Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera, and Their Number Is Influenced By Age, Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases

Extravasated Brain-Reactive Autoantibodies Perturb Neuronal Surface Protein Expression in Alzheimer\u27s Pathology

Background: Increased blood-brain barrier (BBB) permeability is reported in both the neuropathological and in vivo studies in both Alzheimer’s Disease (AD) and age matched cognitively normal, no cognitive impairment (NCI), subjects. Impaired BBB allows various vascular components such as immunoglobulin G (IgG) to extravasate into the brain and specifically bind to various neuronal surface proteins (NSP), also known as brain reactive autoantibodies (BrABs). This interaction is predicted to further enhance deposition of amyloid plaques. Hypothesis: Interaction between extravasated BrABs and its cognate NSPs lower the expression of that NSPs in AD patients. Methods: We selected Western blotting technique to study the expression of various brain proteins and test our hypothesis. Fresh frozen brain samples of AD and NCI subjects were acquired, and total brain protein was extracted using protocol established in Acharya lab. We also identified various NSPs to study the impact of BrABs-NSPs interactions. Additionally, we investigated the expression of amyloid plaques ((amyloid precursor protein (APP)) and apoptosis (Caspase-3) markers. Specific NSPs examined included the alpha7 nicotinic acetylcholine receptor (α7nAChR) and anti-choline acetyltransferase (ChAT). To image the membranes, fluorescent imaging was used initially, which was later switched to chemiluminescence, after much troubleshooting. Results: Most of the work done through these experiments was focused on establishing a thorough Western blot protocol that can be used to reliably perform these experiments. This involved determining the appropriate primary and secondary antibodies concentrations, loading concentrations, and testing different imaging settings to determine the most ideal image-acquisition conditions. Towards the end of the fellowship, we were successful in developing a protocol to further explore our investigation. Using this protocol, we were able to visualize bands for ChAT, α7nAChR, and caspase – 3. Conclusions: Using this protocol further Western blot experiments can be run to study and compare the expression levels of various NSP in AD and control samples for testing our hypothesi

A Preliminary Report: The Hippocampus and Surrounding Temporal Cortex of Patients With Schizophrenia Have Impaired Blood-Brain Barrier

Schizophrenia (SZ) is one of the most severe forms of mental illness, yet mechanisms remain unclear. A widely established brain finding in SZ is hippocampal atrophy, and a coherent explanation similarly is lacking. Epidemiological evidence suggests increased cerebrovascular and cardiovascular complications in SZ independent of lifestyle and medication, pointing to disease-specific pathology. Endothelial cell contributions to blood-brain barrier (BBB) compromise may influence neurovascular unit and peripheral vascular function, and we hypothesize that downstream functional and structural abnormalities may be explained by impaired BBB

Detection of Alzheimer\u27s disease at mild cognitive impairment and disease progression using autoantibodies as blood-based biomarkers

Introduction There is an urgent need to identify biomarkers that can accurately detect and diagnose Alzheimer\u27s disease (AD). Autoantibodies are abundant and ubiquitous in human sera and have been previously demonstrated as disease-specific biomarkers capable of accurately diagnosing mild-moderate stages of AD and Parkinson\u27s disease. Methods Sera from 236 subjects, including 50 mild cognitive impairment (MCI) subjects with confirmed low CSF Aβ42 levels, were screened with human protein microarrays to identify potential biomarkers for MCI. Autoantibody biomarker performance was evaluated using Random Forest and Receiver Operating Characteristic curves. Results Autoantibody biomarkers can differentiate MCI patients from age-matched and gender-matched controls with an overall accuracy, sensitivity, and specificity of 100.0%. They were also capable of differentiating MCI patients from those with mild-moderate AD and other neurologic and non-neurologic controls with high accuracy. Discussion Autoantibodies can be used as noninvasive and effective blood-based biomarkers for early diagnosis and staging of AD

Potential utility of autoantibodies as blood-based biomarkers for early detection and diagnosis of Parkinson’s disease

Introduction There is a great need to identify readily accessible, blood-based biomarkers for Parkinson’s disease (PD) that are useful for accurate early detection and diagnosis. This advancement would allow early patient treatment and enrollment into clinical trials, both of which would greatly facilitate the development of new therapies for PD. Methods Sera from a total of 398 subjects, including 103 early-stage PD subjects derived from the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) study, were screened with human protein microarrays containing 9,486 potential antigen targets to identify autoantibodies potentially useful as biomarkers for PD. A panel of selected autoantibodies with a higher prevalence in early-stage PD was identified and tested using Random Forest for its ability to distinguish early-stage PD subjects from controls and from individuals with other neurodegenerative and non-neurodegenerative diseases. Results Results demonstrate that a panel of selected, blood-borne autoantibody biomarkers can distinguish early-stage PD subjects (90% confidence in diagnosis) from age- and sex-matched controls with an overall accuracy of 87.9%, a sensitivity of 94.1% and specificity of 85.5%. These biomarkers were also capable of differentiating patients with early-stage PD from those with more advanced (mild-moderate) PD with an overall accuracy of 97.5%, and could distinguish subjects with early-stage PD from those with other neurological (e.g., Alzheimer’s disease and multiple sclerosis) and non-neurological (e.g., breast cancer) diseases. Conclusion These results demonstrate, for the first time, that a panel of selected autoantibodies may prove to be useful as effective blood-based biomarkers for the diagnosis of early-stage PD

A Preliminary Report on the Role of Lipoxin A4 in Reinstating the Blood-Brain Barrier Integrity in a Rodent Model of Acute Inflammation with Impaired Cerebrovasculature

Background: The blood-brain barrier (BBB) is responsible for maintaining brain homeostasis and ultimately proper neuronal function. Disruption of the BBB, leading to increased BBB permeability, has been reported in several neurodegenerative diseases, including Alzheimer’s disease (AD) and traumatic brain injury (TBI). Lipoxins (LXs) are a class of arachidonate-derived eicosanoids, which are a class of specialized pro-resolving lipid mediators (SPMs). SPMs are known to inhibit immune response through inhibition of cellular infiltration, downregulation of pro-inflammatory mediators and upregulation of anti-inflammatory mediators. Hence, LXs are recognized as “breaking signals” in the inflammatory process. One form of LXs, Lipoxin A4 (LXA4), has been found to decreased production of proinflammatory mediators, inhibit neutrophils chemotaxis and infiltration to the site of injury, and promote the phagocytic clearance of debris by macrophages. Therefore, LXA4 serves a critical role in resolution of inflammatory process by regulating the activation of monocytes and modulating the generation of reactive oxygen species (ROS). Hypothesis: LXA4 treatment reinstates the BBB integrity in a rodent model of acute BBB breakdown and inflammation. Methods: Nine-month-old female Sprague Dawley rats were given an intravenous (IV) injection of 15 mg/kg lipopolysaccharide (LPS) through the tail for inducing acute inflammation and BBB breach. After three hours, the rats were injected with 9 µg/kg LXA4 or Saline (Vehicle control). Four treatment groups were thus developed: LPS only, LPS/LXA4, LXA4 only, and Saline only. Animals were euthanized at 24 hours of LPS treatment and brain samples were processed for paraffin-embedded sections and immunohistochemistry. Sections comprising of hippocampus and cortical regions were selected for detection of impaired BBB as demonstrated by the extravasation of immunoglobulin G (IgG) and IBA1 (microgliosis marker). The area of the cerebral cortex and number of cortical blood vessels presenting with IgG extravasation were estimated and compared between treatment groups. Similarly, IBA1 immunoreactivity was quantified using Color Deconvolution V9 tool of Aperio ImageScope (Leica BIOSYSTEMS). Results: LXA4 treatment following LPS injection demonstrated decrease in the extent of IgG leak compared to LPS only group. Likewise, we observed significant decrease in microgliosis in LPS-LXA4 group compared to LPS only. Conclusion: These preliminary results demonstrate potential beneficial effects of LXA4 in reinstating BBB integrity and reducing neuroinflammation in rat model of acute BBB breach and inflammation

Evidence That Brain-Reactive Autoantibodies Contribute to Chronic Neuronal Internalization of Exogenous Amyloid-β1-42 and Key Cell Surface Proteins During Alzheimer\u27s Disease Pathogenesis

Blood-brain barrier (BBB) permeability is a recognized early feature of Alzheimer\u27s disease (AD). In the present study, we examined consequences of increased BBB permeability on the development of AD-related pathology by tracking selected leaked plasma components and their interactions with neurons in vivo and in vitro. Histological sections of cortical regions of postmortem AD brains were immunostained to determine the distribution of amyloid-β1-42 (Aβ42), cathepsin D, IgG, GluR2/3, and alpha7 nicotinic acetylcholine receptor (α7nAChR). Results revealed that chronic IgG binding to pyramidal neurons coincided with internalization of Aβ42, IgG, GluR2/3, and α7nAChR as well as lysosomal compartment expansion in these cells in regions of AD pathology. To test possible mechanistic interrelationships of these phenomena, we exposed differentiated SH-SY5Y neuroblastoma cells to exogenous, soluble Aβ42 peptide and serum from AD and control subjects. The rate and extent of Aβ42 internalization in these cells was enhanced by serum containing neuron-binding IgG autoantibodies. This was confirmed by treating cells with individual antibodies specific for α7nAChR, purified IgG from AD or non-AD sera, and sera devoid of IgG, in the presence of 100 nM Aβ42. Initial co-localization of IgG, α7nAChR, and Aβ42 was temporally and spatially linked to early endosomes (Rab11) and later to lysosomes (LAMP-1). Aβ42 internalization was attenuated by treatment with monovalent F(ab) antibody fragments generated from purified IgG from AD serum and then rescued by coupling F(ab) fragments with divalent human anti-Fab. Overall, results suggest that cross-linking of neuron-binding autoantibodies targeting cell surface proteins can accelerate intraneuronal Aβ42 deposition in AD

Prenatal Cocaine Exposure Increases Synaptic Localization of a Neuronal RasGEF, GRASP-1 via Hyperphosphorylation of AMPAR Anchoring Protein, GRIP

Prenatal cocaine exposure causes sustained phosphorylation of the synaptic anchoring protein, glutamate receptor interacting protein (GRIP1/2), preventing synaptic targeting of the GluR2/3-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors (AMPARs; J. Neurosci. 29: 6308–6319, 2009). Because overexpression of GRIP-associated neuronal rasGEF protein (GRASP-1) specifically reduces the synaptic targeting of AMPARs, we hypothesized that prenatal cocaine exposure enhances GRASP-1 synaptic membrane localization leading to hyper-activation of ras family proteins and heightened actin polymerization. Our results show a markedly increased GRIP1-associated GRASP-1 content with approximately 40% reduction in its rasGEF activity in frontal cortices (FCX) of 21-day-old (P21) prenatal cocaine-exposed rats. This cocaine effect is the result of a persistent protein kinase C (PKC)- and downstream Src tyrosine kinase-mediated GRIP phosphorylation. The hyperactivated PKC also increased membrane-associated GRASP-1 and activated small G-proteins RhoA, cdc42/Rac1 and Rap1 as well as filamentous actin (F-actin) levels without an effect on the phosphorylation state of actin. Since increased F-actin facilitates protein transport, our results suggest that increased GRASP-1 synaptic localization in prenatal cocaine-exposed brains is an adaptive response to restoring the synaptic expression of AMPA-GluR2/3. Our earlier data demonstrated that persistent PKC-mediated GRIP phosphorylation reduces GluR2/3 synaptic targeting in prenatal cocaine-exposed brains, we now show that the increased GRIP-associated GRASP-1 may contribute to the reduction in GluR2/3 synaptic expression and AMPAR signaling defects

Prenatal cocaine exposure induces an increased F-actin level which is normalized by protein phosphatase treatment without alteration in phosphorylation state of actin.

<p>(<b>a</b>) Synaptosomes obtained from the frontal cortices (FCX) of P21, prenatal cocaine- or saline-treated rats were treated with alkaline phosphatases, alkaline phosphatases (1 mg/ml) plus phosphatase inhibitors or cytochalasin D (10 µM) <i>in vitro</i>. The reactions were terminated, synaptosomes solubilized, and actin-containing proteins extracted using biotin-conjugated anti-actin antibodies and placed into streptavidin-coated plates. The level of F-actin was determined by rhodamine-conjugated phalloidin and the fluorescence intensity of phalloidin was measured using Beckman multimode plate reader, DX880. <i>n</i> = 6. Data are means ± s.e.m. of the fluorescence intensity. The statistical significance was evaluated by Newman-Keuls multiple comparisons that followed one-way ANOVA. *p<0.01 compared to native GRIP1 from saline-treated group. #p<0.01 compared to dephosphorylated GRIP in cocaine-treated group. +p<0.01 compared with native GRIP1 from respective group. (<b>b</b>) The phosphorylation state of actin was evaluated in synaptosomes derived from the FCX of P21, prenatal cocaine- or saline-treated rats. Total actin was purified by immunoprecipitation with anti-actin and the levels of phosphor-serine (pS), -threonine (pT) and -tyrosine (pY) in the anti-actin immunoprecipitate was determined by Western blotting using phosphoepitope-specific antibodies. <i>n</i> = 4. Data are means ± s.e.m. of the ratios of pS-, pT- or pY-actin to total actin optical intensities. There was no discernible difference noted in any of the actin phosphoepitopes in FCX of the prenatal cocaine- comparing to saline-exposed rats.</p