Fingolimod Rescues Demyelination in a Mouse Model of Krabbe's Disease

Abstract

Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination both in vitro and ex vivo models. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. These in vivo findings corroborate initial in vitro studies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease.

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“…Indeed, FTY720 reduced the oligodendrocyte apoptosis both in vivo [48] and in vitro [43,107,[114][115][116]. In line with this, FTY720 increased the number of proliferating (bromodeoxyuridine/5-bromo-2 -deoxyuridin (BrdU)-expressing) OPCs in the brain [88,106,117], and the expression of the oligodendrocyte lineage marker oligodendrocyte transcription factor (OLIG2) in neural precursors derived from embryonic stem cells (ESCs) [115] and in the brains of Krabbe's disease mice [64], pointing towards an increased survival of oligodendrocytes. Additionally, in EAE rats and in a human oligodendroglioma cell line, FTY720 reduced ceramide levels [118], which are involved in oligodendrocyte metabolism and apoptosis.…”

“…These results point to an effect of FTY720 on the proliferation and differentiation of neurons, possibly due to an increased phosphorylation of ERK 1/2, cAMP-response element binding protein (CREB) and p38 MAPK [107]. However, since FTY720 did not alter the number of calbindin-positive neurons in the cerebellum, Purkinje neurons appear not to be affected by FTY720 [64].…”

“…In contrast, FTY720-treated immortalized astrocytes displayed increased release of the astrocyte-secreted protein GM-CSF [89], whereas LPS-stimulated primary astrocytes decreased GM-CSF release following treatment with FTY720 [65]. No effect of FTY720 on astrocyte reactivity status has been found [46,64,[67][68][69][70] and even an increased number of GFAP-positive cells has been reported in in vivo models [66,90].…”

“…Both in vitro and in vivo studies have shown that FTY720 has inhibitory effects on the pro-inflammatory (M1) microglia phenotype and stimulates the anti-inflammatory (M2) microglia phenotype . In rodent models for several diseases, such as stroke, cuprizone-induced demyelination, MS (experimental autoimmune encephalitis, EAE), familial Alzheimer's disease (AD) and in irradiation-induced injury, FTY720 decreased microglia activation by polarization towards an anti-inflammatory M2 phenotype characterized by the increased expression of markers such as arginase 1 (ARG1) and mannose receptor C-type 1 (MRC1/CD206) [42][43][44] and a decreased microglia M1 state, defined by the expression of allograft inflammatory factor 1 (IBA1), cluster of differentiation 68 (CD68) and lysosome-associated membrane protein 2 (LAMP-2; CD107b/MAC-3) [42,43,[45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64]71] Consistent with these findings, in vitro FTY720 inhibited the expression of M1 microglia markers and promoted the M2 polarization of oxygen-glucose deprivation (OGD)-insulted microglia [55], photothrombotic stroke-derived primary microglia cultures [42] and white matter-derived microglia from an ischemia rat model [43] as well as in the microglia cell line N9 [63], and human and murine primary microglia cultures [65]. Moreover, FTY720 downregulated the mRNA expression of the pro-inflammatory cluster (e.g., C-C motif chemokine ligand (CCL)7, chemokine (C-X-C motif) ligand (CXCL)13 and CCL5) and upregulated genes in the anti-inflammatory cluster (e.g., granulocyte-macrophage colony-stimulating factor (GM-CSF), chitinase 3-like 3 (CHI3L3) and interleukin (IL)-10) in microglia cells from non-obese EAE mice [65].…”

“…Additionally the presence of neurofilament-heavy chain (SMI32)-positive or amyloid precursor protein (APP)-positive axonal spheroids, indicating axonal damage, was profoundly reduced following FTY720 treatment in vivo and in rat primary cultures [49,57,68]. In contrast, neuronal loss was not attenuated by FTY720 after cuprizone-induced demyelination [69], white matter ischemia [43] and in demyelination related to Krabbe's disease [64]. In a rat model of myelin oligodendrocyte glycoprotein (MOG)-induced optic neuritis (MOG-ON), FTY720 reduced axonal damage, but did not prevent the apoptosis of retinal ganglion cells (RGCs) in the optic nerve [57].…”