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.Globoid cell leukodystrophy (GLD; Krabbe’s disease) is an autosomal recessive lysosomal storage disorder. It affects approximately 1 in 100,000 children born each year and often leads to mortality by 2 years of age. GLD results from the lack of metabolic enzyme galactocerebrosidase (GALC).¹ In the absence of GALC, galactocerebroside (GalCer) undergoes alternative catabolism into galactosylsphingosine (alias psychosine) and a fatty acid instead of normal catabolism into its component parts, galactose and ceramide.² Galactosylsphingosine has been shown to cause the terminal pathological changes in the central nervous system (CNS) of individuals affected by GLD: globoid cell formation,^2,3 astroglial cytokine and chemokine secretion,^4–8 and oligodendrocyte death and demyelination.^9–14 In addition to these pathological changes, it is established that monocytes/macrophages are recruited to the CNS early in the disease process and continue to mobilize to the brain until the late stages of disease.^15,16Little is understood, however, about the mechanisms underlying these events, or which cells are involved in eliciting the initial pathological changes.^7,17,18 This study investigated the mechanisms involved in monocyte/macrophage recruitment and cytokine/chemokine up-regulation and secretion, as well as which cell types are the earliest to undergo inflammatory cascades leading to terminal disease.Inflammatory cytokines are up-regulated as a consequence of activation of the innate immune response.^19–22 Given the connection, it is logical to link the innate immune response to inflammation in globoid cell leukodystrophy. Psychosine is a derivative of β-galactosylceramide and a ganglioside; therefore, it could potentially serve as a toll-like receptor (TLR) ligand and induce the up-regulation of cytokine/chemokine secretion and monocyte/macrophage recruitment through the initial activation of a TLR.TLRs are most commonly associated with recognition of specific, exogenously derived recognition of microbial patterns. Examples of these patterns are lipopolysaccharide, peptidoglycan, lipoarabinomannan, and double-stranded RNA.²³ However, TLRs also have known roles in exacerbating inflammation and inflammatory profiles in the brains of other neurodegenerative and demyelinating diseases, such as experimental autoimmune encephalitis (murine model for multiple sclerosis),^24,25 amyotrophic lateral sclerosis,²⁶ and Alzheimer disease.^27,28 Investigating whether TLRs play a role in the initiation of inflammatory signaling pathways in the brains of twitcher mice (the murine model for globoid cell leukodystrophy) is important to understanding how inflammation in GLD is initiated. Elucidating how inflammation is induced in the course of disease will lead to more complete knowledge of the pathogenesis of GLD and the identification of novel therapeutic targets.To date, few studies have been performed that investigate the early events in GLD that lead to the terminal changes associated with the disease. It is known that globoid cell appearance and demyelination occur caudally to rostrally in the twitcher brain,²⁹ but the mechanism has not been studied. Our hypothesis was that TLR up-regulation on perivascular macrophages and/or microglia early in the disease initiates cytokine production and monocyte/macrophage recruitment to brains of affected individuals. After activation, microglia and macrophages activate neighboring astrocytes, which markedly exacerbate cytokine/chemokine secretion and inflammation. This study identifies early morphological changes in resident CNS glial cells and the innate immune system that explain, at least in part, how cells become activated, up-regulate cytokines/chemokines, and recruit monocytes/macrophages.