A simplified method for the preparation of tetanus toxin binding fragment for neurobiology.

The non-toxic binding fragment of tetanus toxin (fragment C) binds avidly to neural tissue and has a growing number of neurobiological uses. Its current utility is limited by both its high commercial cost and the complex procedure for its preparation requiring highly purified tetanus toxin. We have developed a short procedure which prepares fragments of tetanus toxin from crude C. tetani extracts. The resultant proteins are atoxic with molecular sizes and immunological properties closely resembling fragment C. These proteins undergo retrograde axonal and apparent transneuronal transport in a fashion similar to fragment C.

Penetration and internalization of plasma proteins in the human spinal cord.

In animal studies, motoneurons take up plasma proteins including immunoglobulins at their terminals. These proteins are then transported back to cell bodies in the spinal cord. To determine if these processes also occur in humans, we localized several different plasma proteins in autopsied spinal cords from 13 patients without neurological disease. As in animals, plasma proteins are associated with vascular and pial structures. Motoneurons, particularly large cervical and lumbar motoneurons, frequently showed immunoreactivity within their cytoplasm to several plasma proteins. Motoneuron labeling was more consistent with antisera against plasma proteins of lower molecular weights such as IgG, IgA and transferrin, than with antisera against higher molecular weight proteins such as IgM and alpha-2-macroglobulin. Other large neurons without connections outside the blood-brain barrier such as those of Clarke's column also occasionally labeled with antisera against all plasma proteins tested. Our results are compatible with the concept that motoneurons take up and transport plasma proteins. These neurons can be distinguished from cells which internalized extravasated serum proteins before and after death. Uptake of pathogenic antibodies by motoneuron terminals may play a role in the pathogenesis of motoneuron disease.

Enhanced CNS uptake of systemically administered proteins through conjugation with tetanus C-fragment.

No other exogenous protein enters the central nervous system from the circulation as readily as tetanus toxin. We examined the capability of the non-toxic binding fragment of tetanus toxin (C-Fragment) so serve as a vehicle for transport of other proteins into the mouse CNS. Using periodate oxidation of the enzyme horseradish peroxidase (HRP), we synthesized two separate macromolecular complexes, one containing C-fragment and HRP, and the other C-fragment, HRP and a third "test" protein-human IgG. The distribution of C-fragment-HRP was typical of blood borne proteins including native C-fragment, with labeling of all neurons with known projections outside the blood-brain barrier, particularly large spinal motoneurons. C-fragment-HRP conjugates showed superior neuronal labeling to over 100-fold greater quantities of free HRP. Complexes containing C-fragment, HRP and human IgG were internalized by neurons from both intramuscular and intraperitoneal injections. The efficiency of neuronal uptake of IgG in the C-fragment conjugated form was enhanced over 40-fold compared to free IgG. Linkage of a large protein to C-fragment probably leads to enhanced endocytosis of that protein by neuronal terminals projecting outside the blood-brain barrier. C-fragment can serve as a vehicle to allow selected proteins to bypass the barrier and enter the CNS.

Internalization of plasma proteins by cerebellar Purkinje cells.

Animal studies suggest that Purkinje cells internalize proteins from the blood and CSF. This process may relate to the pathogenesis of paraneoplastic cerebellar degeneration in patients with anti-Purkinje cell antibodies. To determine if human Purkinje cells may also internalize plasma proteins, cerebellar tissue was taken from routine autopsies of eight patients without neurologic or neoplastic disease. Several plasma proteins including IgG, IgA, IgM, transferrin, albumin and alpha-2-macroglobulin were detected by immunohistochemistry within the cytoplasm of Purkinje cells. Internalized proteins frequently filled the entire soma and major dendrites, sparing the nucleus. Vascular structures were also immunolabeled, while glia internalized plasma proteins differentially, with oligodendrocytes selectively internalizing transferrin. Purkinje cells were the most numerous and heavily labeled neuronal cell type in spite of their small numerical representation in the cerebellar neuronal population. Our results are compatible with previous animal studies, and suggest that internalization of specific antibodies could contribute to the pathogenesis of Purkinje cell loss in paraneoplastic cerebellar degeneration.