Mark S. Kindy

RAGE mediates amyloid-beta peptide transport across the blood-brain barrier and accumulation in brain.

Amyloid-β peptide (Aβ) interacts with the vasculature to influence Aβ levels in the brain and cerebral blood flow, providing a means of amplifying the Aβ-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Aβ infusion and studies in genetically manipulated mice show that Aβ interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Aβ across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Aβ-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Aβ in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Aβ-vascular interactions, including development of cerebral amyloidosis.

Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors

Brain injury, as occurs in stroke or head trauma, induces a dramatic increase in levels of tumor necrosis factor–α (TNF), but its role in brain injury response is unknown. We generated mice genetically deficient in TNF receptors (TNFR–KO) to determine the role of TNF in brain cell injury responses. Damage to neurons caused by focal cerebral ischemia and epileptic seizures was exacerbated in TNFR–KO mice, indicating that TNF serves a neuroprotective function. Oxidative stress was Increased and levels of an antioxidant enzyme reduced in brain cells of TNFR–KO mice, indicating that TNF protects neurons by stimulating antioxidant pathways. Injury–induced microglial activation was suppressed in TNFR–KO mice, demonstrating a key role for TNF in injury–induced immune response. Drugs that target TNF signaling pathways may prove beneficial in treating stroke and traumatic brain injury.

Neprilysin Gene Transfer Reduces Human Amyloid Pathology in Transgenic Mice

The degenerative process of Alzheimers disease is linked to a shift in the balance between amyloid-β (Aβ) production, clearance, and degradation. Neprilysin has recently been implicated as a major extracellular Aβ degrading enzyme in the brain. However, there has been no direct demonstration that neprilysin antagonizes the deposition of amyloid-β in vivo. To address this issue, a lentiviral vector expressing human neprilysin (Lenti-Nep) was tested in transgenic mouse models of amyloidosis. We show that unilateral intracerebral injection of Lenti-Nep reduced amyloid-β deposits by half relative to the untreated side. Furthermore, Lenti-Nep ameliorated neurodegenerative alterations in the frontal cortex and hippocampus of these transgenic mice. These data further support a role for neprilysin in regulating cerebral amyloid deposition and suggest that gene transfer approaches might have potential for the development of alternative therapies for Alzheimers disease.

Receptor-dependent cell stress and amyloid accumulation in systemic amyloidosis

Accumulation of fibrils composed of amyloid A in tissues resulting in displacement of normal structures and cellular dysfunction is the characteristic feature of systemic amyloidoses. Here we show that RAGE, a multiligand immunoglobulin superfamily cell surface molecule, is a receptor for the amyloidogenic form of serum amyloid A. Interactions between RAGE and amyloid A induced cellular perturbation. In a mouse model, amyloid A accumulation, evidence of cell stress and expression of RAGE were closely linked. Antagonizing RAGE suppressed cell stress and amyloid deposition in mouse spleens. These data indicate that RAGE is a potential target for inhibiting accumulation of amyloid A and for limiting cellular dysfunction induced by amyloid A.

GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism

Glucagon-like peptide-1 (GLP-1) is an endogenous insulinotropic peptide secreted from the gastrointestinal tract in response to food intake. It enhances pancreatic islet β-cell proliferation and glucose-dependent insulin secretion, and lowers blood glucose and food intake in patients with type 2 diabetes mellitus (T2DM). A long-acting GLP-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), is the first of this new class of antihyperglycemia drugs approved to treat T2DM. GLP-1Rs are coupled to the cAMP second messenger pathway and, along with pancreatic cells, are expressed within the nervous system of rodents and humans, where receptor activation elicits neurotrophic actions. We detected GLP-1R mRNA expression in both cultured embryonic primary cerebral cortical and ventral mesencephalic (dopaminergic) neurons. These cells are vulnerable to hypoxia- and 6-hydroxydopamine–induced cell death, respectively. We found that GLP-1 and Ex-4 conferred protection in these cells, but not in cells from Glp1r knockout (-/-) mice. Administration of Ex-4 reduced brain damage and improved functional outcome in a transient middle cerebral artery occlusion stroke model. Ex-4 treatment also protected dopaminergic neurons against degeneration, preserved dopamine levels, and improved motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinsons disease (PD). Our findings demonstrate that Ex-4 can protect neurons against metabolic and oxidative insults, and they provide preclinical support for the therapeutic potential for Ex-4 in the treatment of stroke and PD.

RAGE potentiates Aβ‐induced perturbation of neuronal function in transgenic mice

Receptor for Advanced Glycation Endproducts (RAGE), a multiligand receptor in the immunoglobulin superfamily, functions as a signal‐transducing cell surface acceptor for amyloid‐beta peptide (Aβ). In view of increased neuronal expression of RAGE in Alzheimers disease, a murine model was developed to assess the impact of RAGE in an Aβ‐rich environment, employing transgenics (Tgs) with targeted neuronal overexpression of RAGE and mutant amyloid precursor protein (APP). Double Tgs (mutant APP (mAPP)/RAGE) displayed early abnormalities in spatial learning/memory, accompanied by altered activation of markers of synaptic plasticity and exaggerated neuropathologic findings, before such changes were found in mAPP mice. In contrast, Tg mice bearing a dominant‐negative RAGE construct targeted to neurons crossed with mAPP animals displayed preservation of spatial learning/memory and diminished neuropathologic changes. These data indicate that RAGE is a cofactor for Aβ‐induced neuronal perturbation in a model of Alzheimers‐type pathology, and suggest its potential as a therapeutic target to ameliorate cellular dysfunction.

Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor.

Ischemic and excitotoxic insults to the brain induce rapid production of tumor necrosis factor-α (TNF), but the role of TNF in neuronal responses to brain injury are unclear. Two different TNF receptors (p55 and p75) are expressed in neurons and glia, To understand the role of TNF in brain injury, we generated mice that lack p55, p75, or both receptors, We report that neuronal damage after focal cerebral ischemia—reperfusion is significantly increased in mice lacking p55 receptors (85 ± 7 mm3 infarct volume; mean ± SD) compared with wild-type mice (70 ± 8 mm3) and mice lacking p75 receptors (72 ± 6 mm3). Moreover, mice lacking p55 receptors exhibited increased degeneration of CA3 hippocampal neurons after administration of the excitotoxin kainic acid compared with wild-type mice and mice lacking p75 receptors. When taken together with recent data showing that TNF can prevent apoptosis of cultured neurons exposed to oxidative and metabolic insults, our findings suggest that TNF plays a neuroprotective role after acute brain insults.

High cholesterol-induced neuroinflammation and amyloid precursor protein processing correlate with loss of working memory in mice.

Recent findings suggest that hypercholesterolemia may contribute to the onset of Alzheimer’s disease‐like dementia but the underlying mechanisms remain unknown. In this study, we evaluated the cognitive performance in rodent models of hypercholesterolemia in relation to neuroinflammatory changes and amyloid precursor protein (APP) processing, the two key parameters of Alzheimer’s disease pathogenesis. Groups of normal C57BL/6 and low density lipoprotein receptor (LDLR)‐deficient mice were fed a high fat/cholesterol diet for an 8‐week period and tested for memory in a radial arm maze. It was found that the C57BL/6 mice receiving a high fat diet were deficient in handling an increasing working memory load compared with counterparts receiving a control diet while the hypercholesterolemic LDLR−/− mice showed impaired working memory regardless of diet. Immunohistochemical analysis revealed the presence of activated microglia and astrocytes in the hippocampi from high fat‐fed C57BL/6 mice and LDLR−/− mice. Consistent with a neuroinflammatory response, the hyperlipidemic mice showed increased expression of cytokines/mediators including tumor necrosis factor‐α, interleukin‐1β and ‐6, nitric oxide synthase 2, and cycloxygenase 2. There was also an induced expression of the key APP processing enzyme i.e. β‐site APP cleaving enzyme 1 in both high fat/cholesterol‐fed C57BL/6 and LDLR−/− mice accompanied by an increased generation of C‐terminal fragments of APP. Although ELISA for beta‐amyloid failed to record significant changes in the non‐transgenic mice, a threefold increase in beta‐amyloid 40 accumulation was apparent in a strain of transgenic mice expressing wild‐type human APP on high fat/cholesterol diet. The findings link hypercholesterolemia with cognitive dysfunction potentially mediated by increased neuroinflammation and APP processing in a non‐transgenic mouse model.

Estradiol enhances neurogenesis following ischemic stroke through estrogen receptors α and β

Neurogenesis persists throughout life under normal and degenerative conditions. The adult subventricular zone (SVZ) generates neural stem cells capable of differentiating to neuroblasts and migrating to the site of injury in response to brain insults. In the present study, we investigated whether estradiol increases neurogenesis in the SVZ in an animal model of stroke to potentially promote the ability of the brain to undergo repair. Ovariectomized C57BL/6J mice were implanted with capsules containing either vehicle or 17β‐estradiol, and 1 week later they underwent experimental ischemia. We utilized double‐label immunocytochemistry to identify the phenotype of newborn cells (5‐bromo‐2′‐deoxyuridine‐labeled) with various cellular markers; doublecortin and PSA‐NCAM as the early neuronal marker, NeuN to identify mature neurons, and glial fibrillary acidic protein to identify astrocytes. We report that low physiological levels of estradiol treatment, which exert no effect in the uninjured state, significantly increase the number of newborn neurons in the SVZ following stroke injury. This effect of estradiol is limited to the dorsal region of the SVZ and is absent from the ventral SVZ. The proliferative actions of estradiol are confined to neuronal precursors and do not influence gliosis. Furthermore, we show that both estrogen receptors α and β play pivotal functional roles, insofar as knocking out either of these receptors blocks the ability of estradiol to increase neurogenesis. These findings clearly demonstrate that estradiol stimulates neurogenesis in the adult SVZ, thus potentially facilitating the brain to remodel and repair after injury. J. Comp. Neurol. 500:1064–1075, 2007.

Neprilysin regulates amyloid Beta peptide levels.

That neprilysin (NEP) is a major Aβ peptide-degrading enzyme in vivo is shown by higher Aβ peptide levels in the brain of an NEP knockout mouse. In addition, we show that infusion of an NEP inhibitor, but not inhibitors of other peptidases, into the brains of an APP transgenic mouse elevates Aβ levels. We have investigated the use of NEP as a potential therapeutic agent to prevent the accumulation of Aβ peptides in the brain. Lentivirus expressing NEP was initially used to demonstrate the ability of the enzyme to reduce Aβ levels in a model CHO cell line and to make primary hippocampal neurons resistant to Aβ-mediated neurotoxicity. Injection of NEP-expressing lentivirus, but not inactive NEP-expressing lentivirus, GFP-expressing lentivirus, or vehicle, into the hippocampus of 12–20-mo-old hAPP transgenic mice led to an approx 50% reduction in the number of amyloid plaques. These studies provide the impetus for further investigating of the use of NEP in a gene transfer therapy paradigm to prevent the accumulation of Aβ and prevent or delay the onset of Alzheimer’s disease.