Spontaneous perikaryal neurofilament phosphorylation in the septofimbrial nucleus of the rat

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Spontaneous perikaryal neurofilament phosphorylation in the septofimbrial nucleus of the rat E Klosen and Ph. van den Bosch de Aguilar Laboratoire de Biologie Cellulaire, Uniu~ BANI, Universit~; Catholique de Louvain, Louvain-la-Neuve (Belgium)

(Received 16 December 1991 : Revised version received 18 February 1992; Accepted 18 February 1992) K
Neurofilament phosphorylation; Septofimbrial nucleus: Triangular septal nucleus; Central nervous system; Immunocytochemistry

Phosphorylation of the 200 kDa neurofilament peptide NF-H usually only occurs in axons. We describe the spontaneous presence of phosphorylated NF-H in a population of small spindle-shaped neurons of the rat septofimbrial nucleus. A similar phenomenon has been observed in axotomized neurons and in human neurodegenerative diseases. Our observations, as well as previous studies by other authors, indicate that perikaryal neurofilament phosphorylation is not necessarily linked to pathological conditions.

The neuronal intermediate filament network is composed of three peptides of 68 (NF-L), 160 (NF-M) and 200 kDa (NF-H). During post-translational processing, the two heavier peptides, NF-M and NF-H, are extensively phosphorylated [3]. The effects of this phosphorylation are multiple and range from an increased resistance to proteolysis [14] to a change in apparent molecular weight in SDS-PAGE [3]. In 1983, Sternberger and Sternberger [20] showed that phosphorylated and nonphosphorylated forms of neurofilaments can be distinguished in situ by monoclonal antibodies. Antibodies of this type have since been largely used to study the distribution of phosphorylated and non-phosphorylated neurofilament peptides. These studies, as well as biochemical studies, show that the phosphorylated forms of neurofilament peptides are almost exclusively present in axons. In recent years, several authors have been able to demonstrate the presence of phosphorylated neurofilament peptides in the perikarya ofaxotomized neurons [7, 8, 11, 17] and neurons in the brains of patients with neurodegenerative disorders [5, 12] or congenital malformations [19]. Especially in human or animal motor neuron diseases, perikaryal neurofilament phosphorylation in motor neurons seems to be a common feature [4, 12]. The significance of the presence of phosphorylated neurofilaCorrespondence." E Klosen, Laboratoire de Biologie Cdlulaire, Unite BANI, B~timent Claude Bernard, Place Croix du Sud. 5, B- 1348 Louvain-la-Neuve, Belgium.

ment epitopes in neuronal perikarya in these pathologies or in axotomy has not been established so far~ Spontaneous perikaryal neurofilament phosphorylation has so far only been reported in specific subtypes of sensory neurons: the A type neurons of the spinal and cranial ganglia and the mesencephalic ganglion of the trigeminal nerve [10, 15] and the type II neurons of the spiral ganglion [1, 16]. We report here the spontaneous presence of phosphorylated neurofilaments in the perikarya of neurons in the septofimbrial and triangular septal nuclei. This study includes material from 36 Wistar rats of both sexes and aged between 1 and 32 months. Most of the animals were perfused with 4% paraformaldehyde and embedded in paraffin (n=ll) or Polyethylene Glycol (PEG, n=8). Some animals were anesthetized and decapitated. Their brains were rapidly dissected and fixed by immersion in either Carnoy's (n=2), Bodian's (n--4) or Bouin's (n=2) fixative, and embedded in paraffin. Finally, in a third series of experiments, the animals were perfused with either 4% buffered paraformaldehyde (n-2), Somogyi's (n=6) or Regaud's (n=l) fixative and cryoprotected for cryostat sectioning or processed for vibratome sectioning. Paraffin sections (5-6/~m) and thin PEG sections (3-4 J~m) were processed as slide mounted sections, whereas cryostat, vibratome or thick PEG sections (10-15 j~m) were processed free-floating. Antibodies NEI4 (Boehringer), RT97, 8D8, RSI8 and 147 are directed to phosphorylated NF-H [6]. RT97 and 8D8 cross-react with the microtubule-associated protein

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Fig. 1. A: basket axons around Purkinje cells labelled with antibody 147. B: axonal labelling with antibody 147 in the hippocampus. Cell bodies are completely devoid of labelling, pyr, pyramidal cell layer. C: neuronal cell bodies in the scptofimbrial nucleus labelled with antibody RS18 Iopen arrows). Note the labelled neurites at two poles of the labelled cells (arrows). D: immunolabelling of septofimbrial neurons with antibody N52. S~.'veral labelled cell bodies can be seen a m o n g large labelled neurites, which are probably dendrites (open arrowsl. C o m p a r e these neuritcs with tile predominantly axonal labelling in B. E: Mes V A type neurons labelled with antibody 147. Sonic small labelled C type perikarya arc also observed (open arrow), while some of the larger cell bodies are unlabelled (arrow). The labelling of these neurons appears clearly granular or even reticulated. F: antibody N52 labels all cell bodies in the Mes V very intensely. PEG sections, counterstained with Mayer's hemtllunl (except B). Bur 5(I urn.

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Fig. 2. The distribution of neuronal cell bodies labelled for phosphorylatedNF-H in the caudal septum (stippled area), cc, corpus callosum:t\ fornix: ac, anterior commissure: SFN+septothnbrial nucleus: TSN, triangular septal nucleus.

tau, while NE14, 147 and RSI8 detect only NF-H [13]. Antibody N52 (Boehringer) recognizes phosphatase-insensitive N F - H epitopes [18]. Immunostaining was performed using either indirect immunoperoxidase protocols (DAKO) or streptavidin-biotin systems (Amersham). To check whether labelling of tissue wets due to phosphate-dependent antigens, the sections were dephosphorylated by the method of Sternberger and Sternberger [20] before immunolabelling with antibodies RS18, 147 or 8D8. Phosphate-dependence of the immunolabelling was also assessed by diluting the antibodies in 0.15 M phosphate buffer. Two rats were perfused with ice-cold saline and their brains rapidly dissected. Several brain regions were microdissected and homogenized in Laemmli sample buffer at a ratio of 20 ktl/mg of fresh tissue [9]. Five microliters of this homogenate were run on 6-18% S D S - P A G E gradient gels and transferred to nitrocellulose (Hybond- (', Amersham). Immunolabelling of these Western blots with antibodies RS18, 8D8 and N52 was performed using alcaline phosphatase labelled detection systems. Best results were obtained with Carnoy or Bodian fixatives on paraffin sections and paraformaldehyde o, Somogyi's fixative on free-floating sections. It should be noted that no qualitative labelling differences were observed between the five antibodies directed against phosphorylated N F - H epitopes. The only differences were in the dilution used for each antibody. Thus, the observations described below could be made with any combination of fixative and antibody. Throughout the brain, intensely stained nerve fibers could be seen with antibodies to phosphorylated NF-H. The topography of the staining resembled that of myelin stains, particularly in the cortical areas. While matter was composed of intensely labelled thick axons, whereas gray matter contained many smaller axons criss-crossing in all directions (Fig. 1). In the cerebellum, basket axons around Purkinje cells clearly displayed this axonal label-

ling (Fig. IA). In the mesencephalon, the labelled perikarya of the mesencephalic nucleus of the trlgeminal nerve could easily be detected (Fig. lEt. Antibody N52 labels less axons than the other five antibodies, but recognizes neuronal cell bodies and dendrites in all areas of the brain. However. not all neuronal perikarya are labelled by this antibody, The neurons of the mesencephalic nucleus of the trigeminal nerve were among the most intensely labelled by this antibody (Fig. IF). In the rat forebrain, all five anti-phosphorylated NFH antibodies recognized neuronal cell bodies only m a discrete area of the caudal septum (Fig. 2). This area compriscs the septofimbrial nucleus and the triangular septal nucleus. A most notable concentration of labelled cells was observed around the fornix at its exit fi'om the septal area below the ventral hippocampal commissure. In all other areas of the septum or the rest of the ['orebrain, only labelled axons could be observed with antibodies to phosphorylated NF-H. These septal neurons were also among the most intensely labelled perikarya by antibody N52 to phosphate-independent NF-H epitopes, but this antibody also labelled other neuronal perikarya scattered throughout the septum and the forebrain. One major characteristic of the labelled neurons is their spindle shape (Fig. 1 B). Most of them displayed one thick clearly distinguishable neurite. On some of these neurons, a second neurite, much thinner, could be observed at the opposite pole of the former. These neurons displayed only scant cytoplasm around their nucleus. On paraffin sections, the labelling of the cytoplasm and the dendrite-like neurites seemed homogeneous. On paraformaldehyde fixed PEG seclions, the labelling sometimes appeared fibrillar. After dephosphorylation of the sections by alkaline phosphatase+ the overall immunostaining was clearly reduced (Fig. 3A,B). Nerve fibers were still labelled, but less intensely, indicating that the dephosphorylation was

III

Fig. 3. A: control section, treated with trypsin and incubated with phosphatase buffer without the enzyme. Anti-neurofilament labelling with antibody RSIS. On one neuron the labelled dendrite (arrow}, as well as the labelled axon {arrowheads}, can be seen. Some unlabellcd neurons arc located in the same area (open arrows). B: dephosphorylated section displaying slightly reduced immunolabelling of nerve tibcrs and ~tbsencc of labelled ccll bodies with antibody RS18. The arrov&eads indicate the location of nuclei, which no longer stain with Mayer's hemalun aflcr the dcphosphor>lation. Bodiun's fixative, paraffin sections. Mayer's henmlum counterstain. Bar 20 #m.

probably incomplete. Longer incubations with alkaline phosphatase resulted in extensive damage to the sections. Dephosphorylation abolished the immunostaining of neuronal perikarya in the septofimbrial and triangular septal nuclei. A similar effect could be obtained by diluting the antibodies in 0.15 M phosphate buffer. On Western blots, no differences could be detected between tile different brain regions with any of the three antibodies used (Fig. 4). Most particularly, homogenates from the caudal septum, containing the septofimbrial and triangular scptal nuclei, and the rostral septum, which contains no perikaryal labelling for phosphorylaled N F-H, displayed identical labelling patterns with a major band at 200 kDa. The labelled bands below this 200 kDa band have previously been described as degradation products of neurofilament metabolism [2]. Intensity of tile immunolabelling correlated with the white matter content of the different brain regions. The caudal septum containing also part of the fimbria-fornix had a higher content of NF-H than the rostral septum, which contains only little white matter. The identical resuhs using five different well-characterized monoclonal antibodies are strongly suggestive that the labelling of septofimbrial neurons is due to the presence of phosphorylated neurofilament epitopes in their perikarya, although we cannot rule out a crossreaction with unrelated phosphatase-sensitive molecules. Furthermore, we were able to confirm the presence of N F-H in these neurons with an antibody to phosphataseinsensitive epitopes. Among the molecules anti-neurofilament antibodies

cross-react with, microtubule-associated protein (MAP) tau is probably the best known. While RT97 and 8D8 are known to cross-react with tam RS18 and 147 are completely specifc to nmtrofilaments [13]. Takagi et al. [22] showed that a monoclonal antibody to NF-H also detected an isoform of troponin T in type 1 and 2c muscle fibers. However this protein is restricted to certain muscle fibers. Antibodies 147 and RT97 have been shown to cross-react with Histone HI. However, this cross-reaction results in the labelling of the nucleus and not the cell cytoplasm [23]. Our Western blotting experiments show that any putative cross-reactive molecule in the perikarya of septofimbrial neurons would have to have a molecular wcighl of 200 kDa. Indeed, in homogenates o1" the caudal septum, the labelling pattern is identical to that of other brain regions. Most of this labelling is due to axonal neurofilaments, as it is impossible to prepare perikaryal proteins without any axonal contamination. However, we should have been able to detect a cross-reactive molecule from the perikarya if its molecular weight was different from that of phosphorylated N F-H. Out" observations thus allow us to add these scptofimbrial neurons to the few already described exceptions that present phosphorylated neurotilamcnts in their cell bodies: a type neurons in the spinal ganglion and thc mesencephalic nucleus of the Vth nerve [10, 15] and type II neurons of the spiral ganglion [1~ 16]. Unlike these neurons, septofimbrial neurons are not primary sensory neurons but interncurons. They relay hippocampul mformations to the habenular nuclei and the interpedun-

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SFi MflN CPu mW

-"

I'IO+W

--

SFi MSN CPu

3* 44 kD 29kD

--

---

RS18

8D8

N52

Fig. 4. Western blots of total brain homogenates labelled with antibodies RS 18, 8D8 and N52. The labelling pattern obtained with each antibody is identical in the different brain areas. The asterisk indicates the labelling of tau protein by antibody 8D8. This reaction ts relatively faint compared to the reaction with NF-H. SFi, septofimbrial nucleus: MSN, medial septal nucleus: CPu, caudate+putamen+

c u l a r nucleus t h r o u g h the stria medultaris a n d the fasciculus retroflexus [21]. This s t u d y c o n f i r m s t h a t p h o s p h o r y l a t e d N F - H can be s p o n t a n e o u s l y p r e s e n t in the cell b o d y o f certain neurons. It also shows t h a t this p h e n o m e n o n is n o t necessarily l i n k e d to p a t h o l o g i c a l c o n d i t i o n s . A c o m p a r a t i v e s t u d y o f the s p o n t a n e o u s a n d l e s i o n - i n d u c e d presence o f p h o s p h o r y l a t e d N F - H in n e u r o n a l p e r i k a r y a m a y p r o vide s o m e clues to u n d e r s t a n d this p h e n o m e n o n a n d its m e c h a n i s m . T h e significance o f this p e r i k a r y a t neurofilam e n t p h o s p h o r y l a t i o n , w h e t h e r s p o n t a n e o u s o r lesioninduced, is u n k n o w n so far. The s t u d y o f this p h e n o m e n o n m a y lead to a better u n d e r s t a n d i n g o f the p a t h o genetic m e c h a n i s m s o f the h u m a n n e u r o d e g e n e r a t i v e diseases in which it is observed. T h e a u t h o r s wish to t h a n k Dr. B.H. A n d e r t o n for the gift o f the m o n o c l o n a l a n t i - n e u r o f i l a m e n t a n t i b o d i e s . T h e p h o t o g r a p h i c assistance o f F. D e s n e u x is gratefully a c k n o w l e d g e d . P.K. is the recipient o f a research g r a n t by the M i n i s t r y o f C u l t u r a l Affairs o f the G r a n d - D u c h y o f L u x e m b o u r g . This s t u d y was s u p p o r t e d by F N R S a n d F R F C g r a n t s a n d b y the N a t i o n a l L o t t e r y o f Belgium. This article is p u b l i s h e d within the f r a m e w o r k o f the Commission of European Communities Concerted Act i o n on C e l l u l a r A g i n g a n d Diseases ( E U R A G E ) . 1 Berglund, A.M. and Ryugo, D.K., A monoclonal antibody labels type II neurons of the spiral ganglion, Brain Res., 383 (1986) 327 332. 2 Calvert, R. and Anderton, B.H., In vivo metabolism of mammalian neurofilament polypeptides in developing and adult rat brain, FEBS Lett., 145 (1982) 171-175. 3 Carden, M.J., Schlaepfer, W.W. and Lee, V.M.-Y., The structure,

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