John D. Loike

Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne's or Becker's muscular dystrophy.

A deficiency of the protein dystrophin has recently been shown to be the probable cause of Duchennes muscular dystrophy. We sought to determine the relation between the clinical phenotype and the status of dystrophin in muscle-biopsy specimens from 103 patients with various neuromuscular disorders. We found very low levels (less than 3 percent of normal levels) or no dystrophin in the severe Duchenne phenotype (35 of 38 patients), low concentrations of dystrophin in the intermediate (outlier) phenotype (4 of 7), and dystrophin of abnormal molecular weight in the mild Becker phenotype (12 of 18). Normal levels of dystrophin of normal molecular weight were found in nearly all the patients (38 of 40) with 20 other neuromuscular disorders we studied. These data show the clinical consequences of both quantitative alterations (in Duchennes and intermediate dystrophy) in a single protein. The biochemical assay for dystrophin should prove helpful in delineating myopathies that overlap clinically with Duchennes and Beckers dystrophies, and it shows promise as an accurate diagnostic tool.

Adult mouse astrocytes degrade amyloid-β in vitro and in situ

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-β (Aβ) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Aβ deposition, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions, and cease migration upon interaction with immobilized Aβ1–42. We also show that astrocytes bind and degrade Aβ1–42. Astrocytes plated on Aβ-laden brain sections from a mouse model of AD associate with the Aβ deposits and reduce overall Aβ levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Aβ deposits, indicate a direct role for astrocytes in degradation of Aβ and implicate deficits in astroglial clearance of Aβ in the pathogenesis of AD. Treatments that increase removal of Aβ by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD.

Scavenger receptors in neurobiology and neuropathology: Their role on microglia and other cells of the nervous system

Scavenger receptor class A (SR‐A, CD204), scavenger receptor‐BI (SR‐BI), and CD36 are cell surface proteins that mediate cell adhesion to, and endocytosis of, various native and pathologically modified substances, and participate in intracellular signaling, lipid metabolism, and host defense against bacterial pathogens. Microglia, Mato cells, astrocytes, cerebral microvascular endothelial cells, cerebral arterial smooth muscle cells, and retinal pigment epithelial cells express one or more of these SR. Expression of SR‐A and SR‐BI by microglia is developmentally regulated. Neonatal microglia express SR‐A and SR‐BI, while microglia in normal mouse and human adult brain express neither. Astrocytes in adult brain express SR‐BI. In Alzheimers disease, microglial expression of SR‐A is increased. Such findings, and evidence that SR‐A and SR‐BI mediate adhesion and endocytosis of fibrillar β‐amyloid by microglia and astrocytes, respectively, and that SR‐A, SR‐BI, and CD36 participate in secretion of reactive oxygen species by microglia, suggest roles for these receptors in homeostasis and neuropathology. GLIA 40:195–205, 2002.

Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep

Eukaryotic cells contain two distinct genomes. One is located in the nucleus (nDNA) and is transmitted in a mendelian fashion, whereas the other is located in mitochondria (mtDNA) and is transmitted by maternal inheritance. Cloning of mammals typically has been achieved via nuclear transfer, in which a donor somatic cell is fused by electoporation with a recipient enucleated oocyte. During this whole-cell electrofusion, nDNA as well as mtDNA ought to be transferred to the oocyte. Thus, the cloned progeny should harbour mtDNAs from both the donor and recipient cytoplasms, resulting in heteroplasmy. Although the confirmation of nuclear transfer has been established using somatic cell-specific nDNA markers, no similar analysis of the mtDNA genotype has been reported. We report here the origin of the mtDNA in Dolly, the first animal cloned from an established adult somatic cell line, and in nine other nuclear transfer-derived sheep generated from fetal cells. The mtDNA of each of the ten nuclear-transfer sheep was derived exclusively from recipient enucleated oocytes, with no detectable contribution from the respective somatic donor cells. Thus, although these ten sheep are authentic nuclear clones, they are in fact genetic chimaeras, containing somatic cell-derived nuclear DNA but oocyte-derived mtDNA.

The HMGB1/RAGE axis triggers neutrophil-mediated injury amplification following necrosis

In contrast to microbially triggered inflammation, mechanisms promoting sterile inflammation remain poorly understood. Damage-associated molecular patterns (DAMPs) are considered key inducers of sterile inflammation following cell death, but the relative contribution of specific DAMPs, including high-mobility group box 1 (HMGB1), is ill defined. Due to the postnatal lethality of Hmgb1-knockout mice, the role of HMGB1 in sterile inflammation and disease processes in vivo remains controversial. Here, using conditional ablation strategies, we have demonstrated that epithelial, but not bone marrow-derived, HMGB1 is required for sterile inflammation following injury. Epithelial HMGB1, through its receptor RAGE, triggered recruitment of neutrophils, but not macrophages, toward necrosis. In clinically relevant models of necrosis, HMGB1/RAGE-induced neutrophil recruitment mediated subsequent amplification of injury, depending on the presence of neutrophil elastase. Notably, hepatocyte-specific HMGB1 ablation resulted in 100% survival following lethal acetaminophen intoxication. In contrast to necrosis, HMGB1 ablation did not alter inflammation or mortality in response to TNF- or FAS-mediated apoptosis. In LPS-induced shock, in which HMGB1 was considered a key mediator, HMGB1 ablation did not ameliorate inflammation or lethality, despite efficient reduction of HMGB1 serum levels. Our study establishes HMGB1 as a bona fide and targetable DAMP that selectively triggers a neutrophil-mediated injury amplification loop in the setting of necrosis.

Microglia, Scavenger Receptors, and the Pathogenesis of Alzheimer’s Disease

The senile plaque is the pathological hallmark of Alzheimers disease. Senile plaques are composed of beta amyloid fibrils, associated with activated microglia, astrocytes, and dystrophic neurons. We have recently identified class A scavenger receptors as the main receptors mediating the interaction of microglia with beta amyloid fibrils. Adhesion of microglia to beta amyloid fibrils leads to immobilization of these cells on the fibrils, and induces them to produce reactive oxygen species. We propose that interactions of microglial scavenger receptors with fibrillar beta amyloid may stimulate the microglia to secrete apolipoprotein E and complement proteins, which may further contribute to neurotoxicity and neuronal degeneration. Therefore, microglial scavenger receptors may be novel targets for therapeutic interventions in Alzheimers disease.

Neuromelanin Activates Microglia and Induces Degeneration of Dopaminergic Neurons: Implications for Progression of Parkinson’s Disease

In Parkinson’s disease (PD), there is a progressive loss of neuromelanin (NM)-containing dopamine neurons in substantia nigra (SN) which is associated with microgliosis and presence of extracellular NM. Herein, we have investigated the interplay between microglia and human NM on the degeneration of SN dopaminergic neurons. Although NM particles are phagocytized and degraded by microglia within minutes in vitro, extracellular NM particles induce microglial activation and ensuing production of superoxide, nitric oxide, hydrogen peroxide (H2O2), and pro-inflammatory factors. Furthermore, NM produces, in a microglia-depended manner, neurodegeneration in primary ventral midbrain cultures. Neurodegeneration was effectively attenuated with microglia derived from mice deficient in macrophage antigen complex-1, a microglial integrin receptor involved in the initiation of phagocytosis. Neuronal loss was also attenuated with microglia derived from mice deficient in phagocytic oxidase, a subunit of NADPH oxidase, that is responsible for superoxide and H2O2 production, or apocynin, an NADPH oxidase inhibitor. In vivo, NM injected into rat SN produces microgliosis and a loss of tyrosine hydroxylase neurons. Thus, these results show that extracellular NM can activate microglia, which in turn may induce dopaminergic neurodegeneration in PD. Our study may have far-reaching implications, both pathogenic and therapeutic.

Neurodegeneration and inflammation in Parkinson's disease.

Parkinsons disease (PD) is characterized by the progressive degeneration of dopamine (DA) neurons of the substantia nigra pars compacta (SNpc) accompanied by a buildup of proteinaceous aggregates termed Lewy bodies (LB). In addition to protein aggregation and the loss of DA signaling, PD is also characterized by an active immune response. T-cell infiltration accompanies activated microglial and astrocytic accumulation in and around the SNpc. Although potentially beneficial, microglial activation is most likely responsible for furthering disease pathology and DA neuron degeneration through the release of harmful substances such as pro-inflammatory cytokines, reactive oxidative species and reactive nitrogen species. Activation of the NF-κB death pathway has been shown to occur following microglial activation related release of Cox-2, IL-1β, and Toll-like receptor activation, resulting in increased degeneration of DA neurons of the SNpc. Blockade of microglial activation can lead to DA neuron protection in animal models of PD; however, clinical application of anti-inflammatory drugs has not yielded similar benefits. Future therapeutic designs must take into account the multifactorial nature of PD, including the varied roles of the adaptive and innate immune responses.

Scavenger receptor class B type I (SR-BI) mediates adhesion of neonatal murine microglia to fibrillar β-amyloid

Class A scavenger receptors (SR-A) mediate microglial interaction with fibrillar beta-amyloid (fAbeta). We report here that neonatal microglia from SR-A knockout mice (SR-A-/-) adhere to surface-bound fAbeta, and produce reactive oxygen species (ROS) as efficiently as wildtype microglia; that both wildtype and SR-A-/- microglia express SR-BI; that antibodies against SR-BI do not affect adhesion or ROS production by wildtype microglia, but inhibit adhesion and ROS production of SR-A-/- microglia to immobilized fAbeta by approximately 40%. Adhesion to fAbeta-coated surfaces, and uptake of fAbeta by both wildtype and SR-A-/- microglia was almost completely inhibited by incubation with fucoidan. Thus SR-BI and SR-A mediate similar effector functions in neonatal microglia, which suggests that SR-BI plays as important a role as SR-A, and can maintain the wildtype phenotype in SR-A-/- microglia.

Methamphetamine inhibits antigen processing, presentation, and phagocytosis.

Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.