Computational modelling elucidates the mechanism of ciliary regulation in health and disease

<p>Abstract</p> <p>Background</p> <p>Ciliary dysfunction leads to a number of human pathologies, including primary ciliary dyskinesia, nephronophthisis, situs inversus pathology or infertility. The mechanism of cilia beating regulation is complex and despite extensive experimental characterization remains poorly understood. We develop a detailed systems model for calcium, membrane potential and cyclic nucleotide-dependent ciliary motility regulation.</p> <p>Results</p> <p>The model describes the intimate relationship between calcium and potassium ionic concentrations inside and outside of cilia with membrane voltage and, for the first time, describes a novel type of ciliary excitability which plays the major role in ciliary movement regulation. Our model describes a mechanism that allows ciliary excitation to be robust over a wide physiological range of extracellular ionic concentrations. The model predicts the existence of several dynamic modes of ciliary regulation, such as the generation of intraciliary Ca²⁺spike with amplitude proportional to the degree of membrane depolarization, the ability to maintain stable oscillations, monostable multivibrator regimes, all of which are initiated by variability in ionic concentrations that translate into altered membrane voltage.</p> <p>Conclusions</p> <p>Computational investigation of the model offers several new insights into the underlying molecular mechanisms of ciliary pathologies. According to our analysis, the reported dynamic regulatory modes can be a physiological reaction to alterations in the extracellular environment. However, modification of the dynamic modes, as a result of genetic mutations or environmental conditions, can cause a life threatening pathology.</p

Loss of the tumour suppressor LKB1/STK11 uncovers a leptin-mediated sensitivity mechanism to mitochondrial uncouplers for targeted cancer therapy

Non-small cell lung cancer (NSCLC) constitutes one of the deadliest and most common malignancies. The LKB1/STK11 tumour suppressor is mutated in ∼ 30% of NSCLCs, typically lung adenocarcinomas (LUAD). We implemented zebrafish and human lung organoids as synergistic platforms to pre-clinically screen for metabolic compounds selectively targeting LKB1-deficient tumours. Interestingly, two kinase inhibitors, Piceatannol and Tyrphostin 23, appeared to exert synthetic lethality with LKB1 mutations. Although LKB1 loss alone accelerates energy expenditure, unexpectedly we find that it additionally alters regulation of the key energy homeostasis maintenance player leptin (LEP), further increasing the energetic burden and exposing a vulnerable point; acquired sensitivity to the identified compounds. We show that compound treatment stabilises Hypoxia-inducible factor 1-alpha (HIF1A) by antagonising Von Hippel-Lindau (VHL)-mediated HIF1A ubiquitination, driving LEP hyperactivation. Importantly, we demonstrate that sensitivity to piceatannol/tyrphostin 23 epistatically relies on a HIF1A-LEP-Uncoupling Protein 2 (UCP2) signaling axis lowering cellular energy beyond survival, in already challenged LKB1-deficient cells. Thus, we uncover a pivotal metabolic vulnerability of LKB1-deficient tumours, which may be therapeutically exploited using our identified compounds as mitochondrial uncouplers

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Defining the clinical and molecular spectrum of inherited eye diseases in community settings

Inherited eye diseases are an important contributor to the burden of childhood blindness globally. These conditions are often associated with significant phenotypic and genetic heterogeneity and are extremely difficult to study in a general population setting. This thesis details the study of inherited eye diseases in genetically isolated populations including the North American Amish and rural Pakistani and Palestinian communities. Here, an enrichment of disease-causing founder mutations arising from common ancestry, characteristic marriage patterns and geographical isolation, combined with the often large family sizes typical of families in these regions, enables powerful genomic studies to identify pathogenic sequence variants. As well as providing an important opportunity to learn about the genetic causes of inherited eye diseases, these studies also provide desperately required healthcare benefits for the families and populations involved. Chapter 3 describes studies of oculocutaneous albinism (OCA) in 40 Amish and Pakistani families. Results from comprehensive clinical, genomic and functional studies, initiated by a search for the cause of OCA in a number of Amish families, provide strong evidence for the pathogenicity of two common TYR gene variants [p.(Ser192Tyr) and p.(Arg402Gln)] when inherited in cis. These variants were previously considered gene polymorphisms although this has been heavily debated in many studies, and these variants are currently variably reported and even potentially excluded by clinical testing laboratories. The findings reported in this thesis have important diagnostic implications by helping clarify the contribution of these variants to the OCA phenotype, and by promoting the reporting the TYR p.(Ser192Tyr)/p.(Arg402Gln) in cis haplotype as a pathogenic allele. This will likely increase the molecular diagnoses in albinism patients with missing heritability by 25-50%. This chapter also entails a comprehensive investigation involving genetic studies alongside an exhaustive literature review of all published OCA genetic causes in Pakistani families, including cross-referencing with established genomic databases to evidence the likely causality of each gene variant. Chapter 4 entails clinical and genomic findings in four families with phenotypic features highly suggestive of a ciliopathy disorder. Findings include identification of novel SCAPER and BBS5 variants, enabling a more precise definition of the SCAPER clinical phenotype. This work also consolidates an INPP5E c.1879C>T; p.(Gln627*) variant, a likely pathogenic founder alteration present in Northern Pakistan, as a cause of MORM syndrome. Chapter 5 documents studies of families with rare and ultra-rare inherited ocular diseases in Pakistani and Palestinian communities. This includes consolidating SDHD dysfunction as a cause of mitochondrial disease through investigations of an extended Palestinian family, facilitating a clearer delineation of the variable ocular (and non-ocular) phenotypical features. Alongside this, the identification of novel and known variants in ALDH1A3, FYCO1, TDRD7, CYP1B1, ATOH7, LRP5, FRMD7 and HPS1 in Pakistani communities contributes to an improved knowledge of the genetic spectrum and frequencies of various forms of inherited eye diseases regionally. The comprehensive OCA and BBS datasets provide notably improved knowledge, as well as a centralised repository, of the genetic spectrum and regional frequencies of the molecular causes of these conditions in the Amish and in Pakistan. Ultimately, these findings will greatly facilitate the establishment of robust cost-effective accurate diagnostic genetic testing, clinical management and counselling efforts. Together the work of this thesis describes data of scientific importance, and highlights the immense value of translational community research studies to deliver clinical benefits locally and globally in the field of inherited eye diseases

Mechanics of CSF in Human Ventricular System:Production and Circulation

Cerebrospinal fluid (CSF) is a water-like substance that circulates in a network of interconnected cavities located in the brain. The fluid ensures the brain’s well-being by providing hydromechanical and biochemical support. Moreover, the fluid facilitates neurogenesis (i.e. the birth of neurones); a process essential for embryo development and self-repair in developed brains. Changes in the composition or flow of CSF are linked to the development and progression of various neurological conditions such as hydrocephalus, multiple sclerosis, and Alzheimer’s disease. These conditions have been recognised for over a century; yet there is no definite explanation for their onset nor there is a cure to restore brain health. Hence, medical interventions (e.g. surgery, medication) are used to control the abnormalities and prevent further damage, but they do not address the underlying cause. One reason for the lack of effective treatments is the limited understanding of the CSF system; its production, circulation, and absorption, in healthy and diseased states, and after treatments. In this research, we aim to better understand the CSF system by conducting mathematical and computational modelling. At first, a comprehensive review of the existing literature is provided to bring to attention the controversies and misconceptions related to the system. Further, we discuss the necessity of exploring the system step by step to correct the existing conjectures. We present a novel and comprehensive mathematical model of the organ responsible for CSF production (i.e. choroid plexus (CP)). This model integrates the CP’s biological characteristics into physics and mathematical statements, which are solved using numerical techniques. The simulation predicts parameters such as pressure, concentration, and displacement of the CP, as well as the CSF production rate under different conditions such as ageing. This approach represents the first holistic method for understanding the dynamics of CSF production within the choroid plexus. This research is extended to a preliminary investigation of CSF circulation in a 3D computational model of the brain cavities, constructed from magnetic resonance images (MRI). Simulation results characterise the CSF flow by providing information on pressure gradients, wall shear stresses, and fluid velocitie

Cyanogenesis in the cystic fibrosis pathogen Pseudomonas aeruginosa

Lung disease is the most lethal manifestation of Cystic Fibrosis (CF). The bacterium Pseudomonas aeruginosa is a major pathogen of the CF lung, where it is able to persist for decades. P. aeruginosa is also one of only a few bacterial species capable of producing hydrogen cyanide (HCN). Since cyanide is detectable in the sputum of CF patients, and its presence is negatively correlated with lung function, it has been hypothesised that cyanogenesis in P. aeruginosa may increase virulence in humans. Here, the overall aims have been to explore the clinical relevance of cyanide production in the CF airway and to improve current understanding of the molecular microbiology of cyanogenesis. Ciliary beat frequency (CBF) measurements were performed on nasal brushings and sino-nasal airliquid interface (ALI) cultures obtained from healthy volunteers and CF patients. KCN decreased CBF in healthy nasal brushings (n=6) after 60 min (150µM: 47% fall, p<0.0002; 75µM: 32% fall, P<0.0001). Samples from CF patients (n=3) showed similar results (150µM: 55% fall, P=0.0003). CBF inhibition was not due to loss of cell viability and was reversible. The inhibitory mechanism was independent of ATP levels. KCN also significantly inhibited CBF in ALI cultures, albeit to a lesser extent. Additionally, CBF measurements on ALI cultures treated with culture supernatants from P. aeruginosa mutants implicated cyanide as a key cilio-inhibitory component. In order to uncover novel determinants of cyanogenesis, a high throughput colourimetric cyanide screen was developed. Cyanogenesis values were obtained for 93% of mutants from a comprehensive nonredundant P. aeruginosa transposon library. Additionally, the validity of two severely impaired mutants, PA2196 and PA5339, was confirmed by complementation. Mapping the most severe cyanogenesis outliers onto the P.aeruginosa genome revealed previously unrelated regions of interest. In particular, overrepresentation of genes related to the motile-sessile switch and to the RoxRS system implicated a strong role for these in the regulation of cyanogenesis.Open Acces

Developing Implants for Ophthalmic Drug Delivery and Flow Modulation

Glaucoma is the leading cause of irreversible blindness worldwide. Surgical interventions are frequently necessary to lower the intraocular pressure (IOP) and do so by creating a new channel for aqueous humour to drain into the subconjunctival space. This channel can be formed by performing a glaucoma filtration surgery (GFS) or by implanting a glaucoma drainage device (GDD). However, excessive scarring at the surgical site blocks aqueous outflow, elevates IOP, and results in treatment failure. Drugs injected locally to control scarring rapidly clear from the subconjunctiva, and current implants are susceptible to a foreign body response. This work investigated strategies that could improve the outcomes of these current glaucoma interventions. First, drug-eluting spacers were formulated using established biocompatible materials to prolong drug release in conditions representing the subconjunctival space post-GFS or GDD implantation. Of these formulations, the spacer containing non-ionic surfactant, Brij 98, at a concentration of 1.25% w/v was able to prolong the release of dexamethasone from poly(2-hydroxyethyl methacrylate) pHEMA hydrogels significantly longer (>30 days) than hydrogels containing no surfactant (<7 days) at therapeutically relevant drug concentrations in vitro. Next, engineering principles were applied to inflated elastomeric membranes, which provided novel insights into considerations needed to design a novel ophthalmic drug delivery pump. Pocket geometry and material properties had a significant impact on internal pressure and subsequent pump function. Modelling data supports the feasibility of elastomeric pumps for prolonged subconjunctival drug delivery. Finally, an alternative mechanism of IOP control was investigated. Novel and established hydrogel formulations were evaluated for aqueous permeability and mechanical integrity. Despite evidence to suggest the feasibility of hydrogels to modulate aqueous flow, the in vitro permeability of hydrogel candidates was determined to be too low to maintain optimal IOP. Furthermore, hydrogel permeability tended to negate its mechanical integrity, making them unsuitable candidate materials for GDD development