Identification of agents that protect against inflammatory injury to neurons

A method of screening for an agent that inhibits the effect on a neuron of a neurotoxin from a neurotoxic mononuclear phagocyte activated by β-amyloid or fragments of β-amyloid, wherein said fragments at a concentration of from about 0.001 to about 1 μmole/liter induce significant neuron injury in neuron cultures containing microglia as compared to neuronal injury in cultures lacking microglia, sad method comprising:

• contacting a mononuclear phagocyte with βamyloid or said fragments of β-amyloid effective to activate said mononuclear phagocyte to produce a neurotoxic mononuclear phagocyte, wherein said neurotoxin has a molecular mass of less than 1000 daltons, lacks peptide bonds, and is isolatable in aqueous solution from tissue or tissue culture media in active form;
• separating said neurotoxin from said mononuclear phagocytes;
• contacting tissue or cell culture containing neurons with said neurotoxin produced by said mononuclear phagocyte in an amount effective to produce a disruption of neuron cell metabolism in said neurons, and with an agent suspected of inhibiting the effect of said neurotoxin an a neuron, and
• utilizing a neuron specific marker to identify neurons in said tissue or culture,
• wherein a disruption of neuron cell metabolism as indicated by changes in the neuron-specific marker;
• and further wherein a decrease in disruption of neuron cell metabolism in the presence of said agent compared to a tissue or culture lacking said agent is indicative of an agent that inhibits the effect on a neuron of a neurotoxin from said neurotoxic mononuclear phagocyte.

A method of screening for an agent that inhibits the effect on a neuron of a neurotoxin from a mononuclear phagocyte activated by β-amyloid or fragments of β-amyloid, wherein said fragments at a concentration of from about 0.001 to about 1 μmole/liter induce significant neuron injury in neuron cultures containing microglia as compared to neuronal injury in cultures lacking microglia, said method comprising:

• contacting a mononuclear phagocyte with β-amyloid or said fragments of β-amyloid effective to activate said mononuclear phagocyte to produce a neurotoxin, wherein said neurotoxin has a molecular mass of less than 1000 daltons, lacks peptide bonds, and is isolatable in aqueous solution from tissue or tissue culture media in active form;
• separating said neurotoxin from said mononuclear phagocytes;
• and
• contacting a culture of neurons with an amount of the neurotoxin effective to produce injury or death in the neurons in the presence of an agent suspected of inhibiting the effect of the neurotoxin, when injury or death in the neurons is detected by obtaining an image of the culture;
• and further wherein a decrease in death or injury to neurons as compared to cultures lacking the agent, is indicative of an agent that inhibits the effect on a neuron of a neurotoxin from said activated mononuclear phagocyte.

A method of screening for an agent that inhibits the effect on a neuron of a neurotoxin from a neurotoxic mononuclear phagocyte activated by β-amyloid or fragments of β-amyloid, wherein said fragments at a concentration of from about 0.001 to about 1 μmole/liter induce significant neuron injury in neuron cultures containing microglia as compared to neuronal injury in cultures lacking microglia, said method comprising:

• contacting a mononuclear phagocyte with β-amyloid or said fragments of β-amyloid effective to activate said mononuclear phagocyte to produce a neurotoxic mononuclear phagocyte, wherein said neurotoxin has a molecular mass of less than 1000 daltons, lacks peptide bonds, and is isolatable in aqueous solution from tissue or tissue culture media in active form;
• separating said neurotoxin from said mononuclear phagocytes;
• contacting a tissue or cell culture containing neurons with an amount of said neurotoxin effective to produce injury or death in the neurons in the presence of an agent suspected of inhibiting the effect of the neurotoxin;
• and
• utilizing a neuron specific marker to identify neurons in the tissue or culture,
• wherein injury or death in said neurons is detected by obtaining an image of the tissue or culture;
• and further wherein a decrease in neuron injury or death compared to a tissue or culture lacking the agent is indicative of an agent that inhibits the effect on a neuron of a neurotoxin from said neurotoxic mononuclear phagocyte.

A method of screening for an agent that inhibits the effect on a neuron of a neurotoxin from a mononuclear phagocyte activated by β-amyloid or fragments of β-amyloid, wherein said fragments at a concentration of from about 0.001 to about 1 μmole/liter induce significant neuron injury in neuron cultures containing microglia as compared to neuronal injury in cultures lacking microglia, said method comprising:

• contacting a mononuclear phagocyte with β-amyloid or said fragments of β-amyloid coupled to a solid support, effective to activate said mononuclear phagocyte to produce a neurotoxin, wherein said neurotoxin has a molecular mass of less than 1000 daltons, lacks peptide bonds, and is isolatable in aqueous solution from tissue or tissue culture media in active form;
• separating said neurotoxin from said mononuclear phagocytes;
• and
• contacting a culture of neurons, neuron-like cells or cells expressing an NMDA receptor with an amount of said neurotoxin effective to produce a disruption of normal cell metabolism in said neurons, neuron-like cells or cells expressing an NMDA receptor in the presence of an agent suspected of inhibiting the effect of said neurotoxin,
• wherein a decrease in disruption of normal neuronal cell metabolism in the presence of said agent compared to a culture lacking said agent is indicative of an agent that inhibits the effect on a neuron of a neurotoxin from said activated mononclear phagocyte.

A method of screening for an agent that inhibits the effect on a neuron of a neurotoxin from a mononuclear phagocyte activated by β-amyloid fragments of β-amyloid, wherein, said fragments at a concentration of from about 0.001 to about 1 μmole/liter induce significant neuron injury in neuron culture containing microglia as compared to neuronal injury in culture lacking microglia, said method comprising:

• contacting a mononuclear phagocyte with β-amyloid or said fragments of β-amyloid coupled to a sold support, effective to activate said mononuclear phagocyte to produce a neurotoxin, wherein said neurotoxin has a molecular mass of less than 1000 daltons, lacks peptide bonds, and is isolatable in aqueous solution from tissue or tissue culture media in active form;
• separating said neurotoxin from said mononuclear phagocytes;
• contacting tissue or cell culture containing neurons with said neurotoxin produced by said mononuclear phagocyte in an amount effective to produce a disruption of neuron cell metabolism in said neurons, and with an agent suspected of inhibiting the effect of said neurotoxin on a neuron, and
• utilizing a neuron specific marker to identify neurons in said tissue or culture,
• wherein a disruption of normal cell metabolism is correlated with a disruption of neuron function as indicated by changes in the neuron-specific marker;
• and further
• wherein a decrease in disruption of neuron cell metabolism in the presence of said agent compared to a tissue or culture lacking said agent is indicative of an agent that inhibits the effect on a neuron of a neurotoxin from said activated mononuclear phagocyte.