L-cycloserine slows the clinical and pathological course in mice with globoid cell leukodystrophy (twitcher mice)

Globoid cell leukodystrophy (Krabbe's disease) is an autosomal recessive disease that affects the lysosomal enzyme galactosylceramidase. Galactosylceramidase removes galactose from galactosylceramide and psychosine, which are derived from sphingosine. In the present study, L-cycloserine (an inhibitor of 3-ketodyhydrosphingosine synthase) was administered to the twitcher mouse, an authentic model of globoid cell leukodystrophy. Twitcher mice treated with L-cycloserine had a significantly longer life span and a delayed onset of weight loss than vehicle-injected twitcher mice. Pathological features such as macrophage infiltration and astrocyte gliosis also were less in treated twitcher mice. These results indicate that substrate reduction therapy may have therapeutic value for individuals with residual enzymatic activity, e.g., individuals with late onset disease or individuals with partial enzyme replacement via bone marrow transplantation. In these cases, a reduction in galactosylceramide and psychosine synthesis would enable residual enzymatic activity to keep up with the accumulation of these substrates that would otherwise lead to pathology.     

The twitcher mouse: accumulation of galactosylsphingosine and pathology of the sciatic nerve

Morphological and biochemical changes were investigated in the early developmental stages of sciatic nerve of the twitcher mouse, a murine model of human globoid cell leukodystrophy. The concentration of galactosylsphingosine (psychosine) and the chronological changes of the twitcher mouse peripheral nerve pathology correlated well. Galactosylsphingosine had already accumulated at birth and dramatically increased with age. Characteristic inclusions were observed in Schwann cells and macrophages of the twitcher mouse on the 5th postnatal day. Endoneurial edema developed after 10 postnatal days and the hypomyelination was pronounced at 15-20 postnatal days. These findings suggest that galactosylsphingosine is cytotoxic for myelin-forming cells and is closely related to pathogenetic events in the twitcher mouse.     

TNF-receptor 1 deficiency fails to alter the clinical and pathological course in mice with globoid cell leukodystrophy (twitcher mice) but affords protection following LPS challenge

Twitcher mice have an autosomal recessive mutation in the gene for the lysosomal enzyme galactosylceramidase, which is the same gene that is affected in human globoid cell leukodystrophy (Krabbe's disease). The failure to digest galactosylceramide and psychosine leads to initial pathological changes in oligodendrocytes. Secondary pathological changes that include infiltrating macrophages and other inflammatory responses have been postulated to promote the disease course. TNFalpha levels are elevated in twitcher mice compared to control animals, and studies on another demyelinating disease, experimental allergic encephalomyelitis, indicate that TNF promotes pathogenesis via TNF-receptor 1 (TNF-R1). In the present study, twitcher/TNF-R1 deficient mice were generated, and the clinical and pathological course was compared between these mice and regular twitcher mice. There was no statistical evidence for any differences between these two groups of mice for all clinical (life span, weight loss, onset day of twitching) and pathological (demyelination, astrocyte gliosis, macrophage infiltration) measures that were examined. If mice were administered an intraperitoneal injection of LPS, then twitcher/TNF-R1 deficient mice had a longer [corrected] life span and a decreased [corrected] disruption to the blood-brain barrier compared to regular twitcher mice. These results showed that TNF-R1 is not sufficiently activated to affect the pathological and/or clinical signs during the natural course of this disease. However, when there is a secondary insult, TNF-R1 activation does lead to a significant acceleration of the development of clinical and pathological signs.     

Delayed clinical and pathological signs in twitcher (globoid cell leukodystrophy) mice on a C57BL/6 x CAST/Ei background

Modifier genes may account for the phenotypic variability observed in the late-onset forms of globoid cell leukodystrophy (GCL) in humans. In order to begin a search for modifier genes, the effect of genetic background on the clinical and pathological manifestations of GCL was investigated in twitcher mice. Twitcher mice on a C57BL/6 x CAST/Ei background had an increased life span (61.4 +/- 2.5 vs 37.0 +/- 0.6 days), a delayed onset of tremor (24 vs 21 days), and a delayed decline in walking ability compared to C57BL/6 twitcher mice. Pathologically, C57BL/6 x CAST/Ei twitcher mice had fewer lectin-positive globoid cells, less gliosis, and a greater preservation of myelin compared to C57BL/6 twitcher mice under moribund conditions. Similar concentrations of psychosine, the toxic species that accumulates in GCL, were measured by tandem mass spectrometry between moribund C57BL/6 twitcher mice (286.5 pmol/mg protein), 40-day C57BL/6 x CAST/Ei twitcher mice (276.5 pmol/mg), and moribund C57BL/6 x CAST/Ei twitcher mice (247.0 pmol/mg), suggesting that the milder phenotype in CAST/Ei x C57BL/6 twitcher mice did not correlate with less psychosine. In summary, the introduction of modifier genes from the wild, inbred CAST/Ei strain had a phenotypic effect resulting in a significantly slower disease course.     

The twitcher mouse: accumulation of galactosylsphingosine and pathology of the central nervous system

In the twitcher mouse, a murine model of globoid cell leukodystrophy (GLD), pathological changes of various parts of the central nervous system correlated well with the concentration of galactosylsphingosine (psychosine). The development of GLD lesions was more obvious in tracts with a more rapid progression of myelination. It was suggested that accumulation of galactosylsphingosine subsequent to myelin maturation caused suicidal death of myelin forming cells.     

Abnormality in cultured oligodendrocytes and Schwann cells isolated from the twitcher mouse

Oligodendrocytes and Schwann cells were isolated from the brain and dorsal root ganglia of the twitcher mouse, a murine model of Krabbe’s disease (globoid cell leukodystrophy) and grown in tissue culture. Oligodendrocytes were cultured for up to 22 d in vitro and were immunostained with a galactocerebroside antibody, a specific marker for oligodendrocytes. The control oligodendrocytes developed well-branched processes and membrane sheets, whereas the twitcher oligodendrocyte had wirelike processes with no membrane expansion and progressive degeneration. Schwann cells from the twitcher could not extend their processes as long as normal counter-parts. The amounts of psychosine in the enriched population of oligodendrocytes and Schwann cells from the twitcher mouse are about 50-fold and 70-fold higher, respectively, than those in the control cells. These data suggest that psychosine may play an important role in the progression of abnormal features of oligodendroglial membrane formation and in the absence of process elongation in Schwann cells in the twitcher mutant.     

Biochemical and morphological studies of dorsal root ganglion and its cultured cells from twitcher mouse (murine globoid cell leukodystrophy)

The biochemical pathogenesis of globoid cell leukodystrophy (GLD) (Krabbe disease) was investigated in vitro using the cultured neural cells obtained from dorsal root ganglion (DRG) of twitcher mouse (murine Krabbe disease). Electron microscopic examination of twitcher DRG of 30 days old showed the demyelination and abnormal inclusion bodies, whereas mitochondrial structure appeared to be intact. Cultured neural cells from control mice were well proliferated to form the network processes, while those from twitcher were decreased in cell numbers and showed the vacuolation of cell body, degeneration of processes, and finally died after three weeks. There was no apparent difference of lipid composition between control and twitcher DRG, and the galactocerebroside levels from control were similar to those of twitcher. However, marked accumulation of psychosine was found in the 30-day-old-twitcher DRG. The level of psychosine from twitcher DRG was elevated approximately 70 to 80-fold compared with those of control. These data suggest that neural cells from twitcher mice degenerative with age also in vitro and accumulation of psychosine in twitcher DRG may play an important role for the demyelination in GLD.     

Biochemistry and neuropathology of mice doubly deficient in synthesis and degradation of galactosylceramide

We have generated mice doubly deficient in both synthesis and degradation of galactosylceramide by cross-breeding twitcher mice and galactosylceramide synthase (UDP-galactose:ceramide galactosyltransferase, CGT) knockout mice. The prediction that the phenotype of the doubly deficient mice should be the same as the cgt -/- mice, since the degrading enzyme should not be necessary if the substrate is not synthesized, proved to be only partially correct. In early stages of the disease, the doubly deficient mice (galc -/-, cgt -/-) were essentially indistinguishable from the cgt -/- mice. However, the doubly deficient mice had a much shorter life span than cgt -/- mice. Both galactosylceramide and galactosylsphingosine (psychosine), were undetectable in the brain of the cgt -/- and the doubly deficient mice. The characteristic twitcher pathology was never seen in the galc -/-, cgt -/- mice. However, after 43 days, neuronal pathology was observed in the brainstem and spinal cord. This late neuronal pathology has not been seen in the CGT knockout mice but has been described in some long surviving bone marrow-transplanted twitcher mice. Furthermore, the motor segment of the trigeminal nerve of the galc -/-, cgt -/- mice showed severe degeneration not seen in either twitcher or CGT knockout mice. Thus, the galc -/-, cgt -/- mice, while primarily showing the cgt -/- phenotype as predicted, develop late pathology that is seen only in twitcher mouse and also a unique pathology in the trigeminal nerve. These observations indicate that the functional relationship between galactosylceramidase and galactosylceramide synthase is complex.     

Establishment and characterization of spontaneously immortalized Schwann cells from murine model of globoid cell leukodystrophy (twitcher)

The twitcher mouse is a murine model of human globoid cell leukodystrophy (GLD; Krabbe disease) caused by a genetic defect in the activity of galactosylceramidase (GALC). An accumulation of cytotoxic metabolite, galactosylsphingosine (psychosine), in myelin forming cells (oligodendrocytes and Schwann cells) of the twitcher mouse as well as patients with GLD has been suggested to cause dysfunction of these cells and subsequent demyelination in the central and peripheral nervous system. To investigate further the cellular pathomechanism of GLD, we established spontaneously immortalized Schwann cell lines from the twitcher mouse. Long-term cultures of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of 3-week-old twitcher mice were maintained for 6 months, and spontaneously developed colonies were expanded further and characterized. One of the cell lines, designated TwS1, showed distinct Schwann cell phenotypes, was passaged twice a week and maintained for over 10 months without phenotypic alterations. The TwS1 cells had a nonsense mutation in the GALC genome, and showed markedly reduced GALC activity and elevated psychosine levels. Ultrastructurally, varieties of cytoplasmic inclusions were demonstrated in TwS1 cells. When TwS1 cells were infected with a retrovirus vector encoding GALC, GALC activity was markedly increased and psychosine levels were significantly decreased. These immortalized Schwann cells can be useful in studies on the nervous system lesions in GLD.     

Distribution of freed-serine in vertebrate brains

Freed-serine distribution in vertebrate brains was investigated. In various brain regions of the lower vertebrate species, carp, frog and chick, freed-serine levels were low. On the contrary, in the mammals, mouse, rat and bull, the contents of freed-serine were high in the forebrain (around 400 nmol/g wet weight, and the ratio ofd-serine tol-serine, was d/l = 0.4), and low in the hindbrain. In developing mice,d-serine levels in the cerebrum increased with age and attained the adult level (d/l = 0.40) 8 weeks after birth. In the cerebellum and brain stem, the freed-serine levels increased with age until 2 weeks, followed by a decrease to the adult levels: thed/l ratios remained constant until 2 weeks of age, then decreased to 0.03 in the cerebellum and 0.12 in the brain stem. Freed-serine was shown not to be of microbial origin using germ-free mice. In the rat forebrain,d-serine was evenly distributed in two cerebral regions, namely frontal and occipital lobes. Thed/l ratios in other regions of forebrain, hippocampus and hypothalamus, were comparable to the cerebrum (d/l = 4), while that in the olfactory bulb was lower (d/l = 0.12). In the rat cerebrum, thed-serine content in the grey matter was significantly higher than that in the white matter. The contents of freed-serine in bovine cerebrum and cerebellum were similar to those in other mammalian brains, but thed/l ratio for bovine cerebral grey matter was lower than that for the cerebral white matter. Thed-serine level was discussed in terms ofd-amino-acid oxidase activity.     

Retrovirus-mediated gene transfer and galactocerebrosidase uptake into twitcher glial cells results in appropriate localization and phenotype correction

Galactocerebrosidase (GALC) is deficient in all tissues from human patients and animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The deficiency results in decreased lysosomal catabolism of certain galactolipids including galactosylceramide and psychosine that are synthesized maximally during myelination. According to current theories, the accumulation of psychosine in humans and animals with GLD induces oligodendrocyte degeneration and myelination ceases. Transduction of oligodendrocytes from twitcher mice with a retroviral vector containing the GALC cDNA can correct the enzyme deficiency in these cells. Our data show that twitcher astrocytes and oligodendrocytes can internalize exogenous GALC, as well as donate the enzyme to the mutant glial cells. Antibodies against human GALC localized the GALC antigen in retrovirally transduced cells and cells receiving enzyme via cell to cell secretion and uptake to the lysosomal fraction. In fact immunocytochemical studies in transduced oligodendrocytes revealed that the GALC colocalizes in vesicles lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells with anti-GALC and a marker for oligodendrocytes demonstrated that, upon differentiation, transduced, twitcher oligodendrocytes attained the normal branched process configuration, while untransduced cells show only abnormal morphology. Phenotype correction in mutant oligodendrocytes has also been observed after enzyme transfer. These studies indicate that GALC activity supplied to cultured oligodendrocytes from twitcher mice by different methods can correct the pathological phenotype of these cells.     

MELAS with the mitochondrial DNA 3243 point mutation: a neuropathological study

We performed a neuropathological examination of the central nervous system from seven autopsied patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Five of the seven cases were confirmed to have the mitochondrial DNA (mtDNA) 3243 point mutation. In addition to the changes reported previously, diffuse atrophy of the cerebral and cerebellar cortices, diffuse gliosis of cerebral and cerebellar white matter, and cactus formation of Purkinje cells were observed. Electron microscopy revealed accumulation of mitochondria in the cactus formations. These lesions are common in MELAS with the mtDNA 3243 point mutation, but cannot be explained solely by mitochondrial angiopathy, and suggest that intrinsic mitochondrial malfunction contributes to neuronal damage in MELAS pathology. Moreover, the pathological changes observed in the cerebellum suggest that cerebellar function should be evaluated more carefully at the clinical level. Received: 3 December 1998 / Accepted: 21 April 1999     

Tau-predominant-associated pathology in a sporadic late-onset Hallervorden-Spatz syndrome.

Hallervorden-Spatz syndrome (HSS) is a heterogeneous clinicopathological disorder currently included within the broader title of neurodegeneration with brain iron accumulation (NBIA). The classic histological hallmarks of HSS are axonal spheroids and excessive iron-containing granules accompanied by neuronal loss and gliosis in the globus pallidus and substantia nigra reticulata. In the modern literature, attention has been drawn to the co-occurrence of two other histological markers: Lewy bodies mainly composed of abnormal alpha-synuclein, and neurofibrillary tangles due to hyperphosphorilated tau aggregation. Discrepancies exist regarding the importance of these molecular changes and its relevance for the nosology of HSS. Most authors have emphasized the importance of the Lewy body-like pathology, favoring the inclusion of HSS within the alpha-synucleinopathies. We report on a case of late-onset HSS, with the typical histological findings restricted to the basal ganglia and cerebellum in which tau pathology was exceedingly more abundant than alpha-synuclein pathology. This case contributes to the increasing evidence about the heterogeneity of HSS. We favor the view that the molecular changes and the protein misfolding underlying the Lewy body and tangle formation in HSS/NBIA are secondary to the main pathological process and should not be taken as the basis for its nosological classification.     

Neuropathology in Kearns-Sayre syndrome

Summary The neuropathological changes found at autopsy in a case of Kearns-Sayre syndrome are described. We have previously analyzed the respiratory chain function in isolated muscle mitochondria and also described a large deletion of muscle mitochondrial DNA (mtDNA) in this case. The neuropathological examination revealed prominent neuronal degeneration and gliosis of the basal ganglia and there were bilateral areas of softening and total loss of nerve cells in the lenticular nuclei. The pallidum and caudate nucleus disclosed accumulation of iron-containing pigment. The white matter in the cerebrum, brain stem and cerebellum showed widespread and focally accentuated spongy change due to splitting of myclin lamellae. It is suggested that deficiency of respiratory chain enzymes due to the mtDNA deletion is of pathogenetic importance in the development of the described changes.Supported by Swedish Medical Research Council (Project no. 7122 and 585)     

The "dark cerebellar sign"

We present and discuss the "dark cerebellar sign" in contrast to the well known "white cerebellar sign". The "white cerebellar sign" relates to a normal cerebellum which appears hyperdense in contrast to a pathological hypodense cerebrum on computer tomography (CT). We present a child with a "dark cerebellar sign" characterized by an ischemic or edematous cerebellum which appears hypodense in contrast to a normal relatively dense cerebrum. Isolated infarction of the cerebellum is a rare finding in premature neonates. Even rarer, cerebellar infarction may be observed in children and young adults due to an overdose of tricyclic antidepressants (TCA). The reason for the selective cerebellar infarction with TCA intoxication is still unknown. Our case shows that TCA intoxication should be included in the differential diagnosis of children with a "dark cerebellar sign".     

Pro-thyrotropin-releasing hormone concentrations in the brain of ataxic mice

Concentrations of pro-thyrotropin-releasing hormone (pro-TRH) were studied in the brain of the Weaver ataxic mouse, the Purkinje cell degenerative mouse (pcd-ataxic mouse), the Staggerer ataxic mouse and the C3H mouse. The brain tissue was dissected into 7 parts, e.g., hypothalamus, cerebrum, thalamus, striatum, brain stem, cerebellum and cervical spinal cord. Pro-TRH concentrations in each part of the brain were measured by radioimmunoassay. Pro-TRH concentrations in the brain of Weaver ataxic mice, pcd-ataxic mice and Staggerer ataxic mice were significantly higher in the thalamus, brain stem, cerebellum and cervical spinal cord. Pro-TRH concentrations in the hypothalamus, striatum and cerebrum of ataxic mice did not differ from those of controls. The elution profile of acetic acid extracted cerebellum of ataxic mice on Sephadex G-50 was identical to that of synthetic pro-TRH. These findings suggest that changes in pro-TRH concentrations in the brain may play a pathophysiological role in ataxic mice.     

Neuroinflammation and regeneration in the early stages of Alzheimer''s disease pathology

The initial stages of Alzheimer's disease pathology in the neocortex show upregulation of cell cycle proteins, adhesion and inflammation related factors, indicating the early involvement of inflammatory and regenerating pathways in Alzheimer's disease pathogenesis. These brain changes precede the neurofibrillary pathology and the extensive process of neurodestruction and (astro)gliosis. Amyloid beta deposition, inflammation and regenerative mechanisms are also early pathogenic events in transgenic mouse models harbouring the pathological Alzheimer's disease mutations, while neurodegenerative characteristics are not seen in these models. This review will discuss the relationship between neuroinflammation and neuroregeneration in the early stages of Alzheimer's disease pathogenesis.     

Immunohistological study of globoid cell leukodystrophy

We examined three autopsy cases of globoid cell leukodystrophy (GLD) with different survival, using immunohistochemistry and in situ nick end labeling (ISEL). The white matter lesion was pronounced in the corona radiata, corpus callosum and cerebellar peduncles in three cases, where a spongy state developed, in addition to the neuronal loss in the thalamus, cerebellum and inferior olivary nucleus. Ramified microglia, being immunoreactive for ferritin and HLA-DR alpha, were scattered in the white matter, and some of them also had immunoreactivity for TNF-alpha. Both the small-sized and large-sized globoid cells showed immunoreactivity for ferritin KP-1 and NCAM, while some of the small-sized globoid cells were also immunoreactive for HLA-DR alpha and TNF-alpha. As the survival became longer, the occurrence of the globoid cells decreased, however, they were commonly observed in the corpus callosum and cerebellar peduncle in three cases. T lymphocytes immunoreactive for LCA, UCHL-1 and CD3 were increased around the vessels in the white matter. ISEL stained nuclei of mononuclear cells in the white matter in two cases with short survival, although the cell origin was not verified. ISEL also visualized a few nuclei of the small-sized globoid cells in one case. On the other hand, immunostainings against cell death proteins such as bcl-2 family members and p53 failed to identify any significant changes. These data suggest that the immunological step and to a lesser extent the apoptotic process may partly be involved in the myelin breakdown and glial pathology in GLD, as reported in the twitcher mouse, a murine model of GLD.     

An autopsy case of atypical adrenoleukomyeloneuropathy in childhood

Adrenoleukomyeloneuropathy (ALMN) usually occurs in adulthood, it being extremely rare in childhood. We reported a quite atypical clinical case of ALMN as a variant of adrenoleukodystrophy (ALD). The onset was at 5 years 7 months and ataxia was the major symptom. His condition progressed rapidly to a vegetative state within 1 year. At the age of 11 years and 11 months he died of pneumonia and an autopsy was performed. We herein reported the neuropathological findings in this rare case. The autopsy revealed marked atrophy with diffuse demyelination and astrogliosis throughout the cerebrum, cerebellum and brainstem. Massive degeneration of the pyramidal tracts and loss of neurons were also seen in the spinal cord. The adrenal cortex showed marked atrophy with a striated cytoplasm in ballooned cells. These findings include pathological characteristics of both ALD and adrenomyeloneuropathy (AMN), suggesting ALMN. However, diffuse demyelination with gliosis in the cerebrum and cerebellum is quite atypical for ALMN. They might explain his atypical clinical course, especially the early onset of the disease with ataxia and rapid deterioration.     

An autopsied case of Creutzfeldt‐Jakob disease with mutation in the prion protein gene codon 232 and type 1+2 prion protein

A 68‐year‐old Japanese man gradually showed abnormal behavior and gait disturbance with bradykinesia. Slowly progressive dementia, including memory disturbance and disorientation, was also observed. Cerebral cortical hyperintensity on diffusion‐weighted MRI was observed 6 months after onset. The patient progressed to an akinetic mutism state with mild myoclonus, and atypical periodic sharp‐wave complexes were observed by electroencephalogram 13 months after onset. He was clinically suspected of having atypical CJD and died after 19 months total disease duration. The brain weighed 1160 g and showed mild atrophy of the cerebrum and cerebellum with ventricular dilatation. Spongiform changes with varying vacuole size and gliosis was extensive in the cerebral cortex and basal ganglia. Neuron loss in the cerebral cortex, basal ganglia and thalamus was relatively mild. The cerebellum showed mild spongiform changes of the molecular layer and mild neuron loss in the Purkinje cell layer. PrP immunostaining showed mainly coarse‐type combined with diffuse synaptic‐type PrP deposition in the cerebral gray matter. Some perivacuolar‐type PrP deposition was also present. Numerous plaque‐type PrP depositions were observed in the molecular layer of the cerebellum. Analysis of the PrP gene revealed a methionine‐to‐arginine (Met‐to‐Arg) substitution at codon 232 (M232R) with Met homozygosity at codon 129. Western blot analysis of protease‐resistant PrP indicated type 2 dominant PrP combined with type 1. Genetic CJD with M232R substitution in the PrP gene has only been reported in Japan. Although two clinical phenotypes (rapid‐type and slow‐type) were suggested in the M232R CJD cases (despite the presence of the same PrP genotype), the pathological and molecular backgrounds have not been well understood because there have only been a few autopsied case reports. This is the first case report of M232R CJD presenting with 1 + 2 PrP.