Sim K. Singhrao

Increased Complement Biosynthesis By Microglia and Complement Activation on Neurons in Huntington's Disease ☆

In this study complement activation and biosynthesis have been analysed in the brains of Huntingtons disease (HD) (n = 9) and normal (n = 3) individuals. In HD striatum, neurons, myelin and astrocytes were strongly stained with antibodies to C1q, C4, C3, iC3b-neoepitope and C9-neoepitope. In contrast, no staining for complement components was found in the normal striatum. Marked astrogliosis and microgliosis were observed in all HD caudate and the internal capsule samples but not in normal brain. RT-PCR analysis and in-situ hybridisation were carried out to determine whether complement was synthesised locally by activated glial cells. By RT-PCR, we found that complement activators of the classical pathway C1q C chain, C1r, C4, C3, as well as the complement regulators, C1 inhibitor, clusterin, MCP, DAF, CD59, were all expressed constitutively and at much higher level in HD brains compared to normal brain. Complement anaphylatoxin receptor mRNAs (C5a receptor and C3a receptor) were strongly expressed in HD caudate. In general, we found that the level of complement mRNA in normal control brains was from 2 to 5 fold lower compared to HD striatum. Using in-situ hybridisation, we confirmed that C3 mRNA and C9 mRNA were expressed by reactive microglia in HD internal capsule. We propose that complement produced locally by reactive microglia is activated on the membranes of neurons, contributing to neuronal necrosis but also to proinflammatory activities. Complement opsonins (iC3b) and anaphylatoxins (C3a, C5a) may be involved in the recruitment and stimulation of glial cells and phagocytes bearing specific complement receptors.

Spontaneous Classical Pathway Activation and Deficiency of Membrane Regulators Render Human Neurons Susceptible to Complement Lysis

This study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 microg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation.

The role of complement in disorders of the nervous system.

The complement (C) system plays important roles in host defense but activation at inappropriate sites or to an excessive degree can cause host tissue damage. C has been implicated as a factor in the causation or propagation of tissue injury in numerous diseases. The brain is an immunologically isolated site, sheltered from circulating cells and proteins of the immune system; nevertheless, there is a growing body of evidence implicating C in numerous brain diseases. In this brief article we review the evidence suggesting a role for C in diseases of the central and peripheral nervous system and discuss the possible sources of C at these sites. Some brain cells synthesize C and also express specific receptors; some are exquisitely sensitive to the lytic effects of C. The evidence suggests that C synthesis and activation in the brain are important in immune defense at this site, but may also play a role in brain disease.

Roles of the complement system in human neurodegenerative disorders

Complement is an important component of the innate immune response with the capacity to recognize and clear infectious challenges that invade the CNS through a damaged blood brain barrier. For instance, the membrane attack complex is involved in cytotoxic and cytolytic activities while other smaller fragments lead to cell activation (chemotaxis) and phagocytosis of the intruders. It is noteworthy that there is a growing body of evidence that uncontrolled complement biosynthesis and activation in the CNS can contribute to exacerbate the neuronal loss in several neurodegenerative disorders. We provide here an insightful review of the double-edged sword activities of the local innate complement system in the CNS and discuss further the potential therapeutic avenues of delivering complement inhibitors to control brain inflammation.

Immunocytochemically detectable metallothionein is expressed by astrocytes in the ischaemic human brain

Immunocytochemically detectable metallothionein is expressed by astrocytes in the ischaemic human brain

Alpha-actinin in nemaline bodies in congenital nemaline myopathy: immunological confirmation by light and electron microscopy

To elucidate the protein composition of the nemaline bodies present in the muscle fibres of patients with congenital nemaline myopathy (CNM), we studied muscle biopsies with monoclonal antibodies against alpha-actinin and desmin in combination with a modified Gomori trichrome method. Electron microscopy of immunolabelled resin embedded sections was used for cytochemical localisation of alpha-actinin and desmin. Light microscopy of sections immunolabelled for alpha-actinin showed a cross-striation of the muscle fibres corresponding to the Z band pattern, focal thickening of the Z bands and additional reactivity with a granular pattern corresponding to the presence of nemaline bodies. Labelling of desmin did not show a similar pattern. Electron microscopy confirmed the presence of alpha-actinin in the nemaline bodies and Z bands, whereas desmin was only found in intermediate filaments around the Z bands. Western blots showed single, sharp alpha-actinin bands indistinguishable from normal. Our results provide direct evidence for the presence of alpha-actinin in nemaline bodies and a lack of quantitative or qualitative differences between the alpha-actinin of normal and CNM muscle.

Corpora amylacea could be an indicator of neurodegeneration.

We describe an investigation of corpora amylacea (CA) in the brain tissue of Alzheimers disease (AD) cases and normal ageing controls, using both light (LM) and electron (EM) microscopic techniques. CA populations were shown by routine histological staining of LR White resin sections with methenamine silver and PAS, and were compared with those shown by immunocytochemistry using antibodies to tau, GFAP, tubulin, ubiquitin, β‐amyloid and serum amyloid P component in serial sections. All CA were immunoreactive with anti‐tau and all were unreactive with anti‐β‐amyloid. Most were immunoreactive with anti‐serum amyloid P component, although this was often weak in AD. CA from normal ageing brain were immunoreactive for proteins that are associated with the neuronal cytoskeleton and cell injury. CA from AD brain shared some of these but differed from those in normal ageing brain by being in much larger number and more variable in their immunoreactivity. In all CA, X‐ray microanalysis illustrated the presence of the metallic elements Ca, Fe and Cu. Aluminium, often associated with AD, was not present, even in CA from AD brain. Phosphorus and sulphur, probably from phosphorylated proteins associated with degenerating cytoskeleton elements, were usually detected. In AD brain, the greater numbers of CA and their variable biochemical and elemental composition, when compared with CA in the normal ageing brain, suggests that they may derive from a number of sources both neuronal and glial as a result of the neurodegenerative disease.

The distribution of amyloid plaques in the cerebellum and brain stem in Down's syndrome and Alzheimer's disease: a light microscopical analysis.

SummaryThe distribution and severity of the neuropathological changes in 57 cases of Alzheimers disease, and 11 patients with Downs syndrome were investigated with reference to the cerebellum: A modified silver stain and a monoclonal antibody raised against amyloid β-protein were used to identify amyloid plaques. The highest incidence of amyloid plaques in the cerebellum (93%) was found in the group of patients who developed dementia before 65 years of age. This figure dropped to 56% in those patients with dementia beginning after 75 years. In 37 of these cases the distribution of the pathological changes of the disease were also examined in the brain stem. The severity of the pathological changes in the cerebellum corresponded to the involvement of the brain stem nuclei with connections to the cerebellar cortex. The possibility that the disease process spreads to the cerebellum by involving the fibres from the brain stem is discussed with reference to previous anatomical and neurochemical studies.

A functional role for corpora amylacea based on evidence from complement studies

Few theories have been advanced for the production of corpora amylacea (CA) by the normal ageing brain and by the CNS under various neurological conditions. Proteins derived from neurons and oligodendrocytes are found in CA and to understand their origins brain tissue from patients with Alzheimers disease (AD), multiple sclerosis (MS) and Picks disease (PD) were tested for complement activity. All CA were immunopositive for antisera to classical pathway-specific components, the activation products C3d and the terminal complement complex (TCC), the C3 convertase regulator membrane cofactor protein (MCP) and the fluid phase regulators S-protein and clusterin. CA were immunonegative for the alternative complement pathway proteins and the complement regulators, decay accelerating factor (DAF) and CD59. Western immunoblotting of isolated solubilized CA from the same tissues demonstrated a weak band for MCP but TCC was more easily shown by immunoprecipitation. A filamentous fringe around CA, probably of astrocytic origin, was also immunopositive for complement factors. CA consist of an inert mucopolysaccharide matrix encasing ubiquitinated proteins, resulting from death of and damage to neurons, myelin and oligodendrocytes. A function of CA, therefore, could be to prevent the recognition of these immunogenic proteins by lymphocytes and microglia and thus protect the CNS from further injury.

Regulation of desmosomal cell adhesion in human tumour cells by polyunsaturated fatty acids

Desmosomes are key structures in cell-cell adhesion. In this study we examined the effect of n-6 essential fatty acids on the expression of desmoglein (Dsg), desmosomal cadherin and the formation of desmosomes in E-cadherin negative human breast, colon and lung cancer cells and melanoma cells. Electron microscopy revealed that cells cultured with gamma linolenic acid (GLA) showed increased cell-cell adhesion together with an increase in the formation of desmoglein-containing desmosomes. Western blotting studies of cellular proteins demonstrated that, following culture with fatty acids, Dsg expression was modified, with the greatest increase seen after GLA treatment. Other fatty acids increased Dsg expression, but to a lesser extent. It is concluded that GLA regulates desmosome-mediated cell-cell adhesion in human cancer cells, particularly in cells without E-cadherin.