Localization and activity of transglutaminase, a retinoid-inducible protein, in developing rat spinal cord

The distribution of the retinoid-inducible enzyme, tissue transglutaminase (tTG) in developing rat spinal cord was determined by enzyme assay and immunocytochemistry. tTG activity was at its highest in the forebrain in late foetal development. In hindbrain and spinal cord, elevated activity persisted until after birth. In spinal cord only, a second peak of activity occurred during the first week post partum (P3). tTG was associated with both the cytosolic and particulate tissue fractions throughout spinal cord development, but the particulate component was more prominent in the early postnatal period. tTG was more concentrated during this period in the ventral horn, where the particulate-associated enzyme activity was highest. In spinal cord at 3 days post partum, particulate tTG could be solubilized with lubrol-PX, dithiothreitol and potassium thiocyanate. Both soluble and particulate-associated tTG coeluted with guinea-pig liver transglutaminase C by DEAE-sephacel chromatography. The first peak of tTG activity during late foetal life coincided with the transient localization of the enzyme by immunocytochemistry in vascular endothelia throughout the spinal cord. The second peak of activity at 3 days post partum, by which time vascular immunoreactivity was absent, coincided with the occurrence of small numbers of intensely immunoreactive motor neurones in the ventral horn. Immunoreactive motor neurones were seen predominantly at two levels: the lower thoracic segments and lumbar enlargement. The abnormal appearance of many immunoreactive neurones suggested degenerative changes were occurring. tTG was also present in central canal cluster cells from birth onwards. No neuronal immunoreactivity was seen throughout foetal development. A proportion of motor neurones prepared from E15 spinal cord and grown in coculture with spinal cord astrocytes, were immunoreactive for tTG. All immunoreactive neurones showed signs of degeneration. Addition of myotube-conditioned medium (a source of cholinergic differentiation factor, CDF) reduced the proportion of tTG-immunoreactive neurons in the cultures. Schwann cell-conditioned medium (a source of ciliary neurotrophic factor, CNTF) had a similar but less potent effect on the numbers of immunoreactive neurones. The possibility that tTG is a marker for late, but not early-phase programmed cell death in the developing rat spinal cord is discussed in the light of a proposed role for tTG in the mechanism of natural cell death by apoptosis.