Abstract Alzheimer disease (AD) is associated with increased amyloidogenic processing of amyloid precursor protein (APP) to β-amyloid peptides (Aβ), cholinergic neuron loss with decreased choline ...acetyltransferase (ChAT) activity, and cognitive dysfunction. Both 69-kDa ChAT and 82-kDa ChAT are expressed in cholinergic neurons in human brain and spinal cord with 82-kDa ChAT localized predominantly to neuronal nuclei, suggesting potential alternative functional roles for the enzyme. By gene microarray analysis, we found that 82-kDa ChAT-expressing IMR32 neural cells have altered expression of genes involved in diverse cellular functions. Importantly, genes for several proteins that regulate APP processing along amyloidogenic and non-amyloidogenic pathways are differentially expressed in 82-kDa ChAT-containing cells. The predicted net effect based on observed changes in expression patterns of these genes would be decreased amyloidogenic APP processing with decreased Aβ production. This functional outcome was verified experimentally as a significant decrease in BACE1 protein levels and activity and a concomitant reduction in the release of endogenous Aβ1–42 from neurons cultured from brains of AD-model APP/PS1 transgenic mice. The expression of 82-kDa ChAT in neurons increased levels of GGA3, which is involved in trafficking BACE1 to lysosomes for degradation. shRNA-induced decreases in GGA3 protein levels attenuated the 82-kDa ChAT-mediated decreases in BACE1 protein and activity and Aβ1–42 release. Evidence that 82-kDa ChAT can enhance GGA3 gene expression is shown by enhanced GGA3 gene promoter activity in SN56 neural cells expressing this ChAT protein. These studies indicate a novel relationship between cholinergic neurons and APP processing, with 82-kDa ChAT acting as a negative regulator of Aβ production. This decreased formation of Aβ could result in protection for cholinergic neurons, as well as protection of other cells in the vicinity that are sensitive to increased levels of Aβ. Decreasing levels of 82-kDa ChAT due to increasing age or neurodegeneration could alter the balance towards increasing Aβ production, with this potentiating the decline in function of cholinergic neurons.
Carotid artery disease (CAD) is associated with numerous risk factors, including hypertension, hyperlipidemia, hypercholesterolemia, diabetes mellitus, and smoking. These systemic risk factors do not ...affect the carotid arteries equally in most patients, resulting in asymmetrical bilateral and unilateral CAD. It is unclear if anatomic variations in the carotid arteries predispose an individual to formation of atherosclerotic CAD. We wanted to assess (1) the inter-individual or intra-individual anatomical variations in the carotid arteries and (2) whether anatomical variations predispose the development of atherosclerotic CAD. PubMed and Medline were utilized to identify relevant literature for critical appraisal, summarization and documentation. Inclusion and exclusion criteria were applied to narrow results and articles were critically appraised and analyzed. Evidence suggests that a low outflow/inflow ratio, elevated bifurcation height, and bifurcation angle are associated with increased risk for CAD. Sex and age demonstrated positive correlation with the disease. Additionally, tortuosity and kinking of the carotid arteries may affect the formation of CAD but coiling of the arteries is a natural age-dependent process and does not affect CAD development. This review suggests there are anatomic variations in the carotid arteries that increase the risk of developing carotid artery disease. The most significant risk factors include a low outflow/inflow ratio, increased internal carotid artery tortuosity, elevated bifurcation height, and bifurcation angle.
Chronic Kidney Disease (CKD) affects 10-16% of the US population and its incidence is rising due to increasing prevalence of associated risk factors. Renal replacement therapy is required to treat ...late stage CKD and hemodialysis is the preferred modality for many patients. Vascular access is required for hemodialysis and arteriovenous fistulas (AVF) are currently the gold standard. This review intended to collate current knowledge on AVF outcomes regarding both the patient and fistula. Scopus and Medline were utilized to identify relevant literature. Inclusion and exclusion criteria were applied to narrow search results. Among CKD patients, 33.5-77.4% require a central venous catheter (CVC) before dialysis through a fistula. Many patients (33-51%) use a CVC regardless of AVF creation due to fistula immaturity or failure. There are large variations in AVF creation policies internationally; 16% of American hemodialysis patients use a fistula compared to 72% of German patients. Primary patency and primary AVFs' failure ranges from 60-70% and 20-26%, respectively. AVFs reduce morbidity and mortality in CKD. At present, too many patients are receiving hemodialysis through a CVC. Inadequate referral times for AVF creation can lead to fistula immaturity or failure in the intervention. Many countries are lagging behind recommended AVF creation rates published by the Kidney Disease Outcomes Quality Initiative. There is a paucity of literature concerning when a patient should be referred for AVF creation. It is paramount to have better predictive outcome measures and more clarity as to when patients will benefit from an AVF.
We previously demonstrated that nerve cell lines selected for resistance to amyloid β (Aβ) peptide exhibit elevated aerobic glycolysis in part due to increased expression of pyruvate dehydrogenase ...kinase 1 (PDK1) and lactate dehydrogenase A (LDHA). Here, we show that overexpression of either PDK1 or LDHA in a rat CNS cell line (B12) confers resistance to Aβ and other neurotoxins. Treatment of Aβ-sensitive cells with various toxins resulted in mitochondrial hyperpolarization, immediately followed by rapid depolarization and cell death, events accompanied by increased production of cellular reactive oxygen species (ROS). In contrast, cells expressing either PDK1 or LDHA maintained a lower mitochondrial membrane potential and decreased ROS production with or without exposure to toxins. Additionally, PDK1- and LDHA-overexpressing cells exhibited decreased oxygen consumption but maintained levels of ATP under both normal culture conditions and following Aβ treatment. Interestingly, immunoblot analysis of wild type mouse primary cortical neurons treated with Aβ or cortical tissue extracts from 12-month-old APPswe/PS1dE9 transgenic mice showed decreased expression of LDHA and PDK1 when compared with controls. Additionally, post-mortem brain extracts from patients with Alzheimer disease exhibited a decrease in PDK1 expression compared with nondemented patients. Collectively, these findings indicate that key Warburg effect enzymes play a central role in mediating neuronal resistance to Αβ or other neurotoxins by decreasing mitochondrial activity and subsequent ROS production. Maintenance of PDK1 or LDHA expression in certain regions of the brain may explain why some individuals tolerate high levels of Aβ deposition without developing Alzheimer disease.
Background: Aerobic glycolysis promotes resistance against Aβ toxicity.
Results: Increased LDHA and PDK1 expression attenuates mitochondrial activity and confers resistance to Aβ. These proteins are down-regulated in a transgenic Alzheimer disease (AD) mouse model, and PDK1 is decreased in AD brain.
Conclusion: PDK and LDHA are central mediators of Aβ resistance.
Significance: Drugs that augment aerobic glycolysis may enhance brain cell survival in AD patients.
Abstract
β-Amyloid (Aβ) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages ...release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMϕ) from hematogenous macrophages (hMϕ), as they are morphologically and phenotypically similar when activated. To distinguish between mMϕ and hMϕ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMϕ, but not in mMϕ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMϕ from hMϕ. Mutant mice demonstrated robust increases in Aβ1–42, area of Aβ plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aβ accumulation, paralleled by the accumulation of hMϕ around Aβ plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMϕ around Aβ plaques was age- and sex-dependent and preceded cognitive impairment.
Choline acetyltransferase (ChAT) is essential for cholinergic neuron function as it mediates synthesis of the neurotransmitter acetylcholine. ChAT mutations have been linked to the neuromuscular ...disorder congenital myasthenic syndrome (CMS). One CMS‐related ChAT mutation, V18M, reduces enzyme activity and cellular protein levels, and is positioned within a highly conserved proline‐rich motif with the sequence 14PKLPVPP20. We demonstrate that N‐terminal truncation that includes this proline‐rich motif, as well as mutation of prolines‐17/19 together to alanine (P17A/P19A), dramatically reduces ChAT steady‐state protein levels and cellular activity when expressed in cholinergic SN56 neural cells. The in vitro activity of bacterially expressed recombinant P17A/P19A‐ChAT is also reduced, although this is not caused by changes in protein secondary structure or thermal stability. Treatment of SN56 cells with the proteasome inhibitor MG132 increases cellular P17A/P19A‐ChAT steady‐state protein levels, and by immunoprecipitation we found that ChAT is ubiquitinated and that polyubiquitination of P17A/P19A‐ChAT is increased compared to wild‐type (WT) ChAT. Using a novel fluorescent‐biorthogonal pulse‐chase protocol in SN56 cells, we determined that the protein half‐life of P17A/P19A‐ChAT (2.2 h) is substantially reduced compared to WT‐ChAT (19.7 h). Lastly, we show that two CMS‐related ChAT mutants (V18M and A513T) have enhanced ubiquitination, and that treatment with MG132 can partially restore both the steady‐state protein levels as well as cellular activity of some CMS‐mutant ChAT. These results identify a novel mechanism for regulation of ChAT through the ubiquitin–proteasome system that is influenced by the conserved N‐terminal proline‐rich motif of ChAT and may be implicated in CMS pathology.
Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady‐state protein levels of human 69‐kDa ChAT are regulated by the ubiquitin–proteasome system. Mutation of a highly conserved N‐terminal proline‐rich motif in human 69‐kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)‐mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady‐state protein levels as well as cellular activity of some CMS‐mutant ChAT proteins.
Choline acetyltransferase (ChAT) synthesizes acetylcholine in cholinergic neurons. In this study we find that steady‐state protein levels of human 69‐kDa ChAT are regulated by the ubiquitin–proteasome system. Mutation of a highly conserved N‐terminal proline‐rich motif in human 69‐kDa ChAT reduces both cellular ChAT protein levels, through enhanced ubiquitination and proteasomal degradation, and enzyme activity. Ubiquitination of catalytically deficient congenital myasthenic syndrome (CMS)‐mutant ChAT is increased in cells, and importantly proteasome inhibition partially restores steady‐state protein levels as well as cellular activity of some CMS‐mutant ChAT proteins.
Introduction and aimsSimulation-based learning is an integral part of physician training. It enables acquisition of knowledge, skills and attitudes in a safe, educationally orientated and efficient ...manner. Controlled training environments bridge the gap between the theoretical knowledge acquired in pre-clinical years and the translation of this knowledge to clinical skills required to succeed post-graduation. In Ireland, high-fidelity training centres exist at several medical schools; however, their presence has not heralded an expansive increase in medical student simulation-based learning opportunities. This study aims to assess skill development in a student cohort following the introduction of an emergency medicine simulation competition. The use of competition was predicated on the idea that the imposed stressful environment would mimic the pressure of a hospital environment, thus serving as a proxy for hospital preparedness. The medical students involved do not receive extensive simulation-based training as part of the standard undergraduate curriculum.MethodsStudents (n=32) from the University College Cork College of Medicine & Health received weekly physician-led simulation training for 3 months, culminating in a simulation-based team competition. Individual baseline surveys were conducted at the beginning of training, and on the day of the competition. The survey was composed of questions aimed at both technical and non-technical skills, ranked on a Likert confidence scale from 1 to 5. Results were analyzed using repeated measures ANOVA and were stratified according to medical school year.ResultsThere was a significant increase in student confidence towards both managing a patient‘s airway (3.56 vs. 1.78, F1, 31=69.2, p<0.01) and simulating a rapid sequence induction (2.63 vs. 1.22, F1, 31=36.5, p<0.01) following the competition. In addition, student confidence assuming a leadership role in a trauma situation increased (3.22 vs. 2.72, F1, 31=5.39, p=0.027) as well as their ability to engage in closed-loop communication (4.06 vs. 3.05, F1,31=19.97, p<0.01). Medical school year did not contribute to the increased level of confidence of the competing students.DiscussionThe introduction of a competitive environment coupled with extracurricular simulation training positively impacted medical student confidence in both technical and non-technical skills. These data support a potential benefit of increased exposure to simulation training through extracurricular competition during undergraduate pre-clinical and clinical years.
PDF
