Joshua B. Owen

Insulin in the brain: There and back again

Insulin performs unique functions within the CNS. Produced nearly exclusively by the pancreas, insulin crosses the blood-brain barrier (BBB) using a saturable transporter, affecting feeding and cognition through CNS mechanisms largely independent of glucose utilization. Whereas peripheral insulin acts primarily as a metabolic regulatory hormone, CNS insulin has an array of effects on brain that may more closely resemble the actions of the ancestral insulin molecule. Brain endothelial cells (BECs), the cells that form the vascular BBB and contain the transporter that translocates insulin from blood to brain, are themselves regulated by insulin. The insulin transporter is altered by physiological and pathological factors including hyperglycemia and the diabetic state. The latter can lead to BBB disruption. Pericytes, pluripotent cells in intimate contact with the BECs, protect the integrity of the BBB and its ability to transport insulin. Most of insulins known actions within the CNS are mediated through two canonical pathways, the phosphoinositide-3 kinase (PI3)/Akt and Ras/mitogen activated kinase (MAPK) cascades. Resistance to insulin action within the CNS, sometimes referred to as diabetes mellitus type III, is associated with peripheral insulin resistance, but it is possible that variable hormonal resistance syndromes exist so that resistance at one tissue bed may be independent of that at others. CNS insulin resistance is associated with Alzheimers disease, depression, and impaired baroreceptor gain in pregnancy. These aspects of CNS insulin action and the control of its entry by the BBB are likely only a small part of the story of insulin within the brain.

Lipopolysaccharide alters the blood–brain barrier transport of amyloid β protein: A mechanism for inflammation in the progression of Alzheimer’s disease

Alzheimers disease (AD) brains are characterized by accumulation of amyloid beta protein (Abeta) and neuroinflammation. Increased blood-to-brain influx and decreased brain-to-blood efflux across the blood-brain barrier (BBB) have been proposed as mechanisms for Abeta accumulation. Epidemiological studies suggest that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the progression of AD. We hypothesized that inflammation alters BBB handling of Abeta. Mice treated with lipopolysaccharide (LPS) had increased brain influx and decreased brain efflux of Abeta, recapitulating the findings in AD. Neither influx nor efflux was mediated by LPS acting directly on BBB cells. Increased influx was mediated by a blood-borne factor, indomethacin-independent, blocked by the triglyceride triolein, and not related to expression of the blood-to-brain transporter of Abeta, RAGE. Serum levels of IL-6, IL-10, IL-13, and MCP-1 mirrored changes in Abeta influx. Decreased efflux was blocked by indomethacin and accompanied by decreased protein expression of the brain-to-blood transporter of Abeta, LRP-1. LPS paradoxically increased expression of neuronal LRP-1, a major source of Abeta. Thus, inflammation potentially increases brain levels of Abeta by three mechanisms: increased influx, decreased efflux, and increased neuronal production.

Measurement of oxidized/reduced glutathione ratio.

Glutathione (GSH) is the most abundant antioxidant in aerobic cells, present in micromolar (microM)-concentrations in bodily fluids and in millimolar (mM) concentrations in tissue. GSH is critical for protecting the brain from oxidative stress, acting as a free radical scavenger and inhibitor of lipid peroxidation. GSH also participates in the detoxification of hydrogen peroxide by various glutathione peroxidases. The ratio of reduced GSH to oxidized GSH (GSSG) is an indicator of cellular health, with reduced GSH constituting up to 98% of cellular GSH under normal conditions. However, the GSH/GSSG ratio is reduced in neurodegenerative diseases, such as Parkinsons disease (PD) and Alzheimers disease (AD). Measuring the GSH/GSSG ratio in pathological tissues and experimental models thereof in comparison to the results in controls is an excellent way to assess potential therapeutics efficacy in maintaining cellular redox potential. The availability of UV/Visible instruments equipped with 96-well plate readers as common laboratory equipment has made measuring the GSH/GSSG ratio on multiple samples a manageable procedure.

Proteomics-determined differences in the Concanavalin-A-fractionated proteome of hippocampus and inferior parietal lobule in subjects with Alzheimer’s disease and mild cognitive impairment: Implications for progression of AD

Alzheimers disease (AD) is the most common type of dementia, comprising 60-80% of all reported cases, and currently affects 5.2 million Americans. AD is characterized pathologically by the accumulation of senile plaques (SPs), neurofibrillary tangles (NFTs), and synapse loss. The early stages of memory loss associated with AD have been studied in a condition known as amnestic mild cognitive impairment (MCI), arguably the earliest form of AD. In spite of extensive research across a variety of disciplines, the cause of AD remains elusive. Proteomics techniques have helped to advance knowledge about AD by identifying irregularities in protein expression and post-translational modifications (PTMs) in AD brain. Glycosylation is a less studied PTM with regards to AD and MCI. This PTM is important to study because glycosylation is involved in proper protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles, and these processes are impaired in AD. Concanavalin-A (Con-A) binds to N-linked glycoproteins, but hydrophobic sites on nonglycoproteins are also known to bind Con-A. To our knowledge, the present study is the first to examine Con-A-associated brain proteins in MCI and AD with focus on the hippocampus and inferior parietal lobule (IPL) brain regions. Proteins found in AD hippocampus with altered levels are glutamate dehydrogenase (GDH), glial fibrillary acidic protein (GFAP), tropomyosin 3 (TPM3), Rab GDP-dissociation inhibitor XAP-4 (XAP4), and heat shock protein 90 (HSP90). Proteins found with altered levels in AD IPL are alpha-enolase, gamma-enolase, and XAP-4. MCI hippocampal proteins with altered levels are dihydropyrimidase-2 (DRP2), glucose-regulated protein 78 (GRP-78), protein phosphatase related protein Sds-22 (Sds22), and GFAP and the only protein found with altered levels in MCI IPL was beta-synuclein. These results are discussed with reference to biochemical and pathological alterations in and progression of AD.

Proteomics in animal models of Alzheimer's and Parkinson's diseases

The risk of developing neurodegenerative disorders such as Alzheimers disease (AD) and Parkinsons disease (PD) increases with age. AD and PD are the two most common neurodegenerative diseases that currently affect millions of persons within the United States population. While many clues about the mechanisms of these disorders have been uncovered, to date, the molecular mechanisms associated with the cause of these diseases are not completely understood. Furthermore, there are no available cures or preventive treatments for either disorder. Animal models of AD and PD, though not perfect, offer a means to gain knowledge of the basic biochemistry associated with these disorders and with drug efficacy. The field of proteomics which focuses on identifying the dynamic nature of the protein content expressed within a particular cell, tissue, or organism, has provided many insights into these disturbing disorders. Proteomic studies have revealed many pathways that are associated with disease pathogenesis and that may lead to the development of potential therapeutic targets. This review provides a discussion of key findings from AD and PD proteomics-based studies in various animal models of disease.

Lipopolysaccharide impairs amyloid beta efflux from brain: altered vascular sequestration, cerebrospinal fluid reabsorption, peripheral clearance and transporter function at the blood–brain barrier

BackgroundDefects in the low density lipoprotein receptor-related protein-1 (LRP-1) and p-glycoprotein (Pgp) clearance of amyloid beta (Aβ) from brain are thought to contribute to Alzheimer’s disease (AD). We have recently shown that induction of systemic inflammation by lipopolysaccharide (LPS) results in impaired efflux of Aβ from the brain. The same treatment also impairs Pgp function. Here, our aim is to determine which physiological routes of Aβ clearance are affected following systemic inflammation, including those relying on LRP-1 and Pgp function at the blood–brain barrier.MethodsCD-1 mice aged between 6 and 8 weeks were treated with 3 intraperitoneal injections of 3 mg/kg LPS at 0, 6, and 24 hours and studied at 28 hours. 125I-Aβ1-42 or 125I-alpha-2-macroglobulin injected into the lateral ventricle of the brain (intracerebroventricular (ICV)) or into the jugular vein (intravenous (IV)) was used to quantify LRP-1-dependent partitioning between the brain vasculature and parenchyma and peripheral clearance, respectively. Disappearance of ICV-injected 14 C-inulin from brain was measured to quantify bulk flow of cerebrospinal fluid (CSF). Brain microvascular protein expression of LRP-1 and Pgp was measured by immunoblotting. Endothelial cell localization of LRP-1 was measured by immunofluorescence microscopy. Oxidative modifications to LRP-1 at the brain microvasculature were measured by immunoprecipitation of LRP-1 followed by immunoblotting for 4-hydroxynonenal and 3-nitrotyrosine.ResultsWe found that LPS: caused an LRP-1-dependent redistribution of ICV-injected Aβ from brain parenchyma to brain vasculature and decreased entry into blood; impaired peripheral clearance of IV-injected Aβ; inhibited reabsorption of CSF; did not significantly alter brain microvascular protein levels of LRP-1 or Pgp, or oxidative modifications to LRP-1; and downregulated LRP-1 protein levels and caused LRP-1 mislocalization in cultured brain endothelial cells.ConclusionsThese results suggest that LRP-1 undergoes complex functional regulation following systemic inflammation which may depend on cell type, subcellular location, and post-translational modifications. Our findings that systemic inflammation causes deficits in both Aβ transport and bulk flow like those observed in AD indicate that inflammation could induce and promote the disease.

Oxidative modification to LDL receptor-related protein 1 in hippocampus from subjects with Alzheimer disease: Implications for Aβ accumulation in AD brain

Alzheimer disease (AD) is a neurodegenerative disorder characterized histopathologically by the presence of senile plaques (SPs), neurofibrillary tangles, and synapse loss. The main component of SPs is amyloid-β peptide (Aβ), which has been associated with increased oxidative stress, leading to oxidative modification of proteins and consequently to neurotoxicity and neurodegeneration. Low-density lipoprotein receptor-related protein 1 (LRP1) is the primary moiety responsible for the efflux of Aβ from the brain to the blood across the blood-brain barrier. Impaired brain-to-blood transport of Aβ by LRP1 has been hypothesized to contribute to increased levels of Aβ in AD brain. The cause of LRP1 dysfunction is unknown, but we have hypothesized that Aβ oxidizes LRP1, thus damaging its own transporter. Consistent with this notion, we report in this study a significant increase in the levels of the lipid peroxidation product 4-hydroxy-2-nonenal bound to transmembrane LRP1 in AD hippocampus. In contrast, the levels of LRP1-resident 3-nitrotyrosine did not show a significant increase in AD hippocampus compared to age-matched controls. Based on this study, we propose that Aβ impairs its own efflux from the brain by oxidation of its transporter LRP1, leading to increased Aβ deposition in brain, thereby contributing to subsequent cognitive impairment in AD.

Proteomic identification of nitrated brain proteins in traumatic brain-injured rats treated postinjury with gamma-glutamylcysteine ethyl ester: insights into the role of elevation of glutathione as a potential therapeutic strategy for traumatic brain injury.

Traumatic brain injury (TBI) occurs suddenly and has damaging effects to the brain that are dependent on the severity of insult. Symptoms can be mild, moderate, or severe. Oxidative damage is associated with traumatic brain injury through reactive oxygen/nitrogen species production. One such species, peroxynitrite, is elevated in TBI brain tissue (Orihara et al. [ 2001 ] Forensic Sci. Int. 123:142–149; Deng et al. [ 2007 ] Exp. Neurol. 205:154–165). Peroxynitrite can react with carbon dioxide and decompose to produce NO2 and carbonate radicals, which in turn can lead to 3‐nitrotyrosine, an index of protein nitration. Gamma‐glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma‐glutamylcysteine, an agent that up‐regulates glutathione (GSH) production in brain (Drake et al. [ 2002 ] J. Neurosci. Res. 68:776–784). Many preclinical studies of TBI have employed pretreatment of animals with proposed beneficial agents prior to the injury itself. However, in the real world of TBI, treatment begins postinjury. Hence, insights into agents that improve outcome following injury are desperately needed. This study is one of the first to investigate a potential GSH‐based therapy for TBI postinjury. Protein carbonyls, an index of protein oxidation, were significantly elevated in brain of animals subjected to TBI. However, if, after TBI, GCEE was administered i.p., protein carbonyl levels were significantly reduced. Similarly, 3‐nitrotyrosine levels were elevated in brain following TBI but significantly decreased following TBI if GCEE was administered i.p. Redox proteomics analysis showed that several brain proteins were nitrated after TBI. However, if GCEE was given i.p. following TBI, many of these proteins were protected from nitration. The results are encouraging and are discussed with reference to potential therapeutic strategies for TBI involving elevated GSH.

Rapid Transport of CCL11 across the Blood-Brain Barrier: Regional Variation and Importance of Blood Cells

Increased blood levels of the eotaxin chemokine C-C motif ligand 11 (CCL11) in aging were recently shown to negatively regulate adult hippocampal neurogenesis. How circulating CCL11 could affect the central nervous system (CNS) is not clear, but one possibility is that it can cross the blood-brain barrier (BBB). Here, we show that CCL11 undergoes bidirectional transport across the BBB. Transport of CCL11 from blood into whole brain (influx) showed biphasic kinetics, with a slow phase preceding a rapid phase of uptake. We found that the slow phase was explained by binding of CCL11 to cellular components in blood, whereas the rapid uptake phase was mediated by direct interactions with the BBB. CCL11, even at high doses, did not cause BBB disruption. All brain regions except striatum showed a delayed rapid-uptake phase. Striatum had only an early rapid-uptake phase, which was the fastest of any brain region. We also observed a slow but saturable transport system for CCL11 from brain to blood. C-C motif ligand 3 (CCR3), an important receptor for CCL11, did not facilitate CCL11 transport across the BBB, although high concentrations of a CCR3 inhibitor increased brain uptake without causing BBB disruption. Our results indicate that CCL11 in the circulation can access many regions of the brain outside of the neurogenic niche via transport across the BBB. This suggests that blood-borne CCL11 may have important physiologic functions in the CNS and implicates the BBB as an important regulator of physiologic versus pathologic effects of this chemokine.

The wheat germ agglutinin-fractionated proteome of subjects with Alzheimer's disease and mild cognitive impairment hippocampus and inferior parietal lobule: Implications for disease pathogenesis and progression

Lectin affinity chromatography is a powerful separation technique that fractionates proteins by selectively binding to specific carbohydrate moieties characteristic of protein glycosylation type. Wheat germ agglutinin (WGA) selectively binds terminal N‐acetylglucosamine (O‐GlcNAc) and sialic acid moieties characteristic of O‐linked glycosylation. The current study utilizes WGA affinity chromatography to fractionate proteins from hippocampus and inferior parietal lobule (IPL) from subjects with Alzheimers disease (AD) and arguably its earliest form, mild cognitive impairment (MCI). Proteins identified by proteomics that were fractionated from MCI and AD hippocampus by WGA affinity chromatography with altered levels compared with age‐matched controls included GP96, γ‐enolase, glutamate dehydrogenase, glucosidase IIα, 14‐3‐3ϵ, 14‐3‐3γ, 14‐3‐3ζ, tropomyosin‐2, calmodulin 2, gelsolin, β‐synuclein, α1‐antichymotrypsin, and dimethylguanosine tRNA methyltransferase. Proteins identified by proteomics that were fractionated from MCI and AD IPL by WGA affinity chromatography showing altered levels compared with age‐matched controls included protein disulfide isomerase, calreticulin, and GP96. The proteins described in this study are involved in diverse processes, including glucose metabolism, endoplasmic reticulum (ER) functions, chaperoning, cytoskeletal assembly, and proteolysis, all of which are affected in AD. This study, the first to use proteomics to identify WGA‐fractionated proteins isolated from brains from subjects with MCI and AD, provides additional information about the active proteome of the brain throughout AD progression.