Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis

Recently, the excitatory amino acid neurotransmitter glutamate was implicated in the pathogenesis of a variety of chronic degenerative neurological diseases in humans and animals. This report describes abnormalities in excitatory amino acids in the central nervous system of 18 patients with amyotrophic lateral sclerosis (ALS). The concentration of the excitatory amino acids glutamate and aspartate in the cerebrospinal fluid were increased significantly (p less than 0.01) by 100 to 200% in patients with ALS. Similarly, the concentrations of the excitatory neuropeptide N-acetyl-aspartyl glutamate and its metabolite, N-acetyl-aspartate, were elevated twofold to threefold in the cerebrospinal fluid from the patients. There was no relationship between amino acid concentrations and duration of disease, clinical impairment, or patient age. In the ventral horns of the cervical region of the spinal cord, the level of N-acetyl-aspartyl glutamate and N-acetyl-aspartate was decreased by 60% (p less than 0.05) and 40% (p less than 0.05), respectively, in 8 patients with ALS. Choline acetyltransferase activity was also diminished by 35% in the ventral horn consistent with motor neuron loss. We conclude that excitatory amino acid metabolism is altered in patients with ALS. Based on neurodegenerative disease models, these changes may play a role in motor neuron loss in ALS.     

Autoradiographic localization of thyrotropin-releasing hormone receptors in amyotrophic lateral sclerosis spinal cord

We used quantitative autoradiography to determine the density of thyrotropin-releasing hormone (TRH) receptors in discrete regions of spinal cord from four patients with amyotrophic lateral sclerosis (ALS). The density and distribution of [3H]-3-methyl-histidine-TRH binding to TRH receptors differed from reported values in normal individuals, with fewer TRH receptors in lamina II and lamina IX. The diminished concentration of TRH receptors in lamina IX may reflect the loss of motor neurons in ALS.     

Plasma amino acid levels in patients with amyotrophic lateral sclerosis.

Evidence for a generalized defect in glutamate in patients with amyotrophic lateral sclerosis (ALS), associated with widespread alterations in the central nervous system level of this excitatory amino acid. We measured fasting plasma amino acid in 10 ALS patients and 10 controls matched for age and sex. ALS patients had statistically significant elevations in plasma level of aspartate, glutamate, and glycine. The plasma levels of other amino acids were not significantly different from those found in controls. No correlation between ALS severity or activity and degree of abnormality in amino acids was established.     

Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, we demonstrate a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease. Received: 8 September 1999 / Revised, accepted: 28 October 1999     

Alterations of the blood‐spinal cord barrier in sporadic amyotrophic lateral sclerosis

The blood‐spinal cord barrier (BSCB) of the spinal cord capillary consists of non‐fenestrated endothelial cells with tight junctions, basal laminae, pericytes and astrocyte feet processes, referred to as a “neurovascular unit.” The primary function of the BSCB is the maintenance and control of homeostasis of the spinal cord parenchyma by the selective transport of molecules and cells from the systemic compartment. Dysfunction of the BSCB shows important function in the etiology or progression of several pathological conditions of the spinal cord, including amyotrophic lateral sclerosis (ALS). However, the role of BSCB in the pathogenesis of ALS is still unclear. Here the changes of BSCB in sporadic ALS patients were studied by electron microscopy to determine whether the BSCB is disrupted and involved in the pathogenesis of motor neuron degeneration. A total of 358 and 366 cross‐sectioned capillaries were quantitatively examined in controls and ALS patients, respectively. The frequency of degenerated endothelia and pericytes, vacuolar changes of the cytoplasm in the endothelia and pericytes, and the replication of basement membranes was significantly higher in ALS patients than in the controls (P = 0.0175). The areas of the capillaries with diameters of ≤ 5 µm in the ALS patients were significantly smaller than those in the controls (P = 0.0124). The frequency of collagen fiber content of more than a moderate degree around the perivascular space was significantly higher in the ALS patients compared to the controls (P = 0.048), although there was no significant difference in the mild degree of accumulation of collagen fibers. Thus, the BSCB may be disrupted in sporadic ALS patients due to increased permeability and reduced microcirculation, leading to motor neuron degeneration and to the progression of the disease.     

Alteration in amino acids in motor neurons of the spinal cord in amyotrophic lateral sclerosis.

Little is known concerning the changes of amino acid composition in different regions of the spinal cord in patients with amyotrophic lateral sclerosis (ALS). We performed quantitative amino acid analyses in the posterior funiculus, the lateral corticospinal tract, and the anterior horn of cervical enlargement of the spinal cord from seven ALS patients, and the results were compared with those of seven patients with other neurologic diseases (control A) and seven patients without neurologic diseases (control B). The levels of collagen-associated amino acids, hydroxyproline, proline, glycine, and hydroxylysine, were markedly lower in the lateral corticospinal tract and the anterior horn of ALS patients than in controls A and B. The contents of the acidic amino acids glutamate and aspartate were also significantly decreased in the lateral corticospinal tract and the anterior horn of ALS patients as compared with those of controls A and B. These data suggest that decreased contents of collagen-associated amino acids and excitatory amino acids are related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS.     

Substance P receptors in the human spinal cord: decrease in amyotrophic lateral sclerosis

The distribution of substance P receptors was examined by autoradiography at all levels of the human postmortem spinal cord using the ligand [125I]Bolton-Hunter substance P. Adjacent sections were used to localize substance P-like immunoreactivity by a radioimmunohistochemical technique. In the control spinal cord substance P-like immunoreactivity was found to be highly concentrated in the superficial layers of the dorsal horn, intermediolateral cell columns and lamina X, while lower levels of immunoreactivity were observed in other areas of the grey matter of the spinal cord. In contrast, high densities of substance P binding sites were localized not only to the substantia gelatinosa of the dorsal horn but also to other regions of the grey matter of the spinal cord, particularly in the area of the preganglionic sympathetic neurons in the intermediolateral cell column and in the region of the somatic motor neurons of the ventral horn. In 5 cases of amyotrophic lateral sclerosis we found a marked reduction of substance P binding, especially in the ventral horn associated with the loss of motor neurons. These results suggest a postsynaptic localization of substance P receptors to the motor neurons of the ventral horn in the human spinal cord and a role for substance P in the function of motor neurons.     

Collagen abnormalities in the spinal cord from patients with amyotrophic lateral sclerosis

During the last 10 years, we have demonstrated morphological and biochemical abnormalities of skin extracellular matrices in amyotrophic lateral sclerosis (ALS). However, currently little is known concerning collagen of the spinal cord in ALS. We measured the amount of collagen and characterized collagen at light and electron microscopic levels in posterior funiculus, posterior half of lateral funiculus and anterior horn of cervical enlargement of the spinal cord obtained from ten patients with ALS, 11 patients with other neurologic diseases (control group A), and ten patients without neurologic ones (control group B). In posterior half of lateral funiculus and anterior horn, (1) by light microscopy, there was no significant difference in vessel wall area between ALS patients and control groups A and B; (2) ultrastructurally, collagen bundles were more fragmented and widely separated, and the fibrils were randomly oriented in the perivascular space of capillaries in ALS patients, which were not observed in any areas of control groups or in posterior funiculus of ALS patients; and (3) the collagen contents in ALS were significantly lower (P<0.001 and P<0.001, respectively) than those in control groups A and B. Fragmented and widely separated collagen bundles in the interstitial tissue surrounding capillaries and markedly decreased amount of collagen in posterior half of lateral funiculus and in anterior horn of ALS could be related to the degeneration of the upper and lower motor neurons in the spinal cord in ALS, that is, selective neuronal vulnerability in ALS.     

Lymphocytic infiltration in the spinal cord of patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating systemic atrophy affecting the upper and lower motor neurons. The etiology is unknown, but one theory of pathogenesis supposes that the motor system is affected by abnormal immune responses. We have studied the prevalence and extent of lymphocytic infiltration, previously reported as a rare finding in the ALS spinal cord. Application of monoclonal antibodies against macrophages, T- and B-cells to spinal cords from 48 ALS patients disclosed a cellular mononuclear infiltrate in 38 specimens (79%), intense enough to be revealed by routine neuropathological techniques in 6 of them (12.5%); the remaining 10 cords (21%) exhibited no infiltrates. Since duration and clinical signs of the preceeding illness were the same in cases with and without infiltrates, we consider it unlikely that such infiltrates are entirely secondary to atrophy of the cord. As Wallerian degeneration is not accompanied by infiltrates of lymphocytes, their presence in the cord tracts of our material throws doubt on the conventional view that tract degeneration in ALS is exclusively Wallerian.     

Free amino acid levels in amyotrophic lateral sclerosis

To evaluate reports of abnormal levels of free amino acids (AA) in patients with amyotrophic lateral sclerosis (ALS), we studied serum, cerebrospinal fluid, and urine AA in 12 patients with ALS and 12 controls matched for age, sex, and severity of disability. ALS patients had statistically significant elevations in serum levels of tyrosine, total aromatic AA, and total basic AA. ALS patients also had statistically significant elevations in cerebrospinal fluid of total basic AA, lysine, essential AA, and leucine. The severity of ALS correlated inversely with acidic AA (glutamate and aspartate) and O-phosphoserine in cerebrospinal fluid. Activity of ALS correlated directly with serum aspartate and cerebrospinal fluid alanine. We conclude that subtle abnormalities of AA levels are present in ALS and that these are not due to age, sex, or disability. The pattern of distribution of AA levels differs from that in hepatic or renal disease and suggests defective membrane transport or poor cellular utilization of basic and essential AA in the central nervous system.     

Insulin-like growth factor-1 receptors in human spinal cord: changes in amyotrophic lateral sclerosis

Summary Neurotrophic factors are important for neuronal survival and maintenance in the adult nervous system. The regional distribution of insulin-like growth factor-1 (IGF-1) receptors in human spinal cords from controls and amyotrophic lateral sclerosis (ALS) patients was studied by immunohistochemistry and quantitative autoradiography. When comparing¹²⁵I-IGF-1 binding in the different spinal levels of normal spinal cord the same distribution pattern was found in which the binding was highest in the central canal > dorsal horn > ventral horn > white matter. In the ALS cases although a general upregulation of IGF-1 receptors was observed throughout the spinal cord, significant increases were observed in the cervical and sacral segments compared to controls. IGF-1 receptor immunoreactivity showed a similar pattern to that for¹²⁵I-IGF-1 binding, with immunoreactivity being found in the gray matter of the spinal cord and enhanced immunoreactivity occuring in ALS patients compared to controls. In agreement with the distribution of IGF-1 receptors, IGF-1 immunoreactivity was found within the gray matter of the spinal cord. The cartography of IGF-1 receptors in the normal spinal cord as well as the change of these receptors in diseased spinal cord may be of importance in future treatment strategies of ALS.     

Mitochondrial alterations in the spinal cord of patients with sporadic amyotrophic lateral sclerosis

Little information is available about morphologic changes of mitochondria in sporadic amyotrophic lateral sclerosis (ALS). We examined the anterior horns of the lumbar spinal cord in 14 patients with sporadic ALS and 15 age-matched controls by electron microscopy to illuminate the subject. In the controls, one patient showed occasional swollen mitochondria with markedly increased cristae and marked accumulation of mitochondria in the somata of anterior horn neurons. Another patient had periodic, stubby protrusions on the outer membrane. Among the patients with ALS, 7 showed filamentous structures in the inner compartment of the mitochondria mainly of the somata and only occasionally of the axons. The structures were composed of a stack of multilayered cristae consisting of linear structures on a longitudinal section. Other abnormal structures were periodic transverse processes like rungs of a ladder predominantly in somata and only occasionally in the axons, marked accumulation of mitochondria in the somata, dendrites or proximal axons (axon hillock and initial segment), stubby protrusions on the outer membrane, and swollen mitochondria with markedly increased cristae in the somata. The findings in this study may reflect the metabolic disturbance of mitochondria, probably associated with the pathomechanism of degenerative processes of anterior horn neurons in sporadic ALS.     

Characterizations of heterotopic neurons in the spinal cord of amyotrophic lateral sclerosis patients

This report concerns a comparative immunocytochemical, ultrastructural and morphometric investigation on heterotopic neurons in the white matter of the spinal cords of 19 patients with amyotrophic lateral sclerosis (ALS) and 18 age-matched neurologically normal individuals. The study revealed that the heterotopic neurons were scattered in the white matter, often adjacent to gray matter, that they immunoreacted with the antibody to synaptophysin, and that there were synaptic apparatuses on the surface of their somata and their neuronal processes. Bunina bodies and ubiquitin-positive inclusions such as Lewy body-like inclusions and skein-like inclusions, characteristic of anterior horn neurons of ALS, were present in the cytoplasm of the patients’ heterotopic neurons in the anterior or lateral column of the white matter. These findings suggest that heterotopic neurons in the anterior or lateral column have the characteristics of alpha motor neurons. The average number of heterotopic neurons observed in ALS patients was generally less than in normal subjects. This reduction was correlated with the severity of neuronal loss. The heterotopic neurons in ALS were less susceptible to the degenerative process as compared with spinal cord anterior horn cells. We assume that in this disease the heterotopic neurons may be degenerated and their number diminished after or concomitantly with the depletion of anterior horn neurons. Received: 18 August 1997 / Revised, accepted: 20 October 1997     

Maintained regulation of polyamines in spinal cord from patients with amyotrophic lateral sclerosis

Levels of the polyamines putrescine, spermidine, and spermine were investigated in postmortem spinal cord from seven patients with amyotrophic lateral sclerosis (ALS) and seven control subjects. The method consisted of precolumn derivatization of the polyamines, followed by high-performance liquid chromatography (HPLC) analysis and fluorescence detection. The stability of the polyamines was examined in rat spinal cord during the interval of 0-36 h postmortem. The levels of putrescine, spermidine, and spermine increased by 32%, 15%, and 2%, respectively. Polyamine levels did not differ significantly between the ALS group and the control group, suggesting a maintained regulation of polyamines in the end stage of the disease. However, an effect of gender on the levels of spermidine and spermine was observed. Levels of spermidine and spermine in the ventral horn region of female ALS patients were significantly higher in comparison with the same region of the male ALS group (p<0.05). The female ALS group also presented significantly higher levels of spermidine in comparison with female controls (p<0.05).     

Autophagy in spinal cord motor neurons in sporadic amyotrophic lateral sclerosis

To assess the potential role of autophagy in amyotrophic lateral sclerosis (ALS), lumbar spinal cords in a total of 19 sporadic ALS cases and 27 age-matched controls were investigated. Immunohistochemical analysis using antibodies to the markers of autophagy microtubule-associated protein light chain 3 (LC3) and p62 was performed on samples from 12 ALS and 15 controls. Electron microscopy was performed on samples from 16 ALS and 15 controls, including overlapping cases. In the ALS cases, the somata of normal-appearing and degenerated motor neurons and round bodies were occasionally immunostained for LC3; round bodies and skein-like inclusions were immunostained for p62. By electron microscopy, all 16 ALS patients showed features of autophagy in the cytoplasm of normal-appearing motor neurons and, more frequently, in degenerated motor neurons. Autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane contained sequestered cytoplasmic organelles, such as mitochondria and ribosome-like structures. These autophagy features were also found in close association with the characteristic inclusions of ALS(i.e. round bodies, skein-like inclusions, and Bunina bodies); honeycomb-like structures also occasionally showed autophagy-associated features. Normal-appearing anterior horn neurons in control patients showed no autophagy features. Thus, autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegeneration of motor neurons in sporadic ALS.     

The anterolateral funiculus in the spinal cord in amyotrophic lateral sclerosis

We carried out a morphometric study on the myelinated fibers in the anterolateral funiculus (ALF) and lateral corticospinal tract (LCS) in the cervical segment of the spinal cord of 13 patients with classic amyotrophic lateral sclerosis (ALS), 6 of whom had been on a respirator: 5 age-matched subjects were used as controls. The results obtained revealed that: (1) the fiber-size distributions of the myelinated fibers in the ALF and LCS of the control subjects had peaks at 2 m; (2) there were marked and significant losses of large myelinated fibers in the ALF and LCS of ALS patients; (3) the patients who required respirator support showed more severe degeneration in the ALF than those who required none; and (4) the degree of myelinated fiber loss in the LCS did not correlate with either the illness duration or the history of respirator use.     

Reduced glycine receptor in the spinal cord in amyotrophic lateral sclerosis

Transmitter receptor binding was estimated in the spinal cord of 6 subjects with amyotrophic lateral sclerosis (ALS) and 4 control subjects in assays using 3H-quinuclidinyl benzilate for muscarinic cholinergic receptors, 3H-strychnine for glycinergic receptors, 3H-spiroperidol for dopaminergic receptors, 3H-muscimol for GABAergic receptors, and 3H-dihydroalprenolol for beta-adrenergic receptors. In ALS, glycinergic receptor binding was greatly reduced in the anterior gray matter. This finding may be attributed to loss of large neurons in the anterior gray matter, known to be characteristic of ALS.     

Plasma amino acids percentages in amyotrophic lateral sclerosis patients

Abstract. The aim of the study was to examine plasma amino acids (AA) percentages in amyotrophic lateral sclerosis (ALS) patients. Altered metabolism of AA, especially excitatory AA in ALS, has been reported. The investigation was conducted at the Department of Neurology, University School of Medicine in Lublin. The study comprised 50 patients; 20 persons with ALS and 30 controls. Plasma AA were measured by automated ion-exchange chromatography. The results show significantly lower percentages of plasma tyrosine, valine, methionine, leucine, and isoleucine and significantly higher percentages of plasma glutamine and serine in ALS than in controls. The clinical state significantly influenced the percentage of plasma phenylalanine and alanine. Our study shows significant changes in some plasma AA percentages in ALS; however, excitatory AA percentages did not differ from the control subjects.