We hypothesize that:

1. NLRP3 inflammasome activation contributes to the neuroinflammatory cascade and lesion expansion in cALD  

2. NLRP3 inflammasome effectors would co-localize with lipid crystals.

X-linked adrenoleukodystrophy (ALD) is a neurometabolic disorder characterized by accumulation of saturated very long chain fatty acids (VLCFAs). Up to 60% of ALD males develop a severe form of ALD characterized by progressive, inflammatory brain demyelination (cerebral ALD, cALD). The NLRP3 inflammasome is a pro-inflammatory pathway that can be triggered by saturated fatty acids and culminates in programmed cell death.

We visualized cholesterol crystal deposition and clefts within cells in ALD and control brain tissue using light and polarized microscopy.

We evaluated NLRP3 effector cytokines, IL-18 and IL-1b, in cerebrospinal fluid (CSF) of ALD (n=20) and control (n=9) patients using multiplex assay and in brain tissue using immunohistochemistry (IHC) in ALD (n=2) and control (n=3).

Birefringent crystals and needle-like clefts were visible in cALD lesions, but absent in brain tissue from controls. Clefts were most often identified within microglia (Iba1+) located well behind the leading edge of demyelination.

We found NLRP3 effectors, IL-1β and IL-18, were higher in cALD cerebrospinal fluid compared to non-ALD controls. IL-18-immunoreactivity was absent in the white matter from controls, but present in both intact and demyelinated white matter of cALD. IL-18 immunoreactivity distribution and morphology resembles macrophage/monocyte lineage cells, and co-localizes with Iba1+ microglia with and without needle-like inclusions

Our data provides initial evidence of NLRP3 inflammasome activity in brain and cerebrospinal fluid of cALD patients. This invites further investigation into the role of NLRP3 and cell death pathways in cALD lesions as possible new therapeutic targets to inhibit disease progression.