Innovative approaches to monitor mutant huntingtin and to facilitate its degradation in Huntington's disease models

Huntington’s disease (HD) is a dominant genetic neurodegenerative disease caused by a mutation in the exon 1 of the huntingtin gene. The clinical symptoms, such as motor disturbances (chorea), cognitive decline and psychiatric impairments are usually developed by the patients in mid-life. Mutant huntingtin protein presents an amplification of a polyglutamine repeat at its N-terminus, which induces conformational changes and leads to neurotoxicity, impairment of cell homeostasis and neuronal cell death. The neuropathology of HD is characterized by a progressive degeneration of the brain starting from the striatum and spreading to other regions such as cortex, hypothalamus and cerebellum. In addition to the diffused brain atrophy, HD patients are also affected by multiple peripheral symptoms which contribute to worsening disease progression and eventually lead to death approximately two decades after onset. The mechanisms leading to the toxicity induced by mutant huntingtin are not well understood. However the acquisition of a misfolded conformation and the formation of intracellular inclusions constituted by shorter fragments of the mutant protein are considered important in the neurodegenerative process. In my thesis project I have investigated mechanisms to enhance the cellular degradation of mutant huntingtin. A second focus was on the development of an immunoassay to detect and quantify aggregates in HD models. I analyzed the data obtained form a high through-put screen aimed to identify small molecular weight compounds decreasing mutant huntingtin levels in cells. Among all compounds screened, only inhibitors of heat shock protein 90 (Hsp90) showed a significant effect on mutant huntingtin clearance. I therefore investigated the mechanisms of Hsp90 chaperone inhibition and the reduction of soluble mutant huntigtin levels. Data from biochemical assays demonstrated that mutant huntingtin degradation is enhanced upon compound treatment and that the protein is cleared through the ubiquitin-proteasome system. This was independent from the heat shock response induced after pharmacological Hsp90 inhibition. Co-immunoprecipitation experiments suggested that mutant huntingtin is a client protein of Hsp90. The results were replicated in different cellular models including full length mutant huntingtin expressed from the endogenous locus, thus highlighting the importance of Hsp90 in stabilizing soluble mutant huntingtin and suggesting the possible application of Hsp90 inhibitors as therapies in HD. In the second project I developed a sensitive method to detect mutant protein aggregates in HD models. To this purpose I implemented the already established time resolved fluorescence resonance energy transfer (TR-FRET) based immunoassay for the detection of soluble mutant and wild-type huntingtin. A mixture of either donor or acceptor fluorophore labeled single monoclonal antibody directed against an epitope exposed on the huntingtin aggregate surface was used. This strategy allowed for energy transfer and therefore a measurable TR-FRET signal, only in presence of mutant aggregated protein. I could demonstrate the sensitivity of the bioassay on a microtiter set up both as a single assay and in a duplex combination with the previously developed TR-FRET assay for soluble huntingtin. I applied the TR-FRET for aggregated huntingtin to samples from R6/2 and HdhQ150 mice, expressing exon 1 and full length mutant huntingtin, respectively. In brain homogenates from both models there was an age-dependent, inverse correlation between soluble and aggregated mutant huntingtin. These findings supported the importance of the relation between aggregated and soluble protein in disease progression. Furthermore, I detected the inverse correlation also in peripheral tissues of R6/2 mice where the presence of aggregates was previously demonstrated with other methods. An in-depth analysis of R6/2 samples in a combination of TR-FRET and size exclusion chromatography suggested a differential specificity of the two antibody combinations used for different aggregate populations. The TR-FRET method provides a new means to characterize the aggregation process as well as to test the efficacy of possible disease modifying treatments for HD

Hypothalamic overexpression of mutant huntingtin causes dysregulation of brown adipose tissue.

Expression of mutant huntingtin (htt) protein has been shown to cause metabolic imbalance in animal models of Huntington disease (HD). The pathways involved are not fully understood but dysfunction of both the hypothalamus and brown adipose tissue (BAT) has been implicated. Here we show that targeted expression of mutant HTT in the hypothalamus leads to loss of the A13 dopaminergic cell group located in the zona incerta and reduced mRNA expression of neuropeptide Y1 receptor in the hypothalamus. Furthermore, this is accompanied by downregulation of uncoupling protein 1 expression and PPARγ coactivator-1 alpha in BAT and a rapid body weight gain. Taken together, our data might provide a mechanistic link between expression of mutant HTT, reduced activity of a hypothalamic dopaminergic pathway and dysfunction of BAT and in part explain the development of an obese phenotype in HD mouse models

Detection of Autoantibodies against Recombinant Desmoglein 1 and 3 Molecules in Patients with Pemphigus vulgaris: Correlation with Disease Extent at the Time of Diagnosis and during Follow-Up

The recent availability of cDNA clones for pemphigus antigens has allowed the production of recombinant desmoglein 1 and desmoglein 3 molecules and the development of an ELISA approach in order to determine levels of antibodies to them. The aim of the study was to determine the relationship between autoantibodies levels and the extent of both mucosal and skin lesions in 20 patients with pemphigus vulgaris at the time of diagnosis and during follow-up. For the detection of autoantibodies by ELISA we used the recombinant proteins expressing overlapping sequences with the entire extracellular desmoglein 1 and desmoglein 3 domains. We showed that in presence of mucosal lesions there was a correlation between extension of mucosal involvement and autoantiboidies titres against both desmoglein 1 and desmoglein 3, whereas in presence of skin lesions there was a statistically significant correlation between extension of skin lesions and autoantibodies titres against desmoglein 3, but not against desmoglein 1. A not negligible number of patients showed variations of the desmoglein 3 autoantibodies titre which did not correlate with the severity of both cutaneous and mucosal involvement. Similar results were obtained analyzing autoantibodies titres against desmoglein 1. In conclusion, we believe that the utilization of recombinant desmoglein 1 and desmoglein 3 proteins by ELISA should be used with caution to monitor disease severity and response to therapy, although it remains a high specific test for the initial diagnosis of pemphigus and the identification of a change in the clinical phenotype of this condition

Newly identified climatically and environmentally significant high-latitude dust sources

Dust particles from high latitudes have a potentially large local, regional, and global significance to climate and the environment as short-lived climate forcers, air pollutants, and nutrient sources. Identifying the locations of local dust sources and their emission, transport, and deposition processes is important for understanding the multiple impacts of high-latitude dust (HLD) on the Earth's systems. Here, we identify, describe, and quantify the source intensity (SI) values, which show the potential of soil surfaces for dust emission scaled to values 0 to 1 concerning globally best productive sources, using the Global Sand and Dust Storms Source Base Map (G-SDS-SBM). This includes 64 HLD sources in our collection for the northern (Alaska, Canada, Denmark, Greenland, Iceland, Svalbard, Sweden, and Russia) and southern (Antarctica and Patagonia) high latitudes. Activity from most of these HLD sources shows seasonal character. It is estimated that high-latitude land areas with higher (SI ≥0.5), very high (SI ≥0.7), and the highest potential (SI ≥0.9) for dust emission cover >1 670 000 km2, >560 000 km2, and >240 000 km2, respectively. In the Arctic HLD region (≥60∘ N), land area with SI ≥0.5 is 5.5 % (1 035 059 km2), area with SI ≥0.7 is 2.3 % (440 804 km2), and area with SI ≥0.9 is 1.1 % (208 701 km2). Minimum SI values in the northern HLD region are about 3 orders of magnitude smaller, indicating that the dust sources of this region greatly depend on weather conditions. Our spatial dust source distribution analysis modeling results showed evidence supporting a northern HLD belt, defined as the area north of 50∘ N, with a “transitional HLD-source area” extending at latitudes 50–58∘ N in Eurasia and 50–55∘ N in Canada and a “cold HLD-source area” including areas north of 60∘ N in Eurasia and north of 58∘ N in Canada, with currently “no dust source” area between the HLD and low-latitude dust (LLD) dust belt, except for British Columbia. Using the global atmospheric transport model SILAM, we estimated that 1.0 % of the global dust emission originated from the high-latitude regions. About 57 % of the dust deposition in snow- and ice-covered Arctic regions was from HLD sources. In the southern HLD region, soil surface conditions are favorable for dust emission during the whole year. Climate change can cause a decrease in the duration of snow cover, retreat of glaciers, and an increase in drought, heatwave intensity, and frequency, leading to the increasing frequency of topsoil conditions favorable for dust emission, which increases the probability of dust storms. Our study provides a step forward to improve the representation of HLD in models and to monitor, quantify, and assess the environmental and climate significance of HLD

Genetic and functional characterization of disease associations explains comorbidity

Understanding relationships between diseases, such as comorbidities, has important socio-economic implications, ranging from clinical study design to health care planning. Most studies characterize disease comorbidity using shared genetic origins, ignoring pathway-based commonalities between diseases. In this study, we define the disease pathways using an interactome-based extension of known disease-genes and introduce several measures of functional overlap. The analysis reveals 206 significant links among 94 diseases, giving rise to a highly clustered disease association network. We observe that around 95% of the links in the disease network, though not identified by genetic overlap, are discovered by functional overlap. This disease network portraits rheumatoid arthritis, asthma, atherosclerosis, pulmonary diseases and Crohn's disease as hubs and thus pointing to common inflammatory processes underlying disease pathophysiology. We identify several described associations such as the inverse comorbidity relationship between Alzheimer's disease and neoplasms. Furthermore, we investigate the disruptions in protein interactions by mapping mutations onto the domains involved in the interaction, suggesting hypotheses on the causal link between diseases. Finally, we provide several proof-of-principle examples in which we model the effect of the mutation and the change of the association strength, which could explain the observed comorbidity between diseases caused by the same genetic alterations

The alliance between genetic biobanks and patient organisations: the experience of the telethon network of genetic biobanks

Background: Rare diseases (RDs) are often neglected because they affect a small percentage of the population (6-8 %), which makes research and development of new therapies challenging processes. Easy access to high-quality samples and associated clinical data is therefore a key prerequisite for biomedical research. In this context, Genetic Biobanks are critical to developing basic, translational and clinical research on RDs. The Telethon Network of Genetic Biobanks (TNGB) is aware of the importance of biobanking as a service for patients and has started a dialogue with RD-Patient Organisations via promotion of dedicated meetings and round-tables, as well as by including their representatives on the TNGB Advisory Board. This has enabled the active involvement of POs in drafting biobank policies and procedures, including those concerning ethical issues. Here, we report on our experience with RD-Patient Organisations who have requested the services of existing biobanks belonging to TNGB and describe how these relationships were established, formalised and maintained. Results: The process of patient engagement has proven to be successful both for lay members, who increased their understanding of the complex processes of biobanking, and for professionals, who gained awareness of the needs and expectations of the people involved. This collaboration has resulted in a real interest on the part of Patient Organisations in the biobanking service, which has led to 13 written agreements designed to formalise this process. These agreements enabled the centralisation of rare genetic disease biospecimens and their related data, thus making them available to the scientific community. Conclusions: The TNGB experience has proven to be an example of good practice with regard to patient engagement in biobanking and may serve as a model of collaboration between disease-oriented Biobanks and Patient Organisations. Such collaboration serves to enhance awareness and trust and to encourage the scientific community to address research on RDs

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype–phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first ‘European Forum on Visceral Myopathy’. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy. Graphical Abstract: [Figure not available: see fulltext.

Mobile opportunity against stress: Open study protocol on the effectiveness of a mobile platform for stress self-management in the post-pandemic era

Mobile health platforms have shown promise in the management of various mental health conditions (including stress, anxiety, and depression) and cognitive behavioral strategies emerged as a popular and effective option offered by the platforms. This paper presents the protocol of a study aimed to test the effectiveness of a mobile platform that uses cognitive-behavioral strategies for stress self-management in the Tuscany region (Italy). The mobile app is adapted to the specific needs of each vulnerable population for which it is designed: young and older people, healthcare professionals, entrepreneurs. The app will be evaluated on the following outcomes: (i) perceived susceptibility and severity of the pandemic situation, perceived benefits, and costs of preventive health behaviors, (ii) knowledge about Covid-19 preventive behaviors and negative consequences of social distancing, (iii) stress and psychopathological symptoms (i.e., anxiety, depression, and post-traumatic stress symptoms) and cognitive distortions. If successful, we expect that the platform could give various groups clinical benefits by providing symptom self-monitoring and early intervention, consolidating the number of mental health programs available, and decreasing barriers to treatment-seeking. This population-level approach has the potential to improve mental health outcomes in pandemic periods for many people