SF3B1 hotspot mutations confer sensitivity to PARP inhibition by eliciting a defective replication stress response.

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.

Retinal Changes in Transgenic Mouse Models of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder, the most common form of dementia. AD is characterised by amyloid-ß (Aß) plaques and neurofibrillary tangles (NFT) in the brain, in association with neuronal loss and synaptic failure, causing cognitive deficits. Accurate and early diagnosis is currently unavailable in lifespan, hampering early intervention of potential new treatments. Visual deficits have been well documented in AD patients, and the pathological changes identified in the brain are also believed to be found in the retina, an integral part of the central nervous system. Retinal changes can be detected by real-time non-invasive imaging, due to the transparent nature of the ocular media, potentially allowing an earlier diagnosis as well as monitoring disease progression and treatment outcome. Animal models are essential for AD research, and this review has a focus on retinal changes in various transgenic AD mouse models with retinal imaging and immunohistochemical analysis as well as therapeutic effects in those models. We also discuss the limitations of transgenic AD models in clinical translations.

Dynamic changes in cell size and corresponding cell fate after optic nerve injury.

Identifying disease-specific patterns of retinal cell loss in pathological conditions has been highlighted by the emergence of techniques such as Detection of Apoptotic Retinal Cells and Adaptive Optics confocal Scanning Laser Ophthalmoscopy which have enabled single-cell visualisation in vivo. Cell size has previously been used to stratify Retinal Ganglion Cell (RGC) populations in histological samples of optic neuropathies, and early work in this field suggested that larger RGCs are more susceptible to early loss than smaller RGCs. More recently, however, it has been proposed that RGC soma and axon size may be dynamic and change in response to injury. To address this unresolved controversy, we applied recent advances in maximising information extraction from RGC populations in retinal whole mounts to evaluate the changes in RGC size distribution over time, using three well-established rodent models of optic nerve injury. In contrast to previous studies based on sampling approaches, we examined the whole Brn3a-positive RGC population at multiple time points over the natural history of these models. The morphology of over 4 million RGCs was thus assessed to glean novel insights from this dataset. RGC subpopulations were found to both increase and decrease in size over time, supporting the notion that RGC cell size is dynamic in response to injury. However, this study presents compelling evidence that smaller RGCs are lost more rapidly than larger RGCs despite the dynamism. Finally, using a bootstrap approach, the data strongly suggests that disease-associated changes in RGC spatial distribution and morphology could have potential as novel diagnostic indicators.

Topical recombinant human Nerve growth factor (rh-NGF) is neuroprotective to retinal ganglion cells by targeting secondary degeneration.

Optic neuropathy is a major cause of irreversible blindness worldwide, and no effective treatment is currently available. Secondary degeneration is believed to be the major contributor to retinal ganglion cell (RGC) death, the endpoint of optic neuropathy. Partial optic nerve transection (pONT) is an established model of optic neuropathy. Although the mechanisms of primary and secondary degeneration have been delineated in this model, until now how this is influenced by therapy is not well-understood. In this article, we describe a clinically translatable topical, neuroprotective treatment (recombinant human nerve growth factor, rh-NGF) predominantly targeting secondary degeneration in a pONT rat model. Topical application of rh-NGF twice daily for 3 weeks significantly improves RGC survival as shown by reduced RGC apoptosis in vivo and increased RGC population in the inferior retina, which is predominantly affected in this model by secondary degeneration. Topical rh-NGF also promotes greater axonal survival and inhibits astrocyte activity in the optic nerve. Collectively, these results suggest that topical rh-NGF exhibits neuroprotective effects on retinal neurons via influencing secondary degeneration process. As topical rh-NGF is already involved in early clinical trials, this highlights its potential in multiple indications in patients, including those affected by glaucomatous optic neuropathy.

Topical Curcumin Nanocarriers are Neuroprotective in Eye Disease.

Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5dione) is a polyphenol extracted from turmeric that has long been advocated for the treatment of a variety of conditions including neurodegenerative and inflammatory disorders. Despite this promise, the clinical use of curcumin has been limited by the poor solubility and low bioavailability of this molecule. In this article, we describe a novel nanocarrier formulation comprising Pluronic-F127 stabilised D-α-Tocopherol polyethene glycol 1000 succinate nanoparticles, which were used to successfully solubilize high concentrations (4.3 mg/mL) of curcumin. Characterisation with x-ray diffraction and in vitro release assays localise curcumin to the nanocarrier interior, with each particle measuring <20 nm diameter. Curcumin-loaded nanocarriers (CN) were found to significantly protect against cobalt chloride induced hypoxia and glutamate induced toxicity in vitro, with CN treatment significantly increasing R28 cell viability. Using established glaucoma-related in vivo models of ocular hypertension (OHT) and partial optic nerve transection (pONT), topical application of CN twice-daily for three weeks significantly reduced retinal ganglion cell loss compared to controls. Collectively, these results suggest that our novel topical CN formulation has potential as an effective neuroprotective therapy in glaucoma and other eye diseases with neuronal pathology.

Memantine-Loaded PEGylated Biodegradable Nanoparticles for the Treatment of Glaucoma.

Glaucoma is a multifactorial neurodegenerative disease associated with retinal ganglion cells (RGC) loss. Increasing reports of similarities in glaucoma and other neurodegenerative conditions have led to speculation that therapies for brain neurodegenerative disorders may also have potential as glaucoma therapies. Memantine is an N-methyl-d-aspartate (NMDA) antagonist approved for Alzheimer's disease treatment. Glutamate-induced excitotoxicity is implicated in glaucoma and NMDA receptor antagonism is advocated as a potential strategy for RGC preservation. This study describes the development of a topical formulation of memantine-loaded PLGA-PEG nanoparticles (MEM-NP) and investigates the efficacy of this formulation using a well-established glaucoma model. MEM-NPs <200 nm in diameter and incorporating 4 mg mL-1 of memantine were prepared with 0.35 mg mL-1 localized to the aqueous interior. In vitro assessment indicated sustained release from MEM-NPs and ex vivo ocular permeation studies demonstrated enhanced delivery. MEM-NPs were additionally found to be well tolerated in vitro (human retinoblastoma cells) and in vivo (Draize test). Finally, when applied topically in a rodent model of ocular hypertension for three weeks, MEM-NP eye drops were found to significantly (p < 0.0001) reduce RGC loss. These results suggest that topical MEM-NP is safe, well tolerated, and, most promisingly, neuroprotective in an experimental glaucoma model.

Topical Coenzyme Q10 demonstrates mitochondrial-mediated neuroprotection in a rodent model of ocular hypertension.

Coenzyme Q10 (CoQ10) is a mitochondrial-targeted antioxidant with known neuroprotective activity. Its ocular effects when co-solubilised with α-tocopherol polyethylene glycol succinate (TPGS) were evaluated. In vitro studies confirmed that CoQ10 was significantly protective in different retinal ganglion cell (RGC) models. In vivo studies in Adult Dark Agouti (DA) rats with unilateral surgically-induced ocular hypertension (OHT) treated with either CoQ10/TPGS micelles or TPGS vehicle twice daily for three weeks were performed, following which retinal cell health was assessed in vivo using DARC (Detection of Apoptotic Retinal Cells) and post-mortem with Brn3a histological assessment on whole retinal mounts. CoQ10/TPGS showed a significant neuroprotective effect compared to control with DARC (p<0.05) and Brn3 (p<0.01). Topical CoQ10 appears an effective therapy preventing RGC apoptosis and loss in glaucoma-related models.

Viscoelastic, physical, and bio-degradable properties of dermal scaffolds and related cell behaviour.

Dermal scaffolds promote healing of debilitating skin injuries caused by burns and chronic skin conditions. Currently available products present disadvantages and therefore, there is still a clinical need for developing new dermal substitutes. This study aimed at comparing the viscoelastic, physical and bio-degradable properties of two dermal scaffolds, the collagen-based and clinically well established Integra(®) and a novel fibrin-based dermal scaffold developed at our laboratory called Smart Matrix(®), to further evaluate our previous published findings that suggested a higher influx of cells, reduced wound contraction and less scarring for Smart Matrix(®) when used in vivo. Rheological results showed that Integra(®) (G' = 313.74 kPa) is mechanically stronger than Smart Matrix(®) (G' = 8.26 kPa), due to the presence of the silicone backing layer in Integra(®). Micro-pores were observed on both dermal scaffolds, although nano-pores as well as densely packed nano-fibres were only observed for Smart Matrix(®). Average surface roughness was higher for Smart Matrix(®) (Sa = 114.776 nm) than for Integra(®) (Sa = 75.565 nm). Both scaffolds possess a highly porous structure (80-90%) and display a range of pore micro-sizes that represent the actual in vivo scenario. In vitro proteolytic bio-degradation suggested that Smart Matrix(®) would degrade faster upon implantation in vivo than Integra(®). For both scaffolds, the enzymatic digestion occurs via bulk degradation. These observed differences could affect cell behaviour on both scaffolds. Our results suggest that fine-tuning of scaffolds' viscoelastic, physical and bio-degradable properties can maximise cell behaviour in terms of attachment, proliferation and infiltration, which are essential for tissue repair.

The retina as an early biomarker of neurodegeneration in a rotenone-induced model of Parkinson's disease: evidence for a neuroprotective effect of rosiglitazone in the eye and brain.

Parkinson's Disease (PD) is the second most common neurodegenerative disease worldwide, affecting 1 % of the population over 65 years of age. Dopaminergic cell death in the substantia nigra and accumulation of Lewy bodies are the defining neuropathological hallmarks of the disease. Neuronal death and dysfunction have been reported in other central nervous system regions, including the retina. Symptoms of PD typically manifest only when more than 70 % of dopaminergic cells are lost, and the definitive diagnosis of PD can only be made histologically at post-mortem, with few biomarkers available.In this study, a rotenone-induced rodent model of PD was employed to investigate retinal manifestations in PD and their usefulness in assessing the efficacy of a novel therapeutic intervention with a liposomal formulation of the PPAR-γ (Peroxisome proliferator-activated receptor gamma) agonist rosiglitazone.Retinal assessment was performed using longitudinal in vivo imaging with DARC (detection of apoptosing retinal cells) and OCT (optical coherence tomography) technologies and revealed increased RGCs (Retinal Ganglion Cells) apoptosis and a transient swelling of the retinal layers at day 20 of the rotenone insult. Follow-up of this model demonstrated characteristic histological neurodegenerative changes in the substantia nigra and striatum by day 60, suggesting that retinal changes precede the "traditional" pathological manifestations of PD. The therapeutic effect of systemic administration of different formulations of rosiglitazone was then evaluated, both in the retina and the brain. Of all treatment regimen tested, sustained release administration of liposome-encapsulated rosiglitazone proved to be the most potent therapeutic strategy, as evidenced by its significant neuroprotective effect on retinal neurons at day 20, and on nigrostriatal neurons at day 60, provided convincing evidence for its potential as a treatment for PD.Our results demonstrate significant retinal changes occurring in this model of PD. We show that rosiglitazone can efficiently protect retinal neurons from the rotenone insult, and that systemic administration of liposome-encapsulated rosiglitazone has an enhanced neuroprotective effect on the retina and CNS (Central Nervous System). To our knowledge, this is the first in vivo evidence of RGCs loss and early retinal thickness alterations in a PD model. Together, these findings suggest that retinal changes may be a good surrogate biomarker for PD, which may be used to assess new treatments both experimentally and clinically.

Apoptotic primary normal human dermal fibroblasts for in vitro models of fibrosis.

Recent studies show that apoptosis affects surrounding tissue, playing a role in diseases such as fibrosis, a significant global disease burden. Elucidating the mechanisms by which the different apoptotic cells present during fibrotic wound healing affect their environment would enable development of new therapies. We describe here a simple, rapid, and cost-effective method for inducing apoptosis of primary normal human dermal fibroblasts without affecting the overall cell viability of the population. Such population could be used for in vitro models of fibrotic wound healing in co-culture with other cells involved in this process to study events such as apoptosis-induced proliferation.

A novel multiparameter in vitro model of three-dimensional cell ingress into scaffolds for dermal reconstruction to predict in vivo outcome.

The clinical demand for effective dermal substitutes continues as current commercially available products present limitations. However, there are no definitive in vitro methods to predict in vivo outcomes such as integration, cellularization and contraction, which may help the development of new dermal scaffolds. This study aimed to develop a multiparameter in vitro model of three-dimensional (3D) cell ingress into dermal scaffolds to predict in vivo outcomes of new dermal scaffolds under development. A new dermal scaffold, Smart Matrix, was compared to the scar-forming contractile collagen gel model and to the clinically well-established Integra(®) and Matriderm(®). Parameters studied were cell viability and proliferation, apoptosis, matrix contraction, cell morphology, α-smooth muscle actin, and growth factor expression. Combinatorial evaluation of the results in a scoring matrix showed that Smart Matrix could offer an advantage over existing products. This method would be useful as an international golden scoring matrix to develop new dermal scaffolds that effectively improve the existing products, thus enabling better treatments for burns or chronic wounds.

Analysis of genotype and haplotype effects of ABCB1 (MDR1) polymorphisms in the risk of medically refractory epilepsy in an Indian population.

The transmembrane P-glycoprotein that functions as a drug-efflux transporter coded by ATP-binding cassette, subfamily B, member 1/Multidrug Resistance 1 (ABCB1/MDR1) gene is considered relevant to drug absorption and elimination, with access to the central nervous system. Effects of three ABCB1 single nucleotide polymorphisms (SNPs) in genotypic and haplotypic combination have been evaluated in a south Indian population for risk of pediatric medically refractory epilepsy. The study included age and sex matched medically refractory (N=113) cases and drug responsive epilepsy patients (N=129) as controls, belonging to the same ethnic population recruited from a tertiary referral centre, of Karnataka, Southern India. The genotype frequencies of SNPs c.1236C>T, c.2677G>T/A, and c.3435C>T were determined from genomic DNA of the cases and controls by PCR- RFLP and confirmatory DNA sequencing. 256 normal population samples of the same ethnicity were genotyped for the three loci to check for population stratification. Results indicate that there was no statistically significant difference between allele and genotype frequencies of refractory and drug responsive epilepsy patients. The predicted haplotype frequencies of the three polymorphisms did not show significant difference between cases and controls. The results confirm earlier observations on absence of association of ABCB1 polymorphisms with medically refractory epilepsy.