Absence of Hikeshi, a nuclear transporter for heat-shock protein HSP70, causes infantile hypomyelinating leukoencephalopathy

Abstract

Genetic leukoencephalopathies are a heterogeneous group of central nervous system disorders with white matter involvement. In a Finnish patient, we identified a novel homozygous disease-causing variant in HIKESHI, c.11G>C, p.(Cys4Ser), leading to hypomyelinating leukoencephalopathy with periventricular cysts and vermian atrophy. A founder Ashkenazi-Jewish disease-causing variant recently linked Hikeshi and its heat-shock protective function to leukoencephalopathy. In our patient, clinical features of lower limb spasticity, optic atrophy, nystagmus, and severe developmental delay were similar to reported patients. Additional features included vermian atrophy, epileptic seizures, and an ovarian tumor. Structural modeling and protein analyses revealed that modified interactions inside Hikeshi's hydrophobic pockets induce protein instability. The patient's cells showed impaired nuclear translocation of HSP70 during heat shock, and decreased ERO1-Lα, an endoplasmic reticulum (ER) oxidoreductase. Overall, we show that: (1) the clinical spectrum associated with Hikeshi deficiency extends to leukoencephalopathy with vermian atrophy and epilepsy; (2) the cellular disease process involves both nuclear chaperone and ER functions.

Figure 1

Pedigree, patient's brain MRI at 10 years of age, and consequences of disease-causing variants at protein level. (a) Pedigree of the family: the patient is indicated by filled symbol. (b) Axial view of T2-weighted MR image shows diffuse hypomyelination and (c) axial view of T1-weighted MR image shows cystic changes in periventricular white matter. (d) Sagittal view of T1-weighted MR image shows vermian atrophy. (e) The variant p.(Cys4Ser) found in our patient and p.(Val54Leu) reported in AJ patients reside within N-terminal domain and are conserved in vertebrates. (f) Overview of the asymmetric homodimer arrangement of Hikeshi showing the affected amino acids within NTDs, based on human Hikeshi crystal structure (PDB id 3WVZ). (g) Interactions established by the side chains of wild-type amino acids Cys4 and Val54 within the hydrophobic pocket of monomer A and B. (h) Bonding pattern for p.(Val54Leu) variant: increased number of hydrophobic interactions and loss of wild-type asymmetry of bonding. (i) Interactions induced by p.(Cys4Ser) variant: hydrophobic interactions are lost; Ser4 in monomer A forms a hydrogen bond. Functional domains of Hikeshi: NTD, N-terminal domain; E-loop, extended loop; CTD, C-terminal domain; FLR, flexible linker region. Violet dash lines, hydrophobic bonds. Green dash line, hydrogen bond.

Figure 2

Loss of Hikeshi has consequences in the nucleus and endoplasmic reticulum (ER). (a) Hikeshi is undetectable in patient's fibroblasts harboring p.(Cys4Ser) change. HSC/HSP70 levels are normal in patient's primary fibroblasts. (b) Patient's immortalized fibroblasts are able to induce the heat-shock response by phosphorylating HSF1-S326. (c) Indirect immunostaining of HSC/HSP70 in control and patient's immortalized fibroblasts, in normal conditions and in heat shock. Strong nuclear punctate accumulations appear in control cells, whereas in patient's fibroblasts, the staining is weaker. (d) Screening of ER stress markers indicates a reduction of ERO1-Lα in patient's primary fibroblasts. (e) Relative quantification of Western blot ER stress markers. The error bars show normalized data range between the two controls. (f) Patient's immortalized fibroblasts respond to tunicamycin-induced ER stress in a similar way to control fibroblasts.