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. 2005 Oct 1;106(7):2259-68.
doi: 10.1182/blood-2005-03-1189. Epub 2005 Jun 7.

Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology

Renata Sano  1 , Alessandra Tessitore, Angela Ingrassia, Alessandra d'Azzo
Affiliations

Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology

Renata Sano et al. Blood. .

Abstract

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 G(M1)-gangliosidosis, a neurodegenerative glycosphingolipidosis due to deficiency of lysosomal beta-galactosidase (beta-gal). In this disease, abnormal build up of G(M1)-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-alpha (MIP-1alpha), and MIP-1beta, which chemoattract genetically modified BMCs into the CNS. Mice that underwent bone marrow transplantation showed increased beta-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 beta-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 G(M1)-gangliosidosis, which is characterized by neurodegeneration and neuroinflammation, may influence the response of the CNS to ex vivo gene therapy.

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Figures

Figure 1.
Figure 1.
RNA up-regulation of chemokines in the CNS of Glb1-/- mice. (A) Real-time PCR was performed on total RNA extracted from the cerebellum of Glb1+/+ and Glb1-/- mice at 3 months (formula image) and 4 months (▪) of age. SDF-1α and SDF-1β mRNAs were higher in Glb1-/- mice than in Glb1+/+ mice. The reactions were standardized to the level of GAPDH mRNA. (B) ELISA of SDF-1α protein in Glb1-/- cerebellum extracts showed that relatively more SDF-1α was expressed at 3 months (formula image) than at 4 months (▪) of age. (C-D) RNase protection assay revealed that the β-chemokines RANTES, MIP-1β, MIP-1α, IP-10, MCP-1, and TCA-3 were up-regulated in hindbrain, midbrain, and forebrain regions (HMF); brain stem (BS); cerebellum (CB); and spinal cord of 3- (C) and 4-month-old (D) Glb1-/- mice compared with those of Glb1+/+ controls. The relative levels of RNA induction were normalized against L32 RNA. The amount of chemokines expressed is reported as fold increase over that detected in age-matched Glb1+/+ mice. (E) CSF from 3-month-old Glb1-/- mice contained more white blood cells (WBCs) than did the CSF from PPCA-/- mice or Glb1+/+ littermates. (F) Transmigration assay demonstrated increased monocyte migration toward Glb1-/- CSF than toward Glb1+/+ CSF. Nonspecific cell migration toward serum-free medium was used as a negative control. Data are expressed as mean ± standard deviation (SD).
Figure 2.
Figure 2.
Engraftment of MSCV-β-gal-GFP-transduced BMCs. (A-B) GFP expression in red blood cells (□), platelets (formula image), and WBCs (▪) demonstrated consistent, long-term expression of the transgene as long as 6 months after primary transplantation (1 month, n = 7; 3 months, n = 10; 6 months, n = 6) and as long as 9 months after secondary transplantation (1 month, n = 3; 3 months, n = 3; 6 months, n = 3; 9 months, n = 3). (C-D) Analyses of β-gal activity in systemic tissues of treated mice revealed a significantly higher level of expression of the corrective enzyme after primary (Glb1+/+, n = 7; Glb1-/-, n = 5; 1 month, n = 7; 3 months, n = 8; 6 months, n = 8) and secondary transplantation (Glb1+/+, n = 7; Glb1-/-, n = 3; 1 month, n = 3; 3 months, n = 3; 6 months, n = 3; 9 months, n = 3) compared with Glb1-/- untreated mice. (E) The β-gal activity was higher in the HMF (hindbrain, midbrain, and forebrain), brain stem, cerebellum, and spinal cord of Glb1-/- mice that underwent primary transplantation and in the brainstem, cerebellum, and spinal cord of Glb1-/- mice that underwent secondary transplantation compared with untreated Glb1-/- mice. Data are expressed as mean ± SD; groups were compared by one-way repeated measures analysis of variance (ANOVA). *P < .001 and #P < .05 relative to untreated Glb1-/- littermates at the same age; post hoc Tukey test.
Figure 3.
Figure 3.
Morphologic analyses in the CNS of treated Glb1-/- mice. (A) Cresyl violet-stained tissue sections of the thalamus, cerebellum, and brain stem from a treated Glb1-/- mouse 3 months after transplantation (Glb1-/- BMT) and from age-matched Glb1+/+ and Glb1-/- mice that underwent mock transplantation (β-gal-/- BMTGFP) revealed restoration of tissue morphology in the treated mouse compared with the extensive vacuolation present in the mice that did not undergo transplantation (Glb1-/-) and the Glb1-/- mice that underwent mock transplantation. (B) Immunolabeling of brain stem, cerebellum, and thalamus revealed the presence of numerous β-gal+ cells in Glb1-/- mice 3 months after BMT. The clear punctate staining demonstrated internalization of the corrective enzyme. Immunolabeling with anti-GFP antibody was done in the same tissues to identify cells of hematopoietic origin. (C) Numerous GFP+ cells in various brain regions showed a ramified microglial morphology. Size bars correspond to 25 μm. Images were visualized using an Olympus BX50 microscope equipped with a 40×/0.65 Plan Apochromatic objective lens (Olympus, Melville, NY) and a Three-Shot 11.3 camera (Diagnostic Instruments, Sterling Heights, MI). Images were acquired with Spot Advanced 4.1.1 software (Diagnostic Instruments) and processed with Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA).
Figure 4.
Figure 4.
Identification of BM-derived vector-expressing cells in the CNS of treated Glb1-/- mice. Immunofluorescence analyses were done on single cryostatic sections from the brains of treated Glb1-/- mice. Fluorescent signals from single sections were sequentially acquired and are shown individually and after merging. (A-B) GFP+ cells and β-gal+ cells were identified as microglia by F4/80 immunoreactivity in Glb1-/- mice 3 months after BMT. (C) Overlay of β-gal staining with the neuronal-specific marker NeuN demonstrated colocalization of the 2 signals, a finding that indicates cross-correction of the enzyme-deficient neurons. Size bar corresponds to 50 μm. Image acquisition was performed as described for Figure 3.
Figure 5.
Figure 5.
Attenuation of GM1-ganglioside storage in the brain caused by exogenously delivered β-gal. (A) Thin-layer chromatography of hindbrain, midbrain, and forebrain (HMF), brain stem (BS), and cerebellum (CB) of Glb1+/+, Glb1-/-, and treated Glb1-/- mice 3 months after BMT demonstrated amelioration of GM1-ganglioside storage in the brain stem and cerebellum of the treated mice. The attenuation of GM1-ganglioside accumulation was in agreement with the increment of β-gal activities in those areas (bottom row). GM1-ganglioside was used as a standard. (B) Confocal microscopy with anti-GM1 antibody confirmed less GM1 accumulation in the cerebellum, brain stem, and hippocampus of treated mice (Glb1-/- BMTβ-gal) than in Glb1-/- mice that underwent mock transplantation (Glb1-/- BMTGFP). The arrows indicate cells in which GM1-ganglioside accumulation was most arrested. Size bar corresponds to 50 μm. Image acquisition was performed as described for Figure 3.
Figure 6.
Figure 6.
RNA down-regulation of chemokines in brain regions of Glb1-/- mice. (A-B) RNase protection assays revealed that the β-chemokines RANTES, MIP-1β, MIP-1α, IP-10, MCP-1, and TCA-3 were less activated in the hindbrain, midbrain, and forebrain (HMF); brain stem (BS); cerebellum (CB); and spinal cord of treated Glb1-/- mice (-/-BMT) at 3 (A) and 4 (B) months after transplantation than in those regions of untreated Glb1-/- mice (Glb1-/-). The values were normalized to those observed in Glb1+/+ mice. The relative levels of RNA induction were normalized against L32 RNA. (C) At 3 and 4 months of age, the SDF-1α and SDF-1β mRNA levels in the cerebellum of untreated Glb1-/- mice (SDF-1α, formula image; SDF-1β, □) were higher than those in treated Glb1-/- mice (SDF-1α, ▪; SDF-1β, formula image). (D) The amount of SDF-1α protein in the cerebellum was also lower in treated Glb1-/- mice (▪) than it was in untreated Glb1/- mice (formula image). Data are expressed as mean ± SD.
Figure 7.
Figure 7.
Amelioration of inflammatory response, down-regulation of the UPR effectors, and improvement of neuromotor abilities of Glb1-/- mice after BMT. (A) Immunolabeling using an anti-SDF-1β antibody demonstrated that the level of this chemokine was lower in the thalamus of 6-month-old treated Glb1-/- mice (Glb1-/- BMTβ-gal) than in Glb1-/- mice that underwent mock transplantation (Glb1-/- BMTGFP). Anti-GFAP staining demonstrated attenuation of reactive gliosis in the thalamus of 4-month-old treated Glb1-/- mice. Immunolabeling with anti-F4/80 antibody showed that the proliferation and activation of microglia in the thalamus of 3-month-old treated Glb1-/- mice were reverted to a resting state. Size bar corresponds to 50 μm. (B-C) CHOP and caspase-12 mRNA levels in the hindbrain, midbrain, and forebrain (HMF), brain stem (BS), cerebellum (CB), and spinal cord of treated Glb1-/- mice (▪) were restored to levels that were comparable with those seen in wild-type tissues (formula image). (D) Glb1-/- mice that underwent transplantation (▪) performed better than untreated Glb1-/- mice (formula image) but not as well as wild-type mice (□) on the rotating rod test of motor coordination and balance (n = 15). (E) Similar results were seen on the open-field exploratory activity test (n = 17). Data are expressed as mean ± SD; groups were compared by one-way repeated measures ANOVA. #P < .05 relative to untreated Glb1-/- littermates at the same age; post hoc Tukey test.
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