Immunoreactivity of β-amyloid precursor protein in amyotrophic lateral sclerosis

We investigated the localization and extent of β-amyloid precursor protein (β-APP₆₉₅) immunoreactivity as a sensitive marker for impairment of fast axonal transport in the spinal cords of 21 patients with amyotrophic lateral sclerosis (ALS), paying special attention to anterior horn neurons. Specimens from 18 patients without neurological disease served as controls. Increased β-APP immunoreactivity was frequently recognized in the anterior horns of the ALS patients with short clinical courses or with mild depletion of anterior horn cells, while no β-APP immunoreactivity was demonstrated in those with severe depletion of anterior horn neurons or with long-standing clinical courses. Increased β-APP immunoreactivity in the anterior horn neurons was mainly confined to the perikarya and no immunoreactivity was recognized in the dendrites or proximal axons directly emanating from the somata, except some spheroids (proximal axonal swellings) which showed increased immunoreactivity of β-APP. Increased β-APP immunoreactivity was spotted or focally aggregated in the perikarya of normal-looking large anterior horn neurons, while it was frequently diffuse in that of degenerative neurons such as central chromatolytic cells and or those with simple atrophy. On the other hand, the controls showed no immunostaining with β-APP in the spinal cord. These findings suggest that increased immunoreactivity of β-APP in neuronal perikarya of the anterior horn cells and in some proximal axonal swellings is an early change of ALS, and may be a response of the increased synthesis of β-APP resulting from neuronal damage, or the impairment of fast axonal transport. Received: 27 August 1998 / Revised, accepted: 4 November 1998     

Increase in diameter of the axonal initial segment is an early change in amyotrophic lateral sclerosis

We measured the diameter of the most distal portion of the axonal initial segment, the neuronal size of anterior horn cells, and the length of the axon hillock plus the initial segment (AH+IS) in the lumbar spinal cord in motor neuron disease. Three patients with amyotrophic lateral sclerosis (ALS) and one with lower motor neuron disease (LMND) were compared with 11 controls. Serial plastic sections stained with toluidine blue and electron micrographs were studied. A total of 214 axons directly emanating from the somata (n = 207) and the primary dendrites (n = 7) were observed in the patients. Approximately 19% of the proximal myelinated axons (24 axons out of 155 in ALS, and 17 axons out of 59 in LMND) were swollen at the first internode, and most of the swellings extended to the middle portion of the initial segment. Electron microscopy showed that the swellings of the proximal axons (the initial segment and the first internode) directly connected with their somata consisted mainly of accumulations of 10-nm neurofilaments. The average diameter of the most distal initial segment was markedly larger in ALS (n = 155) (P < 0.0001) and LMND (n = 59) (P < 0.0001) than in the controls (n = 258). Moreover, the average diameter of the most distal portion of even normal-appearing initial segments of the non-swollen axons was larger in ALS (n = 131) (P < 0.0001) than in the controls. The perikarya and axon hillocks connected with the normal-appearing and swollen proximal axons and their dendrites almost always appeared normal. These findings suggest that increase in diameter of the axonal initial segment which reflects the abnormal accumulation of neurofilaments represents an early pathological change in motor neuron disease and that the slow axonal transport of neurofilaments is probably impaired in this portion of the axon at an early stage in the disease process. The average size of the normal-appearing cell bodies from which axons emanated was smaller in ALS (P < 0.0001) and LMND (P < 0.0001) than in the controls. There was no significant difference in the AH+IS length among ALS having normal-appearing initial segment, LMND, and controls.     

Ultrastructure of swollen proximal axons of anterior horn neurons in motor neuron disease

We have investigated ultrastructurally swellings of the proximal axons directly connected with the somata of anterior horn cells in a patient with motor neuron disease. In the distal portion of the initial segment, the swellings consisted predominantly of increased amounts of neurofilaments running nearly parallel to the longitudinal axis together with other cytoplasmic organelles. In the first internode of the myelinated axon, as the swellings became larger, the neurofilaments lost their parallel orientation and became intermingled. Most of the cell bodies connected with the swellings and their dendrites showed no particular changes. The abnormal neurofilamentous accumulations in the proximal axons suggest abnormal axonal transport, which may be pathogenetically associated with motor neuron disease.     

Masses of phosphorylated neurofilaments are associated with abnormal Golgi apparatus of anterior horn neurons of β, β′‐iminodipropionitrile‐intoxicated rats

The Golgi apparatus (GA) of anterior horn neurons of rats chronically intoxicated with β,β′‐iminodipropionitrile (IDPN) in drinking water was examined with an organelle‐specific antibody. The neuropile of the anterior horns contained the typical axonal spheroids associated with IDPN toxicity while the perikarya of approximately one‐third of the neurons contained phosphorylated neurofilaments, which are not found in the perikarya of control rat neurons. By serial or double immunostaining with the SMI‐31 and anti‐MG 160 antibodies, there were no morphological changes of the GA in the majority of neurons including neurons with a mild to moderate degree of neurofilamentous accumulation. However, a few neurons with a massive accumulation of phosphorylated neurofilaments contained abnormal profiles of the GA which consisted of focal clustering, reduction in size and fragmentation. The results suggest that masses of phosphorylated neurofilaments are associated with struc‐tural abnormalities of the GA.     

Swellings of proximal axons in a case of motor neuron disease

Serial semithin sections of lumbar anterior horn cells from a patient with rapidly progressive sporadic motor neuron disease were searched for direct connections between swellings of neuronal processes and perikarya. Focal swellings of the proximal axons were not uncommonly seen to be connected directly to perikarya. These swellings varied in shape and size and some were identified as spheroids. Most of the cell bodies connected with swellings showed otherwise normal architecture. These observations suggest that the focal swellings of proximal axons, particularly the distal portion of the initial segment and the first internode of the myelinated axon, indicate an early pathological change, and may represent functionally and morphologically vulnerable sites.     

Motor unit function during evolution of proximal axonal swellings.

beta,beta'-Iminodipropionitrile (IDPN) impairs axonal transport of neurofilaments; their accumulation leads to the formation of proximal swellings in motor axons. Similar proximal swellings are a feature of some cases of motor neuron disease such as amyotrophic lateral sclerosis (ALS). Motor units in IDPN-treated animals were assessed to determine their relative susceptibilities to impaired function and whether the functional changes resulting from proximal axonal swellings share certain electromyographic features with ALS. Intrinsic properties of medial gastrocnemius motoneurones (MN) and contractile responses of their motor units were examined during the evolution of proximal axonal swellings in cats administered IDPN (50 mg/kg once weekly) for 7, 14 or 35 days. While conduction velocities were significantly decreased in all motor unit types by 35 days, the conduction slowing was greater in fast fatigable (types FF and FI) motor units than in fatigue resistant (types FR and S) motor units. Normal correlations between axonal conduction velocity and MN input resistance (Rin) and the inverse relationship between Rin and rheobase were lost with progression of the neuropathy. Twitch and maximum tetanic tension developed by fast-fatigable motor units declined early in the neuropathy, whereas fatigue-resistant units did not show similar changes until later stages of the intoxication. In some motor units, irregular and abnormal tetanic tensions were elicited by repetitive MN discharge. At 14 and 35 days, a novel, intermediate motor unit response classified as slow and fatigable (SF) was observed. Conduction block, characterized by repetitive MN firing without a corresponding contractile response, was observed in some type FF and S units by 35 days. Morphometric analysis of muscle fiber types showed significant atrophy, particularly in the type I fibers at 14-35 days; the atrophy reversed following cessation of IDPN administration. The influence of proximal axonal swellings on motor unit function in IDPN neuropathy is discussed in terms of reported electrophysiological alterations in motoneurone disease.     

Axonal swellings in the corticospinal tracts in amyotrophic lateral sclerosis

Summary In 2 of 16 cases with sporadic amyotrophic lateral sclerosis (ALS) large numbers of axonal swellings were observed in the corticospinal tracts over a region extending from the posterior limbs of internal capsules to the bulbar pyramids. On electron microscopy, these axonal swellings were seen to consist of accumulations of neurofilaments and altered neuronal organelles (mitochondria and secondary lysosomes). Their morphology differed from the spheroids seen in the anterior horn in ALS.     

Neurofibrillary axonal pathology in aluminum intoxication

Aluminum chloride injected into the cisterna magna of 3-to 4-week-old rabbits is known to produce neurofibrillary changes in the perikarya and dendrites of neurons of the spinal cord and brainstem. This report calls attention to the early appearance and prominence of neurofibrillary swellings in proximal axons of affected neurons. One and 2 days after intoxication, large axonal swellings containing maloriented neurofilaments were present in the ventral horns. Over the next days and weeks, neurofilaments accumulated in the neuronal perikarya and dendrites. Two months after injection, abnormalities in the cell bodies were less apparent but occasional giant axonal swellings persisted in the ventral horn. This sequence of pathological changes, reflecting disorganization of the neuronal cytoskeleton, may result from an increase in synthesis of neurofilament polypeptides, and impairment in integration of these constituents into the neuronal cytoskeleton, or a defect in the intracellular transport of neurofilaments.     

Phosphorylation of neurofilaments is altered in amyotrophic lateral sclerosis

We used a library of monoclonal antibodies (Mab) that distinguish phosphorylated (P+) and non-phosphorylated (P-) neurofilament (NF) epitopes to examine phosphorylation of NF in lower motor neurons of patients with amyotrophic lateral sclerosis (ALS), of neurologically normal controls of different ages, and of patients with central chromatolysis due to injuries to motor root axons. Monoclonal antibodies directed to P+ NF immunostained five to ten times more neuronal perikarya in ALS than in age-matched controls. Spheroids, which are NF containing axonal enlargements, found in significantly greater number in proximal axons in ALS, were also intensely immunostained with Mab to P+ NF. Moreover, anterior root axons in five of eleven cases of ALS reacted only with the Mab to P+ NF, while both P- and P+ NF were present in motor roots from controls. In control groups, the number of neuronal perikarya and spheroids that immunoreacted with the Mab to P+ NF increased moderately with age. Chromatolytic lower motor neurons were recognized by Mab to P+ NF. Our results show that the process of phosphorylation is altered in ALS. We propose that phosphorylation of NF in ALS occurs prematurely and that it is more likely to be associated with an impairment of NF transport than to be part of a chromatolytic reaction of lower motor neurons.     

Poisoning by beta,beta'-iminodipropionitrile (IDPN) in the rat: an experimental model for elective lesions of peripheral motoneurons

Chronic or acute intoxication of rats with beta,beta'-iminodipropionitrile induces characteristic lesions of axons of anterior horn cells. Swellings of proximal axons are associated with disorganization of the cytoskeleton which includes a disorientation of neurofilaments which are segregated in the periphery of the axon, while microtubules and mitochondria are clustered centrally. Slow axonal transport, which is markedly reduced, results in accumulation of neurofilaments in the proximal part of the peripheral motoneuron and distal atrophy. In chronically intoxicated rats, proliferation of Schwann cells with onion bulb formation were observed in the anterior spinal roots after 10 months on diet. In distal sciatic nerve, axonal degeneration was associated with accumulation of neurofilaments, organelles and glycogen. Axonal regeneration occurred in spite of sustained intoxication. The intensity of the lesions induced by IDPN in the proximal part of the axon of the spinal motoneuron are reminiscent of those observed in degenerative motor neuron disease. However, the abnormalities of the myelin sheath and the proliferation of Schwann cells encountered in IDPN-intoxicated rats do not occur in degenerative motor neuron diseases in humans.     

Slow component of axonal transport is impaired in the proximal axon of transgenic mice with a G93A mutant SOD1 gene

The purpose of this study was to determine whether slow axonal transport of neurofilaments (NFs) is impaired in the spinal cord of G93A Cu/Zn superoxide dismutase (SOD1) mutant transgenic mice expressing a relatively low mutant protein (gene copy 10) and, if so, how the impairment occurs in this animal model. Transgenic mice were killed at the ages of 24, 28 and 32 weeks, and the cervical and lumbar spinal cords were examined under an electron microscope. Age-matched non-transgenic wild-type mice served as controls. At 24 weeks (early presymptomatic stage), anterior horn cells were well preserved. The earliest morphological changes were mild vacuolar changes in the neuronal processes, particularly in proximal axons. At 28 weeks (late presymptomatic stage), mild neuronal loss of anterior horn neurons was observed. Vacuolar changes were more prominent in the proximal axons, including swollen axons (spheroids) and neuropils of the anterior horns. Vacuoles in the axons were frequently large enough to occupy almost the entire axonal caliber. The anterior roots were degenerative, showing vacuolar changes and myelin ovoids. Lewy body-like inclusions (LIs) consisting of filaments thicker than NFs (about 1.5 times larger in diameter) were frequently demonstrated in the neuronal processes including swollen axons (spheroids) and occasionally in the somata. At 32 weeks (symptomatic stage), the anterior horns showed a moderate to severe neuronal loss accompanied by prominent astrogliosis. Cord-like swollen axons consisting of accumulated NFs and many neurofilamentous accumulations were frequently observed in the anterior horn. Vacuolar changes were less prominent or disappeared in the neuropils of the anterior horns and the anterior roots, whereas LIs were frequently demonstrated within the neuronal processes including the cord-like swollen axons. In the anterior roots, degenerative changes such as marked fiber loss and frequent myelin ovoids were remarkable. No accumulation of NFs or mitochondrial vacuolation was detected in somata or proximal dendrites at any stage. These findings suggest that the slow component of axonal transport in the proximal axons is impaired at an early stage in this transgenic mouse model, and that the impairment is probably caused by a mechanical impediment of NFs, or by the accumulation of NFs in the proximal axon, as a result of the obstruction of the axonal flow that initially occurs by vacuolar changes, and is later exacerbated by accumulation of LIs.     

PROGRESSIVE AXONOPATHY: AN INHERITED NEUROPATHY OF BOXER DOGS. AN IMMUNOCYTOCHEMICAL STUDY OF THE AXONAL CYTOSKELETON

The distribution of the major axonal cytoskeletal proteins has been determined in lumbar ventral roots and spinal cord of dogs with progressive axonopathy, an inherited neuropathy of boxer dogs. The three neurofilament proteins, and beta-tubulin, actin and fodrin were localized using immunocytochemistry. The majority of swollen axons in the nerve roots contained excessive, disorientated neurofilaments. In about 5% of such fibres the peripheral filaments in the axoplasm were orientated circumferentially and such zones were deficient in tubulin. Many, but not all, spheroids contained increased amounts of actin, often with internal areas of more intense staining. Similar findings were present in axonal swellings in the CNS, although their contents were more variable. The distribution of axonal fodrin in CNS and PNS appeared unaltered. The perikarya of many motor neurons in the spinal cord and brain stem contained phosphorylated neurofilaments. The results support previous suggestions that defects in slow axonal transport are involved in the pathogenesis of this disease.     

The axonal pathology in chronic IDPN intoxication

Neurofilamentous axonal swellings occur in a number of degenerative and toxic disorders of the nervous system. In one of these, experimental intoxication with beta, beta'-iminodiproprionitrile (IDPN), accumulation of neurofilaments has been shown to result from a defect in slow axonal transport. The consequence of this functional abnormality is a series of changes in axonal morphology: Neurofilaments accumulate in the proximal axon; the proximal axon becomes swollen; the distal axon loses volume (axonal atrophy). These studies indicate that axonal atrophy occurs secondary to an impairment of slow axonal transport and suggest that a similar abnormality may underlie the pathological changes in certain other degenerative and toxic diseases of the nervous system.     

Impairment of axonal transport in the axon hillock and the initial segment of anterior horn neurons in transgenic mice with a G93A mutant SOD1 gene

Impaired axonal transport of the fast or slow component has been reported in patients with sporadic amyotrophic lateral sclerosis (ALS), animal models for ALS, and familial ALS-linked mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice. However, little is known about the impairment of axonal transport in mutant SOD1 transgenic mice. This is the first electron microscopic investigation of the axon hillock (AH) and the initial segment (IS) of anterior horn cells in the spinal cord of transgenic mice expressing the G93A mutant human SOD1, and it was launched with a view toward examining whether the axonal transport is impaired in this region. Six transgenic mice were killed at ages ranging from the presymptomatic to symptomatic stages. Six age-matched non-transgenic wild-type mice served as controls. In the non-transgenic mice, 91 AH and IS were observed, but those with increased neurofilaments or mitochondria were rarely found. In the transgenic mice, 95 AH and IS directly emanating from normal-looking large anterior horn cells were seen. AH and IS with increased neurofilaments or, to a lesser extent, increased mitochondria, and round-shaped mitochondria in particular, were more frequently observed, even at the early presymptomatic stage, than in the controls, and the frequency increased with time through the presymptomatic stages. On the other hand, the somata of large motor neurons directly connected with the axons did not exhibit any abnormal accumulation of neurofilaments or mitochondria. These findings suggest that both the slow axonal transport of neurofilaments and the fast axonal transport of mitochondria are impaired in AH and IS before the onset of disease in this animal model.     

Aluminum-induced neurofibrillary changes in axons and dendrites

Summary We examined the chronic effect of aluminum on the rabbit central nervous system (CNS) and documented the occurrence of axonal swellings (spheroids) and dendritic thickening in spinal cord neurons in addition to the accumulation of neurofibrillary material in the perikaryon. The axonal swellings always occurred at the first heminode, and the neurofilaments appeared disorganized, whereas in dendrites the neurofilaments generally retained their longitudinal arrangements. Although neurofibrillary tangles were present in cortical neurons, no axonal swellings were observed. Thickening of segments of apical dendrites proximal to the cell body affected by neurofibrillary changes was present. The axonal swellings resembled those observed in IDPN intoxication, and in amyotrophic lateral sclerosis, and may be useful as a model for studying these diseases.Supported by the Office of Mental Retardation and Developmental Disabilities of the State of New York and the Aluminum Association, Inc.     

Neurofilamentous axonal swellings as a normal finding in the spinal anterior horn of man and other primates

To define the nature and extent of axonal swellings in the normal spinal anterior horn, we studied the spinal cords of patients five days to 83 years of age from a general autopsy population. Axonal swellings were routinely found in the anterior horn of the cervical and lumbosacral spinal cord. The swellings measure 5-50 micron in diameter and are most numerous at the anterior edge of the anterior horn. They first appear about five months of age and appear to increase in number until about 20 years of age, with no increment thereafter. Ultrastructurally, they are filled with neurofilaments and surrounded by a thin myelin sheath. Most are probably aberrant components of motor axons. Identical axonal swellings, in the same anatomical site, were found in the spinal cords of cynomolgus and rhesus monkeys. On the basis of their natural history and morphologic features, they should be distinguished from the neuroaxonal dystrophy of aging. The largest of them resemble the neurofilamentous axonal swellings of early onset motor neuron disease but occur in much smaller numbers. Moreover, location on the proximal axon could not be demonstrated for any of these swellings. An awareness of this normal phenonemon is essential for the interpretation of axonal swellings in the spinal cord.     

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. III. Alterations following single and continuous beta, beta'-iminodipropionitrile administrations.

beta,beta'-Iminodipropionitrile (IDPN) administration produces giant neurofilament-filled axonal swellings in the first proximal internodes of large myelinated sensory and motor fibers without any accompanying axonal degeneration. In the present study, we asked whether proximal giant axonal swellings are sufficient to elicit aberrant neurofilament (NF) phosphorylation in neuronal perikarya. Rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) or tap water (single IDPN exposure) for two days to 7 weeks. Immunoreactivity to phosphorylated NF (pNF) epitopes (using monoclonal antibodies 6-17 and 7-05) was observed in L4 and L5 dorsal root ganglia (DRG) neurons beginning between one and 5 days, corresponding to the development of proximal giant axonal swellings. Quantitation of DRG neurons demonstrated maximal numbers of immunoreactive cell bodies to pNF epitopes (46-51%) by one week. The number of immunostained DRG cells was maintained in animals given continuous IDPN exposure, but declined significantly (P less than 0.001) in rats given a single injection of IDPN to 26 +/- 0.80% and 6 +/- 0.04% at 3 and 5 weeks, respectively. Ventral and dorsal root fibers, which undergo axonal atrophy distal to axonal swellings, showed intense immunoreactivity to pNF epitopes and a marked reduction or a complete lack of immunostaining to antibody 2-135 (directed against non-phosphorylated NF epitopes); pretreatment with alkaline phosphatase reversed this staining pattern. In a separate study, a similar alkaline phosphatase-sensitive lack of staining to antibody 2-135 was also observed in atrophic motor fibers in the DRG 4 weeks following nerve crush. It is suggested that aberrant NF phosphorylation in DRG neuronal cell bodies from IDPN-treated rats arises secondarily to an alteration in a retrogradely transported 'trophic' signal(s) to the neuron due to the presence of giant axonal swellings. Furthermore, pNFs in atrophic axons may correspond to stationary or slowly moving NFs in the axoplasm.     

Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'-iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1-35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5-fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.     

Hereditary canine spinal muscular atrophy.

Hereditary canine spinal muscular atrophy is a newly recognized motor neuron disease occurring in Brittany Spaniels. The clinical manifestations, pattern of inheritance, electrodiagnostic findings, and muscle biopsies have features in common with human spinal muscular atrophy. Neuropathological examination discloses some loss of motor neurons in the spinal cord and brainstem. Many of the surviving motor neurons have neurofibrillary swellings in proximal axons, an abnormality similar to that which occurs early in the course of human amyotrophic lateral sclerosis. These axonal swellings are filled with maloriented skeins of neurofilaments. Since the proteins comprising neurofilaments are carried by slow axonal transport, their accumulation within axons suggest that the swellings may result from impaired slow transport, a hypothesis that can be tested in affected Brittany Spaniels. Hereditary canine spinal muscular atrophy is a new genetic, clinical, and pathological entity, and, at present, it appears to be the best currently available animal model of motor neuron disease.     

Focal accumulation of phosphorylated neurofilaments within anterior horn cell in familial amyotrophic lateral sclerosis

Summary Distribution of phosphorylated neurofilament proteins within anterior horn cells in three cases of familial and six cases of sporadic amyotrophic lateral sclerosis (ALS) and ten control cases were investigated by using a monoclonal antibody. Two distinct staining patterns of perikarya were observed; (1) homogeneous pattern; either the entire or a part of the perikaryon was immunostained homogeneously (homogeneously diffuse or partial pattern); (2) focal pattern: perikarya contained very distinct, inclusion-like focal accumulation of immunoreactive products of various morphologies such as round, ring-shaped, cord-like, tube-like and more irregular shapes. The homogeneous pattern was found in all three groups but was most common in sporadic ALS. On the other hand, the focal pattern was seen almost exclusively in familial ALS. The focal accumulation of neurofilaments appears at least in part to be related to the Lewy body-like hyaline inclusion which is known to contain neurofilaments. In addition, cord-like swellings of neurites in familial ALS also showed focal neurofilament accumulation. These observations suggest that the focal accumulation of phosphorylated neurofilaments is characteristic of familial ALS, although it may not be specific to the entity. The pathological process(es) producing the neurofilamentous abnormality may play an important role in anterior horn cell degeneration in familial ALS.