Clinical, neuroradiological and molecular investigation of Adult-onset Autosomal Dominant LeukoDystrophy (ADLD): dissection of Lamin B1-mediated pathophysiological mechanisms in cellular and mouse models

Autosomal dominant leukodistrophy (ADLD) is a rare, progressive and fatal genetic disease associated with overexpression of the nuclear protein Lamin B1 (LB1). We have collected 2 ADLD families with LB1 gene duplication (Fam-1 and Fam-2), and a third with a variant ADLD (ADLD-TO), carrying a deletion upstream of the LB1 gene but no mutations or duplications. We investigated the clinical phenotype of 3 affected patients from Fam-1. The patients present autonomic dysfunction, which is mostly characterized by cardiovascular and skin noradrenergic failure with preserved cardiovagal function and cholinergic sweat gland innervation. Longitudinal conventional and advanced brain MR studies showed macro- and microstructural, metabolic and bioenergetic alterations, indicative of moderate-severe progressive leukoencephalopathy. Using a custom aCGH assay centered on the LMNB1 gene followed by PCR and sequencing, we defined the duplications of Fam-1 and Fam-2 at nucleotide level. A non-homologous end-joining (NHEJ) mechanism is likely causative of the duplication, although fork-stalling template-switching (FoSTeS) may be involved in Fam-2. In ADLD-TO, using circular chromosome conformation capture (4C), dual luciferase assay, and in vivo mouse enhancer assay, we identified the enhB region, repositioned by the deletion 100 kb upstream from LMNB1 promoter. We hypothesized that the spatial reorganization of the LMNB1 landscape due to the deletion, causes an “enhancer adoption” position effect and an overexpression of LMNB1 gene comparable to duplication. We have established primary cultures of human skin fibroblasts from 3 ADLD patients with LB1 duplication, 1 LB1 duplication carrier, 6 non-carrier siblings, 1 ADLD-TO patient and 9 healthy subjects. Using atomic force microscopy, confocal imaging and electrophysiology, we demonstrated that, in fibroblasts of symptomatic ADLD patients with LB1 duplication, LB1 upregulation is associated with nuclear morphological abnormalities, increased nuclear stiffness and altered nuclear ionic permeability (Ferrera et al, FASEB J, 2014). These results suggest that alteration of LB1 levels alters the mechanobiology of the nucleus, modifying nuclear ionic signaling. We also detected increased nuclear stiffness and morphological abnormalities in primary fibroblasts from ADLD-TO, further supporting the view that LB1 levels modulate nuclear mechanics and architecture. Microarray analysis on primary human skin fibroblasts and whole blood of duplication carriers and non-carriers siblings demonstrated that deregulation of LMNB1 expression induces modified splicing of several genes, likely driven by RAVER2 overexpression, suggesting that an alteration of mRNA processing could be a pathogenic mechanism in ADLD. Finally, we demonstrated that, in primary murine cortical neurons, LB1 regulate neuronal differentiation affecting dendrite and axonal development in concentration-specific manner by affecting definite nuclear signaling pathways. In particular, when overexpressed, LB1 induces apoptosis, impairs axonal outgrowth and alters the localization of nuclear pore complexes (NPCs). Conversely, lamin b1 deficiency reduces dendrite development as a consequence of altered NPCs localization and impaired signaling through ERK. These results suggest that in ADLD neuronal morphology and survival may be altered as a consequence of LB1 overexpression, leading to the disease phenotype.