Understanding why neurons die...

Our aim is to understand why neurons die but glia become reactive within the same molecular milieu during neurodegeneration. The problem is poorly understood and is important at a fundamental level and clinically as there are currently no effective treatments.

Mediators of neurodegeneration

We started a program to isolate mediators of neurodegeneration in which explants of degenerating adult rat brain are co-cultured with embryonic neural explants in collagen gel matrices. Cultured alone, embryonic explants extend axons vigorously over 24-48 hours but die within 24 hours in the nearby presence of adult brain. We blocked this effect by placing agarose beads coated with Sambucus nigra agglutinin (SNA) between the explants and we have used SNA affinity chromatography to identify a family of neurotoxic proteins called F1-4 that are released into the culture medium from adult brain. Rat cortical neurons are killed dose-dependently by nanomolar concentrations of F1 but astrocytes proliferate and up-regulate of glial fibrillary acidic protein. F proteins are released at the injury site and taken up by glia following spinal cord trauma while stereotaxic injection of of F proteins into adult rat brain causes severe neuronal loss and gliosis.

We have identified F proteins in post-mortem Alzheimer's disease (AD) brains and in the cerebrospinal fluid of patients with AD, Parkinson's disease and multiple sclerosis. Serum levels of anti-F antibodies are markedly raised in AD and in adults with Down's syndrome who all later develop AD. Fluorescently labelled F1 is taken up into early endosomes by neurons and astrocytes but in neurons is trafficked to the endoplasmic reticulum. F-proteins bind to a saturable cell surface receptor(s) in primary neurons and astrocytes and our evidence suggests that a proteoglycan receptor(s) is required for uptake while certain GAGs bind to F proteins and prevent their uptake. Although we have not yet mapped the GAG interaction sites a number of consensus heparin binding sites can be predicted which strongly suggests that the interaction is direct. We do not know where the specificity in the system resides: whether there are different cell-type specific receptors for F proteins on neurons compared with astrocytes, or whether the receptor is the same and differences lie in the downstream trafficking pathways following F protein uptake.

The three panels show cultures of mixed cortical neurons and glia which have been exposed to fluorescently labelled F protein for 24 hrs.

The left hand panel shows trafficking of F protein to the endoplasmic reticulum (ER) surrounding the nucleus in neuron, a route that is taken by numerous toxins and viruses. The chromatin (blue) is condensing, the neuron is under ER stress and is dying. In contrast, the middle panel demonstrates endocytosis and cytoplasmic uptake of F protein in glial cells with no ER involvement. The right hand panel shows endocytosed F protein and the resultant differentiation to a reactive GFAP-positive astrocyte.

We are currently epitope mapping these proteins to determine critical sites of activity and to make blocking antibodies against them which could be of therapeutic benefit. Additionally, we are are trying to discover the downstream mechanisms that determine just how these proteins have such contrasting effects in neurons and glia.