Suppression of cathepsins B and L causes a proliferation of lysosomes and the formation of meganeurites in hippocampus

Abstract

Cultured hippocampal slices exhibited prominent ultrastructural features of brain aging after exposure to an inhibitor of cathepsins B and L. Six days of treatment with N-CBZ-L-phenylalanyl-L-alanine-diazomethylketone (ZPAD) resulted in a dramatic increase in the number of lysosomes in the perikarya of neurons and glial cells throughout the slices. Furthermore, lysosomes in CA1 and CA3 pyramidal cells were not restricted to the soma but instead were located throughout dendritic processes. Clusters of lysosomes were commonly found within bulging segments of proximal dendrites that were notable for an absence of microtubules and neurofilaments. Although pyknotic nuclei were sometimes encountered, most of the cells in slices exposed to ZPAD for 6 d appeared relatively normal. Slices given 7 d of recovery contained several unique features, compared with those processed immediately after incubation with the inhibitor. Cell bodies of CA1 neurons were largely cleared of the excess lysosomes but had gained fusiform, somatic extensions that were filled with fused lysosomes and related complex, dense bodies. These appendages, similar in form and content to structures previously referred to as "meganeurites," were not observed in CA3 neurons or granule cells. Because meganeurites were often interposed between cell body and axon, they have the potential to interfere with processes requiring axonal transport. It is suggested that inactivation of cathepsins B and L results in a proliferation of lysosomes and that meganeurite generation provides a means of storing residual catabolic organelles. The accumulated material could be eliminated by pinching off the meganeurite but, at least in some cases, this action would result in axotomy. Reduced cathepsin L activity, increased numbers of lysosomes, and the formation of meganeurites are all reported to occur during brain aging; thus, it is possible that the infusion of ZPAD into cultured slices sets in motion a greatly accelerated gerontological sequence.

Fig. 1.

A, Light micrograph of a semithin section through CA1 of an untreated (control) cultured hippocampal slice maintained in vitro for 16 d. Several pyramidal neurons (P) are shown with their contiguous apical (a) or basal (arrowheads) dendrites. A basophilic organelle (arrow) can be seen clearly in one CA1 neuron. Magnification, 1200×. B, Light micrograph of CA1 neurons from a semithin section obtained from a cultured slice exposed to ZPAD, an inhibitor of cathepsins B and L, from culture day 11–16. The pyramidal cells (P) show many of the same features as those in A, including centrally located nuclei, apical (a) dendrites, and basal (arrowheads) dendrites. Inspection of the cytoplasm of these neurons, however, reveals a dramatic increase in the number of basophilic punctate structures (arrows). Note that many neurons show an asymmetrical distribution of the darkly stained granules in that more are found in the basal portion of their cell bodies. Magnification, 1200×.

Fig. 2.

A, Electron micrograph of a CA1 pyramidal cell from a control cultured hippocampal slice. The nucleus (N) is centrally located and surrounded by numerous cytosolic organelles, including a few lysosomes (arrow). This slice was maintained in culture for 16 d. Magnification, 6200×. B, Electron micrograph of a section of the apical dendrite from a control CA1 pyramidal cell. This portion of the process features prominent microtubules (arrow), smaller neurofilaments, and a synapse (open arrow). Magnification, 32,000×.

Fig. 3.

A, Montage of electron micrographs showing a CA1 neuron from a cultured hippocampal slice exposed to ZPAD for 6 d. Numerous lysosomes (arrows) are located in the apical portion of the perikaryon and proximal apical dendrites. The nucleus (N) appears normal. Magnification, 4700×. B, Enlargement of a portion of the soma. The milieux of some lysosomes contain twisted membranes (large arrow); other lysosomes have vacuoles of various sizes (open arrow). Mitochondria (small arrow) appear normal. Magnification, 26,500×.

Fig. 4.

A, Electron micrograph of the basal portion of a CA1 pyramidal cell soma from a ZPAD-treated slice. Although numerous lysosomes (arrows) encircle the nucleus (N) and are found within the proximal span of a basal dendrite (d), none are observed in the axon hillock (h) and initial segment (is) within this section. Vacuole-containing lysosomes (open arrows) are also evident within the perikaryal cytoplasm. Magnification, 8300×. B, Enlargement of the axon initial segment (is). Note the dense axolemmal undercoating, microtubule bundling, cisternal organelle (arrow), mitochondria (arrowheads), and paucity of lysosomes. Magnification, 15,000×.

Fig. 5.

A, Electron micrograph of a CA3 apical dendrite from a ZPAD-treated slice. Most lysosomes are located either adjacent to the nucleus (N) or found clustered within a bulging segment of the apical dendrite (d). A spine is evident distal to the dendritic bulge (arrow). A satellite cell (S) is present alongside the dendrite. Magnification, 8000×. B, Enlargement of the dendritic bulge. Intact microtubules and neurofilaments are not apparent within the two lysosomal assemblages. Note the presence of several synapses (arrows). Magnification, 15,000×. C, Enlargement of the dendritic spine (sp) noted in A and accompanying mossy fiber (m). Magnification, 22,000×.

Fig. 6.

A, Electron micrograph of a portion of the CA3 field at the junction of the cell body layer and stratum radiatum of a slice treated with ZPAD for 6 d. At the bottom of the field, a row of lysosomes (arrow) is found in an apical dendrite (d). Note that the dendrite also contains vacuolated lysosomes (open arrow). A pyknotic nucleus (P) can be seen adjacent to this dendrite. Membrane-bound cellular processes containing lysosomes and mitochondria are located around the compacted nucleus. Magnification, 13,500×.B, Micrograph of a section of CA3 stratum radiatum from a treated hippocampal slice. A large lysosome (large arrow) fills most of a swollen secondary dendrite. Flanking the lysosome are two spines (small arrows). Magnification, 15,000×.

Fig. 7.

A, Montage of electron micrographs of a CA1 neuron 7 d after the conclusion of ZPAD exposure. Although the nucleus (N), soma, and axon initial segment (is) appear normal, the location of the axon initial segment is distally displaced by a fusiform expansion (arrows) of the axon hillock region (meganeurite). The cytosol of the meganeurite, unlike that of the axon and soma, contains many lysosomes. Magnification, 6200×. B, Enlargement of the indicated portion of the fusiform expansion. Magnification, 18,500×. C, Enlargement of the region containing the axon initial segment. Note that this region contains several mitochondria (arrowheads) and a few lysosomes. Magnification, 18,500×.

Fig. 8.

A, Montage of electron micrographs showing the distal portion of a meganeurite from a treated CA1 pyramidal cell. The length of the meganeurite is more than twice the diameter of the parent soma (data not shown). Note that a columnar organization of lysosomes and FDBs is found within the proximal aspects of the meganeurite (top of field). FDBs at the more distal regions of the process (bottom of field), however, are compacted into larger dense bodies (DB). Magnification, 6200×. B, Enlargement of the more distal segment of the meganeurite. The circular aggregates are composed of FDBs and vacuoles. Note the presence of mitochondria (arrowheads) that appear normal. Magnification, 20,000×. C, Micrograph of a membrane-bound cellular profile filled with FDBs observed in CA1 stratum oriens of a ZPAD-treated slice. Magnification, 13,500×.

Fig. 9.

A, CA1 of a slice maintained in culture for 1 week after ZPAD treatment. An astrocyte (a) is adjacent to several FDB-laden cellular processes. Magnification, 8500×. B, Enlargement of two of the processes. Note the presence of filaments between the two profiles and the discontinuous nature of the membrane encircling the process on the left. Magnification, 18,000×.

Fig. 10.

A, Electron micrograph of a portion of CA1 depicting an apical dendrite (d) of a ZPAD-exposed slice. Clusters of FDBs line up adjacent to an irregular dendritic membrane. Magnification, 19,000×. B, A cellular process filled with FDBs apposes a concavity of a distal CA1 meganeurite. Magnification, 16,000×. C, Lysosomes fill a dendritic protrusion. Note the vesicles containing residual catabolic organelles and whorled bodies (arrow) beside the excrescence. Magnification, 20,000×.

Fig. 11.

Schematic diagram showing a hypothetical sequence of ZPAD-induced alterations on the fine morphology of CA1 neurons. The perikaryal cytosol of a control CA1 pyramidal cell contains only three lysosomes (cell on far left). Six days of exposure to ZPAD, an inhibitor of cathepsins B and L, result in a dramatic expansion of the lysosomal population (second cell in diagram). CA1 neurons allowed to recover for 7 d after ZPAD treatment restore the normal ultrastructure of the soma by sweeping the excess lysosomes and residual bodies into the axon hillock and initial segment (middle neuron). In distal portions of long “meganeurites,” lysosomal bodies are fused into circular aggregates (fourth cell). The last neuron (on thefar right) depicts a hypothesized outcome of this sequence in which subsequent exocytosis of the lysosomal compactions results in pyramidal cell axotomy.