Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation

Abstract

Sandhoff disease is a lysosomal storage disorder characterized by the absence of beta-hexosaminidase and storage of G(M2) ganglioside and related glycolipids in the central nervous system. The glycolipid storage causes severe neurodegeneration through a poorly understood pathogenic mechanism. In symptomatic Sandhoff disease mice, apoptotic neuronal cell death was prominent in the caudal regions of the brain. cDNA microarray analysis to monitor gene expression during neuronal cell death revealed an upregulation of genes related to an inflammatory process dominated by activated microglia. Activated microglial expansion, based on gene expression and histologic analysis, was found to precede massive neuronal death. Extensive microglia activation also was detected in a human case of Sandhoff disease. Bone marrow transplantation of Sandhoff disease mice suppressed both the explosive expansion of activated microglia and the neuronal cell death without detectable decreases in neuronal G(M2) ganglioside storage. These results suggest a mechanism of neurodegeneration that includes a vigorous inflammatory response as an important component. Thus, this lysosomal storage disease has parallels to other neurodegenerative disorders, such as Alzheimer's and prion diseases, where inflammatory processes are believed to participate directly in neuronal cell death.

Figures

Figure 1

Apoptosis in Sandhoff disease mice. (a) Apoptotic cell death in thalamic nucleus of 4-month-old Sandhoff disease mouse was detected by the in situ TUNEL method. (Bar = 20 μm.) (b) Incidence of apoptotic cell death in brainstem including thalamic nuclei (BS) and spinal cord (SC) of Sandhoff disease mice. The TUNEL-positive cells were counted in semisequential coronal sections of brain and spinal cord. Data are mean ± SEM (n = 3–4).

Figure 2

Microglia activation and expansion in Sandhoff disease mice. Immunostaining with anti-F4/80 antibody shows amoeboid microglia in the brainstem of 4-month-old Sandhoff disease mouse (a) and ramified microglia in the brainstem of age-matched control mouse (b). Staining with GSIB4 shows amoeboid microglia in the spinal cord of a 4-month-old Sandhoff disease mouse (c) but not in a control mouse (d). (Bar = 20 μm.) (e) Quantitation of GSIB4-positive microglia in brainstem (BS) and spinal cord (SC) of Sandhoff mice. The GSIB4-positive cells were counted in semisequential sections of brain and spinal cord. Data are mean ± SEM (n = 3–4). (f) TNF-α mRNA expression levels in the spinal cord of Sandhoff disease mice compared with age-matched control mice. (SD). Data are mean ± SEM (n = 3).

Figure 3

Microglia activation and expansion in a Sandhoff disease patient. (a) Hematoxylin and eosin staining of section of thalamus from the Sandhoff disease patient. Arrows indicate neuronal cells with storage of gangliosides. (b) TUNEL staining of the cerebral cortex of the patient. Arrow indicates TUNEL-positive neuronal cells. (c) Immunostaining of thalamic nucleus with anti-CD68 antibody. Arrows indicate CD68-positive microglia. [Bar = 20 μm (a-c).] (d) Immunofluorescent staining of the cerebellum with antiphosphotyrosine antibody. Arrows indicate activated microglia intensely stained with antiphosphotyrosine antibody. (Bar = 50 μm.) (e) Chitotriosidase activity of brain regions from control (C) and from the Sandhoff disease patient (SD). The activity is elevated in the cerebral cortex (CT), thalamic nucleus (TN), and brainstem (BS) of a Sandhoff disease patient. (f) TNF-α mRNA expression levels in control (C) and in the Sandhoff disease patient (SD). The expression levels are elevated in thalamic nucleus (TN) and brainstem (BS) but not in the cerebral cortex (CT) of the Sandhoff disease patient.

Figure 4

Reduction of activated microglia, neuronal apoptosis, but not GM2 storage in bone marrow-transplanted Sandhoff disease mice. Untreated 4-month-old Sandhoff disease mouse [SD (a, c, and e)] and bone marrow-transplanted Sandhoff disease mouse [BMT-SD (b, d, and f)] were analyzed. (a and b) Immunostaining with anti-GM2 ganglioside antibody. (Bar = 50 μm.) (c and d) TUNEL staining of thalamic nuclei. Arrows indicate apoptotic cells. (Bar = 50 μm.) (e and f) Staining with GSIB4. Arrows indicate GSIB4-positive cells. (Bar = 20 μm.)

Figure 5

Model for acute neurodegeneration in GM2 gangliosidoses. In this model, storage of GM2 ganglioside and related glycolipids causes primary neuronal damage. Microglia recognize damaged and dying neurons and remove them by phagocytosis. The inability of the enzyme-deficient microglia to catabolize endocytosed glycolipid leads to their activation and the recruitment of additional microglial precursors from blood. The large expansion of the activated microglial population produces neurotoxic mediators, which provides an additional insult to the neurons already stressed by glycolipid storage, resulting in widespread neuronal apoptosis. BMT may disrupt this pathway through the introduction of normal microglia into the CNS. These normal microglia can effectively remove the neurons damaged by storage and, thus, temporarily suppress the recruitment and expansion of the activated microglial population.