Twitcher Mouse Research Articles

Suppressed UDP-galactose: ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain.

The developmental changes in expression of steady-state mRNA that encode proteins that are important for myelination (myelin basic protein, myelin-associated glycoprotein, proteolipid protein, UDP-galactose: ceramide galactosyltransferase) and glial fibrillary acidic protein were investigated in the brain of the twitcher mouse, a model of human globoid cell leukodystrophy. This disease is caused by a mutation in the gene encoding the lysosomal enzyme, galactosylceramidase, which catalyzes the degradation of the myelin lipid galactosylceramide. Before postnatal day (PND) 20, the levels of myelin protein mRNA were similar in twitcher and normal mice. With progression of demyelination after PND 25-30, myelin protein mRNA levels gradually decreased. The period of maximum expression of the myelin protein genes in twitcher mice was, however, similar to that of normal control mice. mRNA levels for the gene that encodes the enzyme UDP-galactose:ceramide galactosyltransferase which is responsible for catalyzing the final step in galactosylceramide synthesis, was exceptionally down-regulated from the early stages of the disease. The increase of glial fibrillary acidic protein (GFAP) mRNA levels preceded morphological evidence of demyelination.

Suppressed UDP‐galactose: Ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain

Suppressed UDP‐galactose: Ceramide galactosyltransferase and myelin protein mRNA in twitcher mouse brain

Twenty five years of the "psychosine hypothesis": a personal perspective of its history and present status.

Twenty five years ago in 1972, a hypothesis was introduced to explain the pathogenetic mechanism underlying the unusual cellular and biochemical characteristics of globoid cell leukodystrophy (Krabbe disease). It postulated that galactosylsphingosine (psychosine), which cannot be degraded due to the underlying genetic defect, is responsible for the very rapid loss of the oligodendrocytes and the consequent paradoxical analytical finding, the lack of accumulation of the primary substrate, galactosylceramide, in patients' brain. It took nearly ten years before the actual accumulation of psychosine was demonstrated in human Krabbe patients and also in the brain of twitcher mice, an equivalent murine mutant. Meanwhile this "psychosine hypothesis" has been extended to Gaucher disease and then to a more general hypothesis encompassing all sphingolipidoses that the "lyso-derivatives" of the primary sphingolipid substrates of the defective enzymes in respective disorders play a key role in their pathogenesis. Some of these extensions not only remain speculative without conclusive factual evidence but may eventually turn out to be an overstretching. This article attempts, from my personal perspective, at tracing historical development of the "psychosine hypothesis" and examining its current status and possible future directions.

IL-6 and TNF α expression in brains of twitcher, quaking and normal mice

Cytokines have been postulated to play a pathogenic role in twitcher mice, which are an animal model of globoid cell leukodystrophy. In particular, TNF α promotes oligodendrocyte and myelin pathology, and IL-6 expression is induced in astrocyte and microglial cultures that have been incubated with TNF α or myelin debris, respectively. It is unknown whether these cytokines are expressed in twitcher mice. The objectives of the present study were to develop an immunohistochemical method to detect TNF α and IL-6 in the mouse CNS, and then utilize this method to identify the cell types expressing these cytokines, and their spatial distribution, in the brains of normal, twitcher and quaking mice. In normal mice, IL-6 was found in ependymal cells, Bergmann glia, in processes that were adjacent or attached to the ventricles or pial surface, and in lightly stained processes in white matter. These processes were identified to belong to astrocytes and microglia. IL-6 staining was dramatically increased in twitcher mice. Astrocytes, with reactive features, and microglia were labeled in the cerebral cortex, basal ganglia, subcortical white matter, pons, medulla and cerebellar white matter. IL-6-positive reactive astrocytes were less abundant in quaking mice than twitcher mice. Cells expressing TNF α were rare or absent in normal and quaking mice. In twitcher mice, TNF α-positive macrophages were present at a lower concentration in cerebral white matter than in the pons and medulla, which have more advanced demyelination. These data demonstrate that pathological events induce the expressions of TNF α and IL-6 in the CNS of twitcher mice.

Expression of major histocompatibility complex class I antigens in the demyelinating twitcher CNS and PNS

The expression of the major histocompatibility complex Class I antigens (Class I) was investigated in the nervous system of twitcher (C57BL/6J-twi), a murine model of Krabbe disease in humans. Class I mRNA expression was low in wild type and in twitcher mice prior to the onset of demyelination. However, immunoreactivity for Class I antigens was demonstrated in the spindle-shaped cells in the sciatic nerve and in ramified microglia, endothelial cells and Bergmann glia. In twitcher, transcription of Class I mRNA increased significantly with the progression of demyelination and Mac-1+ macrophages/microglia express Class I immunoreactivity. Class I immunoreactivity was also found in CD3+ T-cells that were scattered in demyelinating lesions. CD8+ or CD4+ cells were also found in the demyelinating area. The results of this study indicate that immunoreactivity to Class I antigens is detected in certain cells even in the wild-type mice and that Class I expression is enhanced in the twitcher nervous system paralleling the progression of demyelination. Expression of MHC molecules in non-immunological demyelinating disease such as twitcher may suggest a role of Class I molecules in the progression of demyelination. Alternatively, the expression may be a non-specific cellular response to the breakdown of myelin.

Cloning of the Canine GALC cDNA and Identification of the Mutation Causing Globoid Cell Leukodystrophy in West Highland White and Cairn Terriers

Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3′-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A to C change at 473 is the disease-causing mutation. A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectively and will permit the establishment of a colony of dogs for use in therapy trials.

Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease.

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5' end. It has a 2,278-nucleotide sequence including a 2,004-nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG-->TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6-kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3'-untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Animal models for krabbe disease: The twitcher mouse

Animal models for krabbe disease: The twitcher mouse

Correction of the Galactocerebrosidase Deficiency in Globoid Cell Leukodystrophy-Cultured Cells by SL3-3 Retroviral-Mediated Gene Transfer

Globoid cell leukodystrophy (GCL) or Krabbe disease is an autosomal recessive inherited disease caused by the deficiency of galactocerebrosidase, the lysosomal enzyme responsible for the degradation of galactocerebroside, a major component of myelin. An animal model homologue of GCL is the twitcher mouse. In the present work, using novel recombinant retroviruses harboring the SL3-3 LTR, we have been able to stably correct the galactocerebrosidase deficiency in twitcher mouse TM-2 cells and in primary human fibroblasts from a patient with globoid cell leukodystrophy. These results show the possibility of retroviral-mediated gene therapy for the treatment of GCL.

Murine model of genetic demyelinating disease: the twitcher mouse.

Twitcher mouse is an authentic murine model of human genetic demyelinating disease, globoid cell leukodystrophy (GLD), or Krabbe disease. Since its discovery at the Jackson Laboratory (Bar Harbor, ME) this model has been used extensively for the morphological, biochemical-enzymatic studies to clarify pathogenesis and also for therapeutic manipulation of genetic demyelinating disease in humans. As a result of these studies, now we know that (1) GLD is caused by a deficiency of lysosomal enzyme galactosylceramidase, and a toxic metabolite, psychosine, accumulates in the tissue, including the nervous system, damaging myelin forming cells and resulting in secondary demyelination; (2) morphological features of demyelination and associated cellular reactions in demyelination in this mutant are similar to those seen in autoimmune or toxic demyelination; and (3) with enzyme supplementation provided by bone marrow transplantation, remyelination occurs to some extent in demyelinated fibers in both central and peripheral nervous systems of twitcher mouse.

FT-IR microspectroscopic analysis of diseased white matter brain tissues

In situ FT-IR microspectroscopy has allowed spatially resolved interrogation of different parts of brain tissue. In previous work the spectrrscopic features of normal barin tissue were characterized. The white matter, gray matter and basal ganglia were mapped from appropriate peak area measurements from spectra obtained in a grid pattern. Bands prevalent in white matter were mostly associated with the lipid. These included 2927 and 1469 cm-1 due to CH2 as well as carbonyl at 1740 cm-1. Also 1235 and 1085 cm-1 due to phospholipid and galactocerebroside, respectively (Figs 1and2). Localized chemical changes in the white matter as a result of white matter diseases have been studied. This involved the documentation of localized chemical evidence of demyelination in shiverer mice in which the spectra of white matter lacked the marked contrast between it and gray matter exhibited in the white matter of normal mice (Fig. 3).The twitcher mouse, a model of Krabbe’s desease, was also studied. The purpose in this case was to look for a localized build-up of psychosine in the white matter caused by deficiencies in the enzyme responsible for its breakdown under normal conditions.

Proliferative capacity of oligodendrocytes in the demyelinating twitcher spinal cord.

The proliferative capacity of oligodendrocytes was investigated in the spinal white matter of the twitcher mouse, a murine model of a genetic demyelinating disease globoid cell leukodystrophy (GLD), in which degeneration of oligodendrocytes due to metabolic perturbation has been well documented. In normal mice at 30 and 45 days of age, proliferating cells labeled with 5-bromo-2'-deoxyuridine (BrdU) were scarce, and the majority of BrdU-labeled cells did not immunostain with antibodies for oligodendrocytes, astrocytes, or microglia/macrophages. Only a few cells with markers for oligodendrocytes, carbonic anhydrase (CA), or the Pi form of glutathione-S-transferase (Pi), were labeled with BrdU. In the twitcher spinal cord, total numbers of BrdU-labeled cells were almost 6 times that of the normal littermate mice at 30 days of age, and 28 times at 45 days of age. However, this increase was largely due to an increase of cells labeled with F4/80, a marker for the microglia/macrophages. CA or Pi positive cells only constituted less than 10% of all labeled cells. With progression of demyelination from 30-45 days, total numbers of CA positive or Pi positive oligodendrocytes decreased, but percentages of cells double-labeled with BrdU and CA or Pi remained fairly constant. The results indicated that oligodendrocytes proliferated, to some extent, in the twitcher despite the genetic metabolic defect, and their decrease in number with progression of disease was not due to declined proliferation but rather cellular degeneration as the result of an intrinsic metabolic perturbation.

Differentiation between dysmyelination and demyelination using magnetic resonance diffusional anisotropy

Using magnetic resonance (MR) diffusion-weighted method, we examined the optic and the trigeminal nerves of jimpy and twitcher mice, considered to be animal models of Pelizaeus-Merzbacher disease, hypomyelination disorder, and Krabbe disease, demyelination disorder, respectively. In jimpy mice, diffusional anisotropy of optic nerve did not show a significant difference compared to age-matched control mice, suggesting that diffusional anisotropy does exist in absence of multiple layers of myelin sheath. In twitcher mice, diffusional anisotropy was attenuated remarkably in the optic and trigeminal nerves. Loss of axonal straightness on longitudinal section confirmed by electron microscopy appeared to be the principal explanation for it. It is further suggested that this MR diffusion-weighted imaging method enables us to differentiate hypomyelination from demyelination in vivo.

Spacio-temporal progression of demyelination in twitcher mouse: with clinico-pathological correlation.

The twitcher (twi/twi) is an authentic murine model of human globoid cell leukodystrophy (GLD), caused by a deficiency of galactosylceramidase. Similar to human GLD, the twitcher shows progressive deterioration of neurological function and its neuropathology is characterized by a collection of periodic acid-Schift stain (PAS)-positive macrophages in the areas of demyelination. However, there are some differences in the clinico-pathological aspects between human and murine GLD. We investigated the spacio-temporal progression of neuropathology in the twitcher from postnatal day (PND) 10 to 45. No clinical symptoms or neuropathological changes were apparent in twi/twi until PND 15. Generally, infiltration of macrophages, concomitant with myelin degeneration, was recognized in the cerebellar white matter and the brain stem after PND 20, then in cerebral white matter after PND 25, and in cerebral and cerebellar gray matter after PND 30. The demyelination was very severe in the radix of the 8th and the 5th cranial nerves. The neurological symptoms such as tremor, spasticity and cranial nerve dysfunction were well correlated with the progression of pathological changes. Demyelination progressed in an orderly fashion such that myelin degeneration began 10 to 20 days after the commencement of myelination in any of the given nerve fiber tracts. This suggests that there are no significant differences in the metabolism of galactocerebroside in the myelin and myelin-forming cells in individual nerve fiber tracts throughout the murine brain. Over-expression of glial fibrillary acidic protein was already present before the initiation of obvious demyelination.(ABSTRACT TRUNCATED AT 250 WORDS)

Absence of MHC class ii molecules reduces CNS demyelination, microglial/macrophage infiltration, and twitching in murine globoid cell leukodystrophy

Globoid cell leukodystrophy (GILD) is a severe genetic demyelinating disorder with an increased number of la (immune response antigen) positive brain microglia/ macrophages. To assess the role of aberrant la expression in the central nervous system (CNS), twitcher mice, which represent the murine model for GLD, were mated with la −- transgenic mice. Compared with the la + controls, la − twitcher mice showed a profound reduction in the severity of demyelinating lesions correlated with significantly fewer microglia/macrophages. Most importantly, la − twitcher mice showed significantly reduced twitching compared with la + twitcher mice. In contrast with experimental allergic encephalomyelitis (EAE), there was no significant amount of Inflammatory T cell infiltrates, implying that T cells may not play a predominant role In this disease. These findings may have broad therapeutic implications for Alzheimer's disease, Parkinson's disease, and Huntington's disease, which display enhanced la expression in the CNS without obvious T cell Infiltrates.

Neuropathology of twitcher mice: examination by histochemistry, immunohistochemistry, lectin histochemistry and fourier transform infrared microspectroscopy

The twitcher mouse is an authentic animal model of globoid cell leukodystrophy, which is a genetic disease that affects the lysosomal enzyme galactocerebroside β-galactosidase. This enzyme deficiency causes one of its substrates, galactosylsphingosine (psychosine), to accumulate in myelin-forming cells, which eventually results in their death. In the central nervous system, the death of oligodendrocytes is thought to cause a series of secondary pathological changes. In this study, several techniques were utilized to examine the neuropathology of two different brain regions in the twitcher mouse—the hindbrain and the cerebrum. Neuropathological changes were as follows: (1) demyelination was detected in the hindbrain but not in the cerebrum, (2) a high density of periodic acid-Schiff-positive cells were detected in the hindbrain and to a lesser extent in the cerebrum, (3) astrocyte gliosis was pronounced in both the hindbrain and cerebrum, and (4) macrophages were abundant in both the hindbrain and the cerebrum. We found that Periodic acid-Schiff-positive cells, astrocyte gliosis and macrophage infiltration were present in white and gray matter regions of the cerebrum, while they were generally absent from the granule and molecular layers of the cerebellum. In addition to these studies, we utilized the technique of Fourier transform infrared (FT-IR) microspectroscopy to identify the in situ distribution of psychosine in the brains of twitcher mice. Evidence was obtained that indicates a large accumulation of psychosine in the hindbrain, and to a lesser extent in the white matter of the cerebrum in the twitcher mouse, but not the normal mouse. There was no evidence for the accumulation of psychosine in the molecular layer of the cerebellum from the twitcher or normal mouse. Our conclusions are as follows: (1) pathology is more advanced in the hindbrain compared to the cerebrum, which is likely due to the hindbrain becoming myelinated prior to the cerebrum, (2) demyelination is not necessary for the development of secondary pathological changes, (3) pathology is not limited to white matter in the cerebrum, (4) pathology is not present in all brain regions, i.e. the granule and molecular layers of the cerebellum are devoid of pathological changes, and (5) psychosine accumulates in both the cerebrum and hindbrain, but not in the molecular layer of the cerebellum in the twitcher mouse. This study demonstrates that FT-IR microspectroscopy can be used to correlate chemical changes to histopathological changes in brains from twitcher mice, which suggests that FT-IR microspectroscopy may be a useful tool for studies examining other brain diseases.

Progressive dysfunction of twitcher Schwann cells is evaluated better in vitro than in vivo

The twitcher mutant ( twi / twi) is an authentic murine model of globoid cell leukodystrophy, a genetic demyelinating disorder, where normally formed myelin degenerates due to a metabolic perturbation of myelin forming cells. We investigated the rate of Schwann cell proliferation in vivo and in vitro using thymidine autoradiography to evaluate cellular function of homozygous twitcher ( twi / twi), and heterozygous (+ / twi) and normal littermate (+ /+ ) mice. At day 10 prior to onset of demyelination in twi / twi, the thymidine uptake by Schwann cells from sciatic nerves was similar regardless of genotype. In + / twi or + / +, the rate of proliferation in sciatic nerves sharply declined after day 10 and remained low at day 20 and thereafter. In contrast, in twi / twi nerve, in which myelin degeneration became progressively severe after day 10, Schwann cell proliferation persisted. Reflecting the in vivo status of Schwann cells as described by Clemence et al. [2], thymidine uptake in vitro by Schwann cells from older twi / twi mice was transiently higher for the first 24 h in culture. When the rate of proliferation was determined for Schwann cells 1 to 8 days in vitro (DIV), twi / twi Schwann cells proliferated generally less than of + / twi or + / + and the rate progressively declined with age. The results of this study suggest that progressive dysfunction of twi / twi Schwann cells is better demonstrated in vitro than in vivo. We suggest that in vitro kinetic studies would provide useful methodology to evaluate the Schwann cell function in genetic or hereditary neuropathies in humans, in which intrinsic Schwann cell dysfunction is suspected as cause of demyelination and contribute to the understanding of their pathogenesis.

MHC class II antigen expression and T-cell infiltration in the demyelinating CNS and PNS of the twitcher mouse

The expression of the major histocompatibility complex class II antigens (Ia) was investigated in the central and peripheral nervous systems of the twitcher mouse, an authentic murine model of globoid cell leukodystrophy (Krabbe disease) in humans. In this mutant, demyelination is noted as early as postnatal day 10 in the peripheral nerve and day 20 in the spinal cord. Emergence of Ia antigen expressing cells (Ia + cells) was largely coincident with the initiation of demyelination, suggesting the importance of local factors for the induction of Ia antigens. Ia + cells gradually increased in number with the progression of demyelination, but reached a plateau between postnatal days 30 and 40, then rapidly decreased despite continuous demyelination in both central and peripheral nervous systems. The only exception was in the spinal cord where Ia + cells were numerous even at postnatal day 50. The cells expressing L3T4 antigen (L3T4 + cells), which is a mouse CD4 antigen, and the interleukin-2 receptor antigen expressing cells (IL-2R + cells), also appeared in the regions where Ia + cells were observed. L3T4 + cells were still abundant after P45, when Ia + and IL-2R + cells decreased. Combined autoradiographic and immunocytochemical studies demonstrated mitotic activity in a few Ia + cells. However, low labeling indices of Ia + cells in both cerebrum and sciatic nerve suggest that the increase of Ia + cells was largely due to either induction of Ia antigens on local cells or new recruitment of Ia + cells from the circulation. Expression of Ia antigens associated with an emergence of L3T4 + or IL-2R + cells in the demyelinating lesions may indicate involvement of immunological responses in the pathogenesis of this metabolic demyelinating disorder. Alternatively, these apparent immunological phenomena may be tentative and non-specific local reactions to degenerating tissue components such as myelin. The mechanism(s) regulating the expression of Ia antigens in this genetic metabolic demyelinating disease is yet to be determined.

Hematopoietic cell transplantation in the twitcher mouse. The effects of pretransplant conditioning with graded doses of busulfan.

The effects of congenic hematopoietic cell transplantation (HCT; transplantation of bone marrow and spleen cells) after graded doses of busulfan (BU), a myeloablative but nonimmunosuppressive alkylating agent, were evaluated in the twitcher mouse model of human galactosylceramidase deficiency, a demyelinating sphingolipid storage disease. C57BL/6 twitcher mice (immunophenotype Ly-5.1) were given 10 to 50 mg/kg of BU or total-body irradiation (9.0 Gy) at age nine days and HCT from congenic Ly-5.2 donors 24 hr later. The 30-day post-HCT survival, an indicator of tolerance of the preparative regimen, was at least 83% in twitcher mice given 45 mg/kg or less of BU, was 50% in recipients of 50 mg/kg BU and 75% in TBI-conditioned twitchers. The lifespan of twitcher mice given HCT after 10 or 20 mg/kg of BU was similar to that of untreated twitchers (median survival, 42 days; range, 30-47). In contrast, mice transplanted after 35 to 50 mg/kg of BU had significantly prolonged survival (median, 82 days; range, 56-208) and stabilization of hindlimb paralysis, similar to TBI-conditioned recipients. Post-HCT repopulation by donor Ly-5.2 cells was determined by flow cytometry. Thirty days after HCT, only 11-15% of lymphohematopoietic cells in blood, bone marrow, and spleens were of Ly-5.2 donor origin in twitcher mice transplanted after 10 mg/kg of BU but 60-80% were of Ly-5.2 donor origin in mice transplanted after higher doses of BU. These levels further increased to 70-90% by 90 days after HCT, comparable to that seen after TBI. Levels of galactosylceramidase in livers, spleens, and brains of twitchers transplanted after 35-50 mg/kg of BU or after TBI increased to 30-116% of normal control values by 90 days after HCT. Conditioning for HCT with as little as 35 mg/kg of BU provides minimal peritransplant mortality, rapid and sustained establishment of donor lymphohematopoiesis, replacement of lysosomal hydrolase, and prolonged survival in this murine model of human sphingolipidosis.

Proliferation of microglia/macrophages in the demyelinating CNS and PNS of twitcher mouse

In demyelinating lesions in the central and peripheral nervous systems of twitcher mouse, a murine model of globoid cell leukodystrophy, marked increases of [ 3H]thymidine-labeled elements including Mac-1 immunopositive (Mac-1 +) microglia/macrophages was observed. Their proliferative activities were already pronounced at the postnatal day (P) 20, an early stage of demyelination, peaked at P30 and then declined at P45, at the terminal stage of the disease. Many of the [ 3H]thymidine-labeled Mac-1 + cells had morphological features of ramified microglia. Macrophage-like Mac-1 + cells were less frequently labeled. The results of this study showed that (1) microglia/macrophages proliferated in the genetic demyelinating lesions, (2) many dividing Mac-1 + cells had the shape of ramified microglia and (3) the rate of proliferation declined in later stages when reactive microglia/macrophages were abundant in the lesions. The temporal events of this study suggest that signal(s) from the degenerating myelin or myelin-forming cellslate(s) cellular proliferation and that ramified microglia were one of the principal-dividing elements in the lesion. The biological mechanisms underlying the decline of the proliferative activity of microglia require further studies.