The wobbler mouse: amino acid contents in brain and spinal cord

Reductions in glutamate and aspartate contents, together with increased contents of taurine, have been observed in the autopsied brains and spinal cords of patients who have died with amyotrophic lateral sclerosis (ALS). The wobbler mouse develops an inherited degeneration of motoneurons within the brainstem and spinal cord, and has been proposed as an animal model of ALS. In symptomatic wobbler mice we found brain contents of glutamate, aspartate, and taurine similar to those in unaffected littermates, while brain contents of glutamine were increased, and those of serine and alanine were decreased. Spinal cords of wobbler mice had slightly decreased contents of glutamate, aspartate and glycine compared to normal littermates. Abnormalities of amino acid contents in the nervous system of wobbler mice are dissimilar to those in ALS patients suggesting a different pathogenesis of motoneuron loss.     

Brain amino acid contents are dissimilar in sporadic and Guamanian amyotrophic lateral sclerosis

Amino acid contents were measured in autopsied brains of five Guamanian patients with amyotrophic lateral sclerosis (ALS) or parkinsonism-dementia. Absence of the glutamate deficiency and taurine excess characteristic of sporadic ALS suggest that, despite clinical similarities, Guamanian ALS is a different disorder from sporadic ALS.     

Cognitive dysfunction in patients with amyotrophic lateral sclerosis is associated with spherical or crescent-shaped ubiquitinated intraneuronal inclusions in the parahippocampal gyrus and amygdala, but not in the neostriatum

Skeins or skein-like inclusions, one of the two types of ubiquitinated intraneuronal inclusions in amyotrophic lateral sclerosis (ALS), in the neostriatum are not specific to the disease, but it has not yet been determined whether the other, spherical or crescent-shaped inclusions (SCI) are pathognomonic. To clarify this and also to investigate whether the distribution of SCI in particular brain regions is associated with clinical parameters, we examined the occurrence of SCI in the brains of 24 patients with ALS and 94 controls. SCI in the neostriatum were specifically detected in 54% of the ALS cases, but not in any of the controls. No apparent phenotypic denominator, such as disease duration or the occurrence of dementia, correlated to the distribution of SCI in the neostriatum in ALS cases. On the other hand, the occurrence of SCI in both the second and third layers of the parahippocampal gyrus and amygdala was significantly correlated to the presence of dementia in ALS cases. SCI were distributed in association with each other among the parahippocampal gyrus, dentate gyrus of the hippocampus and amygdala, but not between the spinal anterior horn and any non-motor-associated brain regions. These findings suggest that these particular brain regions might be significantly involved in the neurodegenerative process associated with ALS. The relationship of SCI to either ALS pathogenesis or cognitive dysfunction depends on the brain regions in which they are distributed, and this indicates that the neurodegenerative processes in ALS proceed differentially in particular motor-associated and nonmotor-associated brain regions.     

Brain amino acids and glutathione in progressive supranuclear palsy

We measured amino acid contents in autopsied brains of seven patients with progressive supranuclear palsy (PSP) and in control subjects dying without brain disease. Glutathione was also quantitated in rapidly frozen brains of PSP patients, Parkinson's disease (PD) patients, and controls. In PSP, we found glutamic acid markedly increased in the nucleus accumbens; taurine significantly increased in nucleus accumbens, substantia nigra, and globus pallidus; and gamma-aminobutyric acid significantly increased in nucleus accumbens and putamen. Glycerophosphoethanolamine contents were significantly increased in most regions. Glutathione, which is significantly decreased in substantia nigra in PD, was increased in this brain region in PSP, suggesting that different mechanisms may be responsible for destruction of dopaminergic nigrostriatal neurons in these two disorders.     

Ganglioside patterns in amyotrophic lateral sclerosis brain regions

In a search for evidence of biochemical disorders in regions of postmortem brain other than the motor cortex in amyotrophic lateral sclerosis (ALS), ganglioside patterns were also examined in the frontal, temporal, and parahippocampal gyrus cortex. In 21 ALS brains studied (20 sporadic, 1 familial), abnormal patterns were found in the frontal cortex (81%), temporal cortex (75%), motor cortex (70%), and parahippocampal gyrus cortex (71%). Patterns were established by measuring the percentage distribution of 12 ganglioside species. Two abnormal patterns were detected. One was based on low proportions of GD1b, GT1b, and GQ1b associated with high proportions of GM2 and GD3 (GM1, GD1a, GD2, and GT1a values were normal). The second abnormality was the appearance of Gx. Neither abnormality was seen in the 13 non-ALS control brains. The first, and predominant, abnormality was found in the frontal cortex in 14 brains, and the second was observed in 13 brains; 10 brains showed both abnormalities. These findings thus constitute evidence that the disease process in ALS extends beyond the motor cortex and involves neurons in several brain areas.     

Amyotrophic lateral sclerosis: amino acid levels in plasma and cerebrospinal fluid

Concentrations of glutamic acid have been reported to be elevated in fasting plasma and cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS); glycine concentrations have also been reported to be increased in the CSF of such patients. Autopsy studies have shown glutamate contents to be significantly decreased in brain and spinal cord in ALS. These observations suggested that a systemic abnormality of glutamate metabolism might underlie the pathogenesis of ALS. We report here the findings of our studies of amino acid concentrations in patients with the sporadic form of ALS. Glutamate concentrations were normal in the fasting plasma of a great majority of the patients with ALS. Concentrations of glutamate, aspartate, and glycine were normal in the CSF of all 17 patients examined. beta-N-Methylamino-L-alanine, a plant neurotoxin possibly responsible for causing the Guamanian form of ALS, was not detectable in the plasma or CSF of any of our patients. Our findings do not lend support to the hypothesis that the sporadic form of ALS results from overexcitation of motor neurons by excitatory amino acids.     

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.     

Topographic distribution of brain iron deposition and small cerebrovascular lesions in amyotrophic lateral sclerosis and in frontotemporal lobar degeneration: a post-mortem 7.0-tesla magnetic resonance imaging study with neuropathological correlates

Amyotrophic lateral sclerosis (ALS) is associated with frontotemporal lobar degeneration (FTLD) in 15% of the cases. A neuropathological continuity between ALS and FTLD-TDP is suspected. The present post-mortem 7.0-tesla magnetic resonance imaging (MRI) study compares the topographic distribution of iron (Fe) deposition and the incidence of small cerebrovascular lesions in ALS and in FTLD brains. Seventy-eight post-mortem brains underwent 7.0-tesla MRI. The patients consisted of 12 with ALS, 38 with FTLD, and 28 controls. Three ALS brains had minor FTLD features. Three coronal sections of a cerebral hemisphere were submitted to T2 and T2* MRI sequences. The amount of Fe deposition in the deep brain structures and the number of small cerebrovascular lesions was determined in ALS and the subtypes of FTLD compared to control brains, with neuropathological correlates. A significant increase of Fe deposition was observed in the claustrum, caudate nucleus, globus pallidus, thalamus, and subthalamic nucleus of the FTLD-FUS and FTLD-TDP groups, while in the ALS one, the Fe increase was only observed in the caudate and the subthalamic nuclei. White matter changes were only significantly more severe in the FTLD compared to those in ALS and in controls brains. Cortical micro-bleeds were increased in the frontal and temporal lobes of FTLD as well as of ALS brains compared to controls. Cortical micro-infarcts were, on the other hand, more frequent in the control compared to the ALS and FTLD groups. The present study supports the assumption of a neuropathological continuity between ALS and FTLD and illustrates the favourable vascular risk profile in these diseases.     

Biopterin metabolism in patients with malignant syndrome

We examined the autopsied brains of two parkinsonian patients who had malignant syndrome (MS). Neopterin and biopterin contents, and GTP cyclohydrolase I activity were measured in various region of the brain. We found relatively higher GTP cyclohydrolase I activities in the hypothalamus compared with other regions of the brain from patients with MS. This finding suggested a possible involvement of biopterin metabolism in pathophysiology of MS. This is the first report on biopterin metabolism in the brains of patients with MS.     

Mitochondrial DNA deletion mutation levels are elevated in ALS brains

This study was performed to explore the potential role of mitochondrial DNA mutations in the neurodegenerative process in amyotrophic lateral sclerosis (ALS). Using a semi-quantitative assay, a common mitochondrial DNA deletion mutation (mt DNA4977) was assayed in brain tissue obtained from six sporadic ALS patients and compared to four controls. In each brain, levels of this mutation were measured in a brain region affected by neurodegeneration, the motor cortex (Brodmann area 4), and compared to the temporal cortex (Brodmann area 17). In the ALS but not control brains, levels of mt DNA4977 were an average of more than 30-fold (range 15-250) higher in Brodmann area 4. These results support and extend those of previous studies implying that mitochondria may participate in the neurodegenerative process in ALS.     

Neurotransmitter amino acids in dominantly inherited cerebellar disorders

We measured amino acid contents in the brains of 11 patients with dominantly inherited cerebellar disorders. Despite clinical similarities, three biochemically different disorders were found. One disorder, with demonstrated HLA linkage in one pedigree, was characterized by moderate reduction of aspartate and glutamate contents in cerebellar cortex alone. In a second disorder, aspartate and glutamate contents were reduced markedly in other brain areas as well as in cerebellar cortex. Aspartate and glutamate contents were normal in cerebellar cortex in the third disorder. GABA content in cerebellar cortex and dentate nucleus was reduced in some patients with each disorder, whereas cerebellar taurine content was normal in all patients. Aspartate deficiency in cerebellar cortex did not result from lack of aspartate aminotransferase or pyruvate carboxylase activity. These amino acid abnormalities probably imply loss of specific cerebellar neurons.     

Amino acids, glutathione, and glutathione transferase activity in the brains of patients with Alzheimer's disease

We measured the contents of amino acids and related amino compounds in autopsied brain from 22 patients with Alzheimer's disease (AD) and in cortical biopsy specimens from 2 other patients. The diagnosis of AD was established neuropathologically in all 24 patients by the presence of both neurofibrillary tangles and neuritic plaques in neocortex. The mean contents of gamma-aminobutyric acid (GABA), and of the GABA dipeptide homocarnosine, were significantly reduced in frontal and occipital cortices and in hippocampus of the autopsied brains of AD patients compared to control patients without neurological disease. However, GABA contents were normal in frontal cortex in biopsy samples from 2 patients. Phosphoethanolamine contents were significantly reduced at autopsy in frontal and occipital cortex, and in the substantia innominata. We found no evidence of a deficiency of glutamate, aspartate, or taurine in AD brain, as has been claimed. Glutathione contents and glutathione transferase activities were normal in frontal cortex and substantia innominata. The mechanism of neuronal death in patients with AD is unlikely to involve either insufficient synthesis of glutathione or failure to conjugate free radicals with glutathione.     

Amyotrophic lateral sclerosis and parkinsonism‐dementia complex of the Hohara focus of the Kii Peninsula: A multiple proteinopathy?

The high incidence of amyotrophic lateral sclerosis (ALS) and parkinsonism‐dementia complex (PDC) has been previously known in the Kii Peninsula of Japan and in Guam. Recently, the accumulation of various proteins, such as tau, trans‐activation response DNA binding protein 43 kDa (TDP‐43), and alpha‐synuclein (αSyn), was reported in the brains of patients with ALS/PDC in Guam. To confirm whether similar findings are present in Kii ALS/PDC, we neuropathologically examined the brains and spinal cords of 18 patients with ALS/PDC (clinical diagnoses: eight ALS and 10 PDC) in Hohara Village, which is the eastern focus of Kii ALS. The average age at death was 71.6 years, and 16 patients (88.9%) had a family history of ALS/PDC. Autopsy specimens were immunohistochemically examined with antibodies against four major proteins. Neurofibrillary tangles, including ghost tangles, and tau‐positive astrocytes were distributed widely in all of the brains examined, and TDP‐43‐positive neuronal cytoplasmic inclusions were observed mainly in the limbic system. Synuclein pathology was present in 14 patients (77.8%). These patients were classified into three pathological subtypes according to the most prominent proteinopathy: the tauopathy‐dominant type, the TDP‐43 proteinopathy‐dominant type, and the synucleinopathy‐dominant type. Five patients with severe tau deposition showed clinical features of atypical parkinsonism and dementia with or without motor neuron disease. Eight patients were predominated by phosphorylated TDP‐43 inclusions and clinically showed ALS, and five patients were predominated by synuclein pathology and clinically showed signs of PDC. Based on the common characteristic tau pathology, three subtypes seemed to be pathologically continuous on a spectrum of a single disease. Thus, we conclude that ALS/PDC in the Hohara focus of the Kii Peninsula is a single disease characterized neuropathologically by a multiple proteinopathy, even though the clinical manifestations of the three subtypes differed from each other. It remains unclear whether the coexistence of the three proteinopathies was incidental or pathogenetically related.     

Functional motor compensation in amyotrophic lateral sclerosis

The present study investigated the fMRI correlates of functional compensation/neural reorganization of the motor system in patients with amyotrophic lateral sclerosis (ALS). The hypothesis was that ALS patients would recruit additional brain regions compared with controls in a motor task and that activity in these regions would vary as a function of task difficulty. Patients and controls executed a motor task with two sequences (a simple and a more difficult one) of consecutive button presses. Patients and controls both activated brain regions known to be involved in motor execution and control. Activity in ipsilateral motor areas as well as difficulty-related activity in the left cerebellum could only be observed in patients. The behavioral data indicated that the motor task was much more difficult for patients than for controls. At nearly equal difficulty the observed patterns of hemodynamic activity in controls were very similar to those observed in ALS. The findings suggest that functional compensation in ALS relies on existing resources and mechanisms that are not primarily developed as a consequence of the lesion.     

Decreased receptor-binding sites for kainic acid in brains of patients with Huntington's disease

The specific binding of (3H)kainic acid (KA) to high- and low-affinity receptor sites was assayed in postmortem samples from the brains of patients affected with Huntington's disease (HD) and age-matched controls. Treatment of rat brain with conditions that closely mimic the temperature gradient occurring in postmortem human brain only slightly but not significantly decreased receptor binding by 12 hr after death. In HD brains, total specific binding of (3H)KA was reduced in the caudate nucleus by 51%, putamen by 77%, and frontal cortex by 47%. Specific binding to the high-affinity site was virtually undetectable in the caudate nucleus and was reduced by 90% in the putamen from HD brains. No significant alterations in specific binding of (3H)KA were noted in the insular or temporal cortex, hippocampus, or cerebellum. Thus, losses of KA receptor binding were mainly localized to those regions of the HD brain that are most severely affected by neuronal degeneration, and the high-affinity receptor site appeared more affected.     

Investigation of white matter pathology in ALS and PLS using tract-based spatial statistics

OBJECTIVE: We aimed to investigate differences in fractional anisotropy (FA) between primary lateral sclerosis (PLS) and amyotrophic lateral sclerosis (ALS) and the relationship between FA and disease progression using tract-based spatial statistics (TBSS). METHODS: Two scanners at two different sites were used. Differences in FA between ALS patients and controls scanned in London were investigated. From the results of this analysis, brain regions were selected to test for (i) differences in FA between controls, patients with ALS and patients with PLS scanned in Oxford and (ii) the relationship between FA and disease progression rate in the Oxford patient groups. RESULTS: London ALS patients showed a lower FA than controls in several brain regions. Oxford patients with PLS showed a lower FA than ALS patients and than controls in the body of the corpus callosum and in the white matter adjacent to the right primary motor cortex (PMC), while ALS patients showed reduced FA compared with PLS patients in the white matter adjacent to the superior frontal gyrus. Significant correlations were found between disease progression rate and (i) FA in the white matter adjacent to the PMC in PLS, and (ii) FA along the cortico-spinal tract and in the body of the corpus callosum in ALS. CONCLUSIONS: We described significant FA changes between PLS and ALS, suggesting that these two presentations of motor neuron disease show different features. The significant correlation between FA and disease progression rate in PLS suggests the tissue damage reflected in FA changes contributes to the disease progression rate.     

Cyanobacterial neurotoxin BMAA in ALS and Alzheimer’s disease

Objective – The aim of this study was to screen for and quantify the neurotoxic amino acid β‐N‐methylamino‐l ‐alanine (BMAA) in a cohort of autopsy specimens taken from Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and non‐neurological controls. BMAA is produced by cyanobacteria found in a variety of freshwater, marine, and terrestrial habitats. The possibility of geographically broad human exposure to BMAA had been suggested by the discovery of BMAA in brain tissues of Chamorro patients with ALS/Parkinsonism dementia complex from Guam and more recently in AD patients from North America. These observations warranted an independent study of possible BMAA exposures outside of the Guam ecosystem. Methods – Postmortem brain specimens were taken from neuropathologically confirmed cases of 13 ALS, 12 AD, 8 HD patients, and 12 age‐matched non‐neurological controls. BMAA was quantified using a validated fluorescent HPLC method previously used to detect BMAA in patients from Guam. Tandem mass spectrometric (MS) analysis was carried out to confirm the identification of BMAA in neurological specimens. Results – We detected and quantified BMAA in neuroproteins from postmortem brain tissue of patients from the United States who died with sporadic AD and ALS but not HD. Incidental detections observed in two out of the 24 regions were analyzed from the controls. The concentrations of BMAA were below what had been reported previously in Chamarro ALS/ Parkinsonism dementia complex patients, but demonstrated a twofold range across disease and regional brain area comparisons. The presence of BMAA in these patients was confirmed by triple quadrupole liquid chromatography/mass spectrometry/mass spectrometry. Conclusions – The occurrence of BMAA in North American ALS and AD patients suggests the possibility of a gene/environment interaction, with BMAA triggering neurodegeneration in vulnerable individuals.     

An analysis of the entire SOD1 gene in sporadic ALS

Mutations in the superoxide dismutase 1 gene (SOD1) are associated with familial ALS but the role of SOD1 in sporadic ALS (SALS) is unclear. We therefore sequenced the entire SOD1 gene in 23 patients with SALS. DNA was extracted from frozen pre-frontal cerebral cortex and from blood. The 5 exons, 4 introns and 1 kb upstream and downstream of SOD1 were sequenced. Novel genetic variants were found in 30% (7 of 23) brains and known variants in 91% (21 of 23) brains from patients with SALS. Two novel variants found in SALS patients and not controls were located in the SOD1 promoter and intron 1, with the promoter variant having potential functional implications. A previously described silent variant in exon 5 in one SALS patient appears to abolish an exonic splicing enhancer. All changes found in brain DNA were also found in blood DNA. In conclusion, sequencing the entire SOD1 gene revealed 3 variants in SALS patients that were not detected in controls. Although no unequivocal mutations were found, some of these variants have potential consequences for SALS pathogenesis.     

Altered metabolism of excitatory amino acids, N-acetyl-aspartate and N-acetyl-aspartylglutamate in amyotrophic lateral sclerosis

Since recent studies provided evidence for abnormal glutamate metabolism in amyotrophic lateral sclerosis, we measured amino acid levels in the fasting plasma of 52 ALS patients and an equal number of controls of a similar age. In addition, the content of amino acids, N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG) were measured in spinal cord and brain tissue obtained at autopsy from patients dying of ALS. Results showed significant elevations (by about 70%) in the plasma levels of glutamate in the ALS patients as compared to controls. In contrast, glutamate levels were significantly decreased in all CNS regions studied of ALS patients (by 21–40%), with the greatest changes occurring in the spinal cord. The ratio of glutamine to glutamate was altered significantly in the spinal cord ALS tissue. In addition, reductions in the levels of aspartate (by 32–35%), NAA, and NAAG (by 40–48%) were found in the spinal cord of ALS patients. These results are consistent with a generalized defect in the metabolism of neuroexcitotoxic amino acids. An altered distribution of these compounds may occur between their intracellular and extracellular pools with resultant abnormal potentiation of excitatory transmission mediated by glutamate receptors and selective degeneration of motor neurons.