A method for measuring mitochondrial DNA copy number in pediatric populations

The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of mtDNA to nuclear DNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ∼200 bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress

Statin-induced deficits in memory and learning: A behavioural and electrophysiological investigation

Statins play a crucial role in reducing the risk of death from cardiovascular disease in millions of people worldwide. Recently, data show people taking statins are at increased risk of a number of psychiatric adverse events such as amnesia, anxiety and even aggression. However, there are conflicting epidemiological data and a scarcity of direct experimental evidence that statins can alter neural functioning. This thesis aimed to investigate the effect of statin treatment on memory in an animal model of spatial memory and learning; the Morris Water maze (MWM) using guinea pigs. The behavioural results demonstrate that statins, independent of their musculoskeletal or liver adverse effects, significantly induced deficits in specific aspects of the MWM. Statins at a clinically equivalent dose of 20mg/d did not affect reference memory directly by affecting latency or distance to platform, but resulted in increased thigmotactic activity. Further behavioural investigations using a higher dose and modified protocol showed once again that statins did not affect spatial reference memory; however, statin treatment for six weeks induced deficits in spatial working memory (short-term memory). Mechanisms of memory have been hypothesised to result from changes in synaptic plasticity in the hippocampus. Extracellular field recordings of synaptic transmission in area CA1 of hippocampal slices were conducted to assess the effects of statin application on LTP. Statins significantly reduced the amount of LTP expressed in a dose-dependent manner. Further investigations with methyl-beta-cyclodextrin (MBCD), a compound that sequesters cholesterol from lipid membranes, demonstrated that statins act independently of cholesterol reduction to decrease the expression of LTP. Furthermore, statins did not affect paired pulse facilitation, but induction of LTP reduced the paired pulse ratio (PPR) in statin-treated slices. These results therefore suggest that statins may induce paired pulse depression after the induction of LTP. To assess these findings further and provide clinical consensus, the effect of six weeks of chronic statin administration on LTP was investigated. Hippocampal slices from statin-treated animals showed reduced expression of LTP compared with vehicle treated animals; however, this was not statistically significant. It is possible that behavioural training prior to electrophysiological assessment could have obscured from detection of any deficits in LTP following chronic statin treatment. To further investigate the molecular mechanisms of statin-induced deficits, western blot analysis of GluN1 and GluA1, specific subunits of glutamatergic receptors known to be critically involved in memory and learning were assessed. Statins induced a 1.5 fold increase in GluA1 but did not affect the expression of GluN1. In conclusion, the results of this thesis demonstrate that statins administered to healthy guinea pigs, at clinically relevant doses, can induce specific behavioural deficits in spatial memory. Furthermore, statins attenuated hippocampal LTP (in vitro), independently of their cholesterol-lowering properties. This thesis has taken the first step in providing evidence to suggest how statins may induce deficits in memory and learning, and in the process has led to new understandings of the role of statins in hippocampal synaptic plasticity, memory and learning

Statin-induced deficits in memory and learning: A behavioural and electrophysiological investigation

Statins play a crucial role in reducing the risk of death from cardiovascular disease in millions of people worldwide. Recently, data show people taking statins are at increased risk of a number of psychiatric adverse events such as amnesia, anxiety and even aggression. However, there are conflicting epidemiological data and a scarcity of direct experimental evidence that statins can alter neural functioning. This thesis aimed to investigate the effect of statin treatment on memory in an animal model of spatial memory and learning; the Morris Water maze (MWM) using guinea pigs. The behavioural results demonstrate that statins, independent of their musculoskeletal or liver adverse effects, significantly induced deficits in specific aspects of the MWM. Statins at a clinically equivalent dose of 20mg/d did not affect reference memory directly by affecting latency or distance to platform, but resulted in increased thigmotactic activity. Further behavioural investigations using a higher dose and modified protocol showed once again that statins did not affect spatial reference memory; however, statin treatment for six weeks induced deficits in spatial working memory (short-term memory). Mechanisms of memory have been hypothesised to result from changes in synaptic plasticity in the hippocampus. Extracellular field recordings of synaptic transmission in area CA1 of hippocampal slices were conducted to assess the effects of statin application on LTP. Statins significantly reduced the amount of LTP expressed in a dose-dependent manner. Further investigations with methyl-beta-cyclodextrin (MBCD), a compound that sequesters cholesterol from lipid membranes, demonstrated that statins act independently of cholesterol reduction to decrease the expression of LTP. Furthermore, statins did not affect paired pulse facilitation, but induction of LTP reduced the paired pulse ratio (PPR) in statin-treated slices. These results therefore suggest that statins may induce paired pulse depression after the induction of LTP. To assess these findings further and provide clinical consensus, the effect of six weeks of chronic statin administration on LTP was investigated. Hippocampal slices from statin-treated animals showed reduced expression of LTP compared with vehicle treated animals; however, this was not statistically significant. It is possible that behavioural training prior to electrophysiological assessment could have obscured from detection of any deficits in LTP following chronic statin treatment. To further investigate the molecular mechanisms of statin-induced deficits, western blot analysis of GluN1 and GluA1, specific subunits of glutamatergic receptors known to be critically involved in memory and learning were assessed. Statins induced a 1.5 fold increase in GluA1 but did not affect the expression of GluN1. In conclusion, the results of this thesis demonstrate that statins administered to healthy guinea pigs, at clinically relevant doses, can induce specific behavioural deficits in spatial memory. Furthermore, statins attenuated hippocampal LTP (in vitro), independently of their cholesterol-lowering properties. This thesis has taken the first step in providing evidence to suggest how statins may induce deficits in memory and learning, and in the process has led to new understandings of the role of statins in hippocampal synaptic plasticity, memory and learning

Epistaxis and other haemorrhagic events associated with the smoking cessation medicine varenicline: a case series from two national pharmacovigilance centres

Abstract Purpose To present a case series of haemorrhagic events associated with varenicline identified from the New Zealand (NZ) and Netherlands national pharmacovigilance centres and propose a possible mechanism for these adverse events. Methods Reports of epistaxis and other haemorrhagic events (in all system organ classes excluding gynaecological) associated with varenicline were identified and assessed in both the NZ Intensive Medicines Monitoring Programme (IMMP) and the Netherlands Pharmacovigilance Centre Lareb (Lareb). Additional reports were identified from the World Health Organisation Uppsala Monitoring Centre (WHO-UMC) datasets, and these also underwent causality assessment. Results A total of 30 reports of haemorrhagic events were identified by the NZ IMMP (16 reports) and Lareb (14 reports). Six cases of epistaxis were identified, and four patients had a positive dechallenge on withdrawal of varenicline, suggesting a causal association. Another five reports of gingival bleeding were identified, with three patients having a positive dechallenge. Another patient who experienced haemoptysis while taking varenicline had a positive dechallenge and a positive rechallenge. In the WHO datasets, a further 49 reports of epistaxis, 39 reports of haemoptysis and 21 reports of thrombocytopenia were identified. A plausible mechanism for haemorrhagic events associated with varenicline may be a result of interaction with the serotonin (5-HT) receptor system and transporter. Conclusions This is the first specific investigation of haemorrhagic events associated with varenicline. The results of our assessment of reports identified by two national pharmacovigilance centres suggest that there may be causal relationship between varenicline and these adverse events

Epistaxis and other haemorrhagic events associated with the smoking cessation medicine varenicline:a case series from two national pharmacovigilance centres

Purpose To present a case series of haemorrhagic events associated with varenicline identified from the New Zealand (NZ) and Netherlands national pharmacovigilance centres and propose a possible mechanism for these adverse events. Methods Reports of epistaxis and other haemorrhagic events (in all system organ classes excluding gynaecological) associated with varenicline were identified and assessed in both the NZ Intensive Medicines Monitoring Programme (IMMP) and the Netherlands Pharmacovigilance Centre Lareb (Lareb). Additional reports were identified from the World Health Organisation Uppsala Monitoring Centre (WHO-UMC) datasets, and these also underwent causality assessment. Results A total of 30 reports of haemorrhagic events were identified by the NZ IMMP (16 reports) and Lareb (14 reports). Six cases of epistaxis were identified, and four patients had a positive dechallenge on withdrawal of varenicline, suggesting a causal association. Another five reports of gingival bleeding were identified, with three patients having a positive dechallenge. Another patient who experienced haemoptysis while taking varenicline had a positive dechallenge and a positive rechallenge. In the WHO datasets, a further 49 reports of epistaxis, 39 reports of haemoptysis and 21 reports of thrombocytopenia were identified. A plausible mechanism for haemorrhagic events associated with varenicline may be a result of interaction with the serotonin (5-HT) receptor system and transporter. Conclusions This is the first specific investigation of haemorrhagic events associated with varenicline. The results of our assessment of reports identified by two national pharmacovigilance centres suggest that there may be causal relationship between varenicline and these adverse events

Trafficking of mitochondrial double-stranded RNA from mitochondria to the cytosol.

In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner