A novel neurodegenerative spectrum disorder in patients with MLKL deficiency

Abstract

Mixed lineage kinase domain-like (MLKL) is the main executor of necroptosis, an inflammatory form of programmed cell death. Necroptosis is implicated in combating infections, but also in contributing to numerous other clinical conditions, including cardiovascular diseases and neurodegenerative disorders. Inhibition of necroptosis is therefore of therapeutic interest. Here we report two siblings both of whom over the course of 35 years developed a similar progressive, neurodegenerative spectrum disorder characterized by paresis, ataxia and dysarthria. Magnetic resonance imaging of their central nervous system (CNS) revealed severe global cerebral volume loss and atrophy of the cerebellum and brainstem. These brothers are homozygous for a rare haplotype identified by whole genome sequencing carrying a frameshift variant in MLKL, as well as an in-frame deletion of one amino acid in the adjacent fatty acid 2-hydroxylase (FA2H) gene. Functional studies of patient-derived primary cells demonstrated that the variant in MLKL leads to a deficiency of MLKL protein resulting in impairment of necroptosis. Conversely, shotgun lipidomic analysis of the variant in FA2H shows no impact on either the abundance or the enzymatic activity of the encoded hydroxylase. To our knowledge, this is the first report of complete necroptosis deficiency in humans. The findings may suggest that impaired necroptosis is a novel mechanism of neurodegeneration, promoting a disorder that shares some clinical features with primary progressive multiple sclerosis (PPMS) and other neurodegenerative diseases. Importantly, the necroptotic deficiency does not cause symptoms outside the nervous system, nor does it confer susceptibility to infections. Given the current interest in pharmacological inhibition of necroptosis by targeting MLKL and its associated pathways, this strategy should be developed with caution, with careful consideration of the possible development of adverse neurological effects.

Fig. 1. Axial and sagittal T2-weighted MRI scans of the patients’ brains.

a Axial T2-weighted MRI scans of the patients’ cerebrum. Red arrows denote areas of extensive atrophy. For patient II-2 the red arrow also denotes small, discrete, periventricular T2-hyperintense white matter lesions. b Axial T2-weighted MRI scans of the patients’ brainstem. Red arrows denote atrophy in the pons. c Sagittal T2-weighted MRI scans of the patients. Red arrows denote atrophy in the cerebellum.

Fig. 2. Study family pedigree and FA2H and MLKL rare variant genotype.

a Pedigree of the study family showing the segregation of rare, small nucleotide deletions in FA2H and MLKL. Circles represent women, squares men, the solid symbol represent the clinically affected index patients (brother II-2 and brother II-3, who is the proband), open symbols represent unaffected family members, and dotted symbols indicate heterozygous individuals. A slash through a symbol represents a deceased person; in this family this is the father of the patients, I-1, who passed away in 2015 at the age of 95 years. b FA2H NM_024306.4:c.32_34del variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold are the three nucleotides and corresponding amino acid that are deleted. c MLKL rs561839347 variant genotype of the study family members as determined by Sanger sequencing. Sequences in bold represent the four nucleotides that are deleted. Italicized amino acids are novel residues in the predicted protein due to the frameshift.

Fig. 3. FA2H variant expression and effect on ceramide species hydroxylation.

a Protein level expression of non-variant (NV) FA2H in relation to the FA2H variant carried by the patients studied here (FA2H F11del), variants described in HSP patients (R235C, L77R and Y170X), and the negative control empty vector (EV) in transduced CHO-K1 cells. The molecular weight, in kilodaltons (kDa), of the truncated low-abundance FA2H Y170X protein, relative to the marker, is denoted by the arrow. GAPDH was used as the loading control. b Percentage of hydroxylated hexosylaceramides, relative to the total level of hydroxylated lipids in CHO-K1 cells that were non-transduced (NT), transduced with an empty vector (EV), or transduced to express non-variant (NV) FA2H, the patients’ FA2H protein variant (F11del), or those from hereditary spastic paraplegia patients (R235C, L77R and Y170X). c Percentage of hydroxylated ceramides, relative to the total level of hydroxylated lipids in control and transduced CHO-K1 cells. Error bars indicate the standard error of the mean. NS indicates no significant difference. Asterisks indicate a significant difference (**denotes P < 0.01; ***denotes P < 0.001; ****denotes P < 0.0001).

Fig. 4. MLKL variant expression and effect on necroptosis.

a MLKL protein expression in PBMCs of the sister II-1, unrelated controls C1–C5 and the brothers, II-3 and II-2. The molecular weight, in kilodaltons (kDa), of full-length MLKL and of the predicted, truncated MLKL variant (which was not detectable), relative to the marker, are denoted by the arrows. GAPDH was used as the loading control. b Relative expression of MLKL messenger RNA (mRNA) in PBMCs from controls (sister II-1, and unrelated controls C1–C5; shown in blue) and the patients (brothers II-3 and II-2; shown in red), assessed in triplicate. c MLKL protein expression in unstimulated fibroblasts (−) and fibroblasts stimulated with 1000 ng/ml of IFNγ for 24 h (+). Lysate of HEK-293 cells transiently transfected with the truncated MLKL variant protein was used as a control. d Relative expression and upregulation of MLKL mRNA upon stimulation of fibroblasts from controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) with varying concentrations of IFNγ for 24 h. Three independent experiments were performed. e Proportion of dying fibroblasts from the controls (sister II-1 and unrelated control F1; depicted as blue triangles and diamonds, respectively), and the patients (brothers II-3 and II-2; depicted as red squares and circles, respectively) over an 87-h period. Necroptosis sensitization was achieved by RIPK3 transduction in the presence of 5 ng/ml IFNγ and 50 μM zVAD, and cell death was measured based on the uptake of IncuCyte® Cytotox Red Reagent. Four independent experiments were performed. Blue and dark blue asterisks (*) denote a statistically significant difference (P < 0.05) between II-I and the patients, and F1 and the patients, respectively. f Representative images of the dying fibroblasts (that have taken up the Red Reagent) at the 87-h time point for controls and patients. All images taken are at the same magnification. For all graphs the error bars indicate the standard error of the mean.