Nina Marie Rzechorzek

Adaptive preconditioning in neurological diseases – therapeutic insights from proteostatic perturbations

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.

Maturation of AMPAR Composition and the GABAAR Reversal Potential in hPSC-Derived Cortical Neurons

Rodent-based studies have shown that neurons undergo major developmental changes to ion channel expression and ionic gradients that determine their excitation-inhibition balance. Neurons derived from human pluripotent stem cells theoretically offer the potential to study classical developmental processes in a human-relevant system, although this is currently not well explored. Here, we show that excitatory cortical-patterned neurons derived from multiple human pluripotent stem cell lines exhibit native-like maturation changes in AMPAR composition such that there is an increase in the expression of GluA2(R) subunits. Moreover, we observe a dynamic shift in intracellular Cl− levels, which determines the reversal potential of GABAAR-mediated currents and is influenced by neurotrophic factors. The shift is concomitant with changes in KCC2 and NKCC1 expression. Because some human diseases are thought to involve perturbations to AMPAR GluA2 content and others in the chloride reversal potential, human stem-cell-derived neurons represent a valuable tool for studying these fundamental properties.

Network Analysis Reveals Distinct Clinical Syndromes Underlying Acute Mountain Sickness

Acute mountain sickness (AMS) is a common problem among visitors at high altitude, and may progress to life-threatening pulmonary and cerebral oedema in a minority of cases. International consensus defines AMS as a constellation of subjective, non-specific symptoms. Specifically, headache, sleep disturbance, fatigue and dizziness are given equal diagnostic weighting. Different pathophysiological mechanisms are now thought to underlie headache and sleep disturbance during acute exposure to high altitude. Hence, these symptoms may not belong together as a single syndrome. Using a novel visual analogue scale (VAS), we sought to undertake a systematic exploration of the symptomatology of AMS using an unbiased, data-driven approach originally designed for analysis of gene expression. Symptom scores were collected from 292 subjects during 1110 subject-days at altitudes between 3650 m and 5200 m on Apex expeditions to Bolivia and Kilimanjaro. Three distinct patterns of symptoms were consistently identified. Although fatigue is a ubiquitous finding, sleep disturbance and headache are each commonly reported without the other. The commonest pattern of symptoms was sleep disturbance and fatigue, with little or no headache. In subjects reporting severe headache, 40% did not report sleep disturbance. Sleep disturbance correlates poorly with other symptoms of AMS (Mean Spearman correlation 0.25). These results challenge the accepted paradigm that AMS is a single disease process and describe at least two distinct syndromes following acute ascent to high altitude. This approach to analysing symptom patterns has potential utility in other clinical syndromes.

Hypothermic Preconditioning of Human Cortical Neurons Requires Proteostatic Priming.

Hypothermia is potently neuroprotective but poor mechanistic understanding has restricted its clinical use. Rodent studies indicate that hypothermia can elicit preconditioning, wherein a subtoxic cellular stress confers resistance to an otherwise lethal injury. The molecular basis of this preconditioning remains obscure. Here we explore molecular effects of cooling using functional cortical neurons differentiated from human pluripotent stem cells (hCNs). Mild-to-moderate hypothermia (28–32 °C) induces cold-shock protein expression and mild endoplasmic reticulum (ER) stress in hCNs, with full activation of the unfolded protein response (UPR). Chemical block of a principal UPR pathway mitigates the protective effect of cooling against oxidative stress, whilst pre-cooling neurons abrogates the toxic injury produced by the ER stressor tunicamycin. Cold-stress thus preconditions neurons by upregulating adaptive chaperone-driven pathways of the UPR in a manner that precipitates ER-hormesis. Our findings establish a novel arm of neurocryobiology that could reveal multiple therapeutic targets for acute and chronic neuronal injury.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Hypothermic Preconditioning Reverses Tau Ontogenesis in Human Cortical Neurons and is Mimicked by Protein Phosphatase 2A Inhibition

Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure. Changes in neuronal tau protein are of interest, since tau becomes hyperphosphorylated in injury-resistant, hypothermic brains. Noting inter-species differences in tau isoforms, we have used functional cortical neurons differentiated from human pluripotent stem cells (hCNs) to interrogate tau modulation during hypothermic preconditioning at clinically-relevant temperatures. Key tau developmental transitions (phosphorylation status and splicing shift) are recapitulated during hCN differentiation and subsequently reversed by mild (32 °C) to moderate (28 °C) cooling — conditions which reduce oxidative and excitotoxic stress-mediated injury in hCNs. Blocking a major tau kinase decreases hCN tau phosphorylation and abrogates hypothermic neuroprotection, whilst inhibition of protein phosphatase 2A mimics cooling-induced tau hyperphosphorylation and protects normothermic hCNs from oxidative stress. These findings indicate a possible role for phospho-tau in hypothermic preconditioning, and suggest that cooling drives human tau towards an earlier ontogenic phenotype whilst increasing neuronal resilience to common neurotoxic insults. This work provides a critical step forward in understanding how we might exploit the neuroprotective benefits of cooling without cooling patients.

Diagnosing limb paresis and paralysis in sheep

Paresis and paralysis are uncommon problems in sheep but are likely to prompt farmers to seek veterinary advice. A thorough and logical approach can aid in determining the cause of the problem and highlighting the benefit of veterinary involvement. While this may not necessarily alter the prognosis for an individual animal, it can help in formulating preventive measures and avoid the costs – both in economic and in welfare terms – of misdirected treatment. Distinguishing between central and peripheral lesions is most important, as the relative prognoses are markedly different, and this can often be achieved with minimal equipment. This article describes an approach to performing a neurological examination of the ovine trunk and limbs, the ancillary tests available and the common and important causes of paresis and paralysis in sheep.

Sheep Lung Segmental Delivery Strategy Demonstrates Adenovirus Priming of Local Lung Responses to Bacterial LPS and the Role of Elafin as a Response Modulator

Viral lung infections increase susceptibility to subsequent bacterial infection. We questioned whether local lung administration of recombinant adenoviral vectors in the sheep would alter the susceptibility of the lung to subsequent challenge with bacterial lipopolysaccharide (LPS). We further questioned whether local lung expression of elafin, a locally produced alarm anti-LPS/anti-bacterial molecule, would modulate the challenge response. We established that adenoviral vector treatment primed the lung for an enhanced response to bacterial LPS. Whereas this local effect appeared to be independent of the transgene used (Ad-o-elafin or Ad-GFP), Ad-o-elafin treated sheep demonstrated a more profound lymphopenia in response to local lung administration of LPS. The local influence of elafin in modulating the response to LPS was restricted to maintaining neutrophil myeloperoxidase activity, and levels of alveolar macrophage and neutrophil phagocytosis at higher levels post-LPS. Adenoviral vector-bacterial synergism exists in the ovine lung and elafin expression modulates such synergism both locally and systemically.

Network analysis of canine brain morphometry links tumour risk to oestrogen deficiency and accelerated brain ageing

Background Structural ‘brain age’ is a valuable but complex biomarker for several brain disorders. The dog is an unrivalled comparator for neurological disease modeling, however brain phenotypic diversity among pedigrees creates computational and statistical challenges. Methods We applied unbiased network correlation analysis in dogs to explore complex interactions between brain morphometrics, patient metadata, and neurological disease. Twenty-four parameters measured from each of 286 brain magnetic resonance imaging scans generated 9,438 data points that were used to cluster canine patients according to their brain morphometry profiles. The network was then explored for statistically significant enrichments within breed, sex, age, and diagnostic categories. Findings Morphometric comparisons revealed an advanced ‘aged-brain’ profile in the Boxer breed, consisting of a small brain length, width, and volume, combined with ventriculomegaly. Key features of this profile were paralleled in neutered female dogs which, relative to un-neutered females, had an 11-fold greater risk of developing primary brain tumours. Enrichment analysis confirmed that Boxers and geriatric individuals were enriched for brain tumour diagnoses, despite a lack of geriatric Boxers within the cohort. Interpretation These findings suggest that accelerated brain ageing might contribute to tumour risk in Boxers and may be influenced by oestrogen deficiency — a risk factor for dementia and brain tumours in humans. We propose that morphometric features of brain ageing in dogs, like humans, might better predict neurological disease risk than a patient’s chronological age. Funding Wellcome Trust Integrated Training Fellowship for Veterinarians (096409/Z/11/Z to N.M.R) and an MSD Animal Health Connect Bursary (to O.M.S.).

Hypothermic modulation of human cortical neurons to explore a role for tau protein in neuroprotection

Abstract Background Cooling is the single most effective treatment for acute neuronal injury. Understanding the molecular mechanisms that mediate cooling-induced neuroprotection might yield novel therapeutic targets for neurodegenerative disease. Many of these disorders involve modulation of tau, a protein that is enriched in neurons and becomes hyperphosphorylated in hypothermic, injury-resistant rodent brains as well as in Alzheimers disease. We sought to establish a new model for exploring human tau physiology and its role in neuroprotection in the context of therapeutic hypothermia. Methods Functional cortical neurons (hCNs) were differentiated from three independent human pluripotent stem-cell lines and validated for regional identity and tau status. Matched cultures were incubated at 37°C or clinically targeted temperatures for therapeutic hypothermia (32°C) and suspended animation (28°C). Effects of cooling on tau status and neuronal injury elicited by common neurotoxic stressors were established. Injury experiments were repeated while manipulating tau phosphorylation. Findings hCN differentiation featured transitions in tau status, recapitulating transcriptional and post-translational human in-vivo cortical tau development. Key aspects of this development were reversed by cooling. Notably, cooling induced tau hyperphosphorylation via rapid inhibition of the major tau phosphatase, protein phosphatase 2A (PP2A). Multiplexed injury analysis confirmed that hypothermia robustly protected hCNs from oxidative (100 μM hydrogen peroxide) and excitotoxic (30 μM glutamate) stress (at 28°C, injury was reduced by 78% and 56%, respectively; p Interpretation To our knowledge, this is the first study of human neuronal tau physiology under hypothermic conditions. Although definitive experiments are needed, our findings support previous work linking phospho-tau to neuroprotection. Furthermore, cold-induced tau hyperphosphorylation is a potential trigger for proteostatic priming, a recently discovered mechanism of hypothermic preconditioning in hCNs. Exploiting these cryobiological effects might lead to new treatments for acute and chronic neuronal injury. Funding Wellcome Trust, Anne Rowling Regenerative Neurology Clinic, Euan MacDonald Centre for Motor Neurone Disease Research.