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Determination of psychosine in GLD thymus. A, The GLD thymus lacks GALC activity . The enzyme was active in wild-type (WT) thymus from embryonic day 16 (E16) to P40. Data are expressed as the mean SE from three thymuses per time point. B, C, LC-MS-MS chromatograms showing the absence of psychosine in wild-type thymus (B) and the presence of the neurotoxin in Twitcher thymus (C). Lactosyl-sphingosine was used as an internal standard (IS). D, Quantification of psychosine during the postnatal development of the wild-type and Twitcher thymuses. Data are expressed as the mean SE from two to three thymuses for each time point per genotype. E, The effect of psychosine on thymocytes survival was studied in vitro using wild-type thymocytes from P15 mice. Cultures were exposed to different amounts of the sphingolipid for 1 d before mitochondrial membrane potential was determined as described. Data are expressed as the mean percentage of cell death SE from two cultures per dose.
Source publication
Lysosomal beta-galactosylceramidase deficiency results in demyelination and inflammation in the nervous system causing the neurological Krabbe disease. In the Twitcher mouse model of this disease, we found that neurological symptoms parallel progressive and severe lymphopenia. Although lymphopoiesis is normal before disease onset, primary and secon...
Citations
... Researchers have also described progressive peripheral lymphopenia in the context of other demyelinating diseases including Krabbe disease, a lysosomal beta-galactosylceramidase deficiency that results in severe demyelination. In a mouse model of this disease, severity of neurological symptoms directly correlated with the extent of lymphopenia 81 . ...
Background
The nervous and immune systems interact in a reciprocal manner, both under physiologic and pathologic conditions. Literature spanning various CNS pathologies including brain tumors, stroke, traumatic brain injury and de-myelinating diseases describes a number of associated systemic immunologic changes, particularly in the T-cell compartment. These immunologic changes include severe T-cell lymphopenia, lymphoid organ contraction, and T-cell sequestration within the bone marrow.
Methods
We performed an in-depth systematic review of the literature and discussed pathologies that involve brain insults and systemic immune derangements.
Conclusions
In this review, we propose that the same immunologic changes hereafter termed ‘systemic immune derangements’, are present across CNS pathologies and may represent a novel, systemic mechanism of immune privilege for the CNS. We further demonstrate that systemic immune derangements are transient when associated with isolated insults such as stroke and TBI but persist in the setting of chronic CNS insults such as brain tumors. Systemic immune derangements have vast implications for informed treatment modalities and outcomes of various neurologic pathologies.
... Reduced expression of miR-219 in twitcher mouse oligodendrocyte precursor cells impeded oligodendrocyte maturation. Transfected miR-219 significantly Motor activity (31)(32)(33)(34)(35) Psychosine levels (29,34,36,37) GALC activity (34,38,39) GALC immunohistochemistry (31)(32)(33) Myelination (34,(39)(40)(41) Neuroinflammation (32)(33)(34)39) NMJ staining (42) Neuroinflammation (33,34) Globoid cells (33,41,43,44) Immune cell counts (29) Nerve Conduction (51) reduced the number of apoptotic oligodendrocytes as well as psychosine levels in the differentiated cells (though not back to wild-type levels) (86). The mechanism of action for miR-219 in reducing psychosine levels is not yet clear. ...
... Reduced expression of miR-219 in twitcher mouse oligodendrocyte precursor cells impeded oligodendrocyte maturation. Transfected miR-219 significantly Motor activity (31)(32)(33)(34)(35) Psychosine levels (29,34,36,37) GALC activity (34,38,39) GALC immunohistochemistry (31)(32)(33) Myelination (34,(39)(40)(41) Neuroinflammation (32)(33)(34)39) NMJ staining (42) Neuroinflammation (33,34) Globoid cells (33,41,43,44) Immune cell counts (29) Nerve Conduction (51) reduced the number of apoptotic oligodendrocytes as well as psychosine levels in the differentiated cells (though not back to wild-type levels) (86). The mechanism of action for miR-219 in reducing psychosine levels is not yet clear. ...
... Psychosine-mediated loss of oligodendrocytes and Schwann cells leads to widespread demyelination within both the CNS and PNS, and impacts non-neuronal organs as well. For example, demyelination in the spinal cord leads to thymic atrophy, with significant subsequent decreases in the number of CD4 and CD8 T cells in the Twitcher mice (29), though the clinical importance of this is unclear as the number of T lymphocytes in early or late onset KD in humans have not yet been investigated. Recent research suggests that psychosine's impact on demyelination may also proceed through pathways that are independent of oligodendrocytes and Schwann cells. ...
Krabbe Disease (KD) is an autosomal metabolic disorder that affects both the central and peripheral nervous systems. It is caused by a functional deficiency of the lysosomal enzyme, galactocerebrosidase (GALC), resulting in an accumulation of the toxic metabolite, psychosine. Psychosine accumulation affects many different cellular pathways, leading to severe demyelination. Although there is currently no effective therapy for Krabbe disease, recent gene therapy-based approaches in animal models have indicated a promising outlook for clinical treatment. This review highlights recent findings in the pathogenesis of Krabbe disease, and evaluates AAV-based gene therapy as a promising strategy for treating this devastating pediatric disease.
... Psychosine can accumulate in lipid rafts of myelinating glia in brains and nerves very early in the disease process (White et al., 2009). In fact, psychosine can and does accumulate in a wide range of cell types in KD mice including blood cells and thymus cells (Chuang et al., 2013;Galbiati et al., 2007;Zhu, Lopez-Rosas, Qiu, Van Breemen, & Bongarzone, 2012). An emerging body of work also suggests that psychosine can accumulate in lipid raft organization of neurons and directly induce neuronal apoptosis (Castelvetri et al., 2011;Teixeira et al., 2014) or dysfunction (Castelvetri et al., 2011;White et al., 2009;White et al., 2011), and axonal degeneration due to abnormal neurofilament phosphorylation (Cantuti-Castelvetri et al., 2012), fast axonal transport (Cantuti Castelvetri et al., 2013), and neuromuscular junction function (Cantuti-Castelvetri et al., 2015). ...
Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a lysosomal storage disorder causing extensive demyelination in the central and peripheral nervous systems. GLD is caused by loss‐of‐function mutations in the lysosomal hydrolase, galactosylceramidase (GALC), which catabolizes the myelin sphingolipid galactosylceramide. The pathophysiology of GLD is complex and reflects the expression of GALC in a number of glial and neural cell types in both the central and peripheral nervous systems (CNS and PNS), as well as leukocytes and kidney in the periphery. Over the years, GLD has garnered a wide range of scientific and medical interests, especially as a model system to study gene therapy and novel preclinical therapeutic approaches to treat the spontaneous murine model for GLD. Here, we review recent findings in the field of Krabbe disease, with particular emphasis on novel aspects of GALC physiology, GLD pathophysiology, and therapeutic strategies. GALC catabolizes GalCer. GLD is due to conversion of GalCer to psychosine and poor myelin clearance. Loss of GALC is not corrected by transplanted healthy cells. Hematopoietic stem cell therapy provides healthy macrophages that clear myelin debris.
... Twi mice are indistinguishable from wild-type littermates until PND20-21 and have similar immune response capability as wild-type mice. 48 Neurologic features including tremors and ataxic gait appear around day 20. 49 Evidence of hypomyelination in the PNS also has been reported around PND15-20. ...
Introduction:
Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone.
Methods:
Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 1013 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes.
Results:
When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice.
Conclusion:
Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT.
... We first analyzed Cd11b + myeloid cells from the spleen ( Figure S5A), a primary lymphoid organ that serves as a reservoir for circulating monocytes and resident macrophages (Swirski et al., 2009). Consistent with previous reports (Galbiati et al., 2007), Galc KO spleens were smaller than WT littermates ( Figure S5B), previously attributed to autonomic dysfunction. Interestingly, Galc SC cKO spleens were instead larger, suggesting a CNS-dependent atrophic mechanism in GLD. ...
... A fraction of PBS-homogenate was refrozen and shipped to for analysis by collaborators X.H. and M.G. or intact frozen nerves were sent to D.N. and E.R.B. For the latter, psychosine was quantified according to (Galbiati et al., 2007). ...
Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory “globoid” reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.
... Here, we report for the first time that significant alterations occur in BM SECs of symptomatic twitcher mice at P36, suggesting that such alterations may contribute to HSCT failure in symptomatic KD patients. In addition, our observations may explain the decrease in BM and blood cellularity observed in symptomatic twitcher mice, characterized by a reduced number of BM B220 + B-lymphocytes, SCA-1 + hematopoietic stem cells and GR-1 + granulocytes paralleled by a normal number of Ter119 + erythroid progenitors and peripheral red blood cells [24]. These findings pave the way to further studies aimed at investigating the BM microvasculature in presymptomatic twitcher mice. ...
... Notably, a lower rate of HSC engraftment has been observed in homozygous twitcher P8 recipients compared to heterozygous littermates [13], pointing to the presence of possible defects in the BM microvasculature also in presymptomatic animals. Further studies will be required to assess the impact of GALC deficiency on BM cell types other than SECs whose alteration may contribute to the decreased functionality of the hematopoietic niche [13,24]. ...
Krabbe disease (KD) is an autosomal recessive sphingolipidosis caused by the deficiency of the lysosomal hydrolase β-galactosylceramidase (GALC). Oligodendroglia degeneration and demyelination of the nervous system lead to neurological dysfunctions which are usually lethal by two years of age. At present, the only clinical treatment with any proven efficacy is hematopoietic stem-cell transplantation, which is more effective when administered in the neonatal period to presymptomatic recipients. Bone marrow (BM) sinusoidal endothelial cells (SECs) play a pivotal role in stem cell engraftment and reconstitution of hematopoiesis. Previous observations had shown significant alterations of microvascular endothelial cells in the brain of KD patients and in Galc mutant twitcher mice, an authentic model of the disease. In the present study, we investigated the vascular component of the BM in the femurs of symptomatic homozygous twitcher mice at postnatal day P36. Histological, immunohistochemical, and two-photon microscopy imaging analyses revealed the presence of significant alterations of the diaphyseal BM vasculature, characterized by enlarged, discontinuous, and hemorrhagic SECs that express the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2) but lack platelet/endothelial cell adhesion molecule-1 (CD31) expression. In addition, computer-aided image analysis indicates that twitcher CD31−/VEGFR2+ SECs show a significant increase in lumen size and in the number and size of endothelial gaps compared to BM SECs of wild type littermates. These results suggest that morphofunctional defects in the BM vascular niche may contribute to the limited therapeutic efficacy of hematopoietic stem-cell transplantation in KD patients at symptomatic stages of the disease.
... Beta-adrenergic receptor expression is very limited on immature thymocytes, but increases as thymocytes mature [69,70], while macrophages in the subcapsular cortex and cortico-medullary junction express β2-adrenergic receptors [67]. Numerous experimental and clinical studies support the idea that catecholamines play a role in thymus activity and lymphocyte output [71][72][73][74][75]. Sustained β-adrenergic receptor blockade with propranolol was found to increase both thymocyte proliferation and apoptosis, and to disturb thymocyte differentiation, without altering the relative proportion of circulating CD4+ and CD8+ lymphocytes [75,76]. ...
Although the immune and nervous systems have long been considered independent biological systems, they turn out to mingle and interact extensively. The present review summarizes recent insights into the neural pathways activated by and involved in infection-induced inflammation and discusses potential clinical applications. The simplest activation concerns a reflex action within C-fibers leading to neurogenic inflammation. Low concentrations of pro-inflammatory cytokines or bacterial fragments may also act on these afferent nerve fibers to signal the central nervous system and bring about early fever, hyperalgesia and sickness behavior. In the brain, the preoptic area and the paraventricular hypothalamus are part of a neuronal network mediating sympathetic activation underlying fever while brainstem circuits play a role in the reduction of food intake after systemic exposure to bacterial fragments. A vagally-mediated anti-inflammatory reflex mechanism has been proposed and, in turn, questioned because the major immune organs driving inflammation, such as the spleen, are not innervated by vagal efferent fibers. On the contrary, sympathetic nerves do innervate these organs and modulate immune cell responses, production of inflammatory mediators and bacterial dissemination. Noradrenaline, which is both released by these fibers and often administered during sepsis, along with adrenaline, may exert pro-inflammatory actions through the stimulation of β1 adrenergic receptors, as antagonists of this receptor have been shown to exert anti-inflammatory effects in experimental sepsis.
... The temporal and spatial distribution of these uncorrec-ted cell types as they populate the nervous system could explain the differences in symptom presentation of treated animals compared to uncorrected TWI. While most of the gross motor function appeared to be well protected long term, the cause of death in these animals may be due to dysfunction in the autonomic system or due to immune complications, as previously shown to occur in TWI 42 and supported by EM analysis that found unmyelinated Remak bundles to be poorly protected in TWI treated with AAV or AAV + BMT. ...
We report a global AAV9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe’s disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long term protection of demyelination, neuroinflammation, and motor function. Bone-marrow transplantation, performed in this protocol without immunosuppressive pre-conditioning added minimal benefits to the AAV9-gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving near complete correction of GALC’s metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.
... Samples were extracted by methanol with acetic acid 0.5% (v/v) upon shaking for 1h at room temperature, centrifugation at 15,000 g for 10 min, and the supernatant was collected and analyzed using liquid chromatography tandem mass spectrometry as described previously [15]. Briefly, positive ion electrospray tandem mass spectrometry with selected reaction monitoring was performed using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer equipped with a Shimadzu Nexera UHPLC system. ...
In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe's disease. A model of lipid raft disruption helped explaining psychosine's effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe's disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine's toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders. © 2017 D'Auria et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... Studies with twi mice have shown increased expression of chemoattractant protein (e.g., monocyte chemoattractant protein-1 [MCP-1]) in the brain and increased infiltration of hematogenouslineage cells into the CNS (Wu et al., 2000), suggesting that there are interactions between CNS and PNS immune systems in KD/twi pathophysiology. GALC deficiency in twi mice was also reported to induce functional impairment of hematopoietic stem/progenitor cells , autonomic denervation of lymphoid organs, and thus irreversible thymic atrophy (Galbiati et al., 2007) and altered immune competence. PSY is present in nanomolar concentrations in blood and thymus (Galbiati et al., 2007;Zhu et al., 2012;Chuang et al., 2013) and has been reported to affect survival and inflammatory responses of peripheral blood lymphocytes and peripheral blood mononuclear cells directly (Formichi et al., 2007). ...
... GALC deficiency in twi mice was also reported to induce functional impairment of hematopoietic stem/progenitor cells , autonomic denervation of lymphoid organs, and thus irreversible thymic atrophy (Galbiati et al., 2007) and altered immune competence. PSY is present in nanomolar concentrations in blood and thymus (Galbiati et al., 2007;Zhu et al., 2012;Chuang et al., 2013) and has been reported to affect survival and inflammatory responses of peripheral blood lymphocytes and peripheral blood mononuclear cells directly (Formichi et al., 2007). Overall, these results indicate that GALC deficiency systemically affects immune/inflammatory responses. ...
... PSY is also known to decrease peripheral nerve conduction (Dolcetta et al., 2005) and to increase neuromuscular junctional abnormalities by activation of caspase and repression of Akt activity (Cantuti-Castelvetri et al., 2015). Galbiati et al. (2007) reported autonomic denervation of lymphoid organs leading to immune atrophy in twi mice. Overall, these results provide an evidence of the role of PSY in neuropathy/axonopathy and neuromuscular and immune dysfunctions. ...
Krabbe's disease (KD; also called globoid cell leukodystrophy) is a genetic disorder involving demyelination of the central (CNS) and peripheral (PNS) nervous systems. The disease may be subdivided into three types, an infantile form, which is the most common and severe; a juvenile form; and a rare adult form. KD is an autosomal recessive disorder caused by a deficiency of galactocerebrosidase activity in lysosomes, leading to accumulation of galactoceramide and neurotoxic galactosylsphingosine (psychosine [PSY]) in macrophages (globoid cells) as well as neural cells, especially in oligodendrocytes and Schwann cells. This ultimately results in damage to myelin in both CNS and PNS with associated morbidity and mortality. Accumulation of PSY, a lysolipid with detergent-like properties, over a threshold level could trigger membrane destabilization, leading to cell lysis. Moreover, subthreshold concentrations of PSY trigger cell signaling pathways that induce oxidative stress, mitochondrial dysfunction, apoptosis, inflammation, endothelial/vascular dysfunctions, and neuronal and axonal damage. From the time the “psychosine hypothesis” was proposed, considerable efforts have been made in search of an effective therapy for lowering PSY load with pharmacological, gene, and stem cell approaches to attenuate PSY-induced neurotoxicity. This Review focuses on the recent advances and prospective research for understanding disease mechanisms and therapeutic approaches for KD.
