Clusterin mRNA Distribution in the Rodent and Human Brains

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We, as well as two other groups, have reported on the distribution of clusterin mRNA in the CNS of young adult rats.26,32,38 Transcripts for clusterin were found to be distributed throughout the CNS, although regional differences in their prevalence were readily observed (Fig. 2.1). A strikingly high level of expression was observed in the ependymal lining of the ventricles and the choroid plexus (Figs. 2.1 and 2.2). In keeping with clusterin as a secretion product in other tissues, these results suggest that the protein is secreted locally into the cerebrospinal fluid (CSF), where the demand or turnover rate may be high.

Several neuron-rich cell layers and nuclei contained high levels of clusterin mRNA. These included the pyramidal and granule cell layers of the hippocampal formation, the habenular complex, the hypothalamus, several brainstem nuclei, and some motor neurons in the ventral horns of the spinal cord (Figs. 2.1-2.3). Within most of these areas, much heterogeneity in the hybridization signals was observed over individual neuronal cell bodies. It must be pointed out that the apparent labeling of the dentate gyrus granule cell layer was mostly due to heavily labeled cells located at the edge of the layer, which are thought to represent astrocytes.26,38 A rather homogenous pattern of hybridization was observed in the cerebral cortex, where neurons as well as astrocytes were identified as positive for clusterin mRNA.26,32,38 Other major brain regions like the cerebellum, the basal ganglia, the thalamus, and most of the olfactory bulb displayed an overall low to moderate labeling (Figs. 2.1 and 2.2D,E). Nevertheless, these regions contained some astrocytic and/or neuronal cell bodies showing a strong hybridization signal.26,38

Cell type specificity of clusterin mRNA was determined by in situ hybridization in combination with immunohistochemistry.38 Colocalization of clusterin mRNA with neuron-specific enolase or tyrosine hydroxylase immunoreactivities confirmed neuronal expression of clusterin transcripts in the hippocampal pyramidal layer and hilar region, in subsets of neurons of the substantia nigra, as well as in the red nucleus, the trigeminal motor, facial, somatosensory mesencephalic, and other nuclei.38

In general, white matter gave low intensity signals due to a majority of cell bodies not being labeled.26,32 Moderate to heavy clusterin mRNA concentrations over nonidentified

Fig. 2.1. (opposite) Photomicrographs showing the distribution and levels of clusterin mRNA in selected coronal sections of the adult rat brain. Sections were hybridized with a [35S]-labeled (antisense) riboprobe (for details see Danik et al26). Positive hybridization signals with different intensities are observed in various brain structures. 4V, fourth ventricle; 3, oculomotor nucleus; 7, facial nucleus; CG, central gray; CH, choroid plexus; D3V, dorsal third ventricle; DM, dorsomedial hypothalamic nucleus; gr, granular layer of the cerebellar cortex; GrDG, granular layer of the dentate gyrus; HiF, hippocampal fissure; LHb, lateral habenular nucelus, LS, lateral septum, LV, lateral ventricle; MHb, medial habenular nucleus; Me5, mesencephalic trigeminal nucleus; Mo5, motor trigeminal nucleus; mol, molecular layer of the cerebellar cortex; Pa, paraventricular hypothalamus nucleus; Py, pyramidal cell layer of the hippocampus; R, red nucleus; SFO, subfornical organ; SNC, substantia nigra pars compacta; SO, supraoptic nucleus; Tz, nucleus of trapezoid body; VMH, ventromedial hypothalamic nucleus.

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Fig. 2.2. Cellular distribution of clusterin mRNA at the level of the choroid plexus (A,C), the ventricular ependyma (A,B), the medial habenular nucleus (A,B) and the cerebellar cortex (D,E). (A) Darkfield photomicrograph at the level of the dorsal third ventricle. The choroid plexus shows the strongest signal. Note the robust labeling over the ependymal cell lining of the ventricle. Numerous clusters of clusterin mRNA positive cells are observed in the medial habenular nucleus. High-power brightfield photomicrographs showing hybridization signals at the level of the medial habenular nucleus (B) and the choroid plexus (C). A moderate labeling (arrowheads) is observed over individual cells in the medial habenular nucleus (B). A very dense labeling is clearly seen over all epithelial cells of the choroid plexus (C). Dark (D) and brightfield (E) photomicrographs illustrating the distribution of clusterin mRNA in the cerebellar cortex. Note the presence of clusters of silver grains over the Purkinje cells (arrows). The large arrows in D and E indicate the same cell. CH, choroid plexus; E, ependyma; gr cerebellar granular layer; MHb, medial habenular nucleus. Scale bars = 100 ^m for A and D; 20 (im for B, C and E. (Modified from ref. 26)

Fig. 2.3. Expression of clusterin mRNA in the spinal cord. (A) Autoradiogram showing the distribution of clusterin mRNA in the spinal cord at the level of the fourth lumbar segment. The strongest labeling was clearly seen in the ventral horn, particularly in a-motoneurons of lamina IX and in their vicinity. (B) High-power brightfield photomicrograph showing the high expression of clusterin transcripts over a-motoneurons in lamina IX of the fourth lumbar segment of the spinal cord. Note the lack of significant hybridization signal in these cells after sense strand hybridization (C). IX, lamina IX of the spinal cord. Scale bars = 20 |im for (B) and (C). (Modified from ref. 26)

Fig. 2.3. Expression of clusterin mRNA in the spinal cord. (A) Autoradiogram showing the distribution of clusterin mRNA in the spinal cord at the level of the fourth lumbar segment. The strongest labeling was clearly seen in the ventral horn, particularly in a-motoneurons of lamina IX and in their vicinity. (B) High-power brightfield photomicrograph showing the high expression of clusterin transcripts over a-motoneurons in lamina IX of the fourth lumbar segment of the spinal cord. Note the lack of significant hybridization signal in these cells after sense strand hybridization (C). IX, lamina IX of the spinal cord. Scale bars = 20 |im for (B) and (C). (Modified from ref. 26)

scattered glial cells were noted, however, in various white matter areas including the corpus callosum, the anterior commissure, the fimbria, the stria medullaris of the thalamus, the optic tract, and between cerebellar lobules,26 as well as over radially oriented glia in the spinal cord.32 Strongly positive clusterin mRNA-containing glial cells, presumably astrocytes, were located near the glial limitans and blood vessels.26 Together with its presence in ependymocytes, these localizations are suggestive of a role for clusterin at the CNS-periph-ery interface. To be noted is the absence of labeling over endothelial cells.26 Most brain regions are suspected to contain clusterin-expressing astrocytes. The striatum is the only region where colocalization of clusterin mRNA and glial fibrillary acidic protein (GFAP) immunoreactivity, a marker for fibrous astrocytes, was clearly demonstrated.38 Normal state astrocytic labeling is much weaker than what has been observed for reactive astrocytes in specific brain regions, following an experimental injury.16,22-30 (The reader is referred to Danik et al,26 and Pasinetti et al,38 for a more detailed description of the regions and the different cell types that express clusterin transcripts in the rat brain.)

There is only limited data available on the distribution of clusterin mRNA in the human brain. We have previously reported on the relatively homogenous distribution of transcripts in the temporal cortex of an 81 year old patient without any neurological symptoms who died of a myocardial infarction.17 The distribution pattern was similar to what was seen in the rat cortex except for the presence of larger grain clusters in the former. The significance of this clustering is unknown but may be related to aging. More recently, Pasinetti et al38 have examined brains, used as controls for Alzheimer's disease, in the age range of 76 to 85 years and found similar results for the temporal cortex. These authors have also examined the hippocampal formation of a non-AD case with multi-infarct dementia. As for the rat hippocampus, clusterin mRNA-positive pyramidal neurons were found in all hippoc-ampal fields. In contrast to the rat, clusterin transcripts were more abundant in granule neurons of the human dentate gyrus, although they were less prevalent than in the pyramidal cell layers, and there was no intense labeling of astrocytes along the hilar edge of the layer. Positive astrocytes in the hippocampal molecular layer were also noted. It remains unknown if the differences observed between rat and human are related to factors such as species, age, or the cell response associated with multi-infarction.

The above studies demonstrated that many neuronal as well as glial cell populations constitutively express clusterin transcripts in the normal brain. Basal levels of expression vary considerably, being relatively high in ependymal cells, motoneurons of the spinal cord, and several hypothalamic and brainstem neurons. Although in situ hybridization for clusterin mRNA combined with immunocytochemistry (ICC) for specific glial markers did not show colocalization to oligodendrocytes or microglia in the striatum,39 other brain regions should be examined to confirm the incapacity of these cells to synthesize clusterin transcripts.

The variable concentrations of clusterin mRNA at the cellular level, the diversity of expressor cell types and the distribution pattern of these cells in the CNS together indicate that the role carried out by clusterin in this tissue must be general.

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