Publications by authors named "Adam K Walker"

62 Publications

Riluzole does not ameliorate disease caused by cytoplasmic TDP-43 in a mouse model of amyotrophic lateral sclerosis.

Eur J Neurosci 2021 Sep 26;54(6):6237-6255. Epub 2021 Aug 26.

Centre for Motor Neuron Disease Research, Macquarie Medical School, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, New South Wales, Australia.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease commonly treated with riluzole, a small molecule that may act via modulation of glutamatergic neurotransmission. However, riluzole only modestly extends lifespan for people living with ALS, and its precise mechanisms of action remain unclear. Most ALS cases are characterised by accumulation of cytoplasmic TAR DNA binding protein of 43 kDa (TDP-43), and understanding the effects of riluzole in models that closely recapitulate TDP-43 pathology may provide insights for development of improved therapeutics. We therefore investigated the effects of riluzole in female transgenic mice that inducibly express nuclear localisation sequence (NLS)-deficient human TDP-43 in neurons (NEFH-tTA/tetO-hTDP-43ΔNLS, 'rNLS8', mice). Riluzole treatment from the first day of hTDP-43ΔNLS expression did not alter disease onset, weight loss or performance on multiple motor behavioural tasks. Riluzole treatment also did not alter TDP-43 protein levels, solubility or phosphorylation. Although we identified a significant decrease in GluA2 and GluA3 proteins in the cortex of rNLS8 mice, riluzole did not ameliorate this disease-associated molecular phenotype. Likewise, riluzole did not alter the disease-associated atrophy of hindlimb muscle in rNLS8 mice. Finally, riluzole treatment beginning after disease onset in rNLS8 mice similarly had no effect on progression of late-stage disease or animal survival. Together, we demonstrate specific glutamatergic receptor alterations and muscle fibre-type changes reminiscent of ALS in female rNLS8 mice, but riluzole had no effect on these or any other disease phenotypes. Future targeting of pathways related to accumulation of TDP-43 pathology may be needed to develop better treatments for ALS.
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http://dx.doi.org/10.1111/ejn.15422DOI Listing
September 2021

Unbiased Label-Free Quantitative Proteomics of Cells Expressing Amyotrophic Lateral Sclerosis (ALS) Mutations in Reveals Activation of the Apoptosis Pathway: A Workflow to Screen Pathogenic Gene Mutations.

Front Mol Neurosci 2021 27;14:627740. Epub 2021 Apr 27.

Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia.

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of and models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein-protein and protein-substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.
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http://dx.doi.org/10.3389/fnmol.2021.627740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111008PMC
April 2021

The Anti-Inflammatory Drug Aspirin Does Not Protect Against Chemotherapy-Induced Memory Impairment by Paclitaxel in Mice.

Front Oncol 2020 14;10:564965. Epub 2020 Dec 14.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Inflammation has been proposed to play a causal role in chemobrain which-if true-would represent an opportunity to repurpose existing anti-inflammatory drugs for the prevention and treatment of chemobrain. Here, we show that the chemoagent paclitaxel induces memory impairment and anhedonia in mice within 24 h of treatment cessation, but inflammation is not present until 2 weeks after treatment. We find no evidence of brain inflammation as measured by cytokine analysis at any time point. Furthermore, treating with aspirin to block inflammation did not affect paclitaxel-induced memory impairment. These findings suggest that inflammation may not be responsible for memory impairment induced by paclitaxel. These results contrast with recent findings of a causal role for inflammation in cancer-induced memory deficits in mice that were prevented by treatment with oral aspirin, suggesting that cognitive impairment in cancer patients undergoing treatment may arise from multiple convergent mechanisms.
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http://dx.doi.org/10.3389/fonc.2020.564965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7768078PMC
December 2020

Retraction Notice to "Endoplasmic reticulum stress and induction of the unfolded protein response in human sporadic amyotrophic lateral sclerosis" [Neurobiology of Disease, 30/3 (June 2008) 400 - 407].

Neurobiol Dis 2021 Mar 25;150:105232. Epub 2020 Dec 25.

Howard Florey Institute, University of Melbourne, Parkville, VIC 3010, Australia; Centre for Neuroscience, University of Melbourne, Australia.

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http://dx.doi.org/10.1016/j.nbd.2020.105232DOI Listing
March 2021

Trajectory of change in brain complement factors from neonatal to young adult humans.

J Neurochem 2021 05 15;157(3):479-493. Epub 2020 Dec 15.

Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA.

Immune system components also regulate synapse formation and refinement in neurodevelopment. The complement pathway, associated with cell lysis and phagocytosis, is implicated in synaptic elimination. Aberrant adolescent synaptic pruning may underpin schizophrenia onset; thus, changes in cortical complement activity during human development are of major interest. Complement is genetically linked to schizophrenia via increased C4 copy number variants, but the developmental trajectory of complement expression in the human brain is undetermined. As complement increases during periods of active synaptic engulfment in rodents, we hypothesized that complement expression would increase during postnatal development in humans, particularly during adolescence. Using human postmortem prefrontal cortex, we observed that complement activator (C1QB and C3) transcripts peaked in early neurodevelopment, and were highest in toddlers, declining in teenagers (all ANCOVAs between F = 2.41 -3.325, p = .01-0.05). We found that C4 protein was higher at 1-5 years (H = 16.378, p = .012), whereas C3 protein levels were unchanged with age. The microglial complement receptor subunit CD11b increased in mRNA early in life and peaked in the toddler brain (ANCOVA: pH, F = 4.186, p = .003). Complement inhibitors (CD46 and CD55) increased at school age, but failed to decrease like complement activators (both ANCOVAs, F > 4.4, p < .01). These data suggest the activation of complement in the human prefrontal cortex occurs between 1 and 5 years. We did not find evidence of induction of complement factors during adolescence and instead found increased or sustained levels of complement inhibitor mRNA at maturation. Dysregulation of these typical patterns of complement may predispose the brain to neurodevelopmental disorders such as autism or schizophrenia.
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http://dx.doi.org/10.1111/jnc.15241DOI Listing
May 2021

Mislocalisation of TDP-43 to the cytoplasm causes cortical hyperexcitability and reduced excitatory neurotransmission in the motor cortex.

J Neurochem 2021 05 9;157(4):1300-1315. Epub 2020 Nov 9.

Menzies Institute for Medical Research, College of Health and Medicine, University of Tasmania, Hobart, Tas, Australia.

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease pathologically characterised by mislocalisation of the RNA-binding protein TAR-DNA-binding protein 43 (TDP-43) from the nucleus to the cytoplasm. Changes to neuronal excitability and synapse dysfunction in the motor cortex are early pathological changes occurring in people with ALS and mouse models of disease. To investigate the effect of mislocalised TDP-43 on the function of motor cortex neurons we utilised mouse models that express either human wild-type (TDP-43 ) or nuclear localisation sequence-deficient TDP-43 (TDP-43 ) on an inducible promoter that enriches expression to forebrain neurons. Pathophysiology was investigated through immunohistochemistry and whole-cell patch-clamp electrophysiology. Thirty days expression of TDP-43 in adult mice did not cause any changes in the number of CTIP2-positive neurons in the motor cortex. However, at this time-point, the expression of TDP-43 drives intrinsic hyperexcitability in layer V excitatory neurons of the motor cortex. This hyperexcitability occurs concomitantly with a decrease in excitatory synaptic input to these cells and fluctuations in both directions of ionotropic glutamate receptors. This pathophysiology is not present with TDP-43 expression, demonstrating that the localisation of TDP-43 to the cytoplasm is crucial for the altered excitability phenotype. This study has important implications for the mechanisms of toxicity of one of the most notorious proteins linked to ALS, TDP-43. We provide the first evidence that TDP-43 mislocalisation causes aberrant synaptic function and a hyperexcitability phenotype in the motor cortex, linking some of the earliest dysfunctions to arise in people with ALS to mislocalisation of TDP-43.
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http://dx.doi.org/10.1111/jnc.15214DOI Listing
May 2021

Longitudinal exploration of cancer-related cognitive impairment in patients with newly diagnosed aggressive lymphoma: protocol for a feasibility study.

BMJ Open 2020 09 29;10(9):e038312. Epub 2020 Sep 29.

Cancer Nursing Research Group, Department of Nursing/Centre for Cancer Research, School of Health Sciences/University of Melbourne, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Melbourne, Victoria, Australia.

Introduction: Cancer-related cognitive impairment (CRCI) is a distressing and disabling side-effect of cancer treatments affecting up to 75% of patients. For some patients, their cognitive impairment may be transient, but for a subgroup, these symptoms can be long-standing and have a major impact on the quality of life. This paper describes the protocol for a study: (1) to assess the feasibility of collecting longitudinal data on cognition via self-report, neuropsychological testing, peripheral markers of inflammation and neuroimaging and (2) to explore and describe patterns of cancer-related cognitive impairment over the course of treatment and recovery in patients with newly diagnosed, aggressive lymphoma undergoing standard therapy with curative intent.

Methods And Analysis: This is a prospective, longitudinal, feasibility study in which 30 newly diagnosed, treatment-naive patients with aggressive lymphoma will be recruited over a 12-month period. Patients will complete comprehensive assessments at three time points: baseline (time 1, pre-treatment) and two post-baseline follow-up assessments (time 2, mid-treatment and time 3, 6-8 weeks post-treatment completion). All patients will be assessed for self-reported cognitive difficulties and objective cognitive function using Stroop Colour and Word, Trail Making Test Part A and B, Hopkins Verbal Learning Test-Revised, Controlled Oral Word Association and Digit Span. Blood cell-based inflammatory markers and neuroimaging including a positron emission tomography (PET) with F-labelled fluoro-2-deoxyglucose (F-FDG) and CT (F-FDG-PET/CT) and a MRI will explore potential inflammatory and neuroanatomical or functional mechanisms and biomarkers related to CRCI. The primary intent of analysis will be to assess the feasibility of collecting longitudinal data on cognition using subjective reports and objective tasks from patients during treatment and recovery for lymphoma. These data will inform the design of a larger-scale investigation into the patterns of cognitive change over the course of treatment and recovery, adding to an underexplored area of cancer survivorship research.

Ethics And Dissemination: Ethical approval has been granted by Austin Health Human Rights Ethics Committee (HREC) in Victoria Australia. Peer reviewed publications and conference presentations will report the findings of this novel study.

Trial Registration Number: Australian New Zealand Clinical Trials Registry (ACTRN12619001649101).
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http://dx.doi.org/10.1136/bmjopen-2020-038312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7526311PMC
September 2020

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations.

Mol Neurodegener 2020 09 9;15(1):51. Epub 2020 Sep 9.

Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, 75 Talavera Road NSW, North Ryde, NSW, 2109, Australia.

Background: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response.

Methods: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or HO treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage.

Results: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm.

Conclusions: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.
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http://dx.doi.org/10.1186/s13024-020-00386-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7488163PMC
September 2020

Aspirin and other non-steroidal anti-inflammatory drugs and depression, anxiety, and stress-related disorders following a cancer diagnosis: a nationwide register-based cohort study.

BMC Med 2020 09 9;18(1):238. Epub 2020 Sep 9.

Unit of Integrative Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, 171 77, Stockholm, Sweden.

Background: Cancer patients have a highly increased risk of psychiatric disorders following diagnosis, compared with cancer-free individuals. Inflammation is involved in the development of both cancer and psychiatric disorders. The role of non-steroidal anti-inflammatory drugs (NSAIDs) in the subsequent risk of psychiatric disorders after cancer diagnosis is however unknown.

Methods: We performed a cohort study of all patients diagnosed with a first primary malignancy between July 2006 and December 2013 in Sweden. Cox proportional hazards models were used to assess the association of NSAID use during the year before cancer diagnosis with the risk of depression, anxiety, and stress-related disorders during the first year after cancer diagnosis.

Results: Among 316,904 patients identified, 5613 patients received a diagnosis of depression, anxiety, or stress-related disorders during the year after cancer diagnosis. Compared with no use of NSAIDs, the use of aspirin alone was associated with a lower rate of depression, anxiety, and stress-related disorders (hazard ratio [HR], 0.88; 95% confidence interval [CI], 0.81 to 0.97), whereas the use of non-aspirin NSAIDs alone was associated with a higher rate (HR, 1.24; 95% CI, 1.15 to 1.32), after adjustment for sociodemographic factors, comorbidity, indications for NSAID use, and cancer characteristics. The association of aspirin with reduced rate of depression, anxiety, and stress-related disorders was strongest for current use (HR, 0.84; 95% CI, 0.75 to 0.93), low-dose use (HR, 0.88; 95% CI, 0.80 to 0.98), long-term use (HR, 0.84; 95% CI, 0.76 to 0.94), and among patients with cardiovascular disease (HR, 0.81; 95% CI, 0.68 to 0.95) or breast cancer (HR, 0.74; 95% CI, 0.56 to 0.98).

Conclusion: Pre-diagnostic use of aspirin was associated with a decreased risk of depression, anxiety, and stress-related disorders during the first year following cancer diagnosis.
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http://dx.doi.org/10.1186/s12916-020-01709-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487710PMC
September 2020

Nuclear factor kappa B activation appears weaker in schizophrenia patients with high brain cytokines than in non-schizophrenic controls with high brain cytokines.

J Neuroinflammation 2020 Jul 17;17(1):215. Epub 2020 Jul 17.

Schizophrenia Research Laboratory, Neuroscience Research Australia, Barker Street, Randwick, Sydney, NSW, 2031, Australia.

Background: High inflammation status despite an absence of known infection characterizes a subpopulation of people with schizophrenia who suffer from more severe cognitive deficits, less cortical grey matter, and worse neuropathology. Transcripts encoding factors upstream of nuclear factor kappa B (NF-κB), a major transcriptional activator for the synthesis of pro-inflammatory cytokines, are increased in the frontal cortex in schizophrenia compared to controls. However, the extent to which these changes are disease-specific, restricted to those with schizophrenia and high-neuroinflammatory status, or caused by loss of a key NF-κB inhibitor (HIVEP2) found in schizophrenia brain, has not been tested.

Methods: Post-mortem prefrontal cortex samples were assessed in 141 human brains (69 controls and 72 schizophrenia) and 13 brains of wild-type mice and mice lacking HIVEP2 (6 wild-type, 7 knockout mice). Gene expression of pro-inflammatory cytokines and acute phase protein SERPINA3 was used to categorize high and low neuroinflammation biotype groups in human samples via cluster analysis. Expression of 18 canonical and non-canonical NF-κB pathway genes was assessed by qPCR in human and mouse tissue.

Results: In humans, we found non-canonical upstream activators of NF-κB were generally elevated in individuals with neuroinflammation regardless of diagnosis, supporting NF-κB activation in both controls and people with schizophrenia when cytokine mRNAs are high. However, high neuroinflammation schizophrenia patients had weaker (or absent) transcriptional increases of several canonical upstream activators of NF-κB as compared to the high neuroinflammation controls. HIVEP2 mRNA reduction was specific to patients with schizophrenia who also had high neuroinflammatory status, and we also found decreases in NF-κB transcripts typically induced by activated microglia in mice lacking HIVEP2.

Conclusions: Collectively, our results show that high cortical expression of pro-inflammatory cytokines and low cortical expression of HIVEP2 in a subset of people with schizophrenia is associated with a relatively weak NF-κB transcriptional signature compared to non-schizophrenic controls with high cytokine expression. We speculate that this comparatively milder NF-κB induction may reflect schizophrenia-specific suppression possibly related to HIVEP2 deficiency in the cortex.
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http://dx.doi.org/10.1186/s12974-020-01890-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368759PMC
July 2020

Neurodegenerative disease-associated protein aggregates are poor inducers of the heat shock response in neuronal cells.

J Cell Sci 2020 08 5;133(15). Epub 2020 Aug 5.

Illawarra Health and Medical Research Institute, Wollongong, NSW 2522, Australia

Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1) nor intracellular expression of SOD1 or a pathogenic form of polyglutamine-expanded huntingtin (Htt). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.243709DOI Listing
August 2020

Correction to: the Cysteine (Cys) Residues Cys-6 and Cys-111 in Mutant Superoxide Dismutase 1 (SOD1) A4V Are Required for Induction of Endoplasmic Reticulum Stress in Amyotrophic Lateral Sclerosis.

J Mol Neurosci 2020 09;70(9):1369

Department of Biomedical Sciences Centre for MND Research, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia.

The original version of this article unfortunately contained an error in Fig. 3. The image shown for "C57S" was incorrect.
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http://dx.doi.org/10.1007/s12031-020-01617-5DOI Listing
September 2020

Regional, cellular and species difference of two key neuroinflammatory genes implicated in schizophrenia.

Brain Behav Immun 2020 08 22;88:826-839. Epub 2020 May 22.

Schizophrenia Research Laboratory, Neuroscience Research Australia, Randwick, NSW 2031, Australia; School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia; Department of Neuroscience & Physiology, Upstate Medical University, Syracuse, NY 13210, USA. Electronic address:

The transcription factor nuclear factor kappa B (NF-κB) regulates the expression of many inflammatory genes that are overexpressed in a subset of people with schizophrenia. Transcriptional reduction in one NF-κB inhibitor, Human Immunodeficiency Virus Enhancer Binding Protein 2 (HIVEP2), is found in the brain of patients, aligning with evidence of NF-κB over-activity. Cellular co-expression of HIVEP2 and cytokine transcripts is a prerequisite for a direct effect of HIVEP2 on pro-inflammatory transcription, and we do not know if changes in HIVEP2 and markers of neuroinflammation are occurring in the same brain cell type. We performed in situ hybridisation on postmortem dorsolateral prefrontal cortex tissue to map and compare the expression of HIVEP2 and Serpin Family A Member 3 (SERPINA3), one of the most consistently increased inflammatory genes in schizophrenia, between schizophrenia patients and controls. We find that HIVEP2 expression is neuronal and is decreased in almost all grey matter cortical layers in schizophrenia patients with neuroinflammation, and that SERPINA3 is increased in the dorsolateral prefrontal cortex grey matter and white matter in the same group of patients. We are the first to map the upregulation of SERPINA3 to astrocytes and to some neurons, and find evidence to suggest that blood vessel-associated astrocytes are the main cellular source of SERPINA3 in the schizophrenia cortex. We show that a lack of HIVEP2 in mice does not cause astrocytic upregulation of Serpina3n but does induce its transcription in neurons. We speculate that HIVEP2 downregulation is not a direct cause of astrocytic pro-inflammatory cytokine synthesis in schizophrenia but may contribute to neuronally-mediated neuroinflammation.
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http://dx.doi.org/10.1016/j.bbi.2020.05.055DOI Listing
August 2020

The Cysteine (Cys) Residues Cys-6 and Cys-111 in Mutant Superoxide Dismutase 1 (SOD1) A4V Are Required for Induction of Endoplasmic Reticulum Stress in Amyotrophic Lateral Sclerosis.

J Mol Neurosci 2020 Sep 22;70(9):1357-1368. Epub 2020 May 22.

Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, 2109, Australia.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the degeneration of motor neurons. Between 12 and 20% of inherited cases and approximately 1-2% of all cases are caused by mutations in the gene encoding dismutase 1 (SOD1). Mutant SOD1 A4V (alanine to valine) induces endoplasmic reticulum (ER) stress, which is increasingly implicated as a pathway to motor neuron degeneration and death in ALS. However, it remains unclear how ER stress is induced by mutant SOD1 A4V. Previous studies have established that it is induced early in pathophysiology and it precedes the formation of mutant SOD1 inclusions. SOD1 contains four cysteine residues, two of which form an intra-subunit disulphide bond involving Cys-57 and Cys-146. The remaining two cysteines, Cys-6 and Cys-111, remain unpaired and have been implicated in mutant SOD1 aggregation. In this study, we examined the relationship between the SOD1 A4V cysteine residues and aggregation, ER stress induction and toxicity. We report here that mutation of Cys-6 and Cys-111 in mutant SOD1 A4V, but not Cys-57 or Cys-146, ameliorates ER stress, inclusion formation and apoptosis in neuronal cell lines. These results imply that protein misfolding, induced by Cys-6 and Cys-111, is required for these pathological events in neuronal cells.
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http://dx.doi.org/10.1007/s12031-020-01551-6DOI Listing
September 2020

Workflow for Rapidly Extracting Biological Insights from Complex, Multicondition Proteomics Experiments with WGCNA and PloGO2.

J Proteome Res 2020 07 22;19(7):2898-2906. Epub 2020 May 22.

Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales 2109, Australia.

We describe a useful workflow for characterizing proteomics experiments incorporating many conditions and abundance data using the popular weighted gene correlation network analysis (WGCNA) approach and functional annotation with the PloGO2 R package, the latter of which we have extended and made available to Bioconductor. The approach can use quantitative data from labeled or label-free experiments and was developed to handle multiple files stemming from data partition or multiple pairwise comparisons. The WGCNA approach can similarly produce a potentially large number of clusters of interest, which can also be functionally characterized using PloGO2. Enrichment analysis will identify clusters or subsets of proteins of interest, and the WGCNA network topology scores will produce a ranking of proteins within these clusters or subsets. This can naturally lead to prioritized proteins to be considered for further analysis or as candidates of interest for validation in the context of complex experiments. We demonstrate the use of the package on two published data sets using two different biological systems (plant and human plasma) and proteomics platforms (sequential window acquisition of all theoretical fragment-ion spectra (SWATH) and tandem mass tag (TMT)): an analysis of the effect of drought on rice over time generated using TMT and a pediatric plasma sample data set generated using SWATH. In both, the automated workflow recapitulates key insights or observations of the published papers and provides additional suggestions for further investigation. These findings indicate that the data set analysis using WGCNA combined with the updated PloGO2 package is a powerful method to gain biological insights from complex multifaceted proteomics experiments.
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http://dx.doi.org/10.1021/acs.jproteome.0c00198DOI Listing
July 2020

Genetic and immunopathological analysis of CHCHD10 in Australian amyotrophic lateral sclerosis and frontotemporal dementia and transgenic TDP-43 mice.

J Neurol Neurosurg Psychiatry 2020 02 5;91(2):162-171. Epub 2019 Nov 5.

Macquarie University Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, New South Wales, Australia.

Objective: Since the first report of gene mutations in amyotrophiclateral sclerosis (ALS)/frontotemporaldementia (FTD) patients, genetic variation in has been inconsistently linked to disease. A pathological assessment of the CHCHD10 protein in patient neuronal tissue also remains to be reported. We sought to characterise the genetic and pathological contribution of CHCHD10 to ALS/FTD in Australia.

Methods: Whole-exome and whole-genome sequencing data from 81 familial and 635 sporadic ALS, and 108 sporadic FTD cases, were assessed for genetic variation in . CHCHD10 protein expression was characterised by immunohistochemistry, immunofluorescence and western blotting in control, ALS and/or FTD postmortem tissues and further in a transgenic mouse model of TAR DNA-binding protein 43 (TDP-43) pathology.

Results: No causal, novel or disease-associated variants in were identified in Australian ALS and/or FTD patients. In human brain and spinal cord tissues, CHCHD10 was specifically expressed in neurons. A significant decrease in CHCHD10 protein level was observed in ALS patient spinal cord and FTD patient frontal cortex. In a TDP-43 mouse model with a regulatable nuclear localisation signal (rNLS TDP-43 mouse), CHCHD10 protein levels were unaltered at disease onset and early in disease, but were significantly decreased in cortex in mid-stage disease.

Conclusions: Genetic variation in is not a common cause of ALS/FTD in Australia. However, we showed that in humans, CHCHD10 may play a neuron-specific role and a loss of CHCHD10 function may be linked to ALS and/or FTD. Our data from the rNLS TDP-43 transgenic mice suggest that a decrease in CHCHD10 levels is a late event in aberrant TDP-43-induced ALS/FTD pathogenesis.
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http://dx.doi.org/10.1136/jnnp-2019-321790DOI Listing
February 2020

Proteomics Approaches for Biomarker and Drug Target Discovery in ALS and FTD.

Front Neurosci 2019 11;13:548. Epub 2019 Jun 11.

Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, North Ryde, NSW, Australia.

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.
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http://dx.doi.org/10.3389/fnins.2019.00548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579929PMC
June 2019

The Pathobiology of TDP-43 C-Terminal Fragments in ALS and FTLD.

Front Neurosci 2019 11;13:335. Epub 2019 Apr 11.

Neurodegeneration Pathobiology Laboratory, Queensland Brain Institute, University of Queensland, Brisbane, QLD, Australia.

During neurodegenerative disease, the multifunctional RNA-binding protein TDP-43 undergoes a vast array of post-translational modifications, including phosphorylation, acetylation, and cleavage. Many of these alterations may directly contribute to the pathogenesis of TDP-43 proteinopathies, which include most forms of amyotrophic lateral sclerosis (ALS) and approximately half of all frontotemporal dementia, pathologically identified as frontotemporal lobar degeneration (FTLD) with TDP-43 pathology. However, the relative contributions of the various TDP-43 post-translational modifications to disease remain unclear, and indeed some may be secondary epiphenomena rather than disease-causative. It is therefore critical to determine the involvement of each modification in disease processes to allow the design of targeted treatments. In particular, TDP-43 C-terminal fragments (CTFs) accumulate in the brains of people with ALS and FTLD and are therefore described as a neuropathological signature of these diseases. Remarkably, these TDP-43 CTFs are rarely observed in the spinal cord, even in ALS which involves dramatic degeneration of spinal motor neurons. Therefore, TDP-43 CTFs are not produced non-specifically in the course of all forms of TDP-43-related neurodegeneration, but rather variably arise due to additional factors influenced by regional heterogeneity in the central nervous system. In this review, we summarize how TDP-43 CTFs are generated and degraded by cells, and critique evidence from studies of TDP-43 CTF pathology in human disease tissues, as well as cell and animal models, to analyze the pathophysiological relevance of TDP-43 CTFs to ALS and FTLD. Numerous studies now indicate that, although TDP-43 CTFs are prevalent in ALS and FTLD brains, disease-related pathology is only variably reproduced in TDP-43 CTF cell culture models. Furthermore, TDP-43 CTF expression in both transgenic and viral-mediated models largely fails to induce motor or behavioral dysfunction reminiscent of human disease. We therefore conclude that although TDP-43 CTFs are a hallmark of TDP-43-related neurodegeneration in the brain, they are not a primary cause of ALS or FTLD.
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http://dx.doi.org/10.3389/fnins.2019.00335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470282PMC
April 2019

Mechanisms and Treatment for Cancer- and Chemotherapy-Related Cognitive Impairment in Survivors of Non-CNS Malignancies.

Oncology (Williston Park) 2018 12;32(12):591-8

Up to 70% of survivors of adult-onset, non-central nervous system (CNS) solid tumors report cognitive symptoms, and approximately 30% have impairment on formal neuropsychological testing. The etiology of the impairment is unknown. There is a lack of robust evidence on how to prevent or treat cancer-related cognitive impairment (CRCI). Here, we review the evidence for the putative mechanisms of CRCI by examining clinical and preclinical models, primarily those associated with chemotherapy. Pharmacologic and nonpharmacologic options for treating CRCI are discussed based on the best evidence available. Practical clinical advice for health professionals managing patients with CRCI is also provided.
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December 2018

Low dose aspirin blocks breast cancer-induced cognitive impairment in mice.

PLoS One 2018 10;13(12):e0208593. Epub 2018 Dec 10.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Cancer patients with non-central nervous system tumors often suffer from cognitive impairment. While chemotherapy has long been attributed as the cause of these memory, learning and concentration difficulties, we recently observed cognitive impairment in cancer patients prior to treatment. This suggests the cancer alone may be sufficient to induce cognitive impairment, however the mechanisms are unknown. Here, we show that we can experimentally replicate the clinical phenomenon of cancer-associated cognitive impairment and we identify inflammation as a causal mechanism. We demonstrate that a peripheral tumor is sufficient to induce memory loss. Using an othotopic mouse model of breast cancer, we found that mice with 4T1.2 or EO771 mammary tumors had significantly poorer memory than mice without tumors. Memory impairment was independent of cancer-induced sickness behavior, which was only observed during the later stage of cancer progression in mice with high metastatic burden. Tumor-secreted factors were sufficient to induce memory impairment and pro-inflammatory cytokines were elevated in the plasma of tumor-bearing mice. Oral treatment with low-dose aspirin completely blocked tumor-induced memory impairment without affecting tumor-induced sickness or tumor growth, demonstrating a causal role for inflammation in cognitive impairment. These findings suggest that anti-inflammatories may be a safe and readily translatable strategy that could be used to prevent cancer-associated cognitive impairment in patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0208593PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287899PMC
May 2019

Where There's Smoke, There's Fire-But Who Is Lighting the Match? Bolstering Transcriptional Evidence for the Role of Nuclear Factor-κB in Neuroimmune Activation in Schizophrenia.

Biol Psychiatry 2019 01;85(1):5-7

Schizophrenia Research Laboratory Neuroscience Research Australia, Randwick, New South Wales, Australia; School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia; Department of Neuroscience and Physiology, Upstate Medical University, Syracuse, New York. Electronic address:

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http://dx.doi.org/10.1016/j.biopsych.2018.10.019DOI Listing
January 2019

Circulating epinephrine is not required for chronic stress to enhance metastasis.

Psychoneuroendocrinology 2019 01 15;99:191-195. Epub 2018 Sep 15.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, 3052, Australia; Division of Cancer Surgery, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia; Cousins Center for PNI, UCLA Semel Institute, Jonsson Comprehensive Cancer Center, and UCLA AIDS Institute, University of California Los Angeles, Los Angeles, CA, 90095, USA. Electronic address:

Signaling through β-adrenergic receptors drives cancer progression and β-blockers are being evaluated as a novel therapeutic strategy to prevent metastasis. Orthotopic mouse models of breast cancer show that β-adrenergic signaling induced by chronic stress accelerates metastasis, and that β-adrenergic receptors on tumor cells are critical for this. Endogenous catecholamines are released during chronic stress: norepinephrine from the adrenal medulla and sympathetic nerves, and epinephrine from the adrenal medulla. β-adrenergic receptors are much more sensitive to epinephrine than to norepinephrine. To determine if epinephrine is necessary in the effects of stress on cancer progression, we used a denervation strategy to eliminate circulating epinephrine, and quantified the effect on metastasis. Using both human xenograft and immune-intact murine models of breast cancer, we show that circulating epinephrine is dispensable for the effects of chronic stress on cancer progression. Measured levels of circulating norepinephrine were sufficiently low that they were unlikely to influence β-adrenergic signaling, suggesting a possible role for norepinephrine release from sympathetic nerve terminals.
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http://dx.doi.org/10.1016/j.psyneuen.2018.09.012DOI Listing
January 2019

Leucine competes with kynurenine for blood-to-brain transport and prevents lipopolysaccharide-induced depression-like behavior in mice.

Mol Psychiatry 2019 10 9;24(10):1523-1532. Epub 2018 Jul 9.

Laboratory of Neuroimmunology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Inflammation activates indoleamine 2,3-dioxygenase (IDO) which metabolizes tryptophan into kynurenine. Circulating kynurenine is transported into the brain by the large amino transporter LAT1 at the level of the blood-brain barrier. We hypothesized that administration of leucine that has a high affinity for LAT1 should prevent the entry of kynurenine into the brain and attenuate the formation of neurotoxic kynurenine metabolites. To test whether leucine could prevent inflammation-induced depression-like behavior, mice were treated with lipopolysaccharide (LPS, 0.83 mg/kg IP) or saline and treated with L-leucine (50 mg/kg, IP) or vehicle administered before and 6 h after LPS. Depression-like behavior was measured by increased duration of immobility in the forced swim test and decreased sucrose preference. Leucine decreased brain kynurenine levels, blocked LPS-induced depression-like behavior and had antidepressant-like effects in control mice. Leucine had no effect of its own on sickness behavior and neuroinflammation. To confirm that leucine acts by interfering with the transport of kynurenine into the brain, mice were injected with L-leucine (300 mg/kg, IP) immediately before kynurenine (33 mg/kg IP) and brain kynurenine and depression-like behavior were measured 3 h later. Leucine did prevent the entry of exogenous kynurenine into the brain and abrogated depression-like behavior measured by increased duration of immobility in the forced swim test. Additional experiments using an in vitro model of the blood-brain barrier confirmed that kynurenine competes with leucine at the level of the amino acid transporter LAT1 for brain uptake. These experiments also revealed that efflux was the dominant direction of kynurenine transport and was largely independent of LAT1 and leucine, which explains why leucine could block brain uptake of kynurenine without affecting brain clearance. These findings demonstrate that leucine has antidepressant properties vis-à-vis inflammation-induced depression and one mechanism for this is by blocking the ability of kynurenine to enter the brain.
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http://dx.doi.org/10.1038/s41380-018-0076-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326900PMC
October 2019

Stilbenes from Veratrum maackii Regel Protect against Ethanol-Induced DNA Damage in Mouse Cerebellum and Cerebral Cortex.

ACS Chem Neurosci 2018 07 11;9(7):1616-1624. Epub 2018 May 11.

International Joint Center for Biomedical Innovation, College of Life Sciences , Henan University , Jin Ming Avenue , Kaifeng , Henan 475004 , China.

Ethanol is a principle ingredient of alcoholic beverages with potential neurotoxicity and genotoxicity, and the ethanol-associated oxidative DNA damage in the central nervous system is well documented. Natural source compounds may offer new options to protect the brain against ethanol-induced genotoxicity. Veratrum maackii Regel is a toxic rangeland plant linked to teratogenicity which is also used in traditional Chinese medicine as "Lilu" and is reported to contain a family of compounds called stilbenes that can have positive biological activity. In this study, nine stilbenes were isolated from the aerial parts of V. maackii Regel, and their structures were identified as cis-mulberroside A (1), resveratrol-4,3'- O-β-d-diglucopyranoside (2), mulberroside A (3), gentifolin K (4), resveratrol-3,5- O-β-d-diglucopyranoside (5), oxyresveratrol- 4'- O-β-d-glucopyranoside (6), oxyresveratrol-3- O-β-d-glucopyranoside (7), oxyresveratrol (8), and resveratrol (9) using ESI-MS and NMR techniques. The total concentration of extracted compounds 2-9 was 2.04 mg/g, suggesting that V. maackii Regel is a novel viable source of these compounds. In an in vivo comet assay, compounds 1-9 were observed to decrease DNA damage in mouse cerebellum and cerebral cortex caused by acute ethanol administration. Histological observation also revealed decreased brain injury in mice administered with compounds 1-9 after acute ethanol administration. The protective effects of compound 6 were associated with increasing T-SOD and GSH-PX activities and a decrease in NO and MDA concentrations. These findings suggest that these compounds are potent inhibitors of ethanol-induced brain injury possibly via the inhibition of oxidative stress and may be valuable leads for future therapeutic development.
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http://dx.doi.org/10.1021/acschemneuro.8b00006DOI Listing
July 2018

ERp57 is protective against mutant SOD1-induced cellular pathology in amyotrophic lateral sclerosis.

Hum Mol Genet 2018 04;27(8):1311-1331

Centre for MND Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW 2109, Australia.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder and mutations in superoxide dismutase 1 (SOD1) account for 20% of familial ALS cases. The aetiology of ALS remains unclear, but protein misfolding, endoplasmic reticulum (ER) stress and neuronal apoptosis are implicated. We previously established that protein disulphide isomerase (PDIA1) is protective against ER stress and apoptosis in neuronal cells expressing mutant SOD1, and recently mutations in PDIA1 and related PDI family member endoplasmic reticulum protein 57 (ERp57/PDIA3), were associated with ALS. Here, we examined whether ERp57 is also protective against mutant SOD1 or whether distinct specificity exists amongst individual PDI family members. Neuronal cells co-expressing SOD1 and ERp57 were examined for inclusion formation, ER stress, ubiquitin proteasome system (UPS) dysfunction and apoptosis. Over-expression of ERp57 inhibited inclusion formation, ER stress, UPS dysfunction and apoptosis, whereas silencing of ERp57 expression enhanced mutant SOD1 inclusion formation, ER stress and toxicity, indicating a protective role for ERp57 against SOD1 misfolding. ERp57 also inhibited the formation of mutant SOD1 inclusions and apoptosis in primary cortical neurons, thus confirming results obtained from cell lines. ERp57 partially co-localized with TAR DNA-binding protein-43 (TDP-43)-positive inclusions in spinal cords from sporadic ALS patients, thus linking ERp57 to protein misfolding in human sporadic disease. Our results therefore imply that ERp57 has a protective role against pathological events induced by mutant SOD1 and they link ERp57 to the misfolding of TDP-43. This study therefore has implications for the design of novel therapeutics based on the activities of the PDI family of proteins.
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http://dx.doi.org/10.1093/hmg/ddy041DOI Listing
April 2018

Casein kinase II phosphorylation of cyclin F at serine 621 regulates the Lys48-ubiquitylation E3 ligase activity of the SCF complex.

Open Biol 2017 10;7(10)

Department of Biomedical Sciences, Centre for Motor Neuron Disease Research, Faculty of Medicine and Health Sciences, Macquarie University, 2 Technology Place, North Ryde, NSW 2109, Australia.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3-5 years of diagnosis. Recently, we identified disease-linked mutations in the gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL 2016 , 11253. (doi:10.1038/ncomms11253)). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.
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http://dx.doi.org/10.1098/rsob.170058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666078PMC
October 2017

Defects in optineurin- and myosin VI-mediated cellular trafficking in amyotrophic lateral sclerosis.

Hum Mol Genet 2017 09;26(17):3452

Department of Biomedical Sciences, Faculty of Medicine & Health Sciences, Macquarie University, NSW 2109, Australia.

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http://dx.doi.org/10.1093/hmg/ddx268DOI Listing
September 2017

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

Cell Mol Life Sci 2018 01 29;75(2):335-354. Epub 2017 Aug 29.

Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie University, 2 Technology Place, North Ryde, NSW, 2109, Australia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCF) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.
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http://dx.doi.org/10.1007/s00018-017-2632-8DOI Listing
January 2018

Protein Quality Control and the Amyotrophic Lateral Sclerosis/Frontotemporal Dementia Continuum.

Front Mol Neurosci 2017 10;10:119. Epub 2017 May 10.

Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Macquarie UniversitySydney, NSW, Australia.

Protein homeostasis, or proteostasis, has an important regulatory role in cellular function. Protein quality control mechanisms, including protein folding and protein degradation processes, have a crucial function in post-mitotic neurons. Cellular protein quality control relies on multiple strategies, including molecular chaperones, autophagy, the ubiquitin proteasome system, endoplasmic reticulum (ER)-associated degradation (ERAD) and the formation of stress granules (SGs), to regulate proteostasis. Neurodegenerative diseases are characterized by the presence of misfolded protein aggregates, implying that protein quality control mechanisms are dysfunctional in these conditions. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are now recognized to overlap clinically and pathologically, forming a continuous disease spectrum. In this review article, we detail the evidence for dysregulation of protein quality control mechanisms across the whole ALS-FTD continuum, by discussing the major proteins implicated in ALS and/or FTD. We also discuss possible ways in which protein quality mechanisms could be targeted therapeutically in these disorders and highlight promising protein quality control-based therapeutics for clinical trials.
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http://dx.doi.org/10.3389/fnmol.2017.00119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423993PMC
May 2017

Optimized Method for Untargeted Metabolomics Analysis of MDA-MB-231 Breast Cancer Cells.

Metabolites 2016 Sep 22;6(4). Epub 2016 Sep 22.

Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.

Cancer cells often have dysregulated metabolism, which is largely characterized by the Warburg effect-an increase in glycolytic activity at the expense of oxidative phosphorylation-and increased glutamine utilization. Modern metabolomics tools offer an efficient means to investigate metabolism in cancer cells. Currently, a number of protocols have been described for harvesting adherent cells for metabolomics analysis, but the techniques vary greatly and they lack specificity to particular cancer cell lines with diverse metabolic and structural features. Here we present an optimized method for untargeted metabolomics characterization of MDA-MB-231 triple negative breast cancer cells, which are commonly used to study metastatic breast cancer. We found that an approach that extracted all metabolites in a single step within the culture dish optimally detected both polar and non-polar metabolite classes with higher relative abundance than methods that involved removal of cells from the dish. We show that this method is highly suited to diverse applications, including the characterization of central metabolic flux by stable isotope labelling and differential analysis of cells subjected to specific pharmacological interventions.
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http://dx.doi.org/10.3390/metabo6040030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5192436PMC
September 2016
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