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

Effects of aflatoxin contaminated feed on the fingerlings of tilapia (Oreochromis niloticus Linnaeus, 1758)

Aflatoxin contamination, particularly common in cultured fishes in Asian countries, are considered unsafe both for fish and human health. However, the presence of aflatoxin in cultured fish feed and their effect are still under estimated in Bangladesh. The present study aimed to assess the effects of aflatoxin on growth performance and residues in tilapia (Oreochromis niloticus) fingerlings. Fish feed were treated with several concentration of aflatoxin as 0 ppb (T0, control), 25 ppb (T1), 50 ppb (T2) and 100 ppb (T3) and fed the tilapia fingerlings (n=10) in individual glass aquaria (24×12×12 inch, 105-litre capacity) conditions for 12 weeks. Comparatively higher body length (cm) and weight gain (g) were observed in treatment T0 (1.68 and 4.98) and T1 (1.60 and 5.48) than those of treatment T2 (1.31 and 4.06) and T3 (1.20 and 3.10), respectively. The specific growth rate (SGR) were almost similar in treatment T0 (52%), T1 (51%) and T2 (52%) whereas declined significantly (p<0.05) in T3 (39%). Higher survival rate was also demonstrated in treatment T0 (90%) and T1 (90%) whereas significantly decreased in treatment T2 (60%) and T3 (40%). The residue of aflatoxin was not detected in T0 and T1. On the contrary, the residual effect in tilapia fingerling was evident in T2 and T3 treatment. The findings of the present study revealed that aflatoxin contaminated feed is harmful for the growth performance and survival of O. niloticus fingerlings. Further study is necessary to safeguard the aquaculture production as well as to produce healthy food for human consumption

Universal logic with encoded spin qubits in silicon

Qubits encoded in a decoherence-free subsystem and realized in exchange-coupled silicon quantum dots are promising candidates for fault-tolerant quantum computing. Benefits of this approach include excellent coherence, low control crosstalk, and configurable insensitivity to certain error sources. Key difficulties are that encoded entangling gates require a large number of control pulses and high-yielding quantum dot arrays. Here we show a device made using the single-layer etch-defined gate electrode architecture that achieves both the required functional yield needed for full control and the coherence necessary for thousands of calibrated exchange pulses to be applied. We measure an average two-qubit Clifford fidelity of $97.1 \pm 0.2\%$ with randomized benchmarking. We also use interleaved randomized benchmarking to demonstrate the controlled-NOT gate with $96.3 \pm 0.7\%$ fidelity, SWAP with $99.3 \pm 0.5\%$ fidelity, and a specialized entangling gate that limits spreading of leakage with $93.8 \pm 0.7\%$ fidelity

Loss of Cholinergic Receptor Muscarinic 1 (CHRM1) Protein in the Hippocampus and Temporal Cortex of a Subset of Individuals with Alzheimer\u27s Disease, Parkinson\u27s Disease, or Frontotemporal Dementia: Implications for Patient Survival

BACKGROUND: Dysfunction of cholinergic neurotransmission is a hallmark of Alzheimer\u27s 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\u27s 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 (\u3c50%) 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 (\u3c65-75 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

Muscarinic acetylcholine type 1 receptor constrains neurite outgrowth by inhibiting microtubule polymerization and mitochondrial trafficking in adult sensory neurons

<p><b>Supplementary Figure 1: </b><b>M1R overexpression exhibited spontaneous basal activity in Arrestin recruitment assay</b> A: Graphical representation of the of the Arrestin recruitment assay (TANGO) strategy, TRE-Tet response element. HA-cleavable signal sequence to promote membrane localization, FLAG-epitope tag, TEV- Tobacco Etch Virus cleavage site, V2 tail: C-terminus of the V₂ vasopressin receptor (V₂ tail) to promote arrestin recruitment and tTA- tetracyclin transactivator. B: Scatter plot showing the RLU (Relative Luminescence Units) for the drug treated HTLA cells. N=5 independent experiments. Data represented as Mean±SEM, p value by One-way ANOVA followed by multiple comparison test.<b></b></p><p><b> </b></p><p><b>Supplementary Figure 2</b>: <b>Actin and tubulin cytoskeleton in wild type and Gα12/13 KO cells</b>. A: Confocal immunofluorescent images showing F-actin (phalloidin stained, Left panel) and tubulin (α-Tubulin immunolabelled, right panel) cytoskeleton in wild type and Gα12/13 KO cells. Blue arrow indicates focal adhesions, yellow arrow indicates extended cytoplasmic processes enriched in tubulin. Scale bar: 10µm. B-C: Confocal immunofluorescent images showing F-actin and α-tubulin in GFP-M1R overexpressed wild type and Gα12/13 KO cells. Scale bar: 20µm</p><p><b> </b></p><p><b>Supplementary Figure 3: Effect of carbachol on tubulin cytoskeleton in M1R expressed wild type and Gα12/13 KO cells. </b>Confocal immunofluorescence images showing tubulin cytoskeleton. Yellow arrow indicates crest of the cell with dense pack of tubulin. white and blue arrow indicates elongated cytoplasmic processes enriched in tubulin (white) or GFP-M1R (blue). scale bar: 20µm</p><p> </p><p> </p> <p> </p

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\u27s disease

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\u27s patients was associated with poor survival, whereas similar loss in the hippocampus showed no such association. Mitochondrial dysfunction underlies Alzheimer\u27s 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\u27s 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. 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. 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. 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

Dlc1 mediated cell morphological changes are associated with increased Rac1 activation and interaction with non-muscle myosin heavy chain II-A protein (Myh9)

<p>GFP tagged Dlc1 isform1 was expressed in OC-033 cell line and Imaged for observing cellular morpholgy and protein localization in the cell.</p

Dlc1 mediated cell morphological changes are associated with increased Rac1 activation and interaction with non-muscle myosin heavy chain II-A protein (Myh9)

<p>GFP tagged Dlc1 isform3 was expressed in OC-033 cell line and Imaged for observing cellular morpholgy and protein localization in the cell.</p