Jacqueline O. Gonatas

Superior sensitivity of conjugates of horseradish peroxidase with wheat germ agglutinin for studies of retrograde axonal transport.

We have compared the retrograde axonal transport of horseradish peroxidase (HRP), to the retrograde transport of HRP conjugated with wheat germ agglutinin (WGA). Morphometric studies have shown that WGA-HRP conjugates were 40 times more sensitive than free HRP, in the tracing of retrograde connections from the rat submandibular gland to the superior cervical ganglion. Also, WGA-HRP was more sensitive than free HRP in the tracing of retrograde connections from the rat tongue to the hypoglossal nucleus. Our findings with WGA-HRP are consistent with the observations by Schwab et al. who reported (-125I) WGA is a highly sensitive retrograde tracer (Brain Research 152:145, 1978 (22)).

ULTRASTRUCTURAL AND BIOCHEMICAL OBSERVATIONS ON A CASE OF SYSTEMIC LATE INFANTILE LIPIDOSIS AND ITS RELATIONSHIP TO TAY-SACHS DISEASE AND GARGOYLISM.

1. Electron microscopic and biochemical data in a case of systemic infantile lipidosis and in a case of gargoylism have been presented. 2. In the case of systemic late infantile lipidosis, brain gangliosides were elevated, and thin-layer chromatography and analytical studies revealed a predominance of the G4 fraction. Numerous membranous cytoplasmic bodies (MCB) and large lysosomes were found. Significant differences from Tay-Sachs disease, juvenile lipidosis, and gargoylism have been pointed out, justifying the classification of this case as a separate entity. 3. In the case of gargoylism, elevation of brain gangliosides (G5 and G6) and acid mucopolysaccharides has been documented. Membranous cytoplasmic bodies were noted in formalin fixed tissue.

Cytoarchitecture of the extranuclear and commissural dendrites of hypoglossal nucleus neurons as revealed by conjugates of horseradish peroxidase with cholera toxin

Abstract The ipsilateral extranuclear dendrites of hypoglossal nucleus (HN) neurons of the rat, as revealed by conjugates of horseradish peroxidase (HRP) with cholera toxin (CTHRP), were distributed to four distinct regions: (i) to the reticular formation proper (“external dendrites” of Cajal); (ii) to the solitary nucleus and tract; (iii) to the medial longitudinal fasciculus; and (iv) to the nucleus raphe obscurus. The latter two distributions are described for the first time. They were best revealed by CTHRP; and less adequately by a wheat germ agglutinin HRP conjugate (WGHRP). They could not be demonstrated by free HRP. Furthermore, CTHRP best revealed the distribution of commissural dendrites of HN neurons, the tuftlike structures at the termination of some external dendrites of Cajal, and the few dorsal protoplasmic commissural dendrites coursing dorsal to the central canal and to the contralateral solitary nucleus and the area postrema. To further clarify the distribution of the commissural dendrites of the HN, cholera toxin HRP was injected into the tongue of rats after one hypoglossal nerve had been severed. We found that the retrogradely labeled commissural dendrites crossed the midline horizontally to enter the contralateral HN and the solitary nucleus and tract as well as the reticular formation and the medial longitudinal fasciculus. The method greatly expanded our knowledge of the course and terminations of the commissural dendrites of the “protoplasmic commissure” of Van Gehuchten. We conclude that the cholera toxin HRP conjugate was a sensitive probe for the demonstration of the dendritic arborization of retrogradely labeled neurons.

Dysregulation of Stathmin, a Microtubule-Destabilizing Protein, and Up-Regulation of Hsp25, Hsp27, and the Antioxidant Peroxiredoxin 6 in a Mouse Model of Familial Amyotrophic Lateral Sclerosis

Gain-of-function mutations of the Cu/Zn superoxide dismutase (SOD1) gene cause dominantly inherited familial amyotrophic lateral sclerosis. The identification of differentially regulated proteins in spinal cords of paralyzed mice expressing SOD1(G93A) may contribute to understanding mechanisms of toxicity by mutant SOD1. Protein profiling showed dysregulation of Stathmin with a marked decrease of its most acidic and phosphorylated isoform, and up-regulation of heat shock proteins 25 and 27, peroxiredoxin 6, phosphatidylinositol transfer protein-alpha, apolipoprotein E, and ferritin heavy chain. Stathmin accumulated in the cytoplasm of 30% of spinal cord motor neurons with fragmented Golgi apparatus. Overexpression of Stathmin in HeLa cells was associated with collapse of microtubule networks and Golgi fragmentation. These results, together with the decrease of one Stathmin isoform, suggest a role of the protein in Golgi fragmentation. Mutant SOD1 co-precipitated and co-localized with Hsp25 in neurons and astrocytes. Mutant SOD1 may thus deprive cells of the anti-apoptotic and other protective activities of Hsp25. Astrocytes contained peroxiredoxin 6, a unique nonredundant antioxidant. The up-regulation of peroxiredoxin 6 probably constitutes a defense to oxidative stress induced by SOD1(G93A). Direct effects of SOD1(G93A) or sequential reactions triggered by the mutant may cause the protein changes.

Aggregation of ubiquitin and a mutant ALS-linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus

Transgenic mice that express the G93A mutation of human Cu,Zn superoxide dismutase (SOD1(G93A)), found in familial amyotrophic lateral sclerosis (FALS), showed clinical symptoms and histopathological changes of sporadic ALS, including fragmentation of the neuronal Golgi apparatus (GA). The finding of fragmented neuronal GA in asymptomatic mice, months before the onset of paralysis, suggests that the GA is an early target of the pathological processes causing neuronal degeneration. Transgenic mice expressing human SOD1(G93A) have aggregates of mutant protein and ubiquitin in neuronal and glial cytoplasm; they appeared first in the neuropil and later in the perikarya of motor neurons, where they were adjacent to fragmented GA. The aggregates of SOD1(G93A) appeared in neuronal perikarya of asymptomatic mice containing fragmented GA. The numbers of neurons with deposits of SOD1(G93A) and fragmented GA progressively increased with age. Immuno-electron microscopy using colloidal gold showed labeling of ubiquitin and SOD1 over 13 nm thick cytoplasmic filaments. Spinal cord extracts showed a 20-fold increase of SOD1(G93A) in transgenic mice compared to the wild-type protein in controls. The results suggest a causal relationship between the aggregation of mutant SOD1 and ubiquitin, fragmentation of the Golgi apparatus of motor neurons and neurodegeneration.

Aggregates of mutant protein appear progressively in dendrites, in periaxonal processes of oligodendrocytes, and in neuronal and astrocytic perikarya of mice expressing the SOD1G93A mutation of familial amyotrophic lateral sclerosis

Mice expressing the G93A and other mutations of Cu,Zn superoxide dismutase (SOD1(G93A)) are valid models for the familial form of amyotrophic lateral sclerosis (FALS) with SOD1 mutations and, probably, for sporadic ALS. Adult mice become progressively paralyzed and show most of the histopathological lesions reported in sporadic ALS, i.e. neuronal loss, astrogliosis, ubiquitin and Lewy body-like inclusions, dystrophic axons and fragmentation of the Golgi apparatus (GA) of motor neurons. In transgenic mice, the mutant protein and ubiquitin aggregate within pathological 13 nm thick filaments [Stieber A, Gonatas JO, Gonatas NK. J Neurol Sci 2000;173:53-62]. This immunocytochemical and quantitative study of mice expressing SOD1(G93A) establishes the chronological order and cellular localization of aggregates of SOD1 and their correlation with fragmentation of the GA. Young asymptomatic mice expressing SOD1(G93A) showed aggregates of mutant SOD1 within neurites, prior to the detection of SOD1 in the perikarya of spinal cord motor neurons and astrocytes. Both dendrites and the periaxonal oligodendroglial cytoplasm, surrounding atrophic axons, contained SOD1 as revealed by immunoelectron microscopy The perikarya of a small percentage of spinal cord motor neurons contained both fragmented GA and aggregates of SOD1; however, about 50% of motor neurons with fragmented GA did not contain SOD1 in the perikaryon, suggesting that aggregates of mutant protein may not directly cause fragmentation of the GA. The mechanism of the putative toxic effect by the mutant protein remains to be clarified. The isolation and biochemical characterization of the filamentous aggregates of mutant protein and ubiquitin from spinal cords of transgenic mice expressing mutations of the SOD1 gene may offer clues on pathogenetic mechanisms.

The fragmented neuronal Golgi apparatus in amyotrophic lateral sclerosis includes the trans-Golgi-network: functional implications.

Abstract The Golgi apparatus (GA) of spinal cord motor neurons is fragmented in sporadic amyotrophic lateral sclerosis (ALS), and in asymptomatic and symptomatic transgenic mice expressing the G93A mutation of the gene of the human Cu,Zn superoxide dismutase, found in certain cases of familial ALS (FALS) [Gonatas NK (1994) Am J Pathol 145 : 751–761; Mourelatos Z, et al. (1996) Proc Natl Acad Sci USA 93 : 5472–5477]. A similar fragmentation of the GA has been described in cells treated with microtubule-depolymerizing drugs, where the organelle is functional and contains both Golgi stacks and trans-Golgi network (TGN), the compartment of exit and targeting of proteins processed by the GA. To gain a better definition of the structure of the fragmented neuronal GA in ALS, four cases of sporadic ALS with numerous Bunina bodies in spinal cord motor neurons were stained with antibodies against human TGN and against the lumenal and cytoplasmic domains of MG160, a protein of the medial cisternae of the GA. The fragmented GA was stained with the three antibodies, indicating the presence of both Golgi stacks and TGN. Furthermore, the staining of the fragmented GA by the antiserum against the cytoplasmic domain of MG160 indicates that the fragmentation of the GA is not the result of a terminal and global cytoplasmic lytic event. The Bunina bodies were not stained by the anti-MG160 antibodies, suggesting that they are not derived from the GA. The perikarya of neurons with fragmented GA showed normal immunoreactivity with antibodies against the heavy neurofilament subunit and α-tubulin. However, because of the lack of appropriate antibodies the localization of proteins such as spectrin, ankyrin, centractin and others which link the microtubules with the GA were not done. The findings support the hypothesis that, in ALS, the fragmented neuronal GA is functional. Additional work with animal models of ALS may establish whether the fragmentation of the GA is a sign of early degeneration or a compensatory reaction of the injured motor neuron.

Immunohistochemical application of monoclonal antibodies against myelin basic protein and neurofilament triple protein subunits: advantages over antisera and technical limitations.

Four monoclonal antibodies against guinea pig myelin basic protein (MBP), and four against subunits of bovine neurofilament triplet proteins (NF) were produced and their activity determined by enzyme-linked immunosorbant assay. The specificity and cross-reactivity of these eight monoclonal antibodies and one heterologous antiserum against each of the two central nervous system (CNS) antigens were examined in a histological study using the immunoperoxidase, antibody sandwich technique in rat and human brain tissue. Tissue sections were prepared from paraffin-embedded or fresh brain tissue that had been fixed with one of five different fixatives. The resulting immunoperoxidase labeling was then graded for intensity and examined for artifacts. One monoclonal antibody against MBP and one against NF resulted in labeling that was superior to that given by each of the antisera against their respective antigens. Of the five fixatives tested, a mercuric chloride-formalin solution gave the best preservation of these two antigens in rat and human brain tissue. The mercuric chloride-formalin solution was found to be superior to the other fixatives when immersion fixation was used, and was especially optimal when brains were perfused fixed. Three artifacts were encountered among the various antibody-fixative combinations that produced erroneous, but seemingly specific staining of Purkinje cells, neurons and axons, or astrocytes.

The involvement of the Golgi apparatus in the pathogenesis of amyotrophic lateral sclerosis, Alzheimer’s disease, and ricin intoxication

Abstract Several diseases involving a variety of cells and tissues are associated with defective enzymes of the Golgi apparatus (GA). An intact GA of neurons is crucial for the physiological function of axons and presynaptic terminals since proteins destined for fast axoplasmic transport are processed by the organelle. Despite the obvious importance of the GA of neurons, its function and involvement in pathological reactions have not been studied systematically. The purpose of this paper is to draw attention to the contribution of the neuronal GA in pathology using two paradigms: (1) the involvement of the neuronal GA in the pathogeneses of amyotrophic lateral sclerosis (ALS), in an animal model of ALS, and in Alzheimer’s disease; and (2) the elucidation of a retrograde transport pathway involving the neuronal trans-golgi network, in vitro and in vivo, and the participation of this pathway in the toxicity and/or endocytosis of ricin and other toxic or non-toxic ligands.

A light and electron microscopic study of the intraneuronal transport of horseradish peroxidase and wheat germ agglutinin-peroxidase conjugates in the rat visual system

SummaryThe ability of horseradish peroxidase (HRP) and the lectin wheat germ agglutinin (WGA) covalently conjugated with HRP to label retrogradely dorsal lateral geniculate nucleus (dLGN) neurons, subsequent to injections of either marker into rat striate cortex, was assessed by counting labelled neurons in the dLGN. Rats injected with either marker in concentrations ranging from 0.1 to 100μg/μl of HRP either free or coupled to WGA were perfused 24 h later and their brains incubated using the chromagen tetramethyl benzidine. At high concentrations (2–100μg/μ1), comparable numbers of labelled neurons were observed in the dLGN but at low concentrations (0.1–1.0μg/μ1), WGA-HRP labelled 2–5 times more dLGN neurons than did unconjugated HRP. The sugarN-acetylglucosamine, and free WGA added in excess to WGA-HRP, abolished the retrograde labelling of dLGN neurons.In additional rats, which received striate cortex injections of 100μg/μ1 of either free HRP or HRP coupled to WGA, the injection site was studied with electron microscopy after survivals of 30 min to 24 h. Similar organelles in neuronal perikarya, dendrites and axons were labelled by both markers, with the exception that only rats injected with WGA-HRP had labelled GERL in some of their neurons in striate cortex.It was concluded from these studies that: (1) WGA-HRP is a more sensitive retrograde marker than free HRP at low concentrations in the rat visual system; (2) WGA-HRP binds specifically to moieties with terminalN-acetylglucosamine; and (3) WGA-HRP, but not free HRP, is localized to neuronal GERL of striate cortex subsequent to endocytosis.