despite no effect on blood glucose levels. In DRG of diabetic rats there was suppressed expression of mitochondrial respiratory chain proteins and cytochrome c oxidase activity that was corrected by insulin therapy. Insulin elevates mitochondrial respiratory chain protein expression and function in sensory neurons and this is associated with enhanced neurite outgrowth and protection against indices of neuropathy.

Selective antagonism of muscarinic receptors is neuroprotective in peripheral neuropathy.

Sensory neurons have the capacity to produce, release, and respond to acetylcholine (ACh), but the functional role of cholinergic systems in adult mammalian peripheral sensory nerves has not been established. Here, we have reported that neurite outgrowth from adult sensory neurons that were maintained under subsaturating neurotrophic factor conditions operates under cholinergic constraint that is mediated by muscarinic receptor-dependent regulation of mitochondrial function via AMPK. Sensory neurons from mice lacking the muscarinic ACh type 1 receptor (M1R) exhibited enhanced neurite outgrowth, confirming the role of M1R in tonic suppression of axonal plasticity. M1R-deficient mice made diabetic with streptozotocin were protected from physiological and structural indices of sensory neuropathy. Pharmacological blockade of M1R using specific or selective antagonists, pirenzepine, VU0255035, or muscarinic toxin 7 (MT7) activated AMPK and overcame diabetes-induced mitochondrial dysfunction in vitro and in vivo. These antimuscarinic drugs prevented or reversed indices of peripheral neuropathy, such as depletion of sensory nerve terminals, thermal hypoalgesia, and nerve conduction slowing in diverse rodent models of diabetes. Pirenzepine and MT7 also prevented peripheral neuropathy induced by the chemotherapeutic agents dichloroacetate and paclitaxel or HIV envelope protein gp120. As a variety of antimuscarinic drugs are approved for clinical use against other conditions, prompt translation of this therapeutic approach to clinical trials is feasible.

Mechanisms of Mitochondrial Dysfunction in Alzheimer's Disease.

Mitochondria are the primary source for energy generation in the cell, which manifests itself in the form of the adenosine triphosphate (ATP). Nicotinamide dinucleotide (NADH) molecules are the first to enter the so-called electron transport chain or ETC of the mitochondria. The ETC represents a chain of reducing agents organized into four major protein-metal complexes (I-IV) that utilize the flow of electrons to drive the production of ATP. An additional integral protein that is related to oxidative phosphorylation is ATP synthase, referred to as complex V. Complex V carries out ATP synthesis as a result of the electron flow through the ETC. The coupling of electron flow from NADH to molecular oxygen to the production of ATP represents a process known as oxidative phosphorylation. In this review, we describe mainly the bioenergetic properties of mitochondria, such as those found in the ETC that may be altered in Alzheimer's disease (AD). Increasing evidence points to several mitochondrial functions that are affected in AD. Furthermore, it is becoming apparent that mitochondria are a potential target for treatment in early-stage AD. With growing interest in the mitochondria as a target for AD, it has been hypothesized that deficit in this organelle may be at the heart of the progression of AD itself. The role of mitochondria in AD may be significant and is emerging as a main area of AD research.