Diseases of Polyglutamate Expansion – Basonuclins

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Chemical map of B-sheet content in and around a neuronal inclusion. Left panel: inclusion of Huntington disease brain stained green for huntingtin. Right panel: B-sheet/B-helix ratio determined by synchrotron infrared microspectroscopy. Color scale going from red (highest ratio) to blue (lowest). Correspondence between inclusion and high B-sheet content is obvious.

Our goal is to understand the mechanisms of protein aggregation in neurological diseases caused by polyglutamine expansion and how aggregation causes neuronal lethality. Huntington disease is the most frequent of nine neurological diseases caused by polyglutamine (polyQ) expansion. The expansion occurs in a protein of unknown function named huntingtin, and causes progressive destruction of neurons in the striatum and the cortex. Affected brain regions show characteristic inclusions containing N-terminal fragments of huntingtin bearing the expanded polyQ, co-aggregated with a variety of other proteins.

Misfolding and aggregation of the expanded polyQ are thought to be key pathogenic events in polyQ diseases, although how they cause neuronal toxicity has not yet been established. Recent data have suggested that the mechanism of aggregation may be complex and a variety of polyQ aggregates have been observed in vitro. Using synchrotron based infrared microspectroscopy, we have studied the aggregates present in the brain of patients with adult and juvenile forms of Huntington disease. We have uncovered the existence of putative oligomers and of three kinds of microscopic aggregates, two of which are amyloid. One of the amyloid aggregates is always associated with severe disease and found in the most affected brain regions. This kind of amyloid inclusion may be particularly toxic to neurons.

We are now studying the toxicity (or protective effect) of these aggregates in cell culture models. For this we are using the prion-like ability of the aggregates to propagate in vitro while retaining their original structure. We use as recipient cells, induced pluripotent stem cells (IPS cells). These cells possess either normal or expanded huntingtin and can differentiate into neurons. Study of the mechanism of cell death and of the transcriptome of the cells treated with the different kinds of aggregates will allow us to approach the mechanisms of toxicity of the aggregates on neurons.

Basonuclin 2: a zinc finger protein involved in development and cancer. We have discovered serendipitously basonuclin 2 (BNC2) in 2004. BNC2 is an extremely conserved nuclear protein with three pairs of zinc fingers. There is evidence supporting the existence in the human of many forms of alternatively spliced bnc2 mRNAs. Disruption of the bnc2 gene in mice has shown that lack of the protein causes neonatal death associated with cleft palate and craniofacial abnormalities. This phenotype results from a direct effect of BNC2 on the multiplication of craniofacial mesenchymal cells. In both the human and the mouse, haploinsufficiency of bnc2 causes hypospadias and BNC2 is associated with an increased risk of ovarian cancer.

In our most recent work, we have uncovered new functions of BNC2, and we have shed light on the relation between BNC1 and BNC2. Our findings can be summarized as follows:

BNC2 has a function in the regulation of transcription. We have identified the DNA-binding motif and some of the target genes of this isoform by chromatin immunopurification and massive parallel sequencing.

Paraffin-embedded sections of newborn testes stained with hematoxylin/eosin. (A) wt mouse with quiescent prespermatogonia in the center of the cords. (B) In the homozygous mutant, many prespermatogonia are engaged in mitosis. Arrowheads point to prespermatogonia.

Paraffin-embedded sections of newborn testes stained with hematoxylin/eosin. (A) wt mouse with quiescent prespermatogonia in the center of the cords. (B) In the homozygous mutant, many prespermatogonia are engaged in mitosis. Arrowheads point to prespermatogonia.

We have uncovered the importance of BNC2 in male germ stem cells. We show that prespermatogonia, BNC2 acts as a repressor of both meiosis and mitosis during late embryogenesis. We demonstrate that one of the target genes of BNC2 in prespermatogonia is the methylation protein DNMT3L. We have also demonstrated the importance of BNC2 is the stem cells of hair follicles.

We have shown that, in spite of the resemblance of their zinc fingers and of their coexpression in many cell types, the functions of BNC1 and BNC2 are different since homozygous knockin mice expressing bnc1 instead of bnc2 die at birth with a palatal cleft and craniofacial abnormalities, a phenotype very similar to that of the bnc2-/- mice.

Our project is now to decipher the mechanism of action of BNC2 on stem cells. For this we are identifying the target genes of BNC2 by a variety of techniques.

Funding

  • Association pour la recherche sur le cancer
  • Ligue contre le cancer

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