Mesenchymal Stem Cells Yield Transient Improvements in Motor Function in an Infant Rhesus Macaque with Severe Early-Onset Krabbe Disease.

Krabbe disease, or globoid cell leukodystrophy, is a rare disorder caused by deficient galactosylceramidase activity and loss of myelin-forming oligodendrocytes, resulting in progressive demyelination and severely impaired motor function. Disease symptoms in humans appear within 3-6 months of age (early infantile) and manifest as marked irritability, spasticity, and seizures. The disease is often fatal by the second year of life, with few effective treatment options. Herein we evaluated the therapeutic potential of mesenchymal stem cells (MSCs) administered intracranially to a 1-month-old rhesus macaque diagnosed with severe early-onset Krabbe disease that displayed neurologic and behavioral symptoms similar to those of human patients. The infant was subjected to physical and neurological behavior examinations and nerve conduction velocity tests to assess efficacy, and outcomes were compared with age-matched normal infants and Krabbe-affected rhesus monkeys with late-onset disease. Changes in major blood lymphocyte populations were also monitored to assess host immune cell responses. MSC administration resulted in transient improvements in coordination, ambulation, cognition, and large motor skills, which correlated with increased peripheral nerve conduction velocities and decreased latencies. Improvements also corresponded to transient increases in peripheral blood lymphocyte counts, but secondary challenge failed to elicit allo-antibody production. Nevertheless, white cell and neutrophil counts showed dramatic increases, and CD20+ B cell counts underwent a precipitous decline at late stages of disease progression. Correlative data linking MSC administration to transient improvements in motor function suggest that MSCs should be evaluated further as an experimental therapy for rare neurodegenerative diseases. Stem Cells Translational Medicine 2017;6:99-109.

Neuroimmune mechanisms in Krabbe's disease.

Neuroinflammation, activation of innate immune components of the nervous system followed by an adaptive immune response, is observed in most leukodystrophies and coincides with white matter pathology, disease progression, and morbidity. Despite this, there is a major gap in our knowledge of the contribution of the immune system to disease phenotype. Inflammation in Krabbe's disease has been considered a secondary effect, resulting from cell-autonomous oligodendroglial cell death or myelin loss resulting from psychosine accumulation. However, recent studies have shown immune activation preceding clinical symptoms and white matter pathology. Moreover, the therapeutic effect underlying hematopoietic stem cell transplantation, the only treatment for Krabbe's disease, has been demonstrated to occur via immunomodulation. This Review highlights recent advances in elaboration of the immune cascade involved in Krabbe's disease. Mechanistic insight into the inflammatory pathways participating in myelin and axon loss or preservation may lead to novel therapeutic approaches for this disorder. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Immunological considerations for treating globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD, or Krabbe's disease) is a severe inherited neurodegenerative disease caused by the lack of a lysosomal enzyme, GALC. The disease has been characterized in humans as well as three naturally occurring animal models, murine, canine, and nonhuman primate. Multiple treatment strategies have been explored for GLD, including enzyme replacement therapy, small-molecule pharmacological approaches, gene therapy, and bone marrow transplant. No single therapeutic approach has proved to be entirely effective, and the reason for this is not well understood. It is unclear whether initiation of a neuroinflammatory cascade in GLD precedes demyelination, a hallmark of the disease, but it does precede overt symptoms. This Review explores what is known about the role of inflammation and the immune response in the progression of GLD as well as how various treatment strategies might interplay with innate and adaptive immune responses involved in GLD. The focus of this Review is on GLD, but these concepts may have relevance for other, related diseases. © 2016 Wiley Periodicals, Inc.

A Conserved Circular Network of Coregulated Lipids Modulates Innate Immune Responses.

Lipid composition affects the biophysical properties of membranes that provide a platform for receptor-mediated cellular signaling. To study the regulatory role of membrane lipid composition, we combined genetic perturbations of sphingolipid metabolism with the quantification of diverse steps in Toll-like receptor (TLR) signaling and mass spectrometry-based lipidomics. Membrane lipid composition was broadly affected by these perturbations, revealing a circular network of coregulated sphingolipids and glycerophospholipids. This evolutionarily conserved network architecture simultaneously reflected membrane lipid metabolism, subcellular localization, and adaptation mechanisms. Integration of the diverse TLR-induced inflammatory phenotypes with changes in lipid abundance assigned distinct functional roles to individual lipid species organized across the network. This functional annotation accurately predicted the inflammatory response of cells derived from patients suffering from lipid storage disorders, based solely on their altered membrane lipid composition. The analytical strategy described here empowers the understanding of higher-level organization of membrane lipid function in diverse biological systems.

Innate immune activation in the pathogenesis of a murine model of globoid cell leukodystrophy.

Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded in vitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.

On the role of natural killer cells in neurodegenerative diseases.

Natural killer (NK) cells exert important immunoregulatory functions by releasing several inflammatory molecules, such as IFN-γ and members of chemokines, which include CCL3/MIP-1α and CCL4/MIP-1ß. These cells also express heptahelical receptors, which are coupled to heterotrimeric G proteins that guide them into inflamed and injured tissues. NK cells have been shown to recognize and destroy transformed cells and virally-infected cells, but their roles in neurodegenerative diseases have not been examined in detail. In this review, I will summarize the effects of NK cells in two neurodegenerative diseases, namely multiple sclerosis and globoid cell leukodystrophy. It is hoped that the knowledge obtained from these diseases may facilitate building rational protocols for treating these and other neurodegenerative or autoimmune diseases using NK cells and drugs that activate them as therapeutic tools.

Development and maturation of invariant NKT cells in the presence of lysosomal engulfment.

A defect in invariant NKT (iNKT) cell selection was hypothesized in lysosomal storage disorders (LSD). Accumulation of glycosphingolipids (GSL) in LSD could influence lipid loading and/or presentation causing entrapment of endogenous ligand(s) within storage bodies or competition of the selecting ligand(s) by stored lipids for CD1d binding. However, when we analyzed the iNKT cell compartment in newly tested LSD animal models that accumulate GSL, glycoaminoglycans or both, we observed a defective iNKT cell selection only in animals affected by multiple sulfatase deficiency, in which a generalized aberrant T-cell development, rather than a pure iNKT defect, was present. Mice with single lysosomal enzyme deficiencies had normal iNKT cell development. Thus, GSL/glycoaminoglycans storage and lysosomal engulfment are not sufficient for affecting iNKT cell development. Rather, lipid ligand(s) or storage compounds, which are affected in those LSD lacking mature iNKT cells, might indeed be relevant for iNKT cell selection.

Stem cell-based strategies for treating pediatric disorders of myelin.

The pediatric leukodystrophies comprise a category of disease manifested by neonatal or childhood deficiencies in myelin production or maintenance; these may be due to hereditary defects in one or more genes critical to the initiation of myelination, as in Pelizaeus-Merzbacher Disease, or to enzymatic deficiencies with aberrant substrate accumulation-related dysfunction, as in the lysosomal storage disorders. Despite differences in both phenotype and natural history, these disorders are all essentially manifested by a profound deterioration in neurological function with age. A congenital deficit in forebrain myelination is also noted in children with the periventricular leukomalacia of cerebral palsy, another major source of neurological morbidity. In light of the wide range of disorders to which congenital hypomyelination and/or postnatal demyelination may contribute, and the relative homogeneity of central oligodendrocytes and their progenitors, the pediatric leukodystrophies may be especially attractive targets for cell-based therapeutic strategies. As a result, glial progenitor cells (GPCs), which can give rise to new myelinogenic oligodendrocytes, have become of great interest as potential therapeutic vectors for the restoration of myelin to the hypomyelinated or dysmyelinated childhood CNS. In addition, by distributing themselves throughout the deficient host neuraxis after perinatal allograft, and giving rise to astrocytes as well as oligodendrocytes, glial progenitors appear to be of potential great utility in rectifying enzymatic deficiencies. In this review, we focus on current efforts to develop the use of isolated human GPCs as transplantable agents both for mediating enzymatic restoration to the enzyme-deficient brain and for therapeutic myelination in the disorders of congenital hypomyelination.

Clinical and immunopathologic alterations in rhesus macaques affected with globoid cell leukodystrophy.

Globoid cell leukodystrophy, or Krabbe's disease, is a severe disorder of the central and peripheral nervous system caused by the absence of galactocerebrosidase (GALC) activity. Herein, we describe the clinical, neuropathological, histochemical, and immunohistological features observed in rhesus macaques affected with Krabbe's disease. Clinical signs included pronounced muscle tremors of head and limbs, difficulty ambulating, ataxia, hypermetria, proprioceptive deficits, and respiratory abnormalities. Histopathologically, all animals presented with evidence of demyelination in the peripheral and central nervous systems and accumulation of mononuclear and multinuclear globoid cells in the cerebral and cerebellar white matter associated with severe gliosis. Using immunohistochemistry and multi-label confocal microscopy, it was determined that globoid cells were CD68+, HAM56+, LN5+, CD163+, IBA-1+, and Glut-5+, suggesting that both peripheral blood-derived monocytes/macrophages and resident parenchymal microglia gave rise to globoid cells. Interestingly, many of the globoid cells and parenchymal microglia with a more ameboid morphology expressed HLA-DR, indicating immune activation. Increased expression of iNOS, TNF-alpha, and IL-1 beta were observed in the affected white matter, colocalizing with globoid cells, activated microglia, and astrocytes. Cytokine mRNA levels revealed markedly increased gene expression of CCL2 in the brain of affected macaques. CCL2-expressing cells were detected throughout the affected white matter, colocalizing with GFAP+ cells and astrocytes. Collectively, these data suggest that dysregulation of monocyte/macrophage/microglia and up-regulation of certain cytokines may contribute to the pathogenesis of Krabbe's disease.

Psychosine-induced apoptosis and cytokine activation in immune peripheral cells of Krabbe patients.

Globoid cell leukodystrophy or Krabbe disease (KD), is a hereditary disorder caused by galactosylceramidase deficiency. Progressive accumulation of psychosine is considered to be the critical pathogenetic mechanism of cell death in the Krabbe brain. Psychosine mechanism of action has not been fully elucidated. It seems to induce apoptosis in oligodendrocytes through a mitochondrial pathway and to up-regulate inflammatory cytokines production resulting in oligodendrocyte loss. Our aim was to evaluate the role of psychosine in apoptotic cell death and inflammatory response in a group of patients affected by KD using peripheral blood lymphocytes (PBLs) and peripheral blood mononuclear cells (PBMCs) as a cellular model. PBLs from KP and healthy controls were exposed to 20 microM psychosine and analysed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy. Our results showed that psychosine induces apoptosis in PBLs through a mitochondrial pathway, but the apoptotic response was quite low especially KP. The role of psychosine in the up-regulation of cytokines (TNFalpha, IL8 and MCP1) has been evaluated by ELISA in PBMCs from KP and controls after stimulation with LPS and phytohemagglutinin. Both in basal condition and after LPS stimulation, cells from KP showed a significant increase in TNF-alpha production, reduced MCP1 levels and no modification in IL8. These results indicate that lymphomonocytes from KP had a basal proinflammatory pattern that was amplified by psychosine. In conclusion, the reduced apoptotic response and the atypical cytokine production observed in our experiments, suggest an involvement of inflammatory pattern in immune peripheral cells of KP.

Reconstitution of lymphocyte subpopulations in children with inherited metabolic storage diseases after haematopoietic cell transplantation.

We prospectively evaluated the reconstitution of lymphocyte subpopulations in nine children with lysosomal diseases who underwent 11 allogeneic haematopoietic cell transplants (HCTs) following CD34(+) immunomagnetic enrichment, limited T-cell addback and in vivo B-cell depletion. Absolute lymphocyte count recovery was slow to cross the 5th percentile, occurring at a median of 10 months after HCT in patients with full chimaerism. Natural killer cells represented up to 90% of the total lymphoid population during the first 3 months. CD4(+) lymphocyte recovery occurred 9-18 months after HCT. In most patients, CD8(+) lymphocyte recovery was slow and comparable with that of CD4(+) lymphocytes. The CD4(+)/CD8(+) ratio normalised by 3-7 months after HCT in 50% of the patients. CD8(+) lymphocyte recovery was enhanced in patients with viral reactivation. Reconstitution of B-lymphocytes was particularly delayed in patients treated with rituximab. Declining chimaerism, rejection and viral reactivation were the most common problems in our series. Because of the unique graft manipulation, the pace of lymphocyte reconstitution was particularly slow, suggesting that these patients are at a significantly increased risk of infections for up to 2 years after HCT.

Allogeneic bone marrow stem cell transplantation following CD34+ immunomagnetic enrichment in patients with inherited metabolic storage diseases.

T-cell depletion is an essential step in reducing the risk of graft-versus-host disease (GVHD) in patients with inherited metabolic storage diseases (IMSD) undergoing hematopoietic stem cell transplantation. This goal can be achieved either by selective removal of T cells or by positive selection of CD34+ cells. Large-scale preparations of purified CD34+ cells from bone marrow products have not been extensively described. We report our results with bone marrow CD34+ cell enrichment using the CliniMACS system in eight children with IMSD. The median recovery of positively selected CD34+ cells was 46.2% with a purity of 97.5%, and a residual T cell content of 0.04 x 10(6). A median of 5.5 x 10(6)/kg of CD34+ cells was infused. All patients engrafted at a median time of 12 days and none of the patients developed GVHD. This method is technically feasible and can be successfully used to transplant children with IMSD.

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.

Expression of immune-related molecules is downregulated in twitcher mice following bone marrow transplantation.

Twitcher (twi/twi) is a murine model of a human genetic demyelinating disease, globoid cell leukodystrophy (Krabbe disease). The affected mice usually die before reaching age 45 days, having demyelination associated with extensive glial activation. The twi/twi mice that receive wild-type bone marrow transplantation (BMT) survive up to 3 times longer with improved pathology. We hypothesize that immune-related molecules such as cytokines and chemokines are partly responsible for the demyelination in twi/twi, and that the decrease in the expression of such molecules following BMT contributes to clinico-pathological improvement. Cells expressing TNF-alpha, MCP-1, and MIP-1beta were conspicuous in the twi/twi CNS accompanied by infiltration of Ia+ and CD8+/CD3- hematogenous cells. These cells decreased gradually after BMT TNF-alpha mRNA and mRNA of C-C chemokine families, including MCP-1, IP-10, MIP-1alpha, MIP-1beta, and RANTES, were upregulated in the twi/twi CNS but downregulated gradually following BMT. In twi/twi that survived to 20 wk of age, cells expressing TNF-alpha, MCP-1, MIP-1beta, Ia, or CD8 were hardly detected and pathology was clearly improved. These results are consistent with the hypothesis that cytokine expression in glial cells contributes (to some extent) to the pathogenesis of demyelinating lesions in the twi/twi mice.

IL-6 deficiency allows for enhanced therapeutic value after bone marrow transplantation across a minor histocompatibility barrier in the twitcher (globoid cell leukodystrophy) mouse.

Bone marrow transplantation (BMT) has therapeutic value for twitcher (globoid cell leukodystrophy) mice, which suffer from a genetic deficiency of the lysosomal enzyme galactosylceramidase that leads to progressive demyelination and early death. Preliminary investigations indicated that a semiallogeneic BMT resulted in graft vs. host disease (GVHD) in twitcher mice but not normal mice. Increased production of the cytokine IL-6 has been demonstrated in twitcher mice, and it has been linked with induction of GVHD. We investigated the effects of BMT in twitcher/IL-6 deficient mice and compared these findings with those from transplanted twitcher and control mice. After a semiallogeneic BMT, 11.4% of controls died within few weeks while the rest survived >100 days without GVHD. In contrast, 85% of the transplanted twitcher mice died by 70 days and 65% developed clinical signs of GVHD, e.g., alopecia and weight loss. In transplanted twitcher/IL-6 deficient mice, only 21% died by Day 70, none had alopecia, and 23% had weight loss. There was no difference in the onset day and severity of twitching between twitcher and twitcher/IL-6 deficient mice after BMT. In transplanted twitcher/IL-6 deficient mice, there was improvement of BBB integrity and a decrease in globoid cell number compared with nontransplanted twitcher/IL-6 deficient mice. In summary, these results demonstrate that an underlying pathology like globoid cell leukodystrophy leads to activation of GVHD responses in a donor-host combination that would not normally induce GVHD. Furthermore, IL-6 seems to play a key role because a deficiency of IL-6 results in a better prognosis.

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.

Infiltration of hematogenous lineage cells into the demyelinating central nervous system of twitcher mice.

Infiltration of hematogenous lineage cells into the central nervous system (CNS) was investigated in the twitcher mouse, a murine model of globoid cell leukodystrophy in human. The hematogenous cells were selectively labeled following intraperitoneal injection of rhodamine isothiocyanate (RhIc). The frequency of detecting Rhlc-labeled cells (Rhlc+ cells) in the twitcher CNS varied with age. RhIc+ cells were hardly detected when injection was made prior to the postnatal day (PND) 30. The number of Rhlc' cells increased thereafter peaked at PND 35-38 and declined drastically at PND 40-45. The majority of RhIc+ cells were distributed in white matter of the CNS that correlated well with the areas of demyelination and of increased microglia/macrophage population described in our earlier studies. Almost all Rhlc+ cells were double-labeled with antibody for Mac-1 and also with MHC class II. Some small cells double-labeled with RhIc and antibodies for CD4, CD8, or IL-2R were also identified. By RT-PCR, the expression of monocyte chemoattractant protein- (MCP-1) mRNA increased drastically at PND 30, peaked at PND 35, and decreased gradually after PND 40. This pattern of mRNA changes correlated well with the dynamic pattern of the infiltration of hematogenous cells into the CNS, suggesting a role of chemokine(s) in the cellular infiltration in the twitcher brain. The expression of IL-10 mRNA also increased gradually. IL-10 is a cytokine inhibitory factor and a major regulator in suppressing the inflammatory response. Thus, our results indicated that hematogenous lineage cells infiltrated in the CNS of twitcher mice, and that MCP-1 and IL-10 may play an important role in regulating the cellular recruitment.

Ouchterlony double immunodiffusion method demonstrates absence of ferritin immunoreactivity in visceral organs from nine patients with Niemann-Pick disease type C.

Ouchterlony double immunodiffusion clearly demonstrated absence of ferritin, the principal iron storage protein, in spleen and/or liver extracts from nine patients with Niemann-Pick disease type C (NPC). The patients died from different clinical forms of this disease of still unknown etiology. The absence of ferritin immunoreactivity was shown using two different antisera raised in rabbits against ferritin from human spleen or liver, organs which predominantly contain light chain subunits (L-ferritin). A diagnostic double immunodiffusion assay of ferritin is, therefore, feasible with small amounts of NPC liver tissue, e.g., needle biopsy specimens. Furthermore, SDS-polyacrylamide gel electrophoresis after Coomassie blue staining revealed an almost complete absence of the L-ferritin protein band in crude spleen heat extracts from two NPC patients. The absence of visceral ferritin in all nine patients studied is suggestive of a biochemical abnormality that is as characteristic as the known impairment of cellular trafficking of LDL-derived cholesterol in this complex lysosomal storage disorder. According to recent data a relationship exists between ferritin-dependent lipid peroxidation and oxidative modification of LDL. We suggest that deficiency of the antioxidant ferritin-whatever the nature of this deficiency might be-could lead to uncontrolled LDL oxidation with subsequent multisubstrate lipidosis in NPC disease.

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

Globoid cell leukodystrophy (GLD) is a severe genetic demyelinating disorder with an increased number of Ia (immune response antigen) positive brain microglia/macrophages. To assess the role of aberrant Ia expression in the central nervous system (CNS), twitcher mice, which represent the murine model for GLD, were mated with Ia- transgenic mice. Compared with the Ia+ controls, Ia- twitcher mice showed a profound reduction in the severity of demyelinating lesions correlated with significantly fewer microglia/macrophages. Most importantly, Ia- twitcher mice showed significantly reduced twitching compared with ia+ 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 Ia expression in the CNS without obvious T cell infiltrates.