Sensitization of the UPR by loss of PPP1R15A promotes fibrosis and senescence in IPF.

The unfolded protein response (UPR) is a direct consequence of cellular endoplasmic reticulum (ER) stress and a key disease driving mechanism in IPF. The resolution of the UPR is directed by PPP1R15A (GADD34) and leads to the restoration of normal ribosomal activity. While the role of PPP1R15A has been explored in lung epithelial cells, the role of this UPR resolving factor has yet to be explored in lung mesenchymal cells. The objective of the current study was to determine the expression and role of PPP1R15A in IPF fibroblasts and in a bleomycin-induced lung fibrosis model. A survey of IPF lung tissue revealed that PPP1R15A expression was markedly reduced. Targeting PPP1R15A in primary fibroblasts modulated TGF-ß-induced fibroblast to myofibroblast differentiation and exacerbated pulmonary fibrosis in bleomycin-challenged mice. Interestingly, the loss of PPP1R15A appeared to promote lung fibroblast senescence. Taken together, our findings demonstrate the major role of PPP1R15A in the regulation of lung mesenchymal cells, and regulation of PPP1R15A may represent a novel therapeutic strategy in IPF.

Soluble amyloid beta-containing aggregates are present throughout the brain at early stages of Alzheimer's disease.

Protein aggregation likely plays a key role in the initiation and spreading of Alzheimer's disease pathology through the brain. Soluble aggregates of amyloid beta are believed to play a key role in this process. However, the aggregates present in humans are still poorly characterized due to a lack of suitable methods required for characterizing the low concentration of heterogeneous aggregates present. We have used a variety of biophysical methods to characterize the aggregates present in human Alzheimer's disease brains at Braak stage III. We find soluble amyloid beta-containing aggregates in all regions of the brain up to 200 nm in length, capable of causing an inflammatory response. Rather than aggregates spreading through the brain as disease progresses, it appears that aggregation occurs all over the brain and that different brain regions are at earlier or later stages of the same process, with the later stages causing increased inflammation.

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response.

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post-natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.

Utilizing microphysiological systems and induced pluripotent stem cells for disease modeling: a case study for blood brain barrier research in a pharmaceutical setting.

Microphysiological systems (MPS) may be able to provide the pharmaceutical industry models that can reflect human physiological responses to improve drug discovery and translational outcomes. With lack of efficacy being the primary cause for drug attrition, developing MPS disease models would help researchers identify novel targets, study mechanisms in more physiologically-relevant depth, screen for novel biomarkers and test/optimize various therapeutics (small molecules, nanoparticles and biologics). Furthermore, with advances in inducible pluripotent stem cell technology (iPSC), pharmaceutical companies can access cells from patients to help recreate specific disease phenotypes in MPS platforms. Combining iPSC and MPS technologies will contribute to our understanding of the complexities of neurodegenerative diseases and of the blood brain barrier (BBB) leading to development of enhanced therapeutics.

Prion protein stabilizes amyloid-ß (Aß) oligomers and enhances Aß neurotoxicity in a Drosophila model of Alzheimer's disease.

The cellular prion protein (PrPC) can act as a cell-surface receptor for ß-amyloid (Aß) peptide; however, a role for PrPC in the pathogenesis of Alzheimer's disease (AD) is contested. Here, we expressed a range of Aß isoforms and PrPC in the Drosophila brain. We found that co-expression of Aß and PrPC significantly reduces the lifespan, disrupts circadian rhythms, and increases Aß deposition in the fly brain. In contrast, under the same conditions, expression of Aß or PrPC individually did not lead to these phenotypic changes. In vitro studies revealed that substoichiometric amounts of PrPC trap Aß as oligomeric assemblies and fragment-preformed Aß fibers. The ability of membrane-anchored PrPC to trap Aß as cytotoxic oligomers at the membrane surface and fragment inert Aß fibers suggests a mechanism by which PrPC exacerbates Aß deposition and pathogenic phenotypes in the fly, supporting a role for PrPC in AD. This study provides a second animal model linking PrPC expression with Aß toxicity and supports a role for PrPC in AD pathogenesis. Blocking the interaction of Aß and PrPC represents a potential therapeutic strategy.

Central and peripheral circadian clocks and their role in Alzheimer's disease.

Molecular and cellular oscillations constitute an internal clock that tracks the time of day and permits organisms to optimize their behaviour and metabolism to suit the daily demands they face. The workings of this internal clock become impaired with age. In this review, we discuss whether such age-related impairments in the circadian clock interact with age-related neurodegenerative disorders, such as Alzheimer's disease. Findings from mouse and fly models of Alzheimer's disease have accelerated our understanding of the interaction between neurodegeneration and circadian biology. These models show that neurodegeneration likely impairs circadian rhythms either by damaging the central clock or by blocking its communication with other brain areas and with peripheral tissues. The consequent sleep and metabolic deficits could enhance the susceptibility of the brain to further degenerative processes. Thus, circadian dysfunction might be both a cause and an effect of neurodegeneration. We also discuss the primary role of light in the entrainment of the central clock and describe important, alternative time signals, such as food, that play a role in entraining central and peripheral circadian clocks. Finally, we propose how these recent insights could inform efforts to develop novel therapeutic approaches to re-entrain arrhythmic individuals with neurodegenerative disease.

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant.

We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer's disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases.

CCT complex restricts neuropathogenic protein aggregation via autophagy.

Aberrant protein aggregation is controlled by various chaperones, including CCT (chaperonin containing TCP-1)/TCP-1/TRiC. Mutated CCT4/5 subunits cause sensory neuropathy and CCT5 expression is decreased in Alzheimer's disease. Here, we show that CCT integrity is essential for autophagosome degradation in cells or Drosophila and this phenomenon is orchestrated by the actin cytoskeleton. When autophagic flux is reduced by compromise of individual CCT subunits, various disease-relevant autophagy substrates accumulate and aggregate. The aggregation of proteins like mutant huntingtin, ATXN3 or p62 after CCT2/5/7 depletion is predominantly autophagy dependent, and does not further increase with CCT knockdown in autophagy-defective cells/organisms, implying surprisingly that the effect of loss-of-CCT activity on mutant ATXN3 or huntingtin oligomerization/aggregation is primarily a consequence of autophagy inhibition rather than loss of physiological anti-aggregation activity for these proteins. Thus, our findings reveal an essential partnership between two key components of the proteostasis network and implicate autophagy defects in diseases with compromised CCT complex activity.

Progressive myoclonus epilepsy associated with neuroserpin inclusion bodies (neuroserpinosis).

Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a conformational proteinopathy characterised by neuronal inclusion bodies composed of the serine protease inhibitor (SERPIN), neuroserpin. Presenting clinically as a familial dementia-epilepsy syndrome, the molecular mechanism of the pathogenic abnormalities in neuroserpin has been characterised at atomic resolution. There is a remarkable genotype-phenotype correlation between the degree of molecular destabilisation of the several variants of the neuroserpin protein, their propensity to self-associate and the age of onset of the dementia-epilepsy complex. As with other serpinopathies there appears to be a mix of cell-autonomous toxicity, due to neuronal accumulation of neuroserpin, and non-cell autonomous toxicity, caused by loss of protease inhibition, in this case the dysregulated protease is likely to be tissue plasminogen activator (tPA). FENIB should be considered in cases of progressive myoclonic epilepsy and dementia particularly where there is family history of neuropsychiatric disease.

Flyglow: Single-fly observations of simultaneous molecular and behavioural circadian oscillations in controls and an Alzheimer's model.

Circadian rhythms are essential for health and are frequently disturbed in disease. A full understanding of the causal relationships between behavioural and molecular circadian rhythms requires simultaneous longitudinal observations over time in individual organisms. Current experimental paradigms require the measurement of each rhythm separately across distinct populations of experimental organisms, rendering the comparability of the resulting datasets uncertain. We therefore developed FLYGLOW, an assay using clock gene controlled luciferase expression detected by exquisitely sensitive EM-CCD imaging, to enable simultaneous quantification of parameters including locomotor, sleep consolidation and molecular rhythms in single flies over days/weeks. FLYGLOW combines all the strengths of existing techniques, and also allows powerful multiparametric paired statistics. We found the age-related transition from rhythmicity to arrhythmicity for each parameter occurs unpredictably, with some flies showing loss of one or more rhythms during middle-age. Using single-fly correlation analysis of rhythm robustness and period we demonstrated the independence of the peripheral clock from circadian behaviours in wild type flies as well as in an Alzheimer's model. FLYGLOW is a useful tool for investigating the deterioration of behavioural and molecular rhythms in ageing and neurodegeneration. This approach may be applied more broadly within behavioural neurogenetics research.

Expression of the alternative oxidase mitigates beta-amyloid production and toxicity in model systems.

Mitochondrial dysfunction has been widely associated with the pathology of Alzheimer's disease, but there is no consensus on whether it is a cause or consequence of disease, nor on the precise mechanism(s). We addressed these issues by testing the effects of expressing the alternative oxidase AOX from Ciona intestinalis, in different models of AD pathology. AOX can restore respiratory electron flow when the cytochrome segment of the mitochondrial respiratory chain is inhibited, supporting ATP synthesis, maintaining cellular redox homeostasis and mitigating excess superoxide production at respiratory complexes I and III. In human HEK293-derived cells, AOX expression decreased the production of beta-amyloid peptide resulting from antimycin inhibition of respiratory complex III. Because hydrogen peroxide was neither a direct product nor substrate of AOX, the ability of AOX to mimic antioxidants in this assay must be indirect. In addition, AOX expression was able to partially alleviate the short lifespan of Drosophila models neuronally expressing human beta-amyloid peptides, whilst abrogating the induction of markers of oxidative stress. Our findings support the idea of respiratory chain dysfunction and excess ROS production as both an early step and as a pathologically meaningful target in Alzheimer's disease pathogenesis, supporting the concept of a mitochondrial vicious cycle underlying the disease.

Metabolic changes may precede proteostatic dysfunction in a Drosophila model of amyloid beta peptide toxicity.

Amyloid beta (Aß) peptide aggregation is linked to the initiation of Alzheimer's disease; accordingly, aggregation-prone isoforms of Aß, expressed in the brain, shorten the lifespan of Drosophila melanogaster. However, the lethal effects of Aß are not apparent until after day 15. We used shibire(TS) flies that exhibit a temperature-sensitive paralysis phenotype as a reporter of proteostatic robustness. In this model, we found that increasing age but not Aß expression lowered the flies' permissive temperature, suggesting that Aß did not exert its lethal effects by proteostatic disruption. Instead, we observed that chemical challenges, in particular oxidative stressors, discriminated clearly between young (robust) and old (sensitive) flies. Using nuclear magnetic resonance spectroscopy in combination with multivariate analysis, we compared water-soluble metabolite profiles at various ages in flies expressing Aß in their brains. We observed 2 genotype-linked metabolomic signals, the first reported the presence of any Aß isoform and the second the effects of the lethal Arctic Aß. Lethality was specifically associated with signs of oxidative respiration dysfunction and oxidative stress.

Drosophila melanogaster as a Model for Studies on the Early Stages of Alzheimer's Disease.

Fruit flies (Drosophila melanogaster) have been widely used to study the cellular and molecular basis of human neurodegenerative disease. The biological similarities between the human and the fly have been explored successfully to further investigate the pathological basis of Alzheimer's disease (AD). Here, we discuss transgenic Drosophila models systems and the methodologies that have been employed in the study of AD.

Iron is a specific cofactor for distinct oxidation- and aggregation-dependent Aß toxicity mechanisms in a Drosophila model.

Metals, including iron, are present at high concentrations in amyloid plaques in individuals with Alzheimer's disease, where they are also thought to be cofactors in generating oxidative stress and modulating amyloid formation. In this study, we present data from several Drosophila models of neurodegenerative proteinopathies indicating that the interaction between iron and amyloid beta peptide (Aß) is specific and is not seen for other aggregation-prone polypeptides. The interaction with iron is likely to be important in the dimerisation of Aß and is mediated by three N-terminal histidines. Transgenic fly lines systematically expressing all combinations of His>Ala substitutions in Aß were generated and used to study the pathological role of these residues. Developmental eye phenotypes, longevity and histological examinations indicate that the N-terminal histidines have distinct position-dependent and -independent mechanisms. The former mediate the toxic effects of metals and Aß aggregation under non-oxidising conditions and the latter are relevant under oxidising conditions. Understanding how Aß mediates neurotoxic effects in vivo will help to better target pathological pathways using aggregation blockers and metal-modifying agents.

The pro-domains of neurotrophins, including BDNF, are linked to Alzheimer's disease through a toxic synergy with Aß.

Brain-derived neurotrophic factor (BDNF) has a crucial role in learning and memory by promoting neuronal survival and modulating synaptic connectivity. BDNF levels are lower in the brains of individuals with Alzheimer's disease (AD), suggesting a pathogenic involvement. The Drosophila orthologue of BDNF is the highly conserved Neurotrophin 1 (DNT1). BDNF and DNT1 have the same overall protein structure and can be cleaved, resulting in the conversion of a full-length polypeptide into separate pro- and mature-domains. While the BDNF mature-domain is neuroprotective, the role of the pro-domain is less clear. In flies and mammalian cells, we have identified a synergistic toxic interaction between the amyloid-ß peptide (Aß1-42) and the pro-domains of both DNT1 and BDNF. Specifically, we show that DNT1 pro-domain acquires a neurotoxic activity in the presence of Aß1-42. In contrast, DNT1 mature-domain is protective against Aß1-42 toxicity. Likewise, in SH-SY5Y cell culture, BDNF pro-domain is toxic only in the presence of Aß1-42. Western blots indicate that this synergistic interaction likely results from the Aß1-42-induced upregulation of the BDNF pro-domain receptor p75(NTR). The clinical relevance of these findings is underlined by a greater than thirty fold increase in the ratio of BDNF pro- to mature-domains in the brains of individuals with AD. This unbalanced BDNF pro:mature-domain ratio in patients represents a possible biomarker of AD and may offer a target for therapeutic intervention.

The TRiC/CCT chaperone is implicated in Alzheimer's disease based on patient GWAS and an RNAi screen in Aß-expressing Caenorhabditis elegans.

The human Aß peptide causes progressive paralysis when expressed in the muscles of the nematode worm, C. elegans. We have exploited this model of Aß toxicity by carrying out an RNAi screen to identify genes whose reduced expression modifies the severity of this locomotor phenotype. Our initial finding was that none of the human orthologues of these worm genes is identical with the genome-wide significant GWAS genes reported to date (the "white zone"); moreover there was no identity between worm screen hits and the longer list of GWAS genes which included those with borderline levels of significance (the "grey zone"). This indicates that Aß toxicity should not be considered as equivalent to sporadic AD. To increase the sensitivity of our analysis, we then considered the physical interactors (+1 interactome) of the products of the genes in both the worm and the white+grey zone lists. When we consider these worm and GWAS gene lists we find that 4 of the 60 worm genes have a +1 interactome overlap that is larger than expected by chance. Two of these genes form a chaperonin complex, the third is closely associated with this complex and the fourth gene codes for actin, the major substrate of the same chaperonin.

Insights into amyloid disease from fly models.

The formation of amyloid aggregates is a feature of most, if not all, polypeptide chains. In vivo modelling of this process has been undertaken in the fruitfly Drosophila melanogaster with remarkable success. Models of both neurological and systemic amyloid diseases have been generated and have informed our understanding of disease pathogenesis in two main ways. First, the toxic amyloid species have been at least partially characterized, for example in the case of the Aß (amyloid ß-peptide) associated with Alzheimer's disease. Secondly, the genetic underpinning of model disease-linked phenotypes has been characterized for a number of neurodegenerative disorders. The current challenge is to integrate our understanding of disease-linked processes in the fly with our growing knowledge of human disease, for the benefit of patients.

Oligomer-targeting with a conformational antibody fragment promotes toxicity in Aß-expressing flies.

INTRODUCTION: The self-assembly of Aß peptides into a range of conformationally heterogeneous amyloid states represents a fundamental event in Alzheimer's disease. Within these structures oligomeric intermediates are considered to be particularly pathogenic. To test this hypothesis we have used a conformational targeting approach where particular conformational states, such as oligomers or fibrils, are recognized in vivo by state-specific antibody fragments. RESULTS: We show that oligomer targeting with the KW1 antibody fragment, but not fibril targeting with the B10 antibody fragment, affects toxicity in Aß-expressing Drosophila melanogaster. The effect of KW1 is observed to occur selectively with flies expressing Aß(1-40) and not with those expressing Aß(1-42) or the arctic variant of Aß(1-42) This finding is consistent with the binding preference of KW1 for Aß(1-40) oligomers that has been established in vitro. Strikingly, and in contrast to the previously demonstrated in vitro ability of this antibody fragment to block oligomeric toxicity in long-term potentiation measurements, KW1 promotes toxicity in the flies rather than preventing it. This result shows the crucial importance of the environment in determining the influence of antibody binding on the nature and consequences of the protein misfolding and aggregation. CONCLUSIONS: While our data support to the pathological relevance of oligomers, they highlight the issues to be addressed when developing inhibitory strategies that aim to neutralize these states by means of antagonistic binding agents.

The chaperone domain BRICHOS prevents CNS toxicity of amyloid-ß peptide in Drosophila melanogaster.

Aggregation of the amyloid-ß peptide (Aß) into toxic oligomers and amyloid fibrils is linked to the development of Alzheimer's disease (AD). Mutations of the BRICHOS chaperone domain are associated with amyloid disease and recent in vitro data show that BRICHOS efficiently delays Aß42 oligomerization and fibril formation. We have generated transgenic Drosophila melanogaster flies that express the Aß42 peptide and the BRICHOS domain in the central nervous system (CNS). Co-expression of Aß42 and BRICHOS resulted in delayed Aß42 aggregation and dramatic improvements of both lifespan and locomotor function compared with flies expressing Aß42 alone. Moreover, BRICHOS increased the ratio of soluble:insoluble Aß42 and bound to deposits of Aß42 in the fly brain. Our results show that the BRICHOS domain efficiently reduces the neurotoxic effects of Aß42, although significant Aß42 aggregation is taking place. We propose that BRICHOS-based approaches should be explored with an aim towards the future prevention and treatment of AD.

Retro-inversal of intracellular selected ß-amyloid-interacting peptides: implications for a novel Alzheimer's disease treatment.

The aggregation of ß-amyloid (Aß) into toxic oligomers is a hallmark of Alzheimer's disease pathology. Here we present a novel approach for the development of peptides capable of preventing amyloid aggregation based upon the previous selection of natural all-l peptides that bind Aß1-42. Using an intracellular selection system, successful library members were further screened via competition selection to identify the most effective peptides capable of reducing amyloid levels. To circumvent potential issues arising from stability and protease action for these structures, we have replaced all l residues with d residues and inverted the sequence. These retro-inverso (RI) peptide analogues therefore encompass reversed sequences that maintain the overall topological order of the native peptides. Our results demonstrate that efficacy in blocking and reversing amyloid formation is maintained while introducing desirable properties to the peptides. Thioflavin-T assays, circular dichroism, and oblique angle fluorescence microscopy collectively indicate that RI peptides can reduce amyloid load, while 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays demonstrate modest reductions in cell toxicity. These conclusions are reinforced using Drosophila melanogaster studies to monitor pupal hatching rates and fly locomotor activity in the presence of RI peptides delivered via RI-trans-activating transcriptional activator peptide fusions. We demonstrate that the RI-protein fragment complementation assay approach can be used as a generalized method for deriving Aß-interacting peptides. This approach has subsequently led to several peptide candidates being further explored as potential treatments for Alzheimer's disease.