14 Nov 2001

RNA profiling is increasingly being used in the study of Alzheimer's disease, but interpreting the pretty microarray patterns can be difficult. To name but one problem, individual neurons vary greatly in their degree of pathology. One neuron can be filled with tangles and dying while a neighboring one looks normal, and glial cells have a different set of genes expressed altogether. Add to that vascular cells, and even a microscopic brain sample becomes so heterogeneous that analyzing wholesale gene expression in extracts of ground-up brain may obscure important AD-related changes in either neurons or a particular type of glia.

On Sunday at the Neuroscience meeting, Stephen Ginsberg at New York University, Orangeburg, New York, and Eliot Mufson at Rush-Presbyterian St. Luke's Medical Center, Chicago, presented a way of tackling this problem. They fixed postmortem basal forebrain from 17 elderly people with no cognitive impairment (NCI), mild cognitive impairment (MCI), or Alzheimer's (AD). Since the tissue donors had belonged to a religious order and participated in an observational AD study, extensive information about them was available, including cognitive status, education level, and ApoE status.

The researchers first confirmed that the tissue still contained RNA, then they labeled for an acetylcholine transporter to identify cholinergic neurons (which are known to be particularly damaged in AD), as well as for hyperphosphorylated tau. Next they aspirated, with a micropipette, individual double-labeled neurons and amplified their mRNA using a method called terminal continuation. They did this for 46 single cholinergic basal forebrain neurons.

Then Ginsberg analyzed RNA levels on customized cDNA microarrays. He found that mRNA levels for the synaptic markers synaptophysin and synaptotagmin (but not synapsin 1 or synaptobrevin) were about 30 percent lower in neurons from people with MCI than in normal aging neurons, and were at least 70 percent lower in confirmed AD. Since this is in individual neurons, it does not simply reflect the gradual loss of cholinergic neurons in AD. Correlating synaptic gene expression with mini-mental state exam scores showed a trend for decreasing synaptophysin mRNA with lower test scores. Finally, the scientists showed that only the people with intact cognition had great variability in their levels of synaptophysin expression (indicating either natural variability or a very early, still-presymptomatic decline in some people). Without exception, all neurons form people with MCI or AD had low synpatophysin expression.

The study also detected decreases in mRNA levels of tyrosine kinase B and C, but not of neurotrophin receptors.

Caveats still abound. Translational and post-translational regulation is not reflected in mRNA data, the time between death and tissue fixation varied from three to 16 hours, and comparisons between neurons without, with mild, and with florid neurofibrillary pathology in one individual would have added an interesting wrinkle. However, the study demonstrates that single-cell mRNA analysis can pick up specific gene expression changes in vulnerable neurons and thus provide insight into disease mechanisms early in the development of Alzheimer's.—Gabrielle Strobel

References: Mufson EJ and Ginsberg SD. Single cell and regional cDNA microarray analysis of cholinergic basal forebrain neurons in aged and Alzheimer's disease brain. Soc Neuroscience 2001.

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