Paired twisted filaments a new ultrastructural marker of human pinealomas?

Aria

Neuropathologica

Acta Neuropathol (Berl) (1984) 6 5 : 1 6 3 - 1 6 5

9 Springer-Verlag 1984

Paired Twisted Filaments: A New Ultrastructural Marker of Human Pinealomas?* J. Hassoun 1, B. Devictor 1, D. Gambarelli 1, J.C. Peragut 2, and M. Toga 1 Depts. of 1 Neuropathology and 2 Functional Neurosurgery, Centre Hospitalo-Universitaire de la Timone, Marseille, France

Summary. Paired twisted filaments (PTF) forming helices are described in tumor cells of three human pinealomas. Each filament was 8.11 _+:1.55 nm wide. The maximal width of the helix was 16.62_ 2.62 nm. The periodicity of the constrictions was 26.63 _+4.49 nm. These characteristics appeared original, suggesting protein filaments possibly specific of pinealocytes. The similarities and discrepancies between PTF and Alzheimer's paired helical filaments (PHF) are discussed. Key words: Human pinealomas - Ultrastructure Paired twisted filaments - Paired helical filaments Cytoskeleton

boy; case 2: a 46-year-old woman; case 3: a 44-year-old woman). Histological examination showed classical aspects of pineocytoma in cases I and 3 and pinealoblastoma in case 2. Case 3 has already been published (Hassoun et al. 1983, case 3). Glial fibrillary acidic protein (GFAP)-immunostaining (Histoset, Ortho Diagnostic Systems) was negative in tumor cells of all cases by light microscopy. In each case, tumor fragments were also fixed in 4% glutaraldehyde, postfixed in 1% OsO4, dehydrated, and embedded in Araldite. U1trathin sections connterstained with uranyl acetate and lead citrate were photographed with a Philips EM 300 electron microscope. Quantitative analysis was made on electron microscopic photographs with a Tektronix 4956 deskplot computer and a Tektronix 4051 graphics tablet. In each case, 100 measurements of each parameter were made to calculate the average value and the standard deviation (SD).

Results

Introduction The fine structure of the adult human pineal gland is still unknown. The ultrastructural analysis of pinealomas is an indirect but important approach to such understanding. Several specific ,.markers have been previously reported in four patients (Hassoun et al. 1983). Paired twisted filaments (PTF) were found in two additional pinealomas and in one of the tumors already described in the literature. This new possible marker shows some similarities with paired helical filaments (PHF) of Alzheimer's disease but distinct morphometric characteristics. Materials and Methods A stereotactic biopsy was performed in three patients presenting with a pineal tumor diagnosed on CT scan. (Case 1: a 3-year-old * Supported by a grant from FEGEFLUC (F6d6ration Nationale des Groupements des Entreprises Frangaises darts la Lutte contre le Cancer), Marseille, France

Offprint requests to: Dr. Jacques Hassoun, Laboratoire de Neuropathologie, Facult6 de M6decine, 27 Bd. J. Moulin, F-13385 Marseille C~dex V, France

By electron microscopy, the two new cases showed all the characteristics previously reported: dark and clear cells joined with numerous zonulae adherentes, extensive thin processes, dense-core and clear synaptic-like vesicles, vesicle-crowned rodlets, microtubular sheaves, microtubules, fibrous bodies, and heterogeneous neuromelanin-like inclusions. A complex vacuolar system was typical of the architecture of both tumors. All these criteria have been already described in case 3. An original feature was observed in the three tumors, corresponding to PTF (Fig. 1 a-g). These were arranged in parallel short bundles in the perikarya and cell processes. Each pair was composed of two 8.11-nm (+_ 1.55) wide filaments forming a helix. The periodicity of the constrictions was 26.63 (+_ 4.49) nm. The maximal width of the helix was 16.16 ( + 2.62) nm and the minimal 9.98 (+2.03) nm). P T F formed cristallin-like inclusions on supposed cross sections (Fig. 1 a, g). In some cells, the filaments were found isolated in the perikarya. No direct relationship could be established between P T F and otherwise classical microtubules (20.8 n m + 3.15 in average diameter) or very rare 9-10 nm microfilaments.

Fig. l a - g . Paired twisted filaments (PTF) in pinealoma cells, a x 23,000 (case 1); b x 30,000 (case 3); c x 45,000 (case 2); d x 48,000 (case 1); e x 129,000 (case 1); f x 129,000 (case 2); g x 129,000 (case 1). Helicoidal pattern of PTH is obvious in e, f, and g. See text for details

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Hassoun et al. : Paired Twisted Filaments in Pinealomas

Discussion The ultrastructural study of two new pinealomas confirmed the high specificity of the pattern recently published (Hassoun et al. 1983). PTF constitute a new possible marker since they were present not only in both new cases but also in a previous case where they had not been formerly recognized. Their scarcity and the need for an appropriate search could explain why they ]have escaped current ultrastructural investigations. PTF strikely resembled the PHF described in Alzheimer's disease (Kidd 1963) and in a variety of neurologic diseases (Wisniewski et al. 1979). However, some discrepancies should be emphasized. First, PHF have been only described in neurons. Secondly, the spatial orientation of PTF appeared more regular than that of PHF which generally form waved and extensive tangles in neurons. In contrast, PTF bundles were rather straight and short. Thirdly, in spite of a same helicoidal pattern, the periodicity of the constrictions was quite different: 80 nm for PHF (Wisniewski et al. 1976, 1983), 26.63 nm for PTF. The other parameters, though slightly less in PTF (maximal and minimal width of the helix, width of each filament) were rather similar (see Table 1). The 20-rim microtubules present in great number in the three cases served as a c,onstant reference for morphometric study; their standard dimension (Peters et al. 1976) could exclude the artifacts due to the fixation classically associated with glutaraldehyde and osmic tetroxide. As far as we are aware, PTF have been described neither in mammalian pinealocytes (Welsh and Reiter 1978; Pevet 1979; Vollrath 1981) nor in human fetal pineal gland (Moller 1974) and pineal tumors (Nielsen and Wilson 1975; Herrick and Rubinstein 1979; Kline et al. 1979; Markesberry et al. 1981). Their regular ultrastructural characteristics suggest an original protein molecule perhaps specific of pinealocytes. An electron-immunocytochemical investigation using different anti-neurofilament sera and monoclonal antibodies to antigen related to PHF as described by Wang et al. 1984 could be useful in determining their eventual immunologic specificity. The scarcity of PTF makes any immunoreaction unlikely by light microscopy. At present, PTF does not support the hypothesis of neuronal differentiation of human tumor pinealocytes any more than do vesicle-crowned rodlets or dense-core vesicles.

Table 1. Comparative morphometric characteristics of paired twisted filaments (PTF) and paired helical filaments (PHF) according to Wisniewski et al. (1976, 1983) Filament

PTF PHF

Mean width Max. width 8.11 nm 16.16 nm 9-12 nm 1852 nm

Helix Min. width 9.98 nm 10 nm

Periodicity 26.63 nm 80 nm

Acknowledgements. We are grateful to Mrs. E. Pasquale, Mrs. F. Fabre, and Mr. R. Gochgagarian for their technical assistance.

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Received May 16, 1984 / Accepted July 19, 1984