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Blood, 1 October 2005, Vol. 106, No. 7, pp. 2259-2268. Prepublished online as a Blood First Edition Paper on June 7, 2005; DOI 10.1182/blood-2005-03-1189.
CHEMOKINES Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathologyFrom the Department of Genetics and Tumor Cell Biology, Saint Jude Children's Research Hospital, Memphis, TN.
Bone marrow cells (BMCs) could correct some pathologic conditions of the central nervous system (CNS) if these cells would effectively repopulate the brain. One such condition is GM1-gangliosidosis, a neurodegenerative glycosphingolipidosis due to deficiency of lysosomal  -galactosidase (-gal). In this disease, abnormal build up of GM1-ganglioside in the endoplasmic reticulum of brain cells results in calcium imbalance, induction of an unfolded protein response (UPR), and neuronal apoptosis. These processes are accompanied by the activation/proliferation of microglia and the production of inflammatory cytokines. Here we demonstrate that local neuroinflammation promotes the selective activation of chemokines, such as stromal-cell-derived factor 1 (SDF-1), macrophage inflammatory protein 1- (MIP-1), and MIP-1, which chemoattract genetically modified BMCs into the CNS. Mice that underwent bone marrow transplantation showed increased -gal activity in different brain regions and reduced lysosomal storage. Decreased production of chemokines and effectors of the UPR as well as restoration of neurologic functions accompanied this phenotypic reversion. Our results suggest that -gal-expressing bone marrow (BM)-derived cells selectively migrate to the CNS under a gradient of chemokines and become a source of correcting enzyme to deficient neurons. Thus, a disease condition such as GM1-gangliosidosis, which is characterized by neurodegeneration and neuroinflammation, may influence the response of the CNS to ex vivo gene therapy.
Chemokines comprise a family of about 50 small protein ligands that together with their cognate receptors control leukocyte trafficking during immune surveillance and inflammatory-cell recruitment during host defense.1-3 Besides their known function in the immune system, these molecules have also been implicated in the maintenance of central nervous system (CNS) homeostasis and as a mediator of neuroinflammation.4-8 Two types of interactions primarily control the activity of chemokines in the CNS. The first involves chemokine immobilization by glycosaminoglycans (GAGs), which play a role in the formation of chemokine gradients and trigger localization of leukocyte subpopulations to the site of infection or injury, present at the endothelial surface and the extracellular matrix.9,10 The other interaction entails the tight binding of chemokines to their G-protein-coupled receptors on the surface of leukocytes; this binding activates integrins and promotes their adhesion to the endothelium, followed by their penetration across the endothelial layer into the CNS perivascular space.11-15 In the CNS, chemokines are expressed constitutively at low or negligible levels in astrocytes and microglia, but their expression is rapidly induced by neuroinflammatory conditions16-18 and is mediated by several proinflammatory cytokines (eg, tumor necrosis factor [TNF-], interferon  [IFN-], and tumor growth factor 1 [TGF-1]).19-22 CNS inflammation occurs in several neurodegenerative conditions, including those associated with a lysosomal storage disease (LSD),23-24 and is likely responsible for the recruitment of monocytes and macrophages from peripheral blood. In the murine model of the LSD, GM2-gangliosidosis recruitment of macrophages into the CNS is mediated by the chemokine macrophage inflammatory protein 1- (MIP-1) and has been implicated in the pathogenesis of this disease.25
Genetic defects that alter The mouse model of GM1-gangliosidosis recapitulates the early-onset forms of the disease27 (ie, GM1 and GA1 progressively accumulate in nearly every neuron). Tremor, ataxia, abnormal gait, and gradual deterioration of motor function culminate in the paralysis of the hind limbs.27 Using this model, we recently found that GM1 accumulation in the endoplasmic reticulum (ER) of brain cells and depletion of ER calcium stores stimulate an unfolded protein response (UPR), which in turn, causes neuronal apoptosis.28 The latter process elicits a neuroinflammatory response associated with the activation of inflammatory markers and cytokines, which in turn activate microglia and macrophages at the site of apoptosis.29
Here we demonstrate that neurodegeneration and neuroinflammation in GM1-gangliosidosis mice cause the up-regulation of selected chemokines in specific brain regions. This phenomenon promotes the migration/infiltration of bone marrow cells (BMCs) genetically modified to overexpress
Animals FVB Glb1+/+ mice (2-6 months) were used as BMC donors, and Glb1-/- mice (3-4 weeks) of the same background were used as recipients.27 All procedures were done in accordance with the US Public Health Service Policy on the Human Care and Use of Laboratory Animals. Real-time PCR
Stromal-cell-derived factor 1 ELISA
SDF-1 RNAse protection assay
RNA from midbrain, brain stem, and cerebellum were purified and analyzed for White blood cell count in cerebrospinal fluid and transmigration assay Cerebrospinal fluid (CSF) was collected from Glb1+/+ mice and Glb1-/- mice, cytospun onto a slide, and stained with May-Grünwald-Giemsa for total cell counts. CSF was collected from the cisternea magna of the subarachnoid space (fourth ventricle) and peripheral blood by cardiac puncture. For the transmigration assay, leukocytes from peripheral blood were counted on a hemocytometer. Filtered chemotaxis medium containing the CSF (35 µL) was placed in the lower transwell chamber. Blood cells (1 x 106 cells/100 µL) were placed in the upper chamber; chambers were separated by a 3-µm pore filter (Falcon; Becton Dickinson, San Jose, CA). After incubation for 4 hours at 37°C in 5% CO2 atmosphere, the cells that had migrated to the lower chamber were recovered, cytospun onto a microscope slide, stained with May-Grünwald-Giemsa, and counted. Different cell types were distinguished according to their morphologic characteristics and their relative number was calculated. Vector construction Human Glb1 cDNA was inserted 5' of the internal ribosomal entry site in the MSCV-GFP vector.30,31 An ecotrophic GP+ E86-derived producer-cell line was used. Transduction of BMCs and BMT
BMCs were isolated from femurs and tibias of Glb1+/+ mice and lysed with Gey solution, and nucleated cells were counted on a hemocytometer. BMCs (2 x 106 cells/mL) were prestimulated in Iscoves medium containing 20 ng/mL mouse interleukin-3 (IL-3), 50 ng/mL human IL-6, and mouse stem-cell factor (R&D Systems) for 48 hours and then transduced with a high titer of MSCV-
GFP expression in peripheral-blood samples and
Blood samples (20 µL) were obtained 1, 3, 6, and 9 months after BMT. Fluorescence activity cell sorting (FACS) analyses of erythrocyte, platelet, and lymphocyte content were done. The level of Immunohistochemistry
Mouse brains were either formalin-fixed and paraffin-embedded or flash-frozen in liquid nitrogen and then sectioned for immunohistochemistry. The following primary antibodies and dilutions were used: rabbit anti-human
Thin-layer chromatography of brain ganglioside fractions Total lipids were extracted from mouse brain homogenates. The lipid extract corresponding to 100 µg protein was separated on a thin-layer chromatography plate (Silica gel 60 Å; Whatman, Clifton, NJ). Gangliosides were visualized by resorcinol spray and heating. GM1-ganglioside (TRB Pharma, Campinas, Brazil) was used as the standard. Behavioral testing Three tests were used to ascertain neurologic function: motor coordination and balance, open-field activity, and walking pattern. Glb1+/+ mice, Glb1-/- mice, and Glb1-/- mice that underwent transplantation were tested during the same session to minimize variability. The ability to maintain balance was tested on a standard rotarod apparatus (Stoelting, Wood Dale, IL).33 Motor and exploratory behaviors were assessed in an acrylic open arena.34 Gait abnormalities were determined by painting the mouse paws with nontoxic, washable paint and then placing the mice in a corridor (10 cm x 10.5 cm x 81.5 cm) lined with white paper.35 Statistical analyses Data are expressed as mean ± SD and were evaluated by a one-way analysis of variance (ANOVA) followed by the Tukey test for pairwise comparisons. The analyses were performed using the SPSS/PC+ statistical software (SPSS, Chicago, IL) and the mean differences were considered statistically significant when P values were less than .05.
Up-regulation of chemokines in the brains of Glb1-/- mice
We first tested the cytokine-mediated up-regulation of SDF-1, one of the most potent chemoattractant molecules whose expression is induced in several neurodegenerative and neuroinflammatory conditions.36 Real-time PCR and ELISA analyses of Glb1-/- brain regions revealed up-regulation of SDF-1 Increased white blood cell count in the cerebrospinal fluid compartment of Glb1-/- mice Under normal conditions, some blood-born cells transiently enter the CSF and are released back into the circulation, unless specific stimuli provoke their retention. During CNS inflammation, T cells and monocytes are retained in the subarachnoid space via signals released by the chemokine receptors.38 These migrating cells scavenge invaders and necrotizing tissue debris, thereby helping to repair tissue damage and promote healing.39
At 3 months, the CSF of Glb1-/- mice contained more white blood cells (WBCs) than did that of Glb1+/+ mice. Instead, the number of WBCs in the CSF of PPCA-/- was similar to that of wild-type mice (Figure 1E). Therefore, leukocyte retention in the CSF compartment of Glb1-/- mice is apparently disease specific; this phenomenon usually results from synergistic effects of multiple chemokines. To verify these results, we performed transmigration assays using CSF from 3-month-old Glb1-/- mice and Glb1+/+ mice as a source of chemoattractants and freshly prepared peripheral WBCs as target cells. The number of WBCs that migrated toward the Glb1-/- CSF was double the number that migrated toward Glb1+/+ CSF (Figure 1F). May-Grünwald-Giemsa staining of migrating cells revealed that most transmigrated cells were monocytes (78%). The number of other WBCs (lymphocytes, 12%; neutrophils, 10%) did not differ substantially between Glb1-/- and Glb1+/+ CSF samples. Progeny of retrovirally transduced BMCs efficiently migrate into the CNS of Glb1-/- recipients
We used an ex vivo gene therapy approach to investigate whether increased chemokine expression in Glb1-/- mice would facilitate the recruitment of BMCs into the CNS and whether this approach could be a potential treatment for GM1-gangliosidosis. Wild-type BMCs were transduced with either MSCV-
GFP expression in peripheral blood was paralleled by an increased level of Transplanted BMCs correct neuropathologic features in Glb1-/- mice
After both primary and secondary BMTs,
Enzymatic correction in the CNS reduces GM1 accumulation
To ascertain whether retrovirally transduced BMCs that migrated into the CNS would reverse or retard GM1 accumulation, we measured the amount and distribution of GM1 in different brain regions of the treated mice and compared it with that in age-matched wild-type mice and untreated mice or Glb1-/- mice that underwent mock transplantation (Figure 5). Thin-layer chromatography revealed a clear decrease in the GM1 content in brain stem and cerebellum of treated mice, although the GM1 level in total brain extracts, comprising the hindbrain, midbrain, and forebrain regions, was not substantially different from that in untreated littermates (Figure 5A). The reason for the latter finding was due to the wide heterogeneity of these areas when analyzed together. In fact, immunofluorescence staining of brain sections with anti-GM1 antibody demonstrated a clear reduction of storage in other brain nuclei, such as the hippocampus, of treated Glb1-/- mice compared with mice that underwent mock transplantation (Figure 5B). The decrease in GM1 levels in each brain region correlated with the relative increase in Ex vivo gene therapy reduces the neuroinflammatory response in Glb1-/- mice
Evidence of successful correction of CNS pathology was further provided by the results of RNAse protection assays, which showed decreased levels of chemokine expression at 3 and 4 months after BMT, a time that corresponded to peak expression of several chemokines in untreated mutant mice (Figure 6A-B). The chemokines that displayed the highest activation in Glb1-/- mice (ie, MIP-1
Immunohistochemical labeling with anti-SDF-1 antibody confirmed that considerably fewer SDF-1+ cells were present in the thalamus of treated Glb1-/- mice than in mutant mice that underwent mock transplantation (Figure 7A). SDF1-expressing cells were identified as astrocytes by coimmunostaining with anti-GFAP antibody (available as Supplemental Figure S3). Thus, transplantation of retrovirally transduced BMCs expressing the therapeutic enzyme appeared to reverse chemokine activation. The neuroinflammatory response was also reduced, as demonstrated by fewer F4/80+ microglia in the thalamus (shown as example) of treated mice, while Glb1-/- mice that underwent mock transplantation still exhibited numerous spheroid microglia, a morphologic characteristic of their activated status40 (Figure 7A). Using an anti-GFAP antibody, we also found that treated Glb1-/- mice had greatly attenuated astrogliosis particularly in the thalamus (Figure 7A), which is one of the brain nuclei most affected by this phenotypic abnormality in GM1-gangliosidosis mice.29 Overall, these findings indicate decreased neuroinflammation in the treated Glb1-/- mice.
Ex vivo gene therapy arrests neurodegeneration in Glb1-/- mice Accumulation of GM1 in the ER of Glb1-/- neurons activates a UPR that ultimately results in neuronal apoptosis mediated by the proapoptotic proteins CHOP (CCAAT/enhancer-binding protein [C/EBP]-homologous protein) and caspase-12.28 We therefore tested whether the decreased GM1 content in the brains of treated Glb1-/- mice would affect the expression of these UPR effectors. Real-time PCR analyses of brain regions and spinal cord of treated Glb1-/- mice showed that the level of CHOP mRNA was practically normalized, and that of caspase-12 was drastically reduced 3 months after transplantation (Figure 7B-C). Neuromotor abilities progressively worsen in Glb1-/- mice and culminate in ataxia, tremor, and total paralysis of the hind limbs.27 Behavioral testing demonstrated that Glb1-/- mice have an impaired walking pattern: their gait was slow, and they tended to walk in small, labored, uncoordinated movements. Stride strength was significantly reduced, and the paw print length was increased. In contrast, long-term-treated Glb1-/- mice walked faster, with increased stride length and improved coordination. On a rotarod test of balance, the performance of treated Glb1-/- mice was significantly better than that of untreated mutant mice (Figure 7D). Also in an open-field test of locomotor and exploratory activity, treated mice were significantly more active than untreated mutant mice, and the treated mice left the center square of the open field significantly quicker (Figure 7E). In addition, at 6 months after BMT, treated mice showed increased rearing behavior, which indicated improved strength of the hind limbs.
The CNS is concealed behind the blood-brain barrier (BBB), which impedes the migration of immune cells and the diffusion of plasma proteins,41,42 giving the CNS an immunologically privileged status. The functional integrity of the BBB is altered in inflammatory diseases of the CNS where leukocytes migrate across the BBB and the brain parenchyma16,43 under control of chemical messengers such as proinflammatory cytokines, chemokines, and adhesion molecules.22,44-46 Proinflammatory cytokines stimulate astrocytes, microglia, and endothelial cells to produce chemokines,47 which in turn activate matrix metalloproteinases that degrade extracellular matrix proteins and disrupt the endothelial tight junctions of the microvessels in the brain.48
Here we provide evidence that the neuroinflammation associated with the neurodegenerative condition in GM1-gangliosidosis mice provokes the up-regulation of selected chemokines in specific brain regions. One of these chemokines is SDF-1, a potent chemoattractant for microglia that has been implicated in controlling the formation of glial scars and the restoration of the BBB after injury.49 SDF-1 participates in the recruitment of mononuclear cells into the CNS, and its intrathecal production is elevated in several neuroinflammatory diseases.50 We have also detected activation of numerous The up-regulation of chemokines in the Glb1-/- mice suggests that this disease condition could create a microenvironment within the CNS that would favor the response to ex vivo gene therapy. Ex vivo gene therapy using virally transduced BMCs expressing the therapeutic enzyme is potentially feasible for treating LSDs, because engrafted BMCs can infiltrate and repopulate organs, including the CNS,54 and cross-correct enzyme-deficient cells. This procedure has been implemented with promising results in other animal models of LSDs.23,55-58 It is becoming increasingly clear, however, that individual LSDs respond differently to the same therapeutic paradigm, most likely because of the nature of the enzyme defect, the accumulated products, and their combined effect on cell metabolism.
Animals that received primary and secondary transplants of marked BMCs showed persistent GFP expression in peripheral blood and increased enzyme activity in the systemic organs and the CNS. This finding suggests that gene transfer had efficiently targeted long-term repopulating stem cells. The nuclei most affected by GM1-gangliosidosis,27,28 namely the thalamus, pontine nucleus, cerebellum, and brain stem, were also the sites in which chemokines were up-regulated and the neuronal phenotype was most corrected after transplantation. In these areas, we also found a consistent, elevated number of -gal+ BMCs that also expressed GFP and microglia-specific markers. The restoration of enzyme function in the brain of transplant recipients led to a dramatic decrease in GM1-ganglioside storage and was accompanied by attenuation of the neuroinflammatory response. Most of the disease-activated chemokines, including SDF-1, MIP-1, MIP-1, and IP-10, were down-regulated in response to therapy. Remarkably, the levels of transcription of CHOP and caspase-12, 2 components of the UPR pathways responsible for neuronal apoptosis in Glb1-/- mice,28 were also drastically reduced after BMT. Finally, in the brain of treated mice, -gal-expressing cells persisted long term (up to 9 months after secondary BMT), indicating continuous recruitment of BMCs from circulating precursors. This finding makes it unlikely that radiation-induced tissue damage before BMT was responsible for the recruitment of correcting cells into the CNS. Normalization of the molecular effectors of CNS pathogenesis was paralleled by a strikingly improved gross appearance of the mice at a time when untreated Glb1-/- animals are severely affected by the disease. Tremor, ataxia, rigidity, and inability to walk and eat improved in animals that underwent BMT 6 to 9 months earlier. Overall performance of treated mice improved on behavioral tests assessing motor function, balance, coordination, and exploratory activity. Together, these results indicate that CNS function was, at least in part, rescued by ex vivo gene therapy. It is important to reiterate, however, that not all mice that received transplants experienced the same extent of correction of their neuronal pathology. Differences in neuronal correction may reflect those in the engraftment of HSCs and in stem-cell-specific proviral integration sites, which may influence the transcriptional activity of the provirus.59,60 Curiously, the BMCs with higher transduction efficiency before transplantation usually engrafted better and persisted longer after transplantation into Glb1-/- mice.
These studies underscore the crucial role of chemokine upregulation in the CNS in response to BMC-mediated therapy of neurodegenerative and neuroinflammatory conditions associated with GM1-gangliosidosis. Since PPCA-/- mice did not show altered levels of chemokines in their CNS, this response seems to be characteristic of only some LSDs and underlines differences in pathogenesis that may ultimately impact the efficacy of therapy. Other glycolipid storage diseases, such as Krabbe disease and GM2-gangliosidosis, also involve up-regulation of these molecular effectors.23-25 In the Twitcher mouse, preferential recruitment of hematopoietic cells to the demyelinating areas of the affected brain is most likely caused by activation of MCP-1 and IL-10; in the GM2-gangliosidosis mouse, time-dependent up-regulation of MIP-1 We observed the most extensive improvement of the disease phenotype in treated Glb1-/- mice at the same time points and brain regions that develop the highest levels of chemokine upregulation, namely at 3 to 4 months after transplantation. We believe that the pattern of activation of chemokines coincides or immediately follows the time-dependent progression of neuronal-cell death and neuroinflammation characteristic of this disease. These results directly implicate chemokines in the recruitment of therapeutic BMCs into the brain in GM1-gangliosidosis and possibly other LSDs. If the latter assumption is correct, the positive response to normal BMT in GM2-gangliosidosis mice and the efficient engraftment of lentivirus-transduced BMCs in the mouse model of metachromatic leukodystrophy can also be attributed to up-regulation of chemokines at specific CNS sites.25,57 Further study of CNS chemokines and leukocyte recruitment into the CNS may lead to improved therapeutic strategies for GM1-gangliosidosis and similar neurodegenerative disorders.
We wish to thank Gerard Grosveld for continuous support; Taylor Walker and Elida Gomero for animal maintenance; William J. Martin for help in the collection of the CSF and blood samples; Huimin Hu for help with the immunohistochemistry; Ann Marie Hamilton-Easton and Richard Ashmun for FACS analyses; Tommaso Nastasi and Erik Bonten for technical suggestions; and Angela McArthur and Charlette Hill for editing and formatting the article. R.S. thanks Roberto Giugliani and Janice Coelho for encouragement and support in preparation of her thesis.
Submitted March 24, 2005; accepted May 23, 2005.
Prepublished online as Blood First Edition Paper, June 7, 2005; DOI 10.1182/blood-2005-03-1189.
Supported by National Institutes of Health (NIH) grant RO1-DK52025, the Cancer Center Support Grant CA 21765, the Assisi Foundation of Memphis, and the American Lebanese Syrian Associated Charities (ALSAC). A.d'A. holds an Endowed Chair in Genetics and Gene Therapy from the Jewelry Charity Fund; R.S. was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq, Brazil, and Funda
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Alessandra d'Azzo, Department of Genetics and Tumor Cell Biology, St Jude Children's Research Hospital, 332 N Lauderdale, Memphis, TN 38105; e-mail: sandra.dazzo{at}stjude.org.
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Abstract
Introduction
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