Exome sequencing of individuals with Huntington’s disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset

The age at onset of motor symptoms in Huntington’s disease (HD) is driven by HTT CAG repeat length but modified by other genes. In this study, we used exome sequencing of 683 patients with HD with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We discovered damaging coding variants in candidate modifier genes identified in previous genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier-onset HD. Nuclease activities of purified variants in vitro correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion similar to those observed with complete FAN1 knockout. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD

Protein localisation studies: Where is Huntingtin hiding? [Meeting abstract]

Background The underlying mutation of the neurodegenerative disorder Huntington's disease (HD) is an expanded and unstable CAG repeat in exon 1 of the HD gene. This is translated into an expanded polyglutamine tract at the N terminal of the gene product huntingtin (Htt). Htt has been suggested to have a role in subcellular transport and transcription but the precise function of the Htt protein remains elusive. Htt location and the characteristics of normal and mutant Htt in different subcellular locations and cell types remain unclear yet may give further clues to the normal and mutant functions of this protein. Aims This study has investigated the subcellular localisation of both endogenous and overexpressed Htt in cells in culture and assessed the effects of epidermal growth factor (EGF) on this localisation. Methods HeLa, HEK293, N2A, PC12 and IMR32 cells were grown in culture and studied by either light or confocal laser scanning microscopy. Overexpressed Htt was fluorescently tagged while antibodies spanning the entire length of the Htt protein were employed to detect the endogenous protein. Results While overexpressed Htt remained exclusively cytoplasmic, endogenous Htt appeared to reside in both the nucleus and the cytoplasm of cells cultured under standard conditions. On addition of EGF, an apparent reduction in the nuclear pool of Htt was noted. Conclusions These findings raise the possibility of growth factor mediated movement of the Htt protein from the nucleus to the cytoplasm or possibly growth factor mediated post-translational modification of Htt, rendering it less available for antibody detection. This paves the way for further studies investigating the role of growth factors in HD pathogenesis

Transcriptional dysregulation in Huntington's disease

Huntington’s disease (HD) is an autosomal dominant neurodegeneration which is associated with an expanded CAG repeat in a large gene on chromosome 4p. The protein product of the gene, huntingtin, thus contains an expanded glutamine tract close to its N-terminus. Although the genetic lesion was defined in 1993 (Huntington’s disease collaborative research group, 1993) neither the normal nor pathological function of huntingtin has been defined. Despite the widespread expression of the HD gene in effectively all cell types examined (DiFiglia et al., 1995; Ferrante et al., 1997; Gutekunst et al., 1995; Kosinski et al., 1997) the initial pathology of HD is restricted and specific populations of neurons in the striatum are vulnerable (Vonsattel et al., 1985). The neurons which die first are the medium spiny projection neurons in the caudate and putamen, and those most vulnerable are the enkephalin-containing, dopamine D2 expressing neurons projecting to the globus pallidus pars externa (GPe) (Albin et al., 1990; 1992). The death of neurons is accompanied by extensive reactive gliosis and, at later stages of disease, atrophy occurs in other regions of the basal ganglia and elsewhere with brain weight at death significantly reduced compared to controls (Vonsattel et al., 1985)

Longitudinal analysis of gene expression and behaviour in the HdhQ150 mouse model of Huntington's disease

Substantial transcriptional changes are seen in Huntington's disease (HD) brain and parallel early changes in gene expression are observed in mouse models of HD. Analysis of behaviour in such models also shows substantial deficits in motor, learning and memory tasks. We examined the changes in the transcriptional profile in the HdhQ150 mouse model of HD at 6, 12 and 18 months and correlated these changes with the behavioural tasks the animals had undertaken. Changes in gene expression over time showed a significant enrichment of RNAs altered in abundance that related to cognition in both HdhQ150 and wild-type animals. The most significantly down-regulated mRNA between genotypes over the whole time-course was Htt itself. Other changes between genotypes identified at 6 months related to chromatin organization and structure, whilst at 18 months changes related mainly to intracellular signalling. Correlation of the changes in gene product abundance with phenotypic changes revealed that weight and detection of the opposite position of the platform in the water maze seemed to correlate with the chromatin alterations whereas changes in the rotarod performance related mainly to intracellular signalling and homeostasis. These results implicate alterations in specific molecular pathways that may underpin changes in different behavioural tasks

Identification of novel alternative splicing events in the Huntingtin gene and assessment of the functional consequences using structural protein homology modelling

Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by a pathological CAG trinucleotide repeat expansion in the large multi-exon gene, huntingtin (HTT). Although multiple pathogenic mechanisms have been proposed for HD, there is increasing interest in the RNA processing of the HTT gene. In mammals, most multi-exon genes are alternatively spliced; however, few alternative transcripts have been described for HTT. Given the numerous protein bands detected in mouse and human brain tissue by Western blotting using anti-huntingtin antibodies, we examined whether alternative splicing of HTT may account for some of these fragments. Using RT-PCR in mouse brain, we detected two novel splice variants of Htt that lacked the 111-bp exon 29 (Htt∆ex29) or retained a 57-bp portion of intron 28 (Htt+ 57in28) via use of a cryptic splice site. The alternative transcripts were present in wild-type and homozygous Hdh(Q150/Q150) mouse brain at all ages and in all brain regions and peripheral tissues studied. Differential splicing of Htt∆ex29 was found in the cerebellum of Hdh(Q150/Q150) mice with a significant reduction in transcript levels in mutant animals. In human brain, we detected similar splice variants lacking exons 28 and 29. The ability of alternatively spliced transcripts to encode different protein isoforms with individual functions in the cell, combined with the known role of splicing in disease, renders these novel transcripts of interest in the context of HD pathogenesis

Progetto di recupero e valorizzazione della Cava di Tor Fiscale

Il contributo, corredato dai relativi disegni di progetto, documenta e illustra la soluzione progettuale sperimentale elaborata, come conclusiva della Ricerca Sapienza -finanziata 2012-2013- Responsabile Prof. P.V. Dell'Aira, dal titolo " Sottosuoli urbani La progettazione della 'città che scende'. Tecniche progettuali e realizzative. Identità e qualità spaziale. Comfort ambientale". Il progetto viene presentato attraverso i suoi principali concept informatori: • la relazione tra sopra e sottosuolo • la discenderia, scultura ambientale • le "colonne di luce": landmarks tra sotto e sopra-suolo • il percorso espositivo-narrativo ipogeo • il ridisegno del ponte pedonale • il riuso dell'antico casal