Tiziana Bachetti

PHOX2B mutations and polyalanine expansions correlate with the severity of the respiratory phenotype and associated symptoms in both congenital and late onset Central Hypoventilation syndrome

Congenital Central Hypoventilation syndrome (CCHS (MIM 209880)) is a rare disorder, with fewer than 200 patients currently reported worldwide, characterised by absence of adequate autonomic control of respiration with decreased sensitivity to hypercapnia and hypoxia, in the absence of neuromuscular or lung disease, or an identifiable brain stem lesion.1 Children with CCHS show an adequate ventilation while awake but hypoventilate during sleep. More severely affected children hypoventilate both when awake and during sleep.1 CCHS has been reported in association with several disorders, among which aganglionic megacolon (Hirschsprung disease, HSCR) and tumours of neural crest origin, reflecting a common molecular pathogenesis sustained by defects of one or more genes that control the correct development of neural crest derived cell lineages.1–3 A genetic aetiology has long been hypothesised for CCHS based on recurrence reported in siblings, in half siblings and in affected children born to women with CCHS.2–6 More recently, a generalised autonomic nervous system (ANS) imbalance has been observed among children with CCHS and an increased incidence of ANS dysfunctions (ANSD) reported among relatives of 56 patients with CCHS, as against relatives of 56 matched controls.7 A family transmission study has shown that the risk of developing an ANSD symptom including CCHS, regarded as the most severe expression of ANS imbalance, mainly depends on the genotype at a major locus, while significant residual variants could be due to additional minor genes, modifying loci effects or environmental factors.8 Genes involved in the ANS development, like the RET proto-oncogene, its ligand GDNF , the Endothelin 3 gene, the Brain Derived Neurotrophic Factor ( BDNF ) and the RNX genes, have been tested and a few mutations found, showing no cosegregation with the disease phenotype in CCHS families.9–13 The PHOX2B gene encodes a 314 amino acids …

Autophagy contributes to inflammation in patients with TNFR-associated periodic syndrome (TRAPS)

Objectives Tumour necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is caused by TNFRSF1A mutations, known to induce intracellular retention of the TNFα receptor 1 (TNFR1) protein, defective TNFα-induced apoptosis, and production of reactive oxygen species. As downregulation of autophagy, the main cellular pathway involved in insoluble aggregate elimination, has been observed to increase the inflammatory response, we investigated whether it plays a role in TRAPS pathogenesis. Methods The possible link between TNFRSF1A mutations and inflammation in TRAPS was studied in HEK-293T cells, transfected with expression constructs for wild-type and mutant TNFR1 proteins, and in monocytes derived from patients with TRAPS, by investigating autophagy function, NF-κB activation and interleukin (IL)-1β secretion. Results We found that autophagy is responsible for clearance of wild-type TNFR1, but when TNFR1 is mutated, the autophagy process is defective, probably accounting for mutant TNFR1 accumulation as well as TRAPS-associated induction of NF-κB activity and excessive IL-1β secretion, leading to chronic inflammation. Autophagy inhibition due to TNFR1 mutant proteins can be reversed, as demonstrated by the effects of the antibiotic geldanamycin, which was found to rescue the membrane localisation of mutant TNFR1 proteins, reduce their accumulation and counteract the increased inflammation by decreasing IL-1β secretion. Conclusions Autophagy appears to be an important mechanism in the pathogenesis of TRAPS, an observation that provides a rationale for the most effective therapy in this autoinflammatory disorder. Our findings also suggest that autophagy could be proposed as a novel therapeutic target for TRAPS and possibly other similar diseases.

The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis

Objectives—Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics. Methods and Results—The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo. Conclusion—The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner.

PHOX2B mutations and genetic predisposition to neuroblastoma

Neuroblastoma (NB) is a childhood malignancy originating from neural crest cells, which seldom occurs in association with other neurocristopathies. Owing to the rarity of familial NB cases, only a few linkage data are available and no mutations in candidate genes have been demonstrated up till now. Germline mutations in a small proportion of NB patients have been recently reported in the paired-like homeobox 2B (PHOX2B) gene, suggesting its role in NB predisposition. On the basis of this indication, we screened three Italian families with recurrence of NB and one family with occurrence of ganglioneuroblastoma and isolated Hirschsprung disease for PHOX2B defects. Our analysis did not show any mutation, excluding PHOX2B as the NB susceptibility gene in the families we analysed. Our findings combined with those derived from other PHOX2B mutation screenings and from genome-wide linkage analysis support a remarkable genetic heterogeneity of NB and suggest an oligogenic model of disease transmission. Furthermore, as PHOX2B mutations were mainly observed in some NB families with multifocal and syndromic NB, features that are missing in the families we have studied, we suggest they represent second-site modifications responsible for a specific phenotype rather than causal mutations of a major locus.

In vitro treatments with ceftriaxone promote elimination of mutant glial fibrillary acidic protein and transcription down-regulation

Alexander disease is a rare, untreatable and usually fatal neurodegenerative disorder caused by heterozygous mutations of the glial fibrillary acidic protein (GFAP) gene which ultimately lead to formation of aggregates, containing also alphaB-Crystallin, HSP27, ubiquitin and proteasome components. Recent findings indicate that up-regulation of alphaB-Crystallin in mice carrying GFAP mutations may temper the pathogenesis of the disease. Neuroprotective effects of ceftriaxone have been reported in various animal models and, noteworthy, we have recently shown that the chronic use of ceftriaxone in a patient affected by an adult form of Alexander disease could halt its progression and ameliorate some of the symptoms. Here we show that ceftriaxone is able to reduce the intracytoplasmic aggregates of mutant GFAP in a cellular model of Alexander disease. Underlying mechanisms include mutant GFAP elimination, concurrent with up-regulation of HSP27 and alphaB-Crystallin, polyubiquitination and autophagy. Ceftriaxone has also been shown to modulate the proteasome system, thus decreasing NF-kappaB activation and GFAP promoter transcriptional regulation, which further accounts for the down-modulation of GFAP protein levels. These mechanisms provide previously unknown neuroprotective targets of ceftriaxone and confirm its potential therapeutic role in patients with Alexander disease and other neurodegenerative disorders with astrocyte involvement.

Low amounts of PHOX2B expanded alleles in asymptomatic parents suggest unsuspected recurrence risk in congenital central hypoventilation syndrome.

Heterozygous trinucleotide in frame duplications, leading to expansions of variable lengths of a 20-alanine stretch (polyAla), is the most frequent PHOX2B variant associated with congenital central hypoventilation syndrome (CCHS), a rare neurocristopathy characterized by defective response of the autonomic nervous system to hypoxia and hypercapnia. Sequencing analysis has shown that the vast majority of polyAla expansions arise de novo; while in about 10% of cases, mutations are inherited by one parent who carries either constitutive or somatic mutations. To investigate transmission of PHOX2B mutant alleles from asymptomatic individuals, we have reassessed 44 parental pairs, previously resulted not to carry any mutation, by coupling amplification with FAM-tagged primers and capillary electrophoresis. Low levels of somatic mosaicism were shown in five parents previously undetected, thus increasing the inherited occurrence of the disease from 10% to 25% of the cases. Analysis of the technical detection limits has confirmed a power of resolution much higher for the “FAM” protocol than for the “sequencing” method. These observations are going to have relevant implications on how the carrier status of asymptomatic parents should be assessed and on successive genetic counseling to CCHS families.

PHOX2B-mediated regulation of ALK expression: In vitro identification of a functional relationship between two genes involved in neuroblastoma

Background Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-like Homeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. Methodology/Principal Findings Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. Conclusions/Significance These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.

Tumor necrosis factor receptor-associated periodic syndrome as a model linking autophagy and inflammation in protein aggregation diseases

Autophagy prevents cellular damage by eliminating insoluble aggregates of mutant misfolded proteins, which accumulate under different pathological conditions. Downregulation of autophagy enhances the inflammatory response and thus represents a possible common pathogenic event underlying a number of autoinflammatory syndromes, such as tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS). The pathogenesis of other monogenic or complex disorders that display symptoms of excessive inflammation also involve the autophagy pathway. Studies have shown that TRAPS-associated TNFRSF1A mutations induce cytoplasmic retention of the TNFR1 receptor, defective TNF-induced apoptosis, and production of reactive oxygen species (ROS). Furthermore, autophagy impairment may account for the pathogenic effects of TNFRSF1A mutations, thus inducing inflammation in TRAPS. In this review, we summarize the molecular interactions and functional links between autophagy with regard to nuclear factor-kappa B activation, ROS production, and apoptosis. Furthermore, we propose a complex interplay of these pathways as a model to explain the relationship between mutant protein misfolding and inflammation in genetically determined and aggregation-prone diseases. Accordingly, autophagy function should be investigated in all diseases showing an inflammatory component, and for which the molecular pathogenesis is still unclear.

Transcriptional dysregulation and impairment of PHOX2B auto-regulatory mechanism induced by polyalanine expansion mutations associated with congenital central hypoventilation syndrome.

The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.

Ceftriaxone has a therapeutic role in Alexander disease

Alexander disease (AD) (MIM 203450) is a rare, usually fatal neurodegenerative disorder, involving primarily astroglial cells in the CNS, caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP) (Brenner et al., 2001). It is characterized by dystrophic astrocytes containing intermediate filament aggregates (Rosenthal fibers) (RFs), in combination with myelin abnormalities (Li et al., 2005). Pathogenetic determinants include a toxic gain-of-function of mutated GFAP which causes aggregates and RFs accumulation in astrocytes and an excitotoxicity related to impairment of the buffering capacity of dystrophic astrocytes and of their ability to metabolize extracellular glutamate ([Mignot et al., 2004] and [Sullivan et al., 2007]). AD remains an untreatable genetic disease that severely limits life expectancy in affected individuals. Here we studied the tolerability and therapeutic effects of the chronic use of cycles of ceftriaxone, a beta-lactam antibiotic with neuroprotective effects (Rothstein et al., 2005), in a patient affected by adult AD with a rapidly progressive clinical course. Because AD is rare and its presentation varies it is difficult to evaluate treatments in controlled trials, thus prolonged, longitudinal single-patient studies may be a useful approach to identify the new utilization of drugs in this pathology. The successful clinical outcome related to ceftriaxone reported here in a patient with adult AD highlights the possibility that this β-lactam antibiotic may be useful for other AD patients and, possibly, for other neurodegenerative disorders with astrocyte involvement.