Abstract In neurological disorders, both acute and chronic neural stress can disrupt cellular proteostasis, resulting in the generation of pathological protein. However in most cases, neurons adapt ...to these proteostatic perturbations by activating a range of cellular protective and repair responses, thus maintaining cell function. These interconnected adaptive mechanisms comprise a ‘proteostasis network’ and include the unfolded protein response, the ubiquitin proteasome system and autophagy. Interestingly, several recent studies have shown that these adaptive responses can be stimulated by preconditioning treatments, which confer resistance to a subsequent toxic challenge – the phenomenon known as hormesis. In this review we discuss the impact of adaptive stress responses stimulated in diverse human neuropathologies including Parkinson׳s disease, Wolfram syndrome, brain ischemia, and brain cancer. Further, we examine how these responses and the molecular pathways they recruit might be exploited for therapeutic gain. This article is part of a Special Issue entitled SI:ER stress.
The neuronal ceroid lipofuscinoses (NCLs) are a group of devastating monogenetic lysosomal disorders that affect children and young adults with no cure or effective treatment currently available. One ...of the more severe infantile forms of the disease (INCL or CLN1 disease) is due to mutations in the palmitoyl-protein thioesterase 1 (PPT1) gene and severely reduces the child's lifespan to approximately 9 years of age. In order to better translate the human condition than is possible in mice, we sought to produce a large animal model employing CRISPR/Cas9 gene editing technology. Three PPT1 homozygote sheep were generated by insertion of a disease-causing PPT1 (R151X) human mutation into the orthologous sheep locus. This resulted in a morphological, anatomical and biochemical disease phenotype that closely resembles the human condition. The homozygous sheep were found to have significantly reduced PPT1 enzyme activity and accumulate autofluorescent storage material, as is observed in CLN1 patients. Clinical signs included pronounced behavioral deficits as well as motor deficits and complete loss of vision, with a reduced lifespan of 17 ± 1 months at a humanely defined terminal endpoint. Magnetic resonance imaging (MRI) confirmed a significant decrease in motor cortical volume as well as increased ventricular volume corresponding with observed brain atrophy and a profound reduction in brain mass of 30% at necropsy, similar to alterations observed in human patients. In summary, we have generated the first CRISPR/Cas9 gene edited NCL model. This novel sheep model of CLN1 disease develops biochemical, gross morphological and in vivo brain alterations confirming the efficacy of the targeted modification and potential relevance to the human condition.
The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in ...DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.
The impact of neurological disorders is recognised globally, with one in six people affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to the ...increasing ageing population, and is confounded by the high failure rate of translation from rodent-derived therapeutics to clinically effective human neurological interventions. Improved translation is demonstrated using higher order mammals with more complex/comparable neuroanatomy. These animals effectually span this translational disparity and increase confidence in factors including routes of administration/dosing and ability to scale, such that potential therapeutics will have successful outcomes when moving to patients. Coupled with advancements in genetic engineering to produce genetically tailored models, livestock are increasingly being used to bridge this translational gap.
In order to aid in standardising characterisation of such models, we provide comprehensive neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or lesion(s) for large animal species. We also describe the applicability of these exams in different large animals to help provide a better understanding of the practicalities of cross species neurological disease modelling.
We would encourage the use of these assessments as a reference framework to help standardise neurological clinical scoring of large animal models.
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high‐throughput (HTP) structure determination of proteins from Bacillus ...anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target‐selection strategy combining `low‐hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation‐independent cloning (LIC) and expression systems, employing custom LIC‐PCR, Gateway and In‐Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X‐ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target‐selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
PDF
