Lithium as a possible therapeutic strategy for Cornelia de Lange syndrome

Abstract

Cornelia de Lange Syndrome (CdLS) is a rare developmental disorder affecting a multitude of organs including the central nervous system, inducing a variable neurodevelopmental delay. CdLS malformations derive from the deregulation of developmental pathways, inclusive of the canonical WNT pathway. We have evaluated MRI anomalies and behavioral and neurological clinical manifestations in CdLS patients. Importantly, we observed in our cohort a significant association between behavioral disturbance and structural abnormalities in brain structures of hindbrain embryonic origin. Considering the cumulative evidence on the cohesin-WNT-hindbrain shaping cascade, we have explored possible ameliorative effects of chemical activation of the canonical WNT pathway with lithium chloride in different models: (I) Drosophila melanogaster CdLS model showing a significant rescue of mushroom bodies morphology in the adult flies; (II) mouse neural stem cells restoring physiological levels in proliferation rate and differentiation capabilities toward the neuronal lineage; (III) lymphoblastoid cell lines from CdLS patients and healthy donors restoring cellular proliferation rate and inducing the expression of CyclinD1. This work supports a role for WNT-pathway regulation of CdLS brain and behavioral abnormalities and a consistent phenotype rescue by lithium in experimental models.

Fig. 1. CdLS brain abnormalities in hindbrain-derived structures correlate with cognitive and behavioral alterations.

A MRI data distribution in the cohort of CdLS patients. Purple: MRI negative (35/66); mauve: MRI positive—single anomalies (16/66); pink: MRI positive—multiple anomalies (15/66). B CNS anomalies and intellectual disabilities. Solid mauve: MRI negative; striped mauve: MRI anomalies. C CNS anomalies and behavioral disabilities—autistic traits. Solid purple: MRI negative; striped purple: MRI anomalies; solid mauve: MRI negative; striped mauve: Rhombencephalon derivation; dotted mauve: Prosencephalon derivation. *p < 0.05; * vs MRI negative.

Fig. 2. Lithium rescues mushroom bodies morphology in CdLS D. melanogaster through WNT activation.

A Schematic representation of the drug-treatment protocol. B–D Mushroom bodies of adult animals labeled with anti-FasII are shown in green. B Normal morphology of mushroom bodies was observed in yw controls where α, β, and γ lobes can be distinguished by their distinct projection patterns (arrows). C Abnormal mushroom bodies morphology was observed in Nipped-B⁴⁰⁷ haploinsufficient adults with a twisted structure (arrowhead in the upper brain) or lacking both α lobes (arrowhead in the lower brain) as example. D Rescue of mushroom bodies morphology is shown in Nipped-B⁴⁰⁷ haploinsufficient adults upon treatment with LiCl. E The table reports the total number and percentage of normal or abnormal adult mushroom bodies. Data were analyzed using Fisher’s exact test (***p < 0.005). F–I Histograms show arm (F, G) and en (H, I) gene expression levels in whole flies as 2^-ΔΔCt ± SD. Analyses of yw controls are shown in the panels (F–H) and analyses of the Nipped-B⁴⁰⁷ mutants are shown in the panels (H, I). White: water; dark blue: LiCl 100 mM treatment. ***p < 0.005; * LiCl 100 mM vs H₂O.

Fig. 3. Lithium rescues proliferation and differentiation capabilities in CdLS mouse NSCs through WNT activation.

A Analysis of the effects of PCI34051 and lithium exposure on the proliferation capabilities of the NSCs. White: NSCs treated with DMSO; yellow: NSCs treated with PCI34051; light blue: NSCs treated with DMSO + LiCl; dark green: NSCs treated with PCI34051 and LiCl. *** p < 0.001; $ p < 0.05; * DMSO vs PCI34051; $ PCI34051 vs PCI34051 + LiCl. B Analysis of the effects of Nipbl knockdown and lithium exposure on the proliferation capabilities of the NSCs. White: NSCs treated with AllStar; light green: NSCs treated with siRNA against Nipbl; light blue: NSCs treated with AllStar and LiCl; dark green: NSCs treated with siRNA against Nipbl and LiCl. *** and °°° p < 0.001; * AllStar vs siRNA against Nipbl; ° siRNA against Nipbl vs siRNA against Nipbl + LiCl. C Representative pictures of the neurospheres growing in the well during various treatments as in panel (A). Scale bar: 2 mm. D Representative pictures of the neurospheres growing in the well during various treatments as in panel (B). E Analysis of the effects of PCI34051 and lithium exposure on the gene expression of Ccnd1 in the NSCs. White: NSCs treated with DMSO; yellow: NSCs treated with PCI34051; light blue: NSCs treated with DMSO + LiCl; dark green: NSCs treated with PCI34051 and LiCl. ** p < 0.01; * p < 0.05; ** DMSO + LiCl vs PCI34051; * PCI34051 + LiCl vs PCI34051. F Analysis of the effects of PCI34051 and lithium on the differentiation capabilities of the NSCs. Cells were immunostained for neuron and nuclei detection. White: NSCs treated with DMSO; yellow: NSCs treated with PCI34051; light blue: NSCs treated with LiCl; dark green: NSCs treated with PCI34051 and LiCl. G Comparison of the effects of PCI34051 and PCI34051 + LiCl of NSCs differentiation. ** p < 0.01; * PCI34051 vs PCI34051 + LiCl. H Representative staining of the differentiated NSCs. Differentiated neurons were labeled with β-tubulin III antibody (red) and nuclei were labeled with DAPI (blue). Scale bar: 50 µm.

Fig. 4. Lithium rescues cell survival in CdLS LCLs through WNT activation.

A Proliferation of patient-derived cells (CdLS H₂O, white bar) is reduced compared to the proliferation rate of controls (HD H₂O, white bar). All CdLS lines (striped bar) exposed to lithium chloride (2.5 mM) showed increased proliferation compared to untreated CdLS cells (CdLS H₂O, white bar) and compared to treated HD cells (solid blue bar). B, C TUNEL assay was used to evaluate cytotoxicity in CdLS LCLs compared to healthy donors (HD). B Upon lithium exposure, CdLS cell lines (blue striped bar) show a decrease in cell death compared to untreated CdLS cells (CdLS H₂O, white bar) and treated HD cells (blue solid bar). On the axis are reported: the experimental groups (x-axis), and numbers of TUNEL positive cells at 24 h of lithium exposure, normalized on water/vehicle (y-axis). Bars express mean ± SEM. C Examples of positive TUNEL cells. Images were taken at ×40, while insets display magnification of the white square (×80). Scale bar represents 50 µm. D, E CyclinD1 gene expression was significantly increased upon lithium exposure (2.5 mM) in LCLs, especially in CdLS lines (SMC1A: blue dotted bars, NIPBL: blue oblique bars) except for HDAC8 (blue horizontal bar). D CdLS lines are shown separately. E In pooled CdLS lines data (blue striped bar) CyclinD1 gene expression was increased upon lithium exposure (2.5 mM) compared to controls (blue solid bar). Data are shown as fold change, calculated as 2^−ΔΔCt ± SD. p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.005 (***).