NEWLY IDENTIFIED PHOX2B-REGULATED GENES AS POSSIBLE DRUG TARGETS FOR THE PHARMACOLOGICAL INTERVENTION IN CONGENITAL CENTRAL HYPOVENTILATION SYNDROME (CCHS)

Congenital Central Hypoventilation Syndrome (CCHS, OMIM #209880) is a very rare neonatal neurological disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing (Weese-Mayer et al., 2017). It is often associated with Hirschsprung’s disease (HSCR) and neural crest-derived tumours (i.e. neuroblastoma). Frameshift mutations (5%) and polyalanine triplet expansions (from 4 to 13 residues) (95%) have been detected in the coding region of PHOX2B, a transcription factor required for the development of neurons that regulate the cardiovascular, respiratory and digestive organs, forming the sensory and motor arms of the visceral reflex circuits. Consistent with its role as transcriptional regulator, transcriptional dysregulation might be an important mechanism of CCHS pathogenesis. CCHS is a life-long disorder for which the only treatment option is ventilatory support provided by tracheotomy, nasal mask or diaphragm pacing by phrenic nerve stimulation, as pharmacological respiratory stimulants have proved to be ineffective. A strong limitation to the comprehension of the pathogenesis of CCHS, and the development of new and effective treatment for this disease, is the missing knowledge of target genes regulated by PHOX2B, whose expression may be eventually dysregulated. Very little is known about the genes regulated by PHOX2B. Most of the genes identified so far are regulatory genes that encode for transcription factors and enzymes that control downstream processes involved in the survival and differentiation of specific neural structures, such as TH, DBH (Lo et al., 1999; Adachi et al., 2000), PHOX2A (Flora et al., 2001), TLX2 (Borghini et al.,2006), RET (Bachetti et al., 2005) MSX-1 (Revet et al., 2008), SOX10 (Nagashimada et al., 2012), ALK (Bachetti et al., 2010) and PHOX2B itself (Cargin et al., 2005). The aim of this thesis was to identify new potential pharmacological targets for the development of drugs in order to improve the respiratory symptoms and the quality of life of CCHS patients. In the first part of my project we investigated the molecular mechanisms underlying the recovery of chemosensitivity, observed in two CCHS patients, following the administration of the progestinic desogestrel (Straus et al., 2010). In SK-N-BE(2)C cell clones, stably expressing nuclear progesterone receptor isoforms PR-B and PR-A, we demonstrated that 3-KDG treatment, active metabolite of the desogestrel, reduces the expression of PHOX2B, its target genes as well as PHOX2B +7 alanine expanded protein, by means of a post-transcriptional mechanism. This finding provided the evidence of a direct molecular link between PHOX2B and desogestrel and suggested the possibility that reduction of PHOX2B mutant protein may contribute to the positive effects observed in the two CCHS patients. In the second part of my project we proposed to identify new PHOX2B target genes that can be deregulated in the pathology, in the future perspective that might be potential pharmacological targets, alternative to PHOX2B. In particular, we demonstrated that transcriptional dysregulation and dysfunctions of K+ and Na+ channels activity may contribute to the onset of respiratory problems associated with CCHS. Target directly the de-regulated PHOX2B target genes is an alternative pharmacological strategy to by-pass the effect on PHOX2B in the perspective of rescuing their activity. Since several drugs targeting these proteins are already used in clinics, the potential progress toward a therapeutic intervention to treat CCHS is today more than concrete.