A systematic scoping review of the neurological effects of COVID-19.

BACKGROUND: The global coronavirus 2019 (COVID-19) pandemic began in early 2020, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In mid-2020 the CIAO (Modelling the Pathogenesis of COVID-19 Using the Adverse Outcome Pathway Framework) project was established, bringing together over 75 interdisciplinary scientists worldwide to collaboratively investigate the underlying biological mechanisms of COVID-19 and consolidate the data using the Adverse Outcome Pathway (AOP) Framework. Neurological symptoms such as anosmia and encephalitis have been frequently reported to be associated with infection with SARS-CoV-2. OBJECTIVE: Within CIAO, a working group was formed to conduct a systematic scoping review of COVID-19 and its related neurological symptoms to determine which key events and modulating factors are most commonly reported and to identify knowledge gaps. DESIGN: LitCOVID was used to retrieve 86,075 papers of which 10,244 contained relevant keywords. After title and abstract screening, 2,328 remained and their full texts were reviewed based on predefined inclusion and exclusion criteria. 991 studies fulfilled the inclusion criteria and were retrieved to conduct knowledge synthesis. RESULTS: The majority of publications reported human observational studies. Early key events were less likely to be reported compared to middle and late key events/adverse outcomes. The majority of modulating factors described related to age or sex. Less recognised COVID-19 associated AO or neurological effects of COVID-19 were also identified including multiple sclerosis/demyelination, neurodegeneration/cognitive effects and peripheral neuronal effects. CONCLUSION: There were many methodological and reporting issues noted in the reviewed studies. In particular, publication abstracts would benefit from clearer reporting of the methods and endpoints used and the key findings, to ensure relevant papers are included when systematic reviews are conducted. The information extracted from the scoping review may be useful in understanding the mechanisms of neurological effects of COVID-19 and to further develop or support existing AOPs linking COVID-19 and its neurological key events and adverse outcomes. Further evaluation of the less recognised COVID-19 effects is needed.

The Adverse Outcome Pathway Framework Applied to Neurological Symptoms of COVID-19.

Several reports have shown that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has the potential to also be neurotropic. However, the mechanisms by which SARS-CoV-2 induces neurologic injury, including neurological and/or psychological symptoms, remain unclear. In this review, the available knowledge on the neurobiological mechanisms underlying COVID-19 was organized using the AOP framework. Four AOPs leading to neurological adverse outcomes (AO), anosmia, encephalitis, stroke, and seizure, were developed. Biological key events (KEs) identified to induce these AOs included binding to ACE2, blood-brain barrier (BBB) disruption, hypoxia, neuroinflammation, and oxidative stress. The modularity of AOPs allows the construction of AOP networks to visualize core pathways and recognize neuroinflammation and BBB disruption as shared mechanisms. Furthermore, the impact on the neurological AOPs of COVID-19 by modulating and multiscale factors such as age, psychological stress, nutrition, poverty, and food insecurity was discussed. Organizing the existing knowledge along an AOP framework can represent a valuable tool to understand disease mechanisms and identify data gaps and potentially contribute to treatment, and prevention. This AOP-aligned approach also facilitates synergy between experts from different backgrounds, while the fast-evolving and disruptive nature of COVID-19 emphasizes the need for interdisciplinarity and cross-community research.

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop.

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project "Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework" aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

Phenyl Saligenin Phosphate Disrupts Cell Morphology and the Actin Cytoskeleton in Differentiating H9c2 Cardiomyoblasts and Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocyte Progenitor Cells.

We have previously shown that phenyl saligenin phosphate (PSP), an organophosphorus compound which is classed as a weak inhibitor of acetylcholinesterase, triggered cytotoxicity in mitotic and differentiated H9c2 cardiomyoblasts. The aim of this study was to assess whether sublethal concentrations of PSP could disrupt the morphology of differentiating rat H9c2 cardiomyoblasts and human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells (hiPSC-CMs) and to assess the underlying cytoskeletal changes. PSP-induced changes in protein expression were monitored via Western blotting, immunocytochemistry, and proteomic analysis. PSP-mediated cytotoxicity was determined by measuring MTT reduction, LDH release, and caspase-3 activity. Sublethal exposure to PSP (3 µM) induced morphological changes in differentiating H9c2 cells (7, 9, and 13 days), reflected by reduced numbers of spindle-shaped cells. Moreover, this treatment (7 days) attenuated the expression of the cytoskeletal proteins cardiac troponin I, tropomyosin-1, and α-actin. Further proteomic analysis identified nine proteins (e.g., heat shock protein 90-ß and calumenin) which were down-regulated by PSP exposure in H9c2 cells. To assess the cytotoxic effects of organophosphorus compounds in a human cell model, we determined their effects on human-induced pluripotent stem-cell-derived cardiomyocyte progenitor cells. Chlorpyrifos and diazinon-induced cytotoxicity (48 h) was evident only at concentrations >100 µM. By contrast, PSP exhibited cytotoxicity in hiPSC-CMs at a concentration of 25 µM following 48 h exposure. Finally, sublethal exposure to PSP (3 µM; 7 days) induced morphological changes and decreased the expression of cardiac troponin I, tropomyosin-1, and α-actin in hiPSC-CMs. In summary, our data suggest cardiomyocyte morphology is disrupted in both cell models by sublethal concentrations of PSP via modulation of cytoskeletal protein expression.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

A co-culture nanofibre scaffold model of neural cell degeneration in relevance to Parkinson's disease.

Current therapeutic strategies for Parkinson's disease (PD) aim to delay progression or replace damaged neurons by restoring the original neuronal structures. The poor regenerative capacity of neural tissue highlights the need for the development of cellular environments to model the pathogenesis of PD. In the current work, we have characterised the growth, survival and response to PD mimetics of human SH-SY5Y neuroblastoma and U-87MG glioblastoma cell lines cultured on polyacrylonitrile (PAN) and Jeffamine® doped polyacrylonitrile (PJ) nano-scaffolds. Differentiation induced by a range of agents was evaluated by immunoassays of neural protein biomarkers. PAN and PJ nanofibre scaffolds provided suitable three-dimensional (3D) environment to support the growth, differentiation and network formation of dopaminergic neuron- and astrocyte-like cell populations, respectively. The scaffolds selectively supported the survival and differentiation of both cell populations with prolonged neuronal survival when exposed to PD mimetics in the presence of astrocytes in a co-culture model. Such 3D nanoscaffold-based assays could aid our understanding of the molecular basis of PD mimetic-induced Parkinsonism and the discovery of neuroprotective agents.

Activation of transglutaminase 2 by nerve growth factor in differentiating neuroblastoma cells: A role in cell survival and neurite outgrowth.

NGF (nerve growth factor) and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 transamidase activity by NGF in retinoic acid-induced differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. The role of TG2 in NGF-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with NGF in N2a and SH-SY5Y cells. NGF mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON (Z-ZON-Val-Pro-Leu-OMe; Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-l-valinyl-l-prolinyl-l-leucinmethylester) and R283 (1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride) and by pharmacological inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2), protein kinase B (PKB) and protein kinase C (PKC), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated NGF induced in situ TG2 activity. TG2 inhibition blocked NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth in both cell lines. Together, these results demonstrate that NGF stimulates TG2 transamidase activity via a ERK1/2, PKB and PKC-dependent pathway in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells. Furthermore, NGF-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results suggest a novel and important role of TG2 in the cellular functions of NGF.

ß2-adrenoceptor-induced modulation of transglutaminase 2 transamidase activity in cardiomyoblasts.

Tissue transglutaminase 2 (TG2) is modulated by protein kinase A (PKA) mediated phosphorylation: however, the precise mechanism(s) of its modulation by G-protein coupled receptors coupled to PKA activation are not fully understood. In the current study we investigated the potential regulation of TG2 activity by the ß2-adrenoceptor in rat H9c2 cardiomyoblasts. Transglutaminase transamidation activity was assessed using amine-incorporating and protein cross-linking assays. TG2 phosphorylation was determined via immunoprecipitation and Western blotting. The long acting ß2-adrenoceptor agonist formoterol induced time- and concentration-dependent increases in TG2 transamidation. Increases in TG2 activity were reduced by the TG2 inhibitors Z-DON (Benzyloxycarbonyl-(6-Diazo-5-oxonorleucinyl)-L-valinyl-L-prolinyl-L-leucinmethylester) and R283 ((1,3,dimethyl-2[2-oxo-propyl]thio)imidazole chloride). Responses to formoterol were blocked by pharmacological inhibition of PKA, extracellular signal-regulated kinase 1 and 2 (ERK1/2), or phosphatidylinositol 3-kinase (PI-3K) signalling. Furthermore, the removal of extracellular Ca2+ also attenuated formoterol-induced TG2 activation. Fluorescence microscopy demonstrated TG2-induced biotin-X-cadaverine incorporation into proteins. Formoterol increased the levels of TG2-associated phosphoserine and phosphothreonine, which were blocked by inhibition of PKA, ERK1/2 or PI-3K signalling. Subsequent proteomic analysis identified known (e.g. lactate dehydrogenase A chain) and novel (e.g. Protein S100-A6) protein substrates for TG2. Taken together, the data obtained suggest that ß2-adrenoceptor-induced modulation of TG2 represents a novel paradigm in ß2-adrenoceptor cell signalling, expanding the repertoire of cellular functions responsive to catecholamine stimulation.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells.

The PAC1 receptor and tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. In this study, we investigated the regulation of TG2 activity by the PAC1 receptor in retinoic acid-induced differentiating N2a neuroblastoma cells. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of biotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. The role of TG2 in PAC1 receptor-induced cytoprotection and neurite outgrowth was investigated by monitoring hypoxia-induced cell death and appearance of axonal-like processes, respectively. The amine incorporation and protein crosslinking activity of TG2 increased in a time and concentration-dependent manner following stimulation with pituitary adenylate cyclase-activating polypeptide-27 (PACAP-27). PACAP-27 mediated increases in TG2 activity were abolished by the TG2 inhibitors Z-DON and R283 and by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), protein kinase C (Ro 31-8220), MEK1/2 (PD 98059), and removal of extracellular Ca2+. Fluorescence microscopy demonstrated PACAP-27 induced in situ TG2 activity. TG2 inhibition blocked PACAP-27 induced attenuation of hypoxia-induced cell death and outgrowth of axon-like processes. TG2 activation and cytoprotection were also observed in human SH-SY5Y cells. Together, these results demonstrate that TG2 activity was stimulated downstream of the PAC1 receptor via a multi protein kinase dependent pathway. Furthermore, PAC1 receptor-induced cytoprotection and neurite outgrowth are dependent upon TG2. These results highlight the importance of TG2 in the cellular functions of the PAC1 receptor.

Chlorpyrifos- and chlorpyrifos oxon-induced neurite retraction in pre-differentiated N2a cells is associated with transient hyperphosphorylation of neurofilament heavy chain and ERK 1/2.

Chlorpyrifos (CPF) and CPF-oxon (CPO) are known to inhibit neurite outgrowth but little is known about their ability to induce neurite retraction in differentiating neuronal cells. The aims of this study were to determine the ability of these compounds to destabilize neurites and to identify the key molecular events involved. N2a cells were induced to differentiate for 20h before exposure to CPF or CPO for 2-8h. Fixed cell monolayers labeled with carboxyfluorescein succinimidyl ester or immunofluorescently stained with antibodies to tubulin (B512) or phosphorylated neurofilament heavy chain (Ta51) showed time- and concentration-dependent reductions in numbers and length of axon-like processes compared to the control, respectively, retraction of neurites being observed within 2h of exposure by live cell imaging. Neurofilament disruption was also observed in treated cells stained by indirect immunofluorescence with anti-phosphorylated neurofilament heavy chain (NFH) monoclonal antibody SMI34, while the microtubule network was unaffected. Western blotting analysis revealed transiently increased levels of reactivity of Ta51 after 2h exposure and reduced levels of reactivity of the same antibody following 8h treatment with both compounds, whereas reactivity with antibodies to anti-total NFH or anti-tubulin was not affected. The alteration in NFH phosphorylation at 2h exposure was associated with increased activation of extracellular signal-regulated protein kinase ERK 1/2. However, increased levels of phosphatase activity were observed following 8h exposure. These findings suggest for the first time that organophosphorothionate pesticide-induced neurite retraction in N2a cells is associated with transient increases in NFH phosphorylation and ERK1/2 activation.

A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection.

Sub-lethal concentrations of CdCl2 disrupt cell migration and cytoskeletal proteins in cultured mouse TM4 Sertoli cells.

The aims of this study were to examine the effects of CdCl2 on the viability, migration and cytoskeleton of cultured mouse TM4 Sertoli cells. Time- and concentration-dependent changes were exhibited by the cells but 1 µM CdCl2 was sub-cytotoxic at all time-points. Exposure to 1 and 12 µM CdCl2 for 4 h resulted in disruption of the leading edge, as determined by chemical staining. Cell migration was inhibited by both 1 and 12 µM CdCl2 in a scratch assay monitored by live cell imaging, although exposure to the higher concentration was associated with cell death. Western blotting and immunofluorescence staining indicated that CdCl2 caused a concentration dependent reduction in actin and tubulin levels. Exposure to Cd(2+) also resulted in significant changes in the levels and/or phosphorylation status of the microtubule and microfilament destabilising proteins cofilin and stathmin, suggesting disruption of cytoskeletal dynamics. Given that 1-12 µM Cd(2+) is attainable in vivo, our findings are consistent with the possibility that Cd(2+) induced impairment of testicular development and reproductive health may involve a combination of reduced Sertoli cell migration and impaired Sertoli cell viability depending on the timing, level and duration of exposure.

Phenyl Saligenin Phosphate Induced Caspase-3 and c-Jun N-Terminal Kinase Activation in Cardiomyocyte-Like Cells.

At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. In this study, we have investigated the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on mitotic and differentiated H9c2 cardiomyoblasts. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cytotoxicity was not observed with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 µM). Chlorpyrifos-induced cytotoxicity was only evident at concentrations >100 µM. In marked contrast, PSP displayed pronounced cytotoxicity toward mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2 but not ERK1/2, p38 MAPK, or PKB, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death. The JNK1/2 inhibitor SP 600125 attenuated PSP-induced caspase-3 and JNK1/2 activation, confirming the role of JNK1/2 in PSP-induced cytotoxicity. Fluorescently labeled PSP (dansylated PSP) was used to identify novel PSP binding proteins. Dansylated PSP displayed cytotoxicity toward differentiated H9c2 cells. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 µM) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein ß-1, and nucleolar protein 58 as novel protein targets for PSP. In summary, PSP triggers cytotoxicity in differentiated H9c2 cardiomyoblasts via JNK1/2-mediated activation of caspase-3. Further studies are required to investigate whether the identified novel protein targets of PSP play a role in the cytotoxicity of this OP, which is usually associated with the development of OP-induced delayed neuropathy.

Cardioprotective and cardiotoxic effects of quercetin and two of its in vivo metabolites on differentiated h9c2 cardiomyocytes.

Whilst mitotic rat embryonic cardiomyoblast-derived H9c2 cells have been widely used as a model system to study the protective mechanisms associated with flavonoids, they are not fully differentiated cardiac cells. Hence, the aim of this study was to investigate the cardioprotective and cardiotoxic actions of quercetin and two of its major in vivo metabolites, quercetin 3-glucuronide and 3'-O-methyl quercetin, using differentiated H9c2 cells. The differentiated cardiomyocyte-like phenotype was confirmed by monitoring expression of cardiac troponin 1 after 7 days of culture in reduced serum medium containing 10 nM all-trans retinoic acid. Quercetin-induced cardiotoxicity was assessed by monitoring MTT reduction, lactate dehydrogenase (LDH) release, caspase 3 activity and reactive oxygen species production after prolonged flavonoid exposure (72 hr). Cardiotoxicity was observed with quercetin and 3'-O-methyl quercetin, but not quercetin 3-glucuronide. Cardioprotection was assessed by pre-treating differentiated H9c2 cells with quercetin or its metabolites for 24 hr prior to 2-hr exposure to 600 µM H2 O2, after which oxidative stress-induced cell damage was assessed by measuring MTT reduction and LDH release. Cardioprotection was observed with quercetin and 3'-O-methyl quercetin, but not with quercetin 3-glucuronide. Quercetin attenuated H2 O2 -induced activation of ERK1/2, PKB, p38 MAPK and JNK, but inhibitors of these kinases did not modulate quercetin-induced protection or H2 O2 -induced cell death. In summary, quercetin triggers cardioprotection against oxidative stress-induced cell death and cardiotoxicity after prolonged exposure. Further studies are required to investigate the complex interplay between the numerous signalling pathways that are modulated by quercetin and which may contribute to the cardioprotective and cardiotoxic effects of this important flavonoid.

Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection.

BACKGROUND AND PURPOSE: Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH: H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS: Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (ß-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2 O2 -induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS: TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases.

Diazoxon disrupts the expression and distribution of ßIII-tubulin and MAP 1B in differentiating N2a cells.

This study aimed at assessing the effects of diazoxon (DZO), a major metabolite of the insecticide diazinon (DZ), on key cytoskeletal proteins in differentiating N2a neuroblastoma cells. Initial experiments established that sublethal concentrations of 1, 5 and 10 µM DZO produced profound inhibition of neurite outgrowth. Densitometric scanning of probed immunoblots of N2a cell lysates demonstrated that DZO had no effect on total ß-tubulin levels. However, probing with a monoclonal antibody that recognised specifically the ßIII-tubulin isotype revealed that 10 µM DZO induced a significant reduction in the levels of this particular form. Levels of polyglutamylated tubulin were not altered. Exposure to 10 µM DZO also decreased the expression of microtubule-associated protein 1B (MAP 1B). However, DZO had no effect on the expression of MAP tau. DZO also failed to affect the levels neurofilament light (NFL) and neurofilament medium (NFM) chain levels. Indirect immunofluorescence demonstrated that the staining of neurites in treated cells was weaker than in the controls for ßIII-tubulin. In conclusion, DZO disrupts the microtubule (MT) network affecting the expression and distribution of two specific MT proteins known to be important in neuritogenesis. DZO may contribute to the developmental neurotoxicity seen following exposure to DZ.

Neurodegenerations induced by organophosphorous compounds.

Organophosphorous compounds (OPs) are widely used in agriculture, industry and the home. Though best known for their acute effects when used as pesticides, which target acetylcholinesterase (AChE) activity in neuromuscular junctions and the central nervous system, not all OPs are potent inhibitors of this enzyme. The widespread use of OPs has heightened concern regarding their toxicity in man, with numerous reports linking OPs to various forms of delayed neuropathy encompassing a range of neurodegenerative, psychological and neurobehavioral effects. There is mounting evidence to suggest that sub-acute levels of OPs have the ability to interact directly with a range of target proteins in addition to AChE (i.e., noncholinergic targets), causing major disruption of membrane and protein turnover, protein phosphorylation, mitochondrial dysfunction, oxidative stress and cytoskeletal re-organisation, although the mechanisms involved are not fully understood. However, major advances have been made in the study of one OP binding protein neuropathy target esterase (NTE) in terms of its true physiological role. Additionally, there is increasing evidence for the ability of OPs to cause disruption in a number of metabolic and cell signalling pathways that affect neuronal cell proliferation, differentiation and survival and to interact direct with non-esterase proteins such as tubulin. The aim of this chapter is to review our current understanding of delayed neurotoxicity, to discuss how these molecular events may relate to each other and to suggest possible future directions in mechanistic studies of OP toxicity.

Alterations in the mitochondrial proteome of neuroblastoma cells in response to complex 1 inhibition.

Increasing evidence points to mitochondrial dysfunction in Parkinson's disease (PD) associated with complex I dysfunction, but the exact pathways which lead to cell death have not been resolved. 2D-gel electrophoresis profiles of isolated mitochondria from neuroblastoma cells treated with subcytotoxic concentrations of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a well-characterized complex I inhibitor, were assessed to identify associated targets. Up to 27 differentially expressed proteins were observed, of which 16 were identified using peptide mass fingerprinting. Changes in protein levels were validated by immunoprobing 1D blots, confirming increases in heat shock cognate 71 kDa (Hsc70), 60 kDa heat shock protein (Hsp60), fumarase, glutamate oxaloacetate transaminase 2, ATP synthase subunit d, and voltage-dependent anion-channel 1 (VDAC1). Immunoprobing of 2D blots revealed isoform changes in Hsc70, Hsp60, and VDAC1. Subcytotoxic concentrations of MPTP modulated a host of mitochondrial proteins including chaperones, metabolic enzymes, oxidative phosphorylation-related proteins, an inner mitochondrial protein (mitofilin), and an outer mitochondrial membrane protein (VDAC1). Early changes in chaperones suggest a regulated link between complex 1 inhibition and protein folding. VDAC1, a multifunctional protein, may have a key role in signaling between mitochondria and the rest of the cell prior to cell death. Our work provides new important information of relevance to PD.

Fipronil interferes with the differentiation of mouse N2a neuroblastoma cells.

The purpose of this study was to evaluate the neurotoxic potential of the pesticide fipronil (FIP) towards the differentiation of mouse N2a neuroblastoma cells. At concentrations of 1, 5 and 10 µM that were not cytotoxic, as shown by two different cell viability assays, FIP impaired potently after 24h the development of axon-like processes, with a concentration of 1 µM causing 50% inhibition. Densitometric analysis of immunoblots of extracts of N2a cells exposed to FIP demonstrated that the axon-inhibitory action of the pesticide was not accompanied by significant changes in the levels of total and phosphorylated neurofilament heavy chain (NFH). FIP also induced no alteration in the levels of total and tyrosinated α-tubulin. On the other hand, this pesticide caused severe disruption of the developmentally important ERK 1/2-MAP kinase signal transduction pathway, as evidenced by significant reductions in the activation state of MAPK kinase (MEK 1/2) and, particularly, ERK 1/2. The above data seem to justify very recent concerns that FIP has the capacity to induce developmental neurotoxicity in mammals.

An extracellular transglutaminase is required for apple pollen tube growth.

An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.