The HHQK domain of beta-amyloid provides a structural basis for the immunopathology of Alzheimer's disease.

The beta-amyloid peptide 1-42 (Abeta1-42), a major component of neuritic and core plaques found in Alzheimer's disease, activates microglia to kill neurons. Selective modifications of amino acids near the N terminus of Abeta showed that residues 13-16, the HHQK domain, bind to microglial cells. This same cluster of basic amino acids is also known as a domain with high binding affinity for heparan sulfate. Both Abeta binding to microglia and Abeta induction of microglial killing of neurons were sensitive to heparitinase cleavage and to competition with heparan sulfate, suggesting membrane-associated heparan sulfate mediated plaque-microglia interactions through the HHQK domain. Importantly, small peptides containing HHQK inhibited Abeta1-42 cell binding as well as plaque induction of neurotoxicity in human microglia. In vivo experiments confirmed that the HHQK peptide reduces rat brain inflammation elicited after infusion of Abeta peptides or implantation of native plaque fragments. Strategies which exploit HHQK-like agents may offer a specific therapy to block plaque-induced microgliosis and, in this way, slow the neuronal loss and dementia of Alzheimer's disease.

A strategy for identifying immunosuppressive therapies for Alzheimer disease.

There is increasing evidence that the immune system plays an important role in the pathology of Alzheimer disease (AD). The fundamental steps in this process involve induction of neurotoxic microglia by senile plaques. Recent studies have shown that microglia in contact with isolated plaque fragments secrete neurotoxins that can cause neuronal injury and brain damage typical of AD. In vitro models help to delineate individual steps of this activation cascade by which quiescent microglia become neuron-killing cells. Moreover, such model systems provide rapid screening assays to identify immunosuppressive drugs that might slow brain damage brought on by neurotoxic microglia.

Specific domains of beta-amyloid from Alzheimer plaque elicit neuron killing in human microglia.

Alzheimer's disease (AD) is found to have striking brain inflammation characterized by clusters of reactive microglia that surround senile plaques. A recent study has shown that microglia placed in contact with isolated plaque fragments release neurotoxins. To explore further this process of immunoactivation in AD, we fractionated plaque proteins and tested for the ability to stimulate microglia. Three plaque-derived fractions, each containing full-length native A beta 1-40 or A beta 1-42 peptides, elicited neurotoxin release from microglia. Screening of various synthetic peptides (A beta 1-16, A beta 1-28, A beta 12-28, A beta 25-35, A beta 17-43, A beta 1-40, and A beta 1-42) confirmed that microglia killed neurons only after exposure to nanomolar concentrations of human A beta 1-40 or human A beta 1-42, whereas the rodent A beta 1-40 (5Arg-->Gly, 10Tyr-->Phe 13His-->Arg) was not active. These findings suggested that specific portions of human A beta were necessary for microglia-plaque interactions. When coupled to microspheres, N-terminal portions of human A beta (A beta 1-16, A beta 1-28, A beta 12-28) provided anchoring sites for microglial adherence whereas C-terminal regions did not. Although itself not toxic, the 10-16 domain of human A beta was necessary for both microglial binding and activation. Peptide blockade of microglia-plaque interactions that occur in AD might prevent the immune-driven injury to neurons.

Activated microglia are the principal glial source of thromboxane in the central nervous system.

Thromboxane A2(TxA2) is a potent vasoconstrictor associated with cerebrovascular disease and is thought to be synthesized within tissues of the brain. In order to determine the cellular sources of TxA2 in the central nervous system (CNS), we measured the release of the stable metabolite TxB2 in cultures of mixed or highly enriched populations of brain glia. Using techniques which isolated large numbers of highly enriched microglia and astroglia, we found that only microglia release TxB2. Moreover, microglia, not astroglia, contain the requisite synthetic enzyme thromboxane synthase. Phagocytic signals and lipopolysaccharide are potent stimulants of microglial release of thromboxane, with lesser effects shown by platelet activating factor and substance P. We conclude that microglia, when activated, are the principal source of brain-derived thromboxane and may help to control vascular flow at sites of acute CNS injury.

Study of receptor-mediated neurotoxins released by HIV-1-infected mononuclear phagocytes found in human brain.

Although there is growing evidence that neurotoxic molecules produced by HIV-1-infected mononuclear phagocytes damage neurons, the precise mechanisms of neuronal attack remain uncertain. One class of cytotoxin involves neuronal injury mediated via the NMDA receptor. We examined blood monocytes and brain mononuclear cells isolated at autopsy from HIV-1-infected individuals for the ability to release NMDA-like neuron-killing factors. We found that a neurotoxic amine, NTox, was produced by blood monocytes and by brain mononuclear phagocytes infected with retrovirus. In vivo injections of minute quantities of NTox produced selective damage to hippocampal pyramidal neurons. NTox can be extracted directly from brain tissues infected with HIV-1 and showed structural features similar to wasp and spider venoms. In contrast to NTox, HIV-1 infection did not increase the release of the NMDA excitotoxin quinolinic acid (QUIN) from mononuclear cells. Although we found modest elevations of QUIN in the CSF of HIV-1-infected individuals, the increases were likely attributable to entry through damaged blood-brain barrier. Taken together, our data pinpoint NTox, rather than QUIN, as a major NMDA receptor-directed toxin associated with neuro-AIDS.

Cell surface morphology identifies microglia as a distinct class of mononuclear phagocyte.

To investigate differences among brain-derived microglia and other classes of immune cells, we compared the morphologies and growth properties of mononuclear phagocytes isolated from tissues of the newborn rat. Scanning EM shows that microglia from postnatal rat brain are covered with spines (typically > 20 per cell body) in a distinctive manner which contrasts the smooth surfaces of bone marrow cells and the ruffled surfaces of tissue macrophages from spleen, liver, and peritoneum. The spine-bearing surface of microglia is a specific cell marker, for it does not change with age or after exposure to cytokines or other immunostimulants. Approximately 99% of mononuclear phagocytes cultured from normal adult rat brain are spinous microglia. Five days after injury to rat brain, cells at sites of Wallerian degeneration are essentially all spinous ones while nearly 30% of cells found within areas of infarction or penetrating trauma are invading macrophages. In a similar way, nearly all cells isolated from normal, postmortem adult human brain are spine-bearing microglia (> 99% homogeneity). Brains from patients with amyotrophic lateral sclerosis contain only spinous microglia whereas cells from HIV-1 infected brains include significant numbers of invading ruffled macrophages. Cultured microglia, unlike cultured bone marrow precursors, monocytes, or tissue macrophages, spontaneously develop long, thin processes that extend hundreds of microns in length. Microglia retract these processes after exposure to fetal bovine serum, laminin, or such immunostimulants as recombinant murine interferon gamma (rmIFN gamma) and lipopolysaccharide. Of all types of mononuclear phagocytes tested, only microglia differentiate into quiescent ramified cells when in contact with astrocytes. Thus, microglia represent a unique class of cell maintained, in part, by astroglia as dormant, ramified mononuclear phagocytes in mature CNS. Application of cell surface criteria described here will allow study of distinct populations of mononuclear phagocytes associated with neurologic disorders.

Senile plaques stimulate microglia to release a neurotoxin found in Alzheimer brain.

Senile plaques found in the brains of patients with Alzheimer's disease (AD) are surrounded by clusters of reactive microglia. Isolated human microglia placed in contact with plaques in vitro are activated to release a factor which is toxic to neurons. This same neurotoxin is found in AD brain tissue and causes damage to pyramidal neurons in vivo when infused into rat hippocampus. Highest concentrations of the neurotoxin are in those brain structures most burdened by reactive microglia, suggesting that plaque-activated cells contribute to the neuronal damage and impaired cognition seen in patients with Alzheimer's dementia.

Phagocytic microglia release cytokines and cytotoxins that regulate the survival of astrocytes and neurons in culture.

Numerous studies have now shown that microglia secrete factors which may influence the growth and survival of cells in the CNS. We employed glia-neuron co-cultures to investigate the net effect of soluble products from secretory microglia upon astroglia and neurons following microglial activation by a phagocytic signal. Stimulation of microglia produced soluble factors that both increase the number of astroglia and decrease the number of neurons. The astroglial proliferating activity was blocked when incubated with an interleukin-1 (IL-1) receptor antagonist while the neurotoxic effect was inhibited by a N-methyl-D-aspartate (NMDA) receptor antagonist. Recombinant IL-1 served as a potent mitogen for cultured astroglia and promoted neuron survival by indirect actions upon astrocytes. These observations suggest that reactive microglia mediate both astrogliosis and neuronal injury through the simultaneous release of cell growth factors and poisons. The net effect of secretion products from phagocytic microglia is to diminish neuronal survival.

The impact of microglia-derived cytokines upon gliosis in the CNS.

Injury to the CNS elicits a complex cellular response involving both astrocytes and microglia. Reactive glial populations make up the so-called 'glial scar' that has long been implicated as a barrier to axonal regeneration or as a causal factor in the genesis of epilepsy. Using in vitro models involving highly enriched populations of brain cells we have observed that astroglial growth is regulated in part by an immunomodulatory growth factor, or cytokine, called interleukin-1 (IL-1). A second cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF) serves as a potent microglial mitogen and regulator of the microglial component of the glial scar. Employing cytokines as tools to manipulate reactive gliosis, we found that IL-1 supported neuronal growth by action upon astroglia, while GM-CSF initiated epileptic-like discharges through mechanisms involving reactive microglia. We propose that a 'cytokine network' involving IL-1 and GM-CSF mediates the composition of glial scars at sites of CNS injury; these reactive glia, in turn, influence the survival and function of neighboring neurons.

Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system.

Reactive microglia respond within hours to central nervous system ischemic injury as exhibited by increased surface molecules, including the scavenger receptor. It is at least several days after an insult, however, before these activated mononuclear phagocytes reach a peak of secretory activity with the release of neurotoxins. This period of cytotoxin secretion is associated with a delayed neuronal loss seen in tissues neighboring sites of ischemia. Microglia-suppressing drugs reduce tissue production of neurotoxic factors and improve functional outcome after ischemic injury. Immunosuppressive therapy may offer a means to reduce late neuronal damage associated with stroke.

Reactive mononuclear phagocytes release neurotoxins after ischemic and traumatic injury to the central nervous system.

Reactive microglia and invading macrophages, which appear in brain damaged by stroke or trauma, secrete neuron-killing factors. This release of cytotoxic substances is a delayed process and is not detected until inflammatory cells reach a peak of reactivity by the second day after injury. Proximity to the site of injury and density of mononuclear phagocytes determine in part the amount of neurotoxic activity released by injured tissues. Moreover, drugs that suppress the accumulation of reactive microglia and macrophages also reduce tissue production of neuron poisons. Neurotoxins released by brain inflammatory cells or extracted directly from inflamed tissues are heat-stable, protease-resistant molecules < 500 daltons with actions blocked by N-methyl-D-aspartate (NMDA) receptor antagonists. These molecules are distinguished from free radical intermediates, bind to cation exchange resins, lack carboxyl moieties, and are separated from excitatory amino acids including glutamate or aspartate and from the NMDA receptor-mediated toxin quinolinic acid by ion exchange and reverse phase chromatography. Our data suggest that an unrecognized class of neuron-killing molecules produced by inflammatory cells mediate the delayed neuronal loss associated with stroke and trauma.

The envelope glycoprotein of human immunodeficiency virus type 1 stimulates release of neurotoxins from monocytes.

Mononuclear phagocytes infected with human immunodeficiency virus 1 (HIV-1) produce soluble factors that kill neurons in culture. To define the molecular events that lead to neuron killing, HIV-1 proteins were tested for the ability to trigger release of neurotoxins from human monocytes and lymphocytes. None of the recombinant-derived HIV-1 proteins examined (reverse transcriptase, protease, gag, nef, or gp120) were directly neurotoxic at concentrations from 100 pM to 10 nM. The envelope glycoprotein gp120 did, however, stimulate both isolated human blood monocytes and the monocytoid line THP-1 (but not lymphocytes or the lymphoid cell line H9) to discharge neurotoxic factors. These toxins consisted of heat-stable, protease-resistant molecules (< 500 Da) that copurified with neurotoxins from HIV-1-infected THP-1 cells and were blocked by antagonists to N-methyl-D-aspartate receptors. Release of neurotoxins through gp120 stimulation involved monocytoid CD4 receptors because toxin production could be inhibited either by a monoclonal antibody to the CD4-binding region of gp120 or by soluble CD4 receptors. Alternatively, production of neuron-killing factors could be induced with a peptide from the CD4-binding region of gp120. These data show that the HIV-1 envelope glycoprotein alone can stimulate neurotoxin release by binding to CD4 receptors of mononuclear phagocytes. Such neurotoxic factors may, in turn, contribute to the central nervous system dysfunction associated with HIV-1 by acting on neurons through N-methyl-D-aspartate receptors.

Reactive glia as rivals in regulating neuronal survival.

Reactive gliosis is a response noted after nearly every type of CNS injury and involves both activated microglia and astroglia. Although many investigators believe that reactive glia in some way regulate the survival of injured neurons, the influence of glial elements upon damaged neural tissues remains uncertain. To examine relationships between reactive glia and neurons, secretion products from both microglia and astroglia are tested for their effects upon the survival of cultured neurons. Microglia are found to secrete neurotoxic agents, while astroglia are a source of neuronotrophic factors. Similar patterns of soluble factor production are noted for astroglia-rich or microglia-rich regions of rat neocortex damaged by ischemia. These observations suggest that microglia and astroglia compete for control of neuronal survival. Importantly, microglial neurotoxins might hinder the recovery of neurologic function at sites of inflammation.

Brain glia release factors with opposing actions upon neuronal survival.

Microglia and astroglia have been thought to govern the survival of neurons after damage to the CNS. To investigate these putative glia-neuron relationships, we examined microglia and astroglia secretion products for effects upon growth of cultured neurons. Activated microglia secrete small neurotoxic factors (< 500 Da), while astroglia constitutively release proteins (> 10 kDa) that promote neuronal growth. Proteins released from astroglia, moreover, attenuate microglial toxicity, suggesting that different glial populations have opposing actions upon neuronal survival. Further study shows that neurotoxins from microglia are heat-stable, protease-resistant molecules with biologic activities blocked by NMDA receptor antagonists. Microglial factors, although toxic for chick ciliary neurons and rat spinal cord neurons, did not reduce numbers of oligodendroglia, astroglia, or Schwann cells in culture. The microglial neurotoxins can be distinguished from cytokines, from free radical intermediates, from the excitatory amino acids glutamate or aspartate, and from the NMDA receptor-mediated toxin quinolinic acid. We propose that secretion products from reactive microglia, but not astroglia, endanger surviving neurons after CNS injury by release of a novel class of neuron-killing molecules.

A growth factor from neuronal cell lines stimulates myelin protein synthesis in mammalian brain.

Oligodendroglia growth factor (OGF) is a 16-kDa soluble protein produced by neuronal cell lines. This factor, when incubated with brain glia in culture, selectively stimulates growth of oligodendroglia, the myelin-producing cells of the CNS. OGF infused into the cerebral cortex of the adult rat accelerates the production of myelin proteins as shown by increased specific activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (2',3'-CNPase), by stimulated synthesis of myelin basic protein, and by elevations in levels of myelin proteolipid protein RNA. The ability of OGF to induce myelin protein production in vivo suggests that neuron-secreted growth factors help to regulate myelin formation within the CNS.

Microglial mitogens are produced in the developing and injured mammalian brain.

The central nervous system produces growth factors that stimulate proliferation of ameboid microglia during embryogenesis and after traumatic injury. Two microglial mitogens (MMs) are recovered from the brain of newborn rat. MM1 has an approximate molecular mass of 50 kD and a pI of approximately 6.8; MM2 has a molecular mass of 22 kD and a pI of approximately 5.2. These trypsin-sensitive proteins show specificity of action upon glia in vitro serving as growth factors for ameboid microglia but not astroglia or oligodendroglia. Although the MMs did not stimulate proliferation of blood monocytes or resident peritoneal macrophage, MM1 shows granulocyte macrophage colony-stimulating activity when tested upon bone marrow progenitor cells. Microglial mitogens may help to control brain mononuclear phagocytes in vivo. The MMs first appear in the cerebral cortex of rat during early development with peak levels around embryonic day E-20, a period of microglial proliferation. Microglial mitogens are also produced by traumatized brain of adult rats within 2 d after injury. When infused into the cerebral cortex, MM1 and MM2 elicit large numbers of mononuclear phagocytes at the site of injection. In vitro study shows that astroglia from newborn brain secrete MM2. These observations point to the existence of a regulatory system whereby secretion of proteins from brain glia helps to control neighboring inflammatory responses.

Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1.

Mononuclear phagocytes (microglia, macrophages, and macrophage-like giant cells) are the principal cellular targets for human immunodeficiency virus-1 (HIV-1) in the central nervous system (CNS). Since HIV-1 does not directly infect neurons, the causes for CNS dysfunction in acquired immunodeficiency syndrome (AIDS) remain uncertain. HIV-1-infected human monocytoid cells, but not infected human lymphoid cells, released toxic agents that destroy chick and rat neurons in culture. These neurotoxins were small, heat-stable, protease-resistant molecules that act by way of N-methyl-D-aspartate receptors. Macrophages and microglia infected with HIV-1 may produce neurologic disease through chronic secretion of neurotoxic factors.