Immunocytochemical and ultrastructural studies of upper motor neurons in amyotrophic lateral sclerosis

Summary The pathological alterations in upper motor neurons were investigated in 27 cases of adult-onset sporadic amyotrophic lateral sclerosis (ALS). No signficant cytoskeletal alterations were found in the Betz cells of any of the cases except one, although cytoskeletal pathology was consistently present in lower motor neurons. The one case had severe circumscribed atrophy of the precentral gyrus and, microscopically, had argentophilic intracytoplasmic inclusions in Betz cells and other pyramidal neurons in the primary motor area as eell as in the lower motor neurons. Immunocytochemically these inclusions contained the epitope of phosphorylated neurofilament and ubiquitin and ultrastructurally consisted of granule-associated filaments with neurofilaments. This is the first demonstration of alterations of cytoskeleton and ubiquitination in the giant cells of Betz, an established subset of upper motor neurons in ALS. Thus, although uncommon, cytoskeletal changes can be found in upper motor neurons in some ALS cases.Supported in part by USPHS grants NS24453, HD03110 and ES01704     

Immunohistochemical study of microtubule-associated protein 2 and ubiquitin in chronically aluminum-intoxicated rabbit brain

Summary Experimental neurofibrillary change was produced in rabbit brains by daily subcutaneous aluminum tartrate injection for 40 days. The production of experimental neurofibrillary changes was confirmed by immunostaining with antibodies against neurofilament triplet proteins and the brain tissue was studied immunohistochemically with antibodies against microtubule-associated protein (MAP) 2 and ubiquitin. The hippocampal neurons of the chronically aluminum-intoxicated rabbit brain showed diminished staining of dendrites by anti-MAP2 antibody. The length of anti-MAP2-positive dendrites in hippocampus was significantly shorter than that of the control brain. In the cortex somata of a subset of pyramidal neurons were intensively stained by anti-MAP2 antibody, while the MAP2 immunoreactivity of distal dendrites was diminished. The immunostaining by anti-ubiquitin antibody revealed the positive staining of the neurons bearing experimental neurofibrillary changes in the lower brain stem nuclei. It is speculated that MAP2 dislocation and ubiquitination are accompanying phenomena of the production of experimental neurofibrillary changes in chronically aluminum-intoxicated rabbit brains.Supported in part by grants from Ministry of Education of Japan and the Sandoz Gerontological Research Foundation     

Phosphorylation of rat brain cytoskeletal proteins is increased after orally administered aluminum

In rats, administration of 0.3% aluminum in the drinking water for 4–5 weeks significantly increased the in vivo incorporation of 32-phosphorus (³Pi) into proteins with apparent molecular weights of 300 and 210 kDa in the brainstem and cerebral cortex. The identities of these two phosphoproteins as microtubule-associated protein-2 (MAP-2) and the 200 kDa neurofilament subunit (NF), respectively, were established using immunoprecipitation techniques with monoclonal antibodies. Aluminum treatment did not significantly change the amount of MAP-2 or 200 kDa NF in the cerebral cortex and brainstem. Phosphorylation of MAP-2 in aluminum-treated rats in the brainstem and cerebral cortex was 163 and 155% of control values, respectively. The phosphorylation of 200 kDa NF in the brainstem and cerebral cortex of aluminum-treated rats was 148 and 209% of control values, respectively. These results demonstrate that chronic oral aluminum administration to rats increases the phosphorylation of certain cytoskeletal proteins. This treatment regimen may provide a model system with which the mechanisms and consequences of altered in vivo phosphorylation of cytoskeletal proteins can be studied.     

Early axon and dendritic outgrowth of spinal accessory motor neurons studied with DiI in fixed tissues

We have utilized lateral diffusion of DiI in fixed tissues (Godement et al., '87: Development 101: 697-713) to study early axon and dendritic outgrowth of spinal accessory motor neurons in embryonic rats. Crystals were placed in the central canal of the cervical spinal cord near the ventral commissure in order to label growing accessory axons anterogradely and on the spinal accessory nerve to label somata and dendrites retrogradely. Animals were studied on E11-E13. We show here that it is possible to stain axonal and dendritic processes from the earliest stages of motor neuron differentiation by using DiI. Our results demonstrate that, unlike axons of other cervical motor neurons, accessory axons traverse the lateral region of the embryonic cord, which consists of neuroepithelial endfeet. Thus an affinity for neuroepithelial endfeet could partially explain their unusual intraspinal trajectory. We also show that morphology of the spinal accessory growth cones differs according to position along the accessory nerve pathway. Finally, we show that accessory motor neuron axons are in the region of their target precursors prior to the initiation of dendritic arborization. Use of DiI in fixed tissue allows study of process outgrowth in mammalian spinal cord with detail previously obtainable only in nonmammalian vertebrates.     

Survival motor neuron protein in the nucleolus of mammalian neurons

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutations in the survival motor neuron gene (SMN1). While it has been shown that the SMN protein is involved in spliceosome biogenesis and pre-mRNA splicing, there is increasing evidence indicating that SMN may also perform important functions in the nucleolus. We demonstrate here through the use of a previously characterized polyclonal anti-SMN antibody, abSMN, that the SMN protein shows a striking colocalization with the nucleolar protein, fibrillarin, in both nucleoli and Cajal bodies/gems of primary neurons. Immunoblot analysis with antifibrillarin and two different anti-SMN antibodies reveals that SMN and fibrillarin also cofractionate in the insoluble protein fraction of cultured cell lysates. Immunoprecipitation experiments using whole cell extracts of HeLa cells and cultured neurons revealed that abSMN coprecipitated small amounts of the U3 small nucleolar RNA (snoRNA) previously shown to be associated with fibrillarin in vivo. These studies raise the possibility that SMN may serve a function in rRNA maturation/ribosome synthesis similar to its role in spliceosome biogenesis.     

Sexual dimorphism of pleopod motor neurons in lobster

Sexual dimorphism of the efferent neurons that innervate the pleopods in the first abdominal segment of the lobster was examined with nickel/cobalt backfilling techniques. An average of 28.8 somata (maximum of 31) were stained in the female while the mean number of male efferent somata was 25.8 (maximum of 28). These differences are statistically significant. All except one of these somata are situated close to the base of the ipsilateral first root. They are organized into large anterior (13–21 somata) and small posterior (8–13) clusters. One soma is located on the contralateral side of the ganglion.     

Reversibility of neurofilamentous inclusion formation following repeated sublethal intracisternal inoculums of AlCl3 in New Zealand white rabbits

In this report, we describe the clinical, topographical and immunohistochemical characteristics of neurofilament (NF) inclusion formation induced by the intracisternal inoculation of young adult New Zealand white rabbits at 28-day intervals with 100 g AlCl₃ over the course of 267 days. The ability to recover following cessation of aluminum exposure has also been assessed. The extent of neurofilamentous inclusion formation was proportionate to the cumulative amount of AlCl₃ inoculated and initially consisted of fusiform axonal distention in the ventral spinal cord at day 51 following the initial inoculum. Spinal motor neuron perikaryal inclusions and discrete axonal spheroids were observed at day 107 and supraspinal neurofilamentous pathology by day 156. Perikaryal inclusions were immunoreactive to antibodies recognizing both poorly phosphorylated (SMI 32) and more highly phosphorylated high molecular weight NF (NFH). In contrast, axonal spheroids were intensely immunoreactive at all stages with antibodies recognizing highly phosphorylated NFH and an age-dependent NFH phosphorylation state (SMI 34) with only faint SMI 32 immunoreactivity. Immunoreactivity to an antibody recognizing ubiquitin-protein conjugates did not appear until day 156, whereas inclusions were not immunoreactive to antibodies recognizing either phosphatase-dependent or-independent microtubule-associated protein tau at any stage. Upon withdrawal from further AlCl₃ exposure after intervals of 51, 107 or 156 days following the initial inoculum, clinical recovery ensued in all rabbits. In all but the most severely affected rabbits, perikaryal neurofilamentous inclusions resolved. However, axonal spheroids continued to be prominent. However, axonal spheroids continued to be prominent. These studies demonstrate that the repetitive intracisternal inoculation of AlCl₃ in New Zealand white rabbits induces a reversible process of neurofilamentous inclusion formation that preferentially affects motor neurons, and in which recovery will occur in those inclusions containing an admixture of both poorly and highly phosphorylated NFH.     

Immunocytochemical and ultrastructural studies of hyaline inclusions in sporadic motor neuron disease

Summary We investigated hyaline inclusion bodies (HI) immunocytochemically and ultrastructurally in six cases of sporadic motor neuron disease (MND). All HI contained large amounts of ubiquitin and some HI were stained at the core or the center with anti-neurofilament antibody, with the surrounding halo unstained. No HI were stained with antibodies raised against cytoskeletal proteins such as high-molecular weight microtubule-associated proteins and phosphorylated tau. Ultrastructurally, HI were chiefly composed of filaments measuring about 20 nm in diameter thicker than neurofilaments, and contained fine granules and frequently one or more of four characteristic profiles, i.e., small electron-dense materials resembling Bunina bodies, bundles of tubular filaments measuring approximately 20 nm in diameter, large electron-dense cores, and focal accumulations of randomly arranged neurofilaments. Hyaline inclusions can be regarded as one of the characteristic markers for sporadic MND as well as familial amyotrophic lateral sclerosis. Hyaline inclusions have a markedly heterogeneous ultrastructure and, therefore, differences in immunoreactivity with antineurofilament antibodies are not unexpected.     

Immunocytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis

Neuronal inclusions in lower motor neurons in 23 cases of adult-onset sporadic amyotrophic lateral sclerosis were studied immunocytochemically and ultrastructurally. Monoclonal and polyclonal antiubiquitin antibodies recognized four structures in the neuronal perikarya: (1) all Lewy body-like inclusions in 6 cases with a relatively short clinical course, (2) a small percentage of Bunina bodies in 4 cases with abundant Bunina bodies, (3) ill-defined structures closely associated with Bunina bodies (Bunina body-related structures) in 15 cases, and (4) a focally aggregated meshwork of fine filamentous structures not associated with Bunina bodies in all cases. These four structures were not recognized by the antibodies raised against cytoskeletal proteins (neurofilament, tubulin, microtubule-associated protein 2, and phosphorylated tau). Electron microscopy revealed Lewy body-like inclusions to be accumulations of randomly oriented filaments, approximately 15 nm in diameter, covered by fine granules. Bundles of coated filaments 12 nm in diameter that sometimes formed Bunina body-like structures were also observed in the perikarya. Immunoelectron microscopy showed the reaction product with antiubiquitin to be on the filaments, 15 nm in diameter, of Lewy body-like inclusions. Our study revealed the existence of two types of filaments in lower motor neurons of patients with amyotrophic lateral sclerosis: (1) ubiquitin-positive, granule-associated filaments, approximately 15 nm in diameter, that form Lewy body-like inclusions; and (2) 12 nm coated filaments that may be a candidate for another ubiquitin-positive structure and possibly a precursor of Bunina bodies. These two types of filaments may represent early pathological changes of lower motor neurons in amyotrophic lateral sclerosis.     

Calcitonin gene-related peptide immunoreactivity in spinal spheroids in motor neuron disease

Summary The spinal cords from seven autopsy cases of sporadic motor neuron disease (MND) and two controls were immunohistochemically examined using anti-bodies directed to calcitonin gene-related peptide (CGRP) and to neurofilament proteins (Nf). CGRP immunoreactivity was observed in the posterior horns, especially in the laminae I and II, of all the spinal cords examined. In MND cases in addition, a considerable number of spheroids in the anterior horns were labelled with the antibody. In some spheroids, their entire area was homogeneously immunostained, whereas in others the immunoreactivity was confined to a focal area(s) within the profile of the spheroids and between these two forms of staining several variations of the staining patterns were seen. The anti-Nf intensely and homogeneously labelled all spheroids and there was no appreciable difference in the Nf-immunoreactive pattern, between CGRP-posittive and-negative spheroids. It is possible that the accumulation of CGRP in spheroids may result from entrapment of CGRP during the anterograde axonal transport causing loss of CGRP at the neuromuscular junction and producing weakness and atrophy of the muscles.Supported in part by the Grant-in-Aid for Encouragement of Young Scientists (the Ministry of Education, Science and Culture, Japan) and in part by the Amyotrophic Lateral Sclerosis Society (USA)     

Ultra structure of pre-synaptic input to motor neurons in Onuf''s nucleus: controls and motor neuron disease

Ultrastructural analyses of sphincteric motoneurons in Onuf's nucleus at S2 were undertaken in human spinal cord obtained 3-6 h post-mortem from three subjects with no neurological disease ('controls') and five in which death was due to motor neuron disease (MND). Neurons in specified locations within Onuf's nucleus of control subjects ranged between 17.8 and 71.7 microns diameter (mean 38.6 microns). Analyses of synaptology revealed five ultrastructural classes of presynaptic terminal synapsing with the neuronal surface membrane. When classified by size, vesicle morphology, and synaptic site structure these conformed to the S, F, T, M and C-terminals defining somatic motoneurons. No terminals characteristic of autonomic motoneurons were found. In MND subjects, neurons in Onuf's nucleus at S2 were preserved despite a paucity of neurons in medial and lateral motor nuclei and were of similar size range to those in control subjects. The morphological classes of pre-synaptic terminal found in controls, also characterized sphincteric motoneurons in MND subjects, including the C-type terminal. The presence of C-terminals indicates (i) that sphincteric motoneurons are somatic alpha-motoneurons, and (ii) that hypotheses explaining the survival of sphincteric motoneurons in MND on the basis of Onuf's nucleus being an extension of the pre-ganglionic parasympathetic nucleus, or having intrinsic autonomic properties are incorrect.     

Herpes simplex virus vector-mediated expression of Bcl-2 protects spinal motor neurons from degeneration following root avulsion

Proximal axotomy in adult animals results in delayed death of motor neurons. Features characteristic of both necrosis and apoptosis have been described in motor neurons of the spinal cord following proximal avulsion of the ventral roots. We have previously demonstrated that a genomic herpes simplex virus (HSV)-based vector expressing the anti-apoptotic peptide Bcl-2 protects dopaminergic neurons of the substantia nigra from neurotoxin-induced apoptotic cell death and preserves the neurotransmitter phenotype of those cells. In this study we examined whether the same vector could protect adult rat lumbar motor neurons from cell death following proximal ventral root avulsion. Injection of the Bcl-2-expressing vector 1 week prior to root avulsion increased the survival of lesioned motor neurons, determined by retrograde Fluorogold labeling, by 50%. The Bcl-2-expressing vector did not preserve choline acetyltransferase neurotransmitter phenotype of the lesioned cells. These results shed light on the mechanism of cell death following axonal injury, and have implications for developing an effective treatment for the clinical problem of proximal root avulsion.     

Role of proteasomes in the formation of neurofilamentous inclusions in spinal motor neurons of aluminum‐treated rabbits

We examined the role of the 20S proteasome in pathologic changes, including abnormal aggregation of phosphorylated neurofilaments, of spinal motor nerve cells from aluminum‐treated rabbits. Immunohistochemistry for the 20S proteasome revealed that many lumbar spinal motor neurons without intracytoplasmic neurofilamentous inclusions or with small inclusions were more intensely stained in aluminum‐treated rabbits than in controls, whereas the immunoreactivity was greatly decreased in some enlarged neurons containing large neurofilamentous inclusions. Proteasome activity in whole spinal cord extracts was significantly increased in aluminum‐treated rabbits compared with controls. Furthermore, Western blot analysis indicated that the 20S proteasome degraded non‐phosphorylated high molecular weight neurofilament (neurofilament‐H) protein in vitro. These results suggest that aluminum does not inhibit 20S proteasome activity, and the 20S proteasome degrades neurofilament‐H protein. We propose that abnormal aggregation of phosphorylated neurofilaments is induced directly by aluminum, and is not induced by the proteasome inhibition in the aluminum‐treated rabbits. Proteasome activation might be involved in intracellular proteolysis, especially in the earlier stages of motor neuron degeneration in aluminum‐treated rabbits.     

Distribution and morphological characterization of phosphate-activated glutaminase-immunoreactive neurons in cat visual cortex

Phosphate-activated glutaminase (PAG) is the major enzyme involved in the synthesis of the excitatory neurotransmitter glutamate in cortical neurons of the mammalian cerebral cortex. In this study, the distribution and morphology of glutamatergic neurons in cat visual cortex was monitored through immunocytochemistry for PAG. We first determined the specificity of the anti-rat brain PAG polyclonal antibody for cat brain PAG. We then examined the laminar expression profile and the phenotype of PAG-immunopositive neurons in area 17 and 18 of cat visual cortex. Neuronal cell bodies with moderate to intense PAG immunoreactivity were distributed throughout cortical layers II-VI and near the border with the white matter of both visual areas. The largest and most intensely labeled cells were mainly restricted to cortical layers III and V. Careful examination of the typology of PAG-immunoreactive cells based on the size and shape of the cell body together with the dendritic pattern indicated that the vast majority of these cells were pyramidal neurons. However, PAG immunoreactivity was also observed in a paucity of non-pyramidal neurons in cortical layers IV and VI of both visual areas. To further characterize the PAG-immunopositive neuronal population we performed double-stainings between PAG and three calcium-binding proteins, parvalbumin, calbindin and calretinin, to determine whether GABAergic non-pyramidal cells can express PAG, and neurofilament protein, a marker for a subset of pyramidal neurons in mammalian neocortex. We here present PAG as a neurochemical marker to map excitatory cortical neurons that use the amino acid glutamate as their neurotransmitter in cat visual cortex.     

Glial restricted precursors protect against chronic glutamate neurotoxicity of motor neurons in vitro

We have examined the expression of glutamate transporters in primary and immortalized glial precursors (GRIPs). We subsequently transduced these cells with the GLT1 glutamate transporter and examined the ability of these cells to protect motor neurons in an organotypic spinal cord culture. We show that glial restricted precursors and GRIP-derived astrocytes predominantly express glutamate transporters GLAST and GLT1. Oligodendrocyte differentiation of GRIPs results in downregulation of all glutamate transporter subtypes. Having identified these precursor cells as potential vectors for delivering glutamate transporters to regions of interest, we engineered a line of GRIPS that overexpress the glutamate transporter GLT1. These cells (G3 cells) have a nearly fourfold increase in glutamate transporter expression and at least a twofold increase in the V(max) for glutamate transport. To assess whether G3 seeding can protect motor neurons from chronic glutamate neurotoxicity, G3s were seeded onto rat organotypic spinal cord cultures. These cultures have previously been used extensively to understand pathways involved in chronic glutamate neurotoxicity of motor neurons. After G3 seeding, cells integrated into the culture slice and resulted in levels of glutamate transport sufficient to enhance total glutamate uptake. To test whether neuroprotection was related to glutamate transporter overexpression, we isolated GRIPS from the GLT1 null mouse to serve as controls. The seeding of G3s resulted in a reduction of motor neuron cell death. Hence, we believe that these cells may potentially play a role in cell-based neuroprotection from glutamate excitotoxicity.     

Anterior cingulate pyramidal neurons display altered dendritic branching in depressed suicides

Background

It is hypothesized that mood disorders are accompanied by altered wiring and plasticity in key limbic brain regions such as the anterior cingulate cortex (ACC). To test this hypothesis at the cellular level, we analyzed basilar dendritic arborizations extended by layer VI pyramidal neurons in silver-impregnated postmortem ACC samples from well-characterized depressed suicide subjects (n = 12) and matched sudden-death controls (n = 7).

Methods

One cm³ tissue blocks were stained using a Golgi preparation, cut on a microtome, and mounted on slides. Basilar dendritic arbors from 195 neurons were reconstructed, and the number, length, and diameter of branches were determined at each branch order. The size and number of spines borne by these branches were also assessed.

Results

Third-order branches were significantly reduced in number (24% fewer; p = 0.00262) in depressed suicides compared to controls. The size and average length of these branches, as well as their number of spines/length were unaltered. On average, for each pyramidal neuron analyzed in depressed subjects, the fewer third-order branches resulted in a significant reduction in branch length (28% shorter; p = 0.00976) at this branch order.

Conclusions

These results provide the first evidence of altered cortical dendritic branching in mood disorders. Given that proximal dendritic branches grow during perinatal development, and that they are generally less plastic at maturity than distal segments, we speculate that these differences in dendritic branching may reflect a biological predisposition to depression and suicide.     

Interplay of upper and lower motor neuron degeneration in amyotrophic lateral sclerosis

Objective

We studied motor unit recruitment to test a new method to identify motor unit firing rate (FR) variability.

A survival motor neuron:tetanus toxin fragment C fusion protein for the targeted delivery of SMN protein to neurons

Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene. Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons. The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC). Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies. Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein. The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC. Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface. Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes. While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out.     

Inhibitory connections between motor neurons modify a centrally generated motor pattern in the leech nervous system

Both excitatory and inhibitory motor neurons innervate longitudinal body wall muscles in the leech Hirudo medicinalis. Each inhibitory motor neuron also centrally inhibits the excitatory motor neurons that innervate its same muscle field. This central inhibition is strong, probably monosynaptic, and largely a function of graded membrane potential changes in the inhibitory motor neurons. During leech swimming, both the excitatory and inhibitory motor neurons are rhythmically active. Here, we present evidence that the inhibitory motor neurons phasically inhibit the excitatory motor neurons during swimming, thereby augmenting the amplitude of membrane potential oscillations and the burst intensity of the excitors.