Pharmacology and Therapeutics - Research Publications
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ItemProtein kinase D and Gβγ mediate sustained nociceptive signaling by biased agonists of protease-activated receptor-2(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2019-07-05)Proteases sustain hyperexcitability and pain by cleaving protease-activated receptor-2 (PAR2) on nociceptors through distinct mechanisms. Whereas trypsin induces PAR2 coupling to Gαq, Gαs, and β-arrestins, cathepsin-S (CS) and neutrophil elastase (NE) cleave PAR2 at distinct sites and activate it by biased mechanisms that induce coupling to Gαs, but not to Gαq or β-arrestins. Because proteases activate PAR2 by irreversible cleavage, and activated PAR2 is degraded in lysosomes, sustained extracellular protease-mediated signaling requires mobilization of intact PAR2 from the Golgi apparatus or de novo synthesis of new receptors by incompletely understood mechanisms. We found here that trypsin, CS, and NE stimulate PAR2-dependent activation of protein kinase D (PKD) in the Golgi of HEK293 cells, in which PKD regulates protein trafficking. The proteases stimulated translocation of the PKD activator Gβγ to the Golgi, coinciding with PAR2 mobilization from the Golgi. Proteases also induced translocation of a photoconverted PAR2-Kaede fusion protein from the Golgi to the plasma membrane of KNRK cells. After incubation of HEK293 cells and dorsal root ganglia neurons with CS, NE, or trypsin, PAR2 responsiveness initially declined, consistent with PAR2 cleavage and desensitization, and then gradually recovered. Inhibitors of PKD, Gβγ, and protein translation inhibited recovery of PAR2 responsiveness. PKD and Gβγ inhibitors also attenuated protease-evoked mechanical allodynia in mice. We conclude that proteases that activate PAR2 by canonical and biased mechanisms stimulate PKD in the Golgi; PAR2 mobilization and de novo synthesis repopulate the cell surface with intact receptors and sustain nociceptive signaling by extracellular proteases.
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ItemAltered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord.(Oxford University Press (OUP), 2022-09-14)Aberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce. We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration. We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases. Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development.
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ItemComorbidity characteristics of multiple myeloma patients diagnosed in Finland 2005-2016.(Springer Science and Business Media LLC, 2022-11)Multiple myeloma (MM) patients are predominantly elderly with comorbidities that have an impact on patient mortality and treatment decisions. We previously reported the patient characteristics and overall survival outcomes of the Finnish MM cohort diagnosed between 2005 and 2016 in a nationwide retrospective registry study comprising 3,851 adults. Here, we report detailed comorbidity characteristics for this real-world Finnish MM population at cohort entry and during follow-up. Data on diagnoses and causes of death were obtained from Finnish healthcare data registries and interrogated using various multistate time-to-event models. In the year preceding MM diagnosis, comorbidities (as per Charlson Comorbidity Index definition) were recorded in 38.0% of the cohort, of which 27.9% presented with pre-existing cardiovascular disease (CVD) and 4.8% had suffered a major adverse cardiac event (MACE). At 2 years post-MM diagnosis, cumulative incidence for CVD and MACE more than doubled to 57.1% and 11.4%, respectively, and only 31.9% of the cohort remained CVD-free. Prevalent secondary malignancies were recorded in 16.8% of the patient population at MM diagnosis, with cumulative incidence increasing steadily to 27.5% at 2 years and 33% at 5 years post-diagnosis. The main cause of mortality attributed to MM, CVD, secondary malignancy, or other causes remained stable throughout the follow-up, at an average of 74.2%, 9.4%, 9.8%, and 6.5%, respectively. Prevalence of CVDs and secondary malignancies is high in Finnish patients at MM diagnosis, with older male patients suffering from higher MACE and mortality risk. Proper recording and management of comorbidities alongside novel treatments remain crucial for optimal MM management.
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ItemEditorial: Neuronal Co-transmission(FRONTIERS MEDIA SA, 2019-03-26)
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ItemIron overload and impaired iron handling contribute to the dystrophic pathology in models of Duchenne muscular dystrophy(WILEY, 2022-06)BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.
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ItemLithium administered to pregnant, lactating and neonatal rats: entry into developing brain(BMC, 2021-12-07)BACKGROUND: Little is known about the extent of drug entry into developing brain, when administered to pregnant and lactating women. Lithium is commonly prescribed for bipolar disorder. Here we studied transfer of lithium given to dams, into blood, brain and cerebrospinal fluid (CSF) in embryonic and postnatal animals as well as adults. METHODS: Lithium chloride in a clinically relevant dose (3.2 mg/kg body weight) was injected intraperitoneally into pregnant (E15-18) and lactating dams (birth-P16/17) or directly into postnatal pups (P0-P16/17). Acute treatment involved a single injection; long-term treatment involved twice daily injections for the duration of the experiment. Following terminal anaesthesia blood plasma, CSF and brains were collected. Lithium levels and brain distribution were measured using Laser Ablation Inductively Coupled Plasma-Mass Spectrometry and total lithium levels were confirmed by Inductively Coupled Plasma-Mass Spectrometry. RESULTS: Lithium was detected in blood, CSF and brain of all fetal and postnatal pups following lithium treatment of dams. Its concentration in pups' blood was consistently below that in maternal blood (30-35%) indicating significant protection by the placenta and breast tissue. However, much of the lithium that reached the fetus entered its brain. Levels of lithium in plasma fluctuated in different treatment groups but its concentration in CSF was stable at all ages, in agreement with known stable levels of endogenous ions in CSF. There was no significant increase of lithium transfer into CSF following application of Na+/K+ ATPase inhibitor (digoxin) in vivo, indicating that lithium transfer across choroid plexus epithelium is not likely to be via the Na+/K+ ATPase mechanism, at least early in development. Comparison with passive permeability markers suggested that in acute experiments lithium permeability was less than expected for diffusion but similar in long-term experiments at P2. CONCLUSIONS: Information obtained on the distribution of lithium in developing brain provides a basis for studying possible deleterious effects on brain development and behaviour in offspring of mothers undergoing lithium therapy.
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ItemThe role of the blood-brain barrier in hypertension(WILEY, 2018-03-01)What is the topic of this review? This review highlights the importance of the blood-brain barrier in the context of diseases involving autonomic dysfunction, such as hypertension and heart failure. What advances does it highlight? It highlights the potential role of pro-inflammatory cytokines, leucocytes and angiotensin II in disrupting the blood-brain barrier in cardiovascular diseases. Advances are highlighted in our understanding of neurovascular unit cells, astrocytes and microglia, with a specific emphasis on their pathogenic roles within the brain. The blood-brain barrier (BBB) is a crucial barrier that provides both metabolic and physical protection to an immune-privileged CNS. The BBB has been shown to be disrupted in hypertension. This review addresses the importance of the BBB in maintaining homeostasis in the context of diseases related to autonomic dysfunction, such as hypertension. We highlight the potentially important roles of the immune system and neurovascular unit in the maintenance of the BBB, whereby dysregulation may lead to autonomic dysfunction in diseases such as heart failure and hypertension. Circulating leucocytes and factors such as angiotensin II and pro-inflammatory cytokines are thought ultimately to downregulate endothelial tight junction proteins that are a crucial component of the BBB. The specific mechanisms underlying BBB disruption and their role in contributing to autonomic dysfunction are not yet fully understood but are a growing area of interest. A greater understanding of these systems and advances in our knowledge of the molecular mechanisms causing BBB disruption will allow for the development of future therapeutic interventions in the treatment of autonomic imbalance associated with diseases such as heart failure and hypertension.
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ItemType-I interferons in Parkinson's disease: innate inflammatory response drives fate of neurons in model of degenerative brain disorder: An editorial comment on 'Type-I interferons mediate the neuroinflammatory response and neurotoxicity induced by rotenone'(WILEY, 2017-04)Read the commented article 'Type-I interferons mediate the neuroinflammatory response and neurotoxicity induced by rotenone' on page 75.
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ItemType-I interferons mediate the neuroinflammatory response and neurotoxicity induced by rotenone(WILEY, 2017-04)Evidence from post-mortem human brains, animal studies and cell culture models has implicated neuroinflammation in the aetiology of chronic neuropathologies including Alzheimer's and Parkinson's diseases. Although the neuroinflammatory response is considered detrimental in contributing to these pathologies, the underlying mechanisms are still not well understood. The type-I interferons (IFNs) have been well characterised in the periphery and are known to initiate/modulate the immune response. Recently, they have been implicated in ageing and we have also demonstrated increased type-I IFN expression in post-mortem human Alzheimer's and Parkinson's disease brains. We hypothesise that the type-I IFNs are key drivers of the damaging, self-perpetuating pro-inflammatory response that contributes to these chronic neuropathologies. In support of this, we have recently confirmed in models of Alzheimer's and Parkinson's disease that mice lacking the type-I IFN receptor (IFNAR1), display an attenuated neuroinflammatory response with subsequent neuroprotection. To further investigate type-I IFN-mediated neuroinflammation and the specific CNS cell types involved, this study treated primary cultured wild-type and IFNAR1-/- neurons or mixed glia with the mitochondrial complex I inhibitor, rotenone. Wild-type neurons and glia treated with 3 nM and 25 nM rotenone, respectively, exhibited a pro-inflammatory response, including increased type-I IFN expression that was attenuated in cells lacking IFNAR1. Reduced type-I IFN signalling in IFNAR1-/- neurons also conferred protection against caspase-3-mediated rotenone-induced cell death. Further, this reduced pro-inflammatory response in the IFNAR1-/- glia subsequently diminished their neurotoxic effects to wild-type neurons. In support of this, we confirmed that therapeutically targeting the type-I IFN glial response to rotenone through a specific IFNAR1 blocking monoclonal antibody was neuroprotective. Our data has confirmed that both neurons and glia contribute to the pro-inflammatory response induced by rotenone with attenuation of this response beneficial in reducing neuronal cell death. Read the Editorial Comment for this article on page 9.