Sequestration of Muscarinic Cholinergic Receptors in Permeabilized Neuroblastoma Cells

The feasibility of using a permeabilized preparation of human SH-SY-5Y neuroblastoma cells for studies of muscarinic acetylcholine receptor (mAChR) sequestration has been evaluated. Exposure of cells permeabilized with digitonin, streptolysin-O, or the Α-toxin from Staphylococcus aureus to oxotremorine-M (Oxo-M) for 30 min resulted in a 25–30% reduction in the number of cell surface mAChRs, as monitored by the loss of N [ 3 H]methyl- scopolamine ([ 3 H]NMS) binding sites. The corresponding value for intact cells was 40%. For cells permeabilized with 20 Μ M digitonin, the Oxo-M-mediated reduction in [ 3 H]NMS binding was time ( t 1/2 ∼ 5 min) and concentration (EC 50 ∼ 10 Μ M ) dependent and was agonist specific (Oxo M > bethanechol = arecoline = pilocarpine). In contrast, no reduction in total mAChR number, as monitored by the binding of [ 3 H]quinuclidinyl benzilate, occurred following Oxo-M treatment. The loss of [ 3 H]NMS sites observed in the presence of Oxo-M was unaffected by omission of either ATP or Ca 2+ , both of which are required for stimulated phosphoinositide hydrolysis, but could be inhibited by the inclusion of guanosine 5′- O -(2-thiodiphosphate). mAChRs sequestered in response to Oxo-M addition were unmasked when the cells were permeabilized in the presence of higher concentrations of digitonin (80 Μ M ). The results indicate (a) that permeabilized SH-SY-5Y cells support an agonist-induced sequestration of mAChRs, the magnitude of which is ∼ 65–70% of that observed for intact cells, (b) that when internalized, mAChRs are located in a cellular compartment to which [ 3 H]NMS has only a limited access despite the removal of the plasma membrane barrier, and (c) that the production of phosphoinositide-derived second messengers is not a prerequisite for mAChR sequestration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65736/1/j.1471-4159.1994.62051795.x.pd

Proteomic Analysis of Hippocampal Dentate Granule Cells in Frontotemporal Lobar Degeneration: Application of Laser Capture Technology

Frontotemporal lobar degeneration (FTLD) is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U). While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins. Moreover, these inclusions suggest altered expression, processing, or degradation of proteins during FTLD-U pathogenesis. Thus, one approach to understanding disease mechanisms is to delineate the molecular changes in protein composition in FTLD-U brain. Using a combined approach consisting of laser capture microdissection (LCM) and high-resolution liquid chromatography-tandem mass spectrometry (LC–MS/MS), we identified 1252 proteins in hippocampal dentate granule cells excised from three post-mortem FTLD-U and three unaffected control cases processed in parallel. Additionally, we employed a labeling-free quantification technique to compare the abundance of the identified proteins between FTLD-U and control cases. Quantification revealed 54 proteins with selective enrichment in FTLD-U, including TAR–DNA binding protein 43 (TDP-43), a recently identified component of ubiquitinated inclusions. Moreover, 19 proteins were selectively decreased in FTLD-U. Subsequent immunohistochemical analysis of TDP-43 and three additional protein candidates suggests that our proteomic profiling of FTLD-U dentate granule cells reveals both inclusion-associated proteins and non-aggregated disease-specific proteins. Application of LCM is a valuable tool in the molecular analysis of complex tissues, and its application in the proteomic characterization of neurodegenerative disorders such as FTLD-U may be used to identify proteins altered in disease

Molecules of senescent glial cells differentiate Alzheimer's disease from ageing

BACKGROUND: Ageing is a major risk factor for Alzheimer's disease (AD), which is accompanied by cellular senescence and thousands of transcriptional changes in the brain. OBJECTIVES: To identify the biomarkers in the cerebrospinal fluid (CSF) that could help differentiate healthy ageing from neurodegenerative processes. METHODS: Cellular senescence and ageing-related biomarkers were assessed in primary astrocytes and postmortem brains by immunoblotting and immunohistochemistry. The biomarkers were measured in CSF samples from the China Ageing and Neurodegenerative Disorder Initiative cohort using Elisa and the multiplex Luminex platform. RESULTS: The cyclin-dependent kinase inhibitors p16/p21-positive senescent cells in human postmortem brains were predominantly astrocytes and oligodendrocyte lineage cells, which accumulated in AD brains. CCL2, YKL-40, HGF, MIF, S100B, TSP2, LCN2 and serpinA3 are biomarkers closely related to human glial senescence. Moreover, we discovered that most of these molecules, which were upregulated in senescent glial cells, were significantly elevated in the AD brain. Notably, CSF YKL-40 (β=0.5412, p<0.0001) levels were markedly elevated with age in healthy older individuals, whereas HGF (β=0.2732, p=0.0001), MIF (β=0.33714, p=0.0017) and TSP2 (β=0.1996, p=0.0297) levels were more susceptible to age in older individuals with AD pathology. We revealed that YKL-40, TSP2 and serpinA3 were useful biomarkers for discriminating patients with AD from CN individuals and non-AD patients. DISCUSSION: Our findings demonstrated the different patterns of CSF biomarkers related to senescent glial cells between normal ageing and AD, implicating these biomarkers could identify the road node in healthy path off to neurodegeneration and improve the accuracy of clinical AD diagnosis, which would help promote healthy ageing

Familial Parkinson's Disease-associated L166P Mutation Disrupts DJ-1 Protein Folding and Function

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinson's disease. Here we report that DJ-1 is a dimeric protein that exhibits protease activity but no chaperone activity. The protease activity was abolished by mutation of Cys-106 to Ala, suggesting that DJ-1 functions as a cysteine protease. Our studies revealed that the Parkinson's disease-linked L166P mutation impaired the intrinsic folding propensity of DJ-1 protein, resulting in a spontaneously unfolded structure that was incapable of forming a homodimer with itself or a heterodimer with wild-type DJ-1. Correlating with the disruption of DJ-1 structure, the L166P mutation abolished the catalytic function of DJ-1. Furthermore, as a result of protein misfolding, the L166P mutant DJ-1 was selectively polyubiquitinated and rapidly degraded by the proteasome. Together these findings provide insights into the molecular mechanism by which loss-of-function mutations in DJ-1 lead to Parkinson's disease

Large-scale proteomic analysis of human brain identifies proteins associated with cognitive trajectory in advanced age

In advanced age, some individuals maintain a stable cognitive trajectory while others experience a rapid decline. Such variation in cognitive trajectory is only partially explained by traditional neurodegenerative pathologies. Hence, to identify new processes underlying variation in cognitive trajectory, we perform an unbiased proteome-wide association study of cognitive trajectory in a discovery (n = 104) and replication cohort (n = 39) of initially cognitively unimpaired, longitudinally assessed older-adult brain donors. We find 579 proteins associated with cognitive trajectory after meta-analysis. Notably, we present evidence for increased neuronal mitochondrial activities in cognitive stability regardless of the burden of traditional neuropathologies. Furthermore, we provide additional evidence for increased synaptic abundance and decreased inflammation and apoptosis in cognitive stability. Importantly, we nominate proteins associated with cognitive trajectory, particularly the 38 proteins that act independently of neuropathologies and are also hub proteins of protein co-expression networks, as promising targets for future mechanistic studies of cognitive trajectory.Accelerating Medicine Partnership for AD [U01AG046161, U01 AG061357]; Emory Alzheimer's Disease Research Center [P50 AG025688]; NINDS Emory Neuroscience Core [P30 NS055077]; intramural program of the National Institute on Aging (NIA); Alzheimer's Association; Alzheimer's Research UK; Michael J. Fox Foundation for Parkinson's Research; Weston Brain Institute Biomarkers Across Neurodegenerative Diseases Grant [11060]; National Institute of Neurological Disorders and Stroke [U24 NS072026]; National Institute on Aging [P30 AG19610]; Arizona Department of Health Services [211002]; Arizona Biomedical Research Commission [4001, 0011, 05-901, 1001]; [R01 AG056533]; [R01 AG053960]; [U01 MH115484]; [I01 BX003853]; [IK2 BX001820]; [R01 AG061800]; [R01 AG057911]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Effects of APOE Genotype on Brain Proteomic Network and Cell Type Changes in Alzheimer's Disease

Polymorphic alleles in the apolipoprotein E (APOE) gene are the main genetic determinants of late-onset Alzheimer's disease (AD) risk. Individuals carrying the APOE E4 allele are at increased risk to develop AD compared to those carrying the more common E3 allele, whereas those carrying the E2 allele are at decreased risk for developing AD. How ApoE isoforms influence risk for AD remains unclear. To help fill this gap in knowledge, we performed a comparative unbiased mass spectrometry-based proteomic analysis of post-mortem brain cortical tissues from pathologically-defined AD or control cases of different APOE genotypes. Control cases (n = 10) were homozygous for the common E3 allele, whereas AD cases (n = 24) were equally distributed among E2/3, E3/3, and E4/4 genotypes. We used differential protein expression and co-expression analytical approaches to assess how changes in the brain proteome are related to APOE genotype. We observed similar levels of amyloid-β, but reduced levels of neurofibrillary tau, in E2/3 brains compared to E3/3 and E4/4 AD brains. Weighted co-expression network analysis revealed 33 modules of co-expressed proteins, 12 of which were significantly different by APOE genotype in AD. The modules that were significantly different by APOE genotype were associated with synaptic transmission and inflammation, among other biological processes. Deconvolution and analysis of brain cell type changes revealed that the E2 allele suppressed homeostatic and disease-associated cell type changes in astrocytes, microglia, oligodendroglia, and endothelia. The E2 allele-specific effect on brain cell type changes was validated in a separate cohort of 130 brains. Our systems-level proteomic analyses of AD brain reveal alterations in the brain proteome and brain cell types associated with allelic variants in APOE, and suggest further areas for investigation into the upstream mechanisms that drive ApoE-associated risk for AD

Discovery of FERM domain protein-protein interaction inhibitors for MSN and CD44 as a potential therapeutic approach for Alzheimer\u27s disease.

Proteomic studies have identified moesin (MSN), a protein containing a four-point-one, ezrin, radixin, moesin (FERM) domain, and the receptor CD44 as hub proteins found within a coexpression module strongly linked to Alzheimer\u27s disease (AD) traits and microglia. These proteins are more abundant in Alzheimer\u27s patient brains, and their levels are positively correlated with cognitive decline, amyloid plaque deposition, and neurofibrillary tangle burden. The MSN FERM domain interacts with the phospholipid phosphatidylinositol 4,5-bisphosphate (PI

First presentation with neuropsychiatric symptoms in autosomal dominant Alzheimer\u27s disease: The Dominantly Inherited Alzheimer\u27s Network Study

Behavioural changes and neuropsychiatric symptoms (NPS) commonly occur in Alzheimer’s disease (AD) but may not be recognised as AD-related when they are the presenting feature. NPS are important as they are associated with greater functional impairment, poorer quality of life, accelerated cognitive decline and worsened caregiver burden.1 Autosomal dominant AD (ADAD), although \u3c 1% of total AD cases, provides a valuable opportunity to study the clinical heterogeneity of AD. The young age at onset reduces the prevalence of age-related comorbid pathologies and the near 100% penetrance of pathogenic mutations reduces the likelihood of misdiagnosis.2 Anxiety and depression commonly occur in ADAD family members, with increased levels of depression having been found among predementia female mutation carriers.3 Subsequent studies, however, have shown that anxiety and/or depression are common regardless of mutation status, occurring in almost one in three at-risk individuals, with one study reporting a higher rate of depression in non-carriers (17%) than asymptomatic carriers (5%).4 5 Despite the high frequency of NPS in ADAD families, relatively little is known about the proportion of ADAD cases who present with predominantly behavioural symptoms. Our aims were to assess the first reported clinical change in symptomatic ADAD, to compare presentations across genotypes, and to compare cognitive performance between behavioural and cognitive-led presentations