Long-lasting aberrant tubulovesicular membrane inclusions accumulate in developing motoneurons after a sublethal excitotoxic insult: a possible model for neuronal pathology in neurodegenerative disease

Abstract

We have previously shown that chronic treatment of chick embryos [from embryonic day 5 (E5) to E9] with NMDA rescues spinal cord motoneurons (MNs) from programmed cell death. In this situation, MNs exhibit a reduced vulnerability to acute excitotoxic lesions and downregulate NMDA and AMPA-kainate receptors. Here, we report that this treatment results in long-lasting sublethal structural changes in MNs. In Nissl-stained sections from the spinal cord of NMDA-treated embryos, MNs display an area adjacent to an eccentrically positioned nucleus in which basophilia is excluded. Ultrastructurally, MNs accumulate tubulovesicular structures surrounded by Golgi stacks. Thiamine pyrophosphatase but not acid phosphatase was detected inside the tubulovesicular structures, which are resistant to disruption by brefeldin A or monensin. Immunocytochemistry reveals changes in the content and distribution of calcitonin gene-related peptide, the KDEL receptor, the early endosomal marker EEA1, and the recycling endosome marker Rab11, indicating that a dysfunction in membrane trafficking and protein sorting occurs in these MNs. FM1-43, a marker of the endocytic pathway, strongly accumulates in MNs from isolated spinal cords after chronic NMDA treatment. Changes in the distribution of cystatin C and presenilin-1 and an accumulation of amyloid precursor protein and beta-amyloid product were also observed in NMDA-treated MNs. None of these alterations involve an interruption of MN-target (muscle) connections, as detected by the retrograde tracing of MNs with cholera toxin B subunit. These results demonstrate that chronic NMDA treatment induces severe changes in the motoneuronal endomembrane system that may be related to some neuropathological alterations described in human MN disease.

Fig. 1.

Changes in MN numbers (mean ± SEM) in the lumbar lateral motor column of E9 and E10 chick embryos after treatment with either saline or different regimens of NMDA. NMDA was administered on E8 only, daily from E6 to E8, daily from E5 to E8, or on E5 and E6 (in the case of embryos killed on E9); and daily from E5 to E9 (in the case of embryos killed on E10). Doses of NMDA were 0.5 mg on E5 and E6 and 0.25 mg on E7, E8, and E9. Numbers inparentheses indicate sample sizes. *p < 0.01, **p < 0.001 versus saline (Student's t test).

Fig. 2.

Ultrastructural micrographs from MNs of E10 NMDA-treated embryos. A shows that the core of the Nissl-excluded region (marked with a continuousline) is formed by an interconnected network of membrane-bounded tubules (arrows) and vesicles, some of them filled with an electrodense material; this complex is peripherally surrounded by Golgi stacks (dashed lines) and is located in a juxtanuclear area of the MN; endoplasmic reticulum (ER), which is mainly located outside of this region, at the periphery of cell body, displays a dilated profile.B shows a high magnification of tubulovesicular structures partially filled with electrodense material (arrow). C, TPPase cytochemistry displays a positive reaction in NMDA-induced tubulovesicular structures (delimitated by a continuous line) and in the most external stacks of Golgi apparatus (G, delimitated by a dashed line). N,Nucleus; M, mitochondrion. Scale bars: A, 2.30 μm; B, 1.15 μm; C, 1.80 μm.

Fig. 3.

Ultrastructural changes in MNs from NMDA-treated embryos induced by brefeldin A (A) or monensin (B). A, brefeldin A induces a profound disorganization of Golgi apparatus, which disappears at the boundary of Nissl-excluded region (dashed lines); tubulovesicular structures (arrows) remain unaltered.B, Monesin treatment induces an alteration of Golgi apparatus (G) that is transformed to smooth membraned vacuoles; no changes are seen in tubulovesicular structures (arrows) inside the Nissl-excluded region (dashed lines). m, Mitochondria; N,nucleus. Scale bar (in A): A, 2.30 μm;B, 2.90 μm.

Fig. 4.

Morphological and immunocytochemical characterization of Nissl-excluded region in E16 MNs. A,A′, Thionin-stained paraffin sections to demonstrate that, after chronic NMDA treatment, MNs display an area adjacent to the nucleus in which basophilia or Nissl substance is excluded (asterisk in A′); compareA′ with the normal pattern of Nissl staining in MNs of saline-treated embryos (A). B,B′, The CGRP immunostaining (green) in normal MNs shows a fine dotted pattern distributed in the entire MN cell body (B), which corresponds to Golgi stacks and secretory vesicles; conversely, in MNs from NMDA-treated embryos, CGRP immunolabeling shows a redistribution, forming a circle that surrounds the juxtanuclear Nissl-excluded region (asterisk in B′); the sections were counterstained with propidium iodide (red). C–D′, Double fluorescent immunolabeling to demonstrate the distribution of KDELr (green) and CGRP (red);C shows that, in normal MNs, KDELr is located in structures surrounding CGRP-immunopositive Golgi stacks;C′ is a digital enlargement of C; in NMDA-treated embryos the redistribution of CGRP-positive Golgi stacks involves a relocation of the KDELr-positive ERGIC compartment out of the CGRP-immunopositive circle (D);D′ is a digital enlargement of D, where the cis–trans polarization of Golgi apparatus, deduced from the KDELr location, is indicated with anarrow. E, E′, Double fluorescent immunolabeling for cystatin C (green) and CGRP (red); cystatin C-immunopositive granules are homogeneously distributed in the cytoplasm of normal MNs (E); by contrast, in NMDA-treated embryos, cystatin C granules are concentrated inside the Nissl-excluded region (asterisk in E′) surrounded by a CGRP-positive boundary. F–I, Sections from spinal cord of chronically treated NMDA embryos labeled with CGRP antibody (red), and WGA (green inF), or antibodies against APP (green in G), βAP (green in H), and Rab11 (a recycling endosome marker, green inI); all of these markers accumulate inside the Nissl-excluded region (asterisk), which is delimitated by CGRP immunostaining. J is from the spinal cord of an NMDA-treated embryo in which MNs were retrogradely labeled with muscle-injected CTB; the section was double-immunolabeled to demonstrate CGRP (red) and CTB (green). Note that CTB colocalizes in part with CGRP but does not accumulate inside the Nissl-excluded area (asterisk). K is a section from an isolated spinal cord of an NMDA-treated embryo incubated with FM1-43 showing the accumulation of this endocytic tracer inside the Nissl-excluded region (*). L–N, Triple fluorescent labeling to demonstrate CGRP (red), PS1 (green), and DNA (DAPI staining,blue). In normal MNs, PS1 immunoreactivity is not detectable (L); however, after NMDA treatment, PS1 immunoreactivity is markedly increased inside the Nissl-excluded region (arrows in L′). PS1 immunoreactivity exhibits a positive ring inside the Nissl-excluded region shown in M and N(asterisk). N, Nucleus. Scale bars:A–B′, C, D–H, J, K, 10 μm; C′, 5 μm; D′, 3 μm; I, 15 μm;L, 30 μm; L′, 35 μm; M, N, 20 μm.

Fig. 5.

Neurofibrilar staining by means of silver impregnation (A, B) reveals a reorganization of cytoskeleton around NER in MNs of NMDA-treated embryos. Compare the MN from an E19 saline-treated embryo (A) with that from an E19 NMDA-treated embryo (B). A similar result is obtained after immunostaining of phosphorylated neurofilament proteins (C). N, Nucleus; * indicates the core of NER. Scale bar, 25 μm.

Fig. 6.

Ultrastructure of MN cytoplasm of E18 (A,B) and E20 (C) embryos previously treated with NMDA showing the evolution of tubulovesicular structures to autophagic-like vacuoles.A, By E18 tubulovesicular structures (delimited bydashed lines) become more electrodense, and they are enfolded by new membrane profiles, as seen in a high magnification inB; note the accumulation of vesicles intermixed with the electrodense structures. C, By E20 many multilamellar structures and autophagic bodies, some of them engulfing portions of cytoplasm and mitochondria (arrows), are present.N, Nucleus; m, mitochondrion;ER, endoplasmic reticulum. Scale bars: A, 3.60 μm; B, 0.90 μm; C, 1.80 μm.