Understanding the pathogenesis of engineered stone-associated silicosis: The effect of particle chemistry on the lung cell response.

BACKGROUND AND OBJECTIVE: The resurgence of severe and progressive silicosis among engineered stone benchtop industry workers is a global health crisis. We investigated the link between the physico-chemical characteristics of engineered stone dust and lung cell responses to understand components that pose the greatest risk. METHODS: Respirable dust from 50 resin-based engineered stones, 3 natural stones and 2 non-resin-based materials was generated and analysed for mineralogy, morphology, metals, resin, particle size and charge. Human alveolar epithelial cells and macrophages were exposed in vitro to dust and assessed for cytotoxicity and inflammation. Principal component analysis and stepwise linear regression were used to explore the relationship between engineered stone components and the cellular response. RESULTS: Cutting engineered stone generated fine particles of <600 nm. Crystalline silica was the main component with metal elements such as Ti, Cu, Co and Fe also present. There was some evidence to suggest differences in cytotoxicity (p = 0.061) and IL-6 (p = 0.084) between dust samples. However, IL-8 (CXCL8) and TNF-α levels in macrophages were clearly variable (p < 0.05). Quartz explained 11% of the variance (p = 0.019) in macrophage inflammation while Co and Al accounted for 32% of the variance (p < 0.001) in macrophage toxicity, suggesting that crystalline silica only partly explains the cell response. Two of the reduced-silica, non-engineered stone products induced considerable inflammation in macrophages. CONCLUSION: These data suggest that silica is not the only component of concern in these products, highlighting the caution required as alternative materials are produced in an effort to reduce disease risk.

The aryl hydrocarbon receptor pathway is a marker of lung cell activation but does not play a central pathologic role in engineered stone-associated silicosis.

Engineered stone-associated silicosis is characterised by a rapid progression of fibrosis linked to a shorter duration of exposure. To date, there is lack of information about molecular pathways that regulates disease development and the aggressiveness of this form of silicosis. Therefore, we compared transcriptome responses to different engineered stone samples and standard silica. We then identified and further tested a stone dust specific pathway (aryl hydrocarbon receptor [AhR]) in relation to mitigation of adverse lung cell responses. Cells (epithelial cells, A549; macrophages, THP-1) were exposed to two different benchtop stone samples, standard silica and vehicle control, followed by RNA sequencing analysis. Bioinformatics analyses were conducted, and the expression of dysregulated AhR pathway genes resulting from engineered stone exposure was then correlated with cytokine responses. Finally, we inhibited AhR pathway in cells pretreated with AhR antagonist and observed how this impacted cell cytotoxicity and inflammation. Through transcriptome analysis, we identified the AhR pathway genes (CYP1A1, CYP1B1 and TIPARP) that showed differential expression that was unique to engineered stones and common between both cell types. The expression of these genes was positively correlated with interleukin-8 production in A549 and THP-1 cells. However, we only observed a mild effect of AhR pathway inhibition on engineered stone dust induced cytokine responses. Given the dual roles of AhR pathway in physiological and pathological processes, our data showed that expression of AhR target genes could be markers for assessing toxicity of engineered stones; however, AhR pathway might not play a significant pathologic role in engineered stone-associated silicosis.

Development and characterisation of a rat model that exhibits both metabolic dysfunction and neurodegeneration seen in type 2 diabetes.

Accurate modelling type 2 diabetes and diabetic complications in rodents has proven a challenge, largely as a result of the long-time course of disease development in humans. In the present study, we aimed to develop and comprehensively characterise a new rodent model of type 2 diabetes. To do this, we fed Sprague-Dawley rats a high fat/high sugar diet (HFD) to induce obesity and dyslipidaemia. After 3 weeks, we s.c. implanted osmotic mini pumps to enable a 14 day, slow infusion of streptozotocin (STZ; lower dose = 100 mg kg-1 ; higher dose = 120 mg kg-1 ) to dose-dependently reduce pancreatic beta cell mass. After removing the mini pumps, we monitored animals for 4 months using a battery of tests to assess both metabolic and neurodegenerative changes across time. Our data demonstrate the combination of the HFD and lower dose STZ leads to induction of early-stage type 2 diabetes defined by moderate hyperglycaemia, hyperinsulinaemia and impaired glucose tolerance, at the same time as the retention of an obese phenotype. By contrast, combining the HFD and higher dose STZ leads to induction of later-stage type 2 diabetes defined by frank hyperglycaemia, hypoinsulinaemia (but not insulin depletion) and severely impaired glucose tolerance, at the same time as retaining an obese phenotype. Regardless of dose of STZ (and level of hyperglycaemia), all diabetic rats exhibited signs of peripheral neurodegeneration in the skin and muscle. Thus, this model recapitulates many of the complex metabolic disturbances seen in type 2 diabetes and provides an excellent platform for investigating the pathophysiological mechanisms that lead to diabetic complications such as peripheral neuropathy. KEY POINTS: Type 2 diabetes is a major health concern and markedly increases risk cardiovascular and neurodegenerative diseases. Accurate modelling of type 2 diabetes is a major challenge and has impeded our ability to understand the mechanisms that contribute to complications of type 2 diabetes. We have developed a method of inducing different stages of type 2 diabetes using a high fat/high sugar diet and 14 day infusion of streptozotocin to dose-dependently destroy pancreatic beta cell mass. Over 4 months, we comprehensively characterised these animals and confirmed that they develop sustained metabolic dysfunction and progressive peripheral neurodegeneration as seen in type 2 diabetes. This new model will improve our ability to investigate the pathophysiological mechanisms that link type 2 diabetes with complications such as neurodegeneration.

Effects of chemical composition on the lung cell response to coal particles: Implications for coal workers' pneumoconiosis.

BACKGROUND AND OBJECTIVE: Coal mine dust has a complex and heterogeneous chemical composition. It has been suggested that coal particle chemistry plays a critical role in determining the pathogenesis of coal workers' pneumoconiosis (CWP). In this study, we aimed to establish the association between the detrimental cellular response and the chemical composition of coal particles. METHODS: We sourced 19 real-world coal samples. Samples were crushed prior to use to minimize the impact of particle size on the response and to ensure the particles were respirable. Key chemical components and inorganic compounds were quantified in the coal samples. The cytotoxic, inflammatory and pro-fibrotic responses in epithelial cells, macrophages and fibroblasts were assessed following 24 h of exposure to coal particles. Principal component analysis (PCA) and stepwise regression were used to determine which chemical components of the coal particles were associated with the cell response. RESULTS: The cytotoxic, inflammatory and pro-fibrotic response varied considerably between coal samples. There was a high level of collinearity in the cell responses and between the chemical compounds within the coal samples. PCA identified three factors that explained 75% of the variance in the cell response. Stepwise multiple regression analysis identified K2 O (p <0.001) and Fe2 O3 (p = 0.011) as significant predictors of cytotoxicity and cytokine production, respectively. CONCLUSION: Our data clearly demonstrate that the detrimental cellular effects of exposure to coal mine dusts are highly dependent on particle chemistry. This has implications for understanding the pathogenesis of CWP.

Adverse effects of prenatal exposure to residential dust on post-natal brain development.

BACKGROUND: Previous studies have shown an association between prenatal exposure to particulate matter (PM) and adverse brain development. However, it is unclear whether gestational exposure to community-sampled residential PM has an impact on the developing brain. OBJECTIVES: We aimed to test whether in utero exposure to PM from residential roof spaces (ceiling voids) alters critical foetal neurodevelopmental processes. METHODS: Pregnant C57BL/6 mice were intranasally exposed to 100 µg of roof space particles (~5 mg kg-1) in 50 µl of saline, or saline alone under light methoxyflurane anaesthesia, throughout mid-to-late gestation. At 2 weeks post-natal age, pups were sacrificed and assessed for body and brain growth. The brain tissue was collected and examined for a range of neurodevelopmental markers for synaptogenesis, synaptic plasticity, gliogenic events and myelination by immunohistochemistry. RESULTS: Gestational exposure to roof space PM reduced post-natal body and brain weights. There was no significant effect of roof space PM exposure on synaptogenesis, synaptic plasticity or astrocyte density. However, PM exposure caused increased myelin load in the white matter and elevated microglial density which was dependent on the PM sample. These effects were found to be consistent between male and female mice. CONCLUSIONS: Our data suggest that exposure to residential roof space PM during pregnancy impairs somatic growth and causes neuropathological changes in the developing brain.

Knockout of Amyloid ß Protein Precursor (APP) Expression Alters Synaptogenesis, Neurite Branching and Axonal Morphology of Hippocampal Neurons.

The function of the ß-A4 amyloid protein precursor (APP) of Alzheimer's disease (AD) remains unclear. APP has a number of putative roles in neuronal differentiation, survival, synaptogenesis and cell adhesion. In this study, we examined the development of axons, dendrites and synapses in cultures of hippocampus neutrons derived from APP knockout (KO) mice. We report that loss of APP function reduces the branching of cultured hippocampal neurons, resulting in reduced synapse formation. Using a compartmentalised culture approach, we found reduced axonal outgrowth in cultured hippocampal neurons and we also identified abnormal growth characteristics of isolated hippocampal neuron axons. Although APP has previously been suggested to play an important role in promoting cell adhesion, we surprisingly found that APPKO hippocampal neurons adhered more strongly to a poly-L-lysine substrate and their neurites displayed an increased density of focal adhesion puncta. The findings suggest that the function of APP has an important role in both dendritic and axonal growth and that endogenous APP may regulate substrate adhesion of hippocampal neurons. The results may explain neuronal and synaptic morphological abnormalities in APPKO mice and the presence of abnormal APP expression in dystrophic neurites around amyloid deposits in AD.

Amyloid ß precursor protein regulates neuron survival and maturation in the adult mouse brain.

The amyloid-ß precursor protein (APP) is a transmembrane protein that is widely expressed within the central nervous system (CNS). While the pathogenic dysfunction of this protein has been extensively studied in the context of Alzheimer's disease, its normal function is poorly understood, and reports have often appeared contradictory. In this study we have examined the role of APP in regulating neurogenesis in the adult mouse brain by comparing neural stem cell proliferation, as well as new neuron number and morphology between APP knockout mice and C57bl6 controls. Short-term EdU administration revealed that the number of proliferating EdU+ neural progenitor cells and the number of PSA-NCAM+ neuroblasts produced in the SVZ and dentate gyrus were not affected by the life-long absence of APP. However, by labelling newborn cells with EdU and then following their fate over-time, we determined that ~48% more newly generated EdU+ NeuN+ neurons accumulated in the granule cell layer of the olfactory bulb and ~57% more in the dentate gyrus of young adult APP knockout mice relative to C57bl6 controls. Furthermore, proportionally fewer of the adult-born olfactory bulb granule neurons were calretinin+. To determine whether APP was having an effect on neuronal maturation, we administered tamoxifen to young adult Nestin-CreERT2::Rosa26-YFP and Nestin-CreERT2::Rosa26-YFP::APP-knockout mice, fluorescently labelling ~80% of newborn (EdU+) NeuN+ dentate granule neurons formed between P75 and P105. Our analysis of their morphology revealed that neurons added to the hippocampus of APP knockout mice have shorter dendritic arbors and only half the number of branch points as those generated in C57bl6 mice. We conclude that APP reduces the survival of newborn neurons in the olfactory bulb and hippocampus, but that it does not influence all neuronal subtypes equally. Additionally, APP influences dentate granule neuron maturation, acting as a robust regulator of dendritic extension and arborisation.

Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER-Golgi transport is a novel therapeutic target in ALS.

Can Maternal Exposure to Air Pollution Affect Post-Natal Liver Development?

Emerging evidence suggests that inhalation of particulate matter (PM) can have direct adverse effects on liver function. Early life is a time of particular vulnerability to the effects of air pollution. On that basis, we tested whether in utero exposure to residential PM has an impact on the developing liver. Pregnant mice (C57BL/6J) were intranasally administered 100 µg of PM sampled from residential roof spaces (~5 mg/kg) on gestational days 13.5, 15.5, and 17.5. The pups were euthanized at two weeks of age, and liver tissue was collected to analyse hepatic metabolism (glycogen storage and lipid level), cellular responses (oxidative stress, inflammation, and fibrosis), and genotoxicity using a range of biochemical assays, histological staining, ELISA, and qPCR. We did not observe pronounced effects of environmentally sampled PM on the developing liver when examining hepatic metabolism and cellular response. However, we did find evidence of liver genomic DNA damage in response to in utero exposure to PM. This effect varied depending on the PM sample. These data suggest that in utero exposure to real-world PM during mid-late pregnancy has limited impacts on post-natal liver development.

Do Microglia Default on Network Maintenance in Alzheimer's Disease?

Although the cause of Alzheimer's disease (AD) remains unknown, a number of new findings suggest that the immune system may play a critical role in the early stages of the disease. Genome-wide association studies have identified a wide array of risk-associated genes for AD, many of which are associated with abnormal functioning of immune cells. Microglia are the brain's immune cells. They play an important role in maintaining the brain's extracellular environment, including clearance of aggregated proteins such as amyloid-ß (Aß). Recent studies suggest that microglia play a more active role in the brain than initially considered. Specifically, microglia provide trophic support to neurons and also regulate synapses. Microglial regulation of neuronal activity may have important consequences for AD. In this article we review the function of microglia in AD and examine the possible relationship between microglial dysfunction and network abnormalities, which occur very early in disease pathogenesis.

Axonal degeneration, distal collateral branching and neuromuscular junction architecture alterations occur prior to symptom onset in the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Degeneration of the distal axon and neuromuscular junction (NMJ) is considered a key and early feature of the pathology that accompanies motor neuron loss in people with amyotrophic lateral sclerosis (ALS). The mutant SOD1(G93A) mouse replicates many features of the disease, however the sequence of events resulting in degeneration of the neuromuscular circuitry remains unknown. Furthermore, despite widespread degenerative neuronal pathology throughout the spinal cord in this model, hindlimb motor function is lost before forelimb function. We investigated axons and NMJs in the hindlimb (gastrocnemius) and forelimb (extensor) muscles in the high copy number mutant SOD1(G93A)xYFP (yellow fluorescent protein) mouse. We found that distal axonal and NMJ alterations were present prior to previously reported functional symptom onset in this strain. Indeed, increased branch complexity as well as colocalisation between pre- and post-synaptic markers indicated widespread early axonal and NMJ alterations in the hindlimb. Immunohistochemical analysis demonstrated that the colocalisation of the scaffolding proteins nestin, LRP-4, dystrophin and rapsyn were diminished before post-synaptic receptors in the gastrocnemius, and the degree of loss differed between proteins. Analysis of the forelimb muscle revealed axonal and NMJ degeneration at a late, post symptomatic stage, as well as novel differences in NMJ morphology, with reduced complexity. Furthermore, post-synaptic scaffolding proteins were preserved in the forelimb compared with the hindlimb. Analysis of protein levels indicated an increase in LRP-4, dystrophin and rapsyn in post symptomatic skeletal muscle that may suggest ongoing attempts at repair. This study indicates that axonal and NMJ degeneration in the SOD1 model of ALS is a complex and evolving sequence of events. We provide evidence that YFP can detect morphological and plastic alterations in the SOD1(G93A) mouse, and that the pre- and post-synaptic integrity of the NMJ plays an important role in the pathogenic mechanisms of ALS.

Identifying the primary site of pathogenesis in amyotrophic lateral sclerosis - vulnerability of lower motor neurons to proximal excitotoxicity.

There is a desperate need for targeted therapeutic interventions that slow the progression of amyotrophic lateral sclerosis (ALS). ALS is a disorder with heterogeneous onset, which then leads to common final pathways involving multiple neuronal compartments that span both the central and peripheral nervous system. It is believed that excitotoxic mechanisms might play an important role in motor neuron death in ALS. However, little is known about the mechanisms by which excitotoxicity might lead to the neuromuscular junction degeneration that characterizes ALS, or about the site at which this excitotoxic cascade is initiated. Using a novel compartmentalised model of site-specific excitotoxin exposure in lower motor neurons in vitro, we found that spinal motor neurons are vulnerable to somatodendritic, but not axonal, excitotoxin exposure. Thus, we developed a model of somatodendritic excitotoxicity in vivo using osmotic mini pumps in Thy-1-YFP mice. We demonstrated that in vivo cell body excitotoxin exposure leads to significant motor neuron death and neuromuscular junction (NMJ) retraction. Using confocal real-time live imaging of the gastrocnemius muscle, we found that NMJ remodelling preceded excitotoxin-induced NMJ degeneration. These findings suggest that excitotoxicity in the spinal cord of individuals with ALS might result in a die-forward mechanism of motor neuron death from the cell body outward, leading to initial distal plasticity, followed by subsequent pathology and degeneration.

Excitotoxin-induced caspase-3 activation and microtubule disintegration in axons is inhibited by taxol.

BACKGROUND: Axon degeneration, a key pathological event in many neurodegenerative diseases and injury, can be induced by somatodendritic excitotoxin exposure. It is currently unclear, however, whether excitotoxin-induced axon degeneration is mechanistically similar to Wallerian degeneration, which occurs following axon transection, but does not involve axonal caspase activation. RESULTS: We have used mouse primary cortical neurons at 9 days in vitro, in a compartmented culture model that allows separation of the axon from the soma, to examine the pathological cascade of excitotoxin-induced axon degeneration. Excitotoxicity induced by chronic exposure to kainic acid, resulted in axonal fragmentation, which was associated with activation of caspase-3 in the axonal compartment. To examine the role of microtubules in these events, the microtubule-stabilizing agent, taxol, was added to either the axonal or somatodendritic compartment. Our results demonstrated that microtubule stabilization of axons resulted in a significant reduction in the number of fragmented axons following excitotoxin exposure. Interestingly, taxol exposure to either the somatodendritic or axonal compartment resulted in reduced caspase-3 activation in axons, suggesting that caspase activation is a downstream event of microtubule destabilization and involves signalling from the cell soma. CONCLUSION: These data suggest that excitotoxin-induced axon degeneration shows some mechanistic differences to Wallerian degeneration, and that microtubule stabilization may assist in protecting nerve cells from excitotoxic effects.

Microfluidic primary culture model of the lower motor neuron-neuromuscular junction circuit.

Modelling the complex process of neuromuscular signalling is key to understanding not only normal circuit function but also importantly the mechanisms underpinning a range of degenerative diseases. We describe a novel in vitro model of the lower motor neuron-neuromuscular junction circuit, incorporating primary spinal motor neurons, supporting glia and skeletal muscle. This culture model is designed to spatially mimic the unique anatomical and cellular interactions of this circuit in compartmented microfluidic devices, such that the glial cells are located with motor neuron cell bodies in the cell body chamber and motor neuron axons extend to a distal chamber containing skeletal muscle cells whilst simultaneously allowing targeted intervention. This model is suitable for use in conjunction with a range of downstream experimental approaches and could also be modified to utilise other cellular sources including appropriate immortal cell lines, cells derived from transgenic models of disease and also patient derived stem cells.

Degeneration of axons in spinal white matter in G93A mSOD1 mouse characterized by NFL and α-internexin immunoreactivity.

Axonal degeneration is a prominent feature of amyotrophic lateral sclerosis (ALS) both in lower motor nerves as well as descending white matter axons in the spinal cord of human patients. Although the pathology of lower motor axonal degeneration has been described in both human ALS and related transgenic animal models, few studies have examined the pathological features of descending axon degeneration, particularly in mouse models of ALS. We have examined the degeneration of white matter tracts in the G93A mutant superoxide dismutase-1 (mSOD1+) mouse spinal cord white matter from 12 weeks of age to end-stage disease. In a G93A mSOD1 mouse model where green fluorescent protein was expressed in neurons (mSOD1+/GFP+), degeneration of white matter tracts was present from the ventral to dorsolateral funiculi. This pattern of axonal pathology occurred from 16 weeks of age. However, the dorsal funiculus, the site of the major corticospinal tract in mice, showed relatively less degeneration. Immunohistochemical analysis demonstrated that the neurofilament light chain (NFL) and neuronal intermediate filament protein alpha-internexin accumulated in axon swellings in the spinal white matter. Increased levels of alpha-internexin protein, in mSOD1+ mouse spinal cord tissue, were demonstrated by Western blotting. In contrast, degenerating axons did not show obvious accumulations of neurofilament medium and heavy chain proteins (NFM and NFH). These data suggest that white matter degeneration in this mouse model of ALS is widespread and involves a specific molecular signature, particularly the accumulation of NFL and alpha-internexin proteins.