A Chronic Increase in Blood-Brain Barrier Permeability Facilitates Intraneuronal Deposition of Exogenous Bloodborne Amyloid-Beta1-42 Peptide in the Brain and Leads to Alzheimer's Disease-Relevant Cognitive Changes in a Mouse Model.

Background: Increased blood-brain barrier (BBB) permeability and amyloid-ß (Aß) peptides (especially Aß1-42) (Aß42) have been linked to Alzheimer's disease (AD) pathogenesis, but the nature of their involvement in AD-related neuropathological changes leading to cognitive changes remains poorly understood. Objective: To test the hypothesis that chronic extravasation of bloodborne Aß42 peptide and brain-reactive autoantibodies and their entry into the brain parenchyma via a permeable BBB contribute to AD-related pathological changes and cognitive changes in a mouse model. Methods: The BBB was rendered chronically permeable through repeated injections of Pertussis toxin (PT), and soluble monomeric, fluorescein isothiocyanate (FITC)-labeled or unlabeled Aß42 was injected into the tail-vein of 10-month-old male CD1 mice at designated intervals spanning ∼3 months. Acquisition of learned behaviors and long-term retention were assessed via a battery of cognitive and behavioral tests and linked to neuropathological changes. Results: Mice injected with both PT and Aß42 demonstrated a preferential deficit in the capacity for long-term retention and an increased susceptibility to interference in selective attention compared to mice exposed to PT or saline only. Immunohistochemical analyses revealed increased BBB permeability and entry of bloodborne Aß42 and immunoglobulin G (IgG) into the brain parenchyma, selective neuronal binding of IgG and neuronal accumulation of Aß42 in animals injected with both PT and Aß42 compared to controls. Conclusion: Results highlight the potential synergistic role of BBB compromise and the influx of bloodborne Aß42 into the brain in both the initiation and progression of neuropathologic and cognitive changes associated with AD.

Novel treatment strategy of targeting epigenetic dysregulation in pancreatic neuroendocrine tumors.

BACKGROUND: Epigenetic dysregulation is an integral step in the progression of pancreatic neuroendocrine tumors. We hypothesized that tumor suppressor repression by DNA methyltransferase 1 in pancreatic neuroendocrine tumors could be targeted with epigenetic treatment. METHODS: Resected pancreatic neuroendocrine tumors from 32 patients were stained for DNA methyltransferase 1 and scored. Human (BON1) and murine (STC) pancreatic neuroendocrine tumor cells were treated with DNA methyltransferase 1 inhibitor 5-azacytidine and chemotherapeutic agents 5-fluorouracil and temozolomide. Cell proliferation assay and tumor suppressor gene analysis were performed with qRT-PCR and Clarion S microarray. Tumor measurements were compared in a murine treatment model. RESULTS: High DNA methyltransferase scores were associated with high Ki-67 (6.7% vs 70.6% P < .01), mitotic rate (0.0% vs 31.3%), and grade (20.0% vs 80.4%, P < .01). Treatment with 5-azacytidine and chemotherapy resulted in a reduction of cell proliferation compared to chemotherapy alone in BON1 (77.3% vs 53.1%, P < .001) and STC (73.4% vs 34.2%, P < .001). Treatment with 5-azacytidine and chemotherapy resulted in upregulation of tumor suppressors CDKN1A (7.6 rel. fold, P < .001), BRCA2 (4.3 rel. fold, P < .001), and CDH1 (6.0 rel. fold, P = .026) in BON1 and CDKN1a (14.5 rel. fold, P < .001) and CDH (17.5 rel. fold, P < .001) in STC. In microarray, 5-azacytidine drove global genetic changes in combination treatment. In vivo tumors treated with chemotherapy measured 88.6 ± 19.54 mm3 vs 52.89 ± 10.51 mm3 in those treated with combination therapy (P = .009). CONCLUSION: Epigenetic dysregulation with DNA methyltransferase 1 is associated with pancreatic neuroendocrine tumors and is a potential targetable strategy. 5-azacytidine and chemotherapy in combination can reduce cell proliferation, upregulate silenced tumor suppressor genes, and decrease in vivo tumors in pancreatic neuroendocrine tumors.

Cerebral Microvascular Pathology Is a Common Endophenotype Between Traumatic Brain Injury, Cardiovascular Disease, and Dementia: A Hypothesis and Review.

Traumatic brain injury (TBI) exposure has been associated with an increased risk of age-related cognitive decline or dementia in multiple epidemiological studies. Current therapeutic interventions in the field of Brain Injury Medicine focus largely on episodic symptom management during the chronic phase of TBI recovery, rather than targeting specific underlying pathological processes. This approach may be especially problematic for secondary age-related cognitive decline or dementia following TBI exposure. Although there are likely multiple pathophysiological mechanisms involved, a growing body of literature demonstrates that cerebral microvascular pathology is a common endophenotype across the spectrum of TBI severity. Similarly, a combination of pre-clinical and clinical research over the past two decades has implicated cerebral microvascular pathology in the initiation and progression of multiple neurodegenerative diseases, including Alzheimer's disease and other dementias. We hypothesize that cerebral microvascular pathology is a common endophenotype between TBI, cardiovascular disease (CVD), and dementia, which can be targeted through modifiable cardiovascular risk factor reductions during the chronic phase of TBI recovery. We posit that our hypothesis is supported by the currently available scientific literature, as detailed in our review.

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

Though hippocampal volume reduction is a pathological hallmark of schizophrenia, the molecular pathway(s) responsible for this degeneration remains unknown. Recent reports have suggested the potential role of impaired blood-brain barrier (BBB) function in schizophrenia pathogenesis. However, direct evidence demonstrating an impaired BBB function is missing. In this preliminary study, we used immunohistochemistry and serum immunoglobulin G (IgG) antibodies to investigate the state of BBB function in formalin-fixed postmortem samples from the hippocampus and surrounding temporal cortex of patients with schizophrenia (n = 25) and controls without schizophrenia (n = 27) matched for age, sex, and race. Since a functional BBB prevents the extravasation of IgGs, detection of IgGs in the parenchyma is used as direct evidence of BBB breakdown. We also developed a semi-quantitative approach to quantify the extent of IgG leak and therein BBB breach. Analysis of our immunohistochemistry data demonstrated a significantly higher incidence of IgG leak in patients with schizophrenia compared to controls. Further, BBB permeability was significantly higher in advanced-age patients with schizophrenia than both advanced-age controls and middle-aged patients with schizophrenia. Male patients with schizophrenia also demonstrated a significant increase in IgG permeability compared to control males. Interestingly, the extravasated IgGs also demonstrated selective immunoreactivity for neurons. Based on these observations, we suggest that BBB dysfunction and IgG autoantibodies could be two key missing pathoetiological links underwriting schizophrenia hippocampal damage.

Evidence that Brain-Reactive Autoantibodies Contribute to Chronic Neuronal Internalization of Exogenous Amyloid-ß1-42 and Key Cell Surface Proteins During Alzheimer's Disease Pathogenesis.

Blood-brain barrier (BBB) permeability is a recognized early feature of Alzheimer's 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-ß1-42 (Aß42), cathepsin D, IgG, GluR2/3, and alpha7 nicotinic acetylcholine receptor (α7nAChR). Results revealed that chronic IgG binding to pyramidal neurons coincided with internalization of Aß42, 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 Aß42 peptide and serum from AD and control subjects. The rate and extent of Aß42 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 Aß42. Initial co-localization of IgG, α7nAChR, and Aß42 was temporally and spatially linked to early endosomes (Rab11) and later to lysosomes (LAMP-1). Aß42 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 Aß42 deposition in AD.

Autoantibodies as diagnostic biomarkers for the detection and subtyping of multiple sclerosis.

The goal of this preliminary proof-of-concept study was to use human protein microarrays to identify blood-based autoantibody biomarkers capable of diagnosing multiple sclerosis (MS). Using sera from 112 subjects, including 51 MS subjects, autoantibody biomarkers effectively differentiated MS subjects from age- and gender-matched normal and breast cancer controls with 95.0% and 100% overall accuracy, but not from subjects with Parkinson's disease. Autoantibody biomarkers were also useful in distinguishing subjects with the relapsing-remitting form of MS from those with the secondary progressive subtype. These results demonstrate that autoantibodies can be used as noninvasive blood-based biomarkers for the detection and subtyping of MS.

Retinal pathology is associated with increased blood-retina barrier permeability in a diabetic and hypercholesterolaemic pig model: Beneficial effects of the LpPLA2 inhibitor Darapladib.

Using a porcine model of diabetes mellitus and hypercholesterolaemia, we previously showed that diabetes mellitus and hypercholesterolaemia is associated with a chronic increase in blood-brain barrier permeability in the cerebral cortex, leading to selective binding of immunoglobulin G and deposition of amyloid-beta1-42 peptide in pyramidal neurons. Treatment with Darapladib (GlaxoSmithKline, SB480848), an inhibitor of lipoprotein-associated phospholipase-A2, alleviated these effects. Here, investigation of the effects of chronic diabetes mellitus and hypercholesterolaemia on the pig retina revealed a corresponding increased permeability of the blood-retina barrier coupled with a leak of plasma components into the retina, alterations in retinal architecture, selective IgG binding to neurons in the ganglion cell layer, thinning of retinal layers due to cell loss and increased glial fibrillary acidic protein expression in Müller cells, all of which were curtailed by treatment with Darapladib. These findings suggest that chronic diabetes mellitus and hypercholesterolaemia induces increased blood-retina barrier permeability that may be linked to altered expression of blood-retina barrier-associated tight junction proteins, claudin and occludin, leading to structural changes in the retina consistent with diabetic retinopathy. Additionally, results suggest that drugs with vascular anti-inflammatory properties, such as Darapladib, may have beneficial effects on eye diseases strongly linked to vascular abnormalities such as diabetic retinopathy and age-related macular degeneration.

Mechanical stress regulates transport in a compliant 3D model of the blood-brain barrier.

Transport of fluid and solutes is tightly controlled within the brain, where vasculature exhibits a blood-brain barrier and there is no organized lymphatic network facilitating waste transport from the interstitial space. Here, using a compliant, three-dimensional co-culture model of the blood-brain barrier, we show that mechanical stimuli exerted by blood flow mediate both the permeability of the endothelial barrier and waste transport along the basement membrane. Application of both shear stress and cyclic strain facilitates tight junction formation in the endothelial monolayer, with and without the presence of astrocyte endfeet in the surrounding matrix. We use both dextran perfusion and TEER measurements to assess the initiation and maintenance of the endothelial barrier, and microparticle image velocimetry to characterize the fluid dynamics within the in vitro vessels. Application of pulsatile flow to the in vitro vessels induces pulsatile strain to the vascular wall, providing an opportunity to investigate stretch-induced transport along the basement membrane. We find that a pulsatile wave speed of approximately 1 mm/s with Womersley number of 0.004 facilitates retrograde transport of high molecular weight dextran along the basement membrane between the basal endothelium and surrounding astrocytes. Together, these findings indicate that the mechanical stress exerted by blood flow is an important regulator of transport both across and along the walls of cerebral microvasculature.

Breakdown of the Cerebrovasculature and Blood-Brain Barrier: A Mechanistic Link Between Diabetes Mellitus and Alzheimer's Disease.

Alzheimer's disease (AD) and diabetes mellitus (DM) are among the most pervasive and devastating disorders that afflict people throughout the world. Although typically associated with older demographics, recent epidemiologic studies have reported parallel trends in decreasing age of onset and increasing incidence of these conditions. Promising research continues to implicate the cerebrovasculature and blood-brain barrier (BBB) as playing key roles in AD pathoetiology. Similarly, complications accompanying DM, such as diabetic nephropathy/retinopathy, cardiovascular disease, and stroke, have been rooted in vascular compromise. Not surprisingly, DM is now considered a major risk factor for AD. The purpose of this review is to highlight investigations into the role of the cerebrovasculature in the development and progression of AD. We give particular attention to studies on humans and a variety of animal model systems that have demonstrated a link between BBB dysfunction and pathological changes in the brain consistent with aging and AD. Together, these studies suggest that the vascular complications associated with chronic, poorly managed DM can lead to subclinical BBB breakdown that precedes and drives the pathological changes progressing to symptomatic AD, providing a common mechanistic thread connecting these two disorders. Furthermore, this emphasizes the need to focus on the vasculature as a potential therapeutic target with the intent of limiting BBB breakdown involved in disease initiation and progression. In conclusion, AD may be more than just an associated comorbidity of DM, and instead another manifestation of the underlying vascular pathology that is common to both.

Detection of Alzheimer's 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's 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's disease. METHODS: Sera from 236 subjects, including 50 mild cognitive impairment (MCI) subjects with confirmed low CSF Aß42 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.

Age-dependent increase of blood-brain barrier permeability and neuron-binding autoantibodies in S100B knockout mice.

S100B is a calcium-sensor protein that impacts multiple signal transduction pathways. It is widely considered to be an important biomarker for several neuronal diseases as well as blood-brain barrier (BBB) breakdown. In this report, we demonstrate a BBB deficiency in mice that lack S100B through detection of leaked Immunoglobulin G (IgG) in the brain parenchyma. IgG leaks and IgG-binding to selected neurons were observed in S100B knockout (S100BKO) mice at 6 months of age but not at 3 months. By 9 months, IgG leaks persisted and the density of IgG-bound neurons increased significantly. These results reveal a chronic increase in BBB permeability upon aging in S100BKO mice for the first time. Moreover, coincident with the increase in IgG-bound neurons, autoantibodies targeting brain proteins were detected in the serum via western blots. These events were concurrent with compromise of neurons, increase of activated microglia and lack of astrocytic activation as evidenced by decreased expression of microtubule-associated protein type 2 (MAP2), elevated number of CD68 positive cells and unaltered expression of glial fibrillary acidic protein (GFAP) respectively. Results suggest a key role for S100B in maintaining BBB functional integrity and, further, propose the S100BKO mouse as a valuable model system to explore the link between chronic functional compromise of the BBB, generation of brain-reactive autoantibodies and neuronal dysfunctions.

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.

Utility of autoantibodies as biomarkers for diagnosis and staging of neurodegenerative diseases.

Autoantibodies are self-reactive antibodies that have been widely implicated as causal agents of autoimmune diseases. They are found in the blood of all human sera, regardless of age, gender, or the presence or absence of disease. While the underlying reason for their ubiquity remains unknown, it has been hypothesized that they participate in the clearance of blood-borne cell and tissue debris generated in both healthy and diseased individuals on a daily basis. Although much evidence supports this debris clearance role, recent studies also suggest a causal role for autoantibodies in disease. This chapter first presents well-known examples of autoimmune diseases that emphasize a direct causal role for autoantibodies and then discusses the veritable explosion of evidence now supporting their involvement in a wide variety of other diseases, including cancers and several types of neurological and neurodegenerative diseases. Lastly, translational strategies that take advantage of the "cause and/or effect" role of autoantibodies and recent technological advancements in their detection to exploit autoantibodies as sensitive and specific biomarkers useful for the detection and diagnosis of disease are outlined. Their use in the diagnosis and staging of Alzheimer's and Parkinson's diseases is presented, and future applications in clinical medicine and basic science are highlighted.

Sevoflurane and Isoflurane induce structural changes in brain vascular endothelial cells and increase blood-brain barrier permeability: Possible link to postoperative delirium and cognitive decline.

A large percentage of patients subjected to general anesthesia at 65 years and older exhibit postoperative delirium (POD). Here, we test the hypothesis that inhaled anesthetics (IAs), such as Sevoflurane and Isoflurane, act directly on brain vascular endothelial cells (BVECs) to increase blood-brain barrier (BBB) permeability, thereby contributing to POD. Rats of young (3-5 months), middle (10-12 months) and old (17-19 months) ages were anesthetized with Sevoflurane or Isoflurane for 3h. After exposure, some were euthanized immediately; others were allowed to recover for 24h before sacrifice. Immunohistochemistry was employed to monitor the extent of BBB breach, and scanning electron microscopy (SEM) was used to examine changes in the luminal surfaces of BVECs. Quantitative immunohistochemistry revealed increased BBB permeability in older animals treated with Sevoflurane, but not Isoflurane. Extravasated immunoglobulin G showed selective affinity for pyramidal neurons. SEM demonstrated marked flattening of the luminal surfaces of BVECs in anesthetic-treated rats. Results suggest an aging-linked BBB compromise resulting from exposure to Sevoflurane. Changes in the luminal surface topology of BVECs indicate a direct effect on the plasma membrane, which may weaken or disrupt their BBB-associated tight junctions. Disruption of brain homeostasis due to plasma influx into the brain parenchyma and binding of plasma components (e.g., immunoglobulins) to neurons may contribute to POD. We propose that, in the elderly, exposure to some IAs can cause BBB compromise that disrupts brain homeostasis, perturbs neuronal function and thereby contributes to POD. If unresolved, this may progress to postoperative cognitive decline and later dementia.

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.

Diabetes and hypercholesterolemia increase blood-brain barrier permeability and brain amyloid deposition: beneficial effects of the LpPLA2 inhibitor darapladib.

Diabetes mellitus (DM) and hypercholesterolemia (HC) have emerged as major risk factors for Alzheimer's disease, highlighting the importance of vascular health to normal brain functioning. Our previous study showed that DM and HC favor the development of advanced coronary atherosclerosis in a porcine model, and that treatment with darapladib, an inhibitor of lipoprotein-associated phospholipase A2, blocks atherosclerosis progression and improves animal alertness and activity levels. In the present study, we examined the effects of DM and HC on the permeability of the blood-brain barrier (BBB) using immunoglobulin G (IgG) as a biomarker. DMHC increased BBB permeability and the leak of microvascular IgG into the brain interstitium, which was bound preferentially to pyramidal neurons in the cerebral cortex. We also examined the effects of DMHC on the brain deposition of amyloid peptide (Aß42), a well-known pathological feature of Alzheimer's disease. Nearly all detectable Aß42 was contained within cortical pyramidal neurons and DMHC increased the density of Aß42-loaded neurons. Treatment of DMHC animals with darapladib reduced the amount of IgG-immunopositive material that leaked into the brain as well as the density of Aß42-containing neurons. Overall, these results suggest that a prolonged state of DMHC may have chronic deleterious effects on the functional integrity of the BBB and that, in this DMHC pig model, darapladib reduces BBB permeability. Also, the preferential binding of IgG and coincident accumulation of Aß42 in the same neurons suggests a mechanistic link between the leak of IgG through the BBB and intraneuronal deposition of Aß42 in the brain.

Plant lectin can target receptors containing sialic acid, exemplified by podoplanin, to inhibit transformed cell growth and migration.

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Neuronal PAD4 expression and protein citrullination: possible role in production of autoantibodies associated with neurodegenerative disease.

Peptidyl arginine deiminases (PADs) catalyze a post-translational protein modification reaction called citrullination, where arginine is converted to citrulline. This modification has been linked to the pathogenesis of autoimmune diseases including rheumatoid arthritis (RA). More recently, several studies have suggested that Alzheimer's disease (AD), a devastating neurodegenerative disorder, may have an autoimmune component. In the present study, we have investigated the possibility that expression of PADs and protein citrullination plays a role in the production of brain-reactive autoantibodies that may contribute to Alzheimer's-related brain pathology. Here, we report the selective expression of the PAD isoforms, PAD2 and PAD4, in astrocytes and neurons, respectively, and the concomitant accumulation of citrullinated proteins within PAD4-expressing cells, including neurons of the hippocampus and cerebral cortex. Expression of PADs and citrullinated proteins is prominent in brain regions engaged in neurodegenerative changes typical for AD pathology. Furthermore, we also demonstrate that the pentatricopeptide repeat domain2 (PTCD2) protein, an antigen target of a prominent AD diagnostic autoantibody, is present in a citrullinated form in AD brains. Our results suggest that disease-associated neuronal loss results in the release of cellular contents, including citrullinated proteins, into the brain interstitium. We propose that these citrullinated proteins and their degradation fragments enter into the blood and lymphatic circulation, and some are capable of eliciting an immune response that results in the production of autoantibodies. The long-term and progressive nature of AD and other neurodegenerative diseases results in chronic exposure of the immune system to these citrullinated products and may drive the continual production of autoantibodies.

Brain-reactive autoantibodies prevalent in human sera increase intraneuronal amyloid-ß(1-42) deposition.

Previous studies have reported immunoglobulin-positive neurons in Alzheimer's disease (AD) brains, an observation indicative of blood-brain barrier (BBB) breakdown. Recently, we demonstrated the nearly ubiquitous presence of brain-reactive autoantibodies in human sera. The significance of these observations to AD pathology is unknown. Here, we show that IgG-immunopositive neurons are abundant in brain regions exhibiting AD pathology, including intraneuronal amyloid-ß(42) (Aß(42)) and amyloid plaques, and confirm by western analysis that brain-reactive autoantibodies are nearly ubiquitous in human serum. To investigate a possible interrelationship between neuronal antibody binding and Aß pathology, we tested the effects of human serum autoantibodies on the intraneuronal deposition of soluble Aß(42) peptide in adult mouse neurons in vitro (organotypic brain slice cultures). Binding of human autoantibodies to mouse neurons dramatically increased the rate and extent of intraneuronal Aß(42) accumulation in the mouse cerebral cortex and hippocampus. Additionally, individual sera exhibited variable potency related to their capacity to enhance intraneuronal Aß(42) peptide accumulation and immunolabel neurons in AD brain sections. Replacement of human sera with antibodies targeting abundant neuronal surface proteins resulted in a comparable enhancement of Aß(42) accumulation in mouse neurons. Overall, results suggest that brain-reactive autoantibodies are ubiquitous in the blood and that a defective BBB allows these antibodies to access the brain interstitium, bind to neuronal surfaces and enhance intraneuronal deposition of Aß(42) in AD brains. Thus, in the context of BBB compromise, brain-reactive autoantibodies may be an important risk factor for the initiation and/or progression of AD as well as other neurodegenerative diseases.