Annika Vaarmann

PGC-1{alpha} and PGC-1{beta} regulate mitochondrial density in neurons.

Recent studies indicate that regulation of cellular oxidative capacity through enhancing mitochondrial biogenesis may be beneficial for neuronal recovery and survival in human neurodegenerative disorders. The peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) has been shown to be a master regulator of mitochondrial biogenesis and cellular energy metabolism in muscle and liver. The aim of our study was to establish whether PGC-1α and PGC-1β control mitochondrial density also in neurons and if these coactivators could be up-regulated by deacetylation. The results demonstrate that PGC-1α and PGC-1β control mitochondrial capacity in an additive and independent manner. This effect was observed in all studied subtypes of neurons, in cortical, midbrain, and cerebellar granule neurons. We also observed that endogenous neuronal PGC-1α but not PGC-1β could be activated through its repressor domain by suppressing it. Results demonstrate also that overexpression of SIRT1 deacetylase or suppression of GCN5 acetyltransferase activates transcriptional activity of PGC-1α in neurons and increases mitochondrial density. These effects were mediated exclusively via PGC-1α, since overexpression of SIRT1 or suppression of GCN5 was ineffective where PGC-1α was suppressed by short hairpin RNA. Moreover, the results demonstrate that overexpression of PGC-1β or PGC-1α or activation of the latter by SIRT1 protected neurons from mutant α-synuclein- or mutant huntingtin-induced mitochondrial loss. These evidences demonstrate that activation or overexpression of the PGC-1 family of coactivators could be used to compensate for neuronal mitochondrial loss and suggest that therapeutic agents activating PGC-1 would be valuable for treating neurodegenerative diseases in which mitochondrial dysfunction and oxidative damage play an important pathogenic role.

Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy.

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.

Novel and sensitive high-performance liquid chromatographic method based on electrochemical coulometric array detection for simultaneous determination of catecholamines, kynurenine and indole derivatives of tryptophan.

A novel and simple method has been developed for the simultaneous quantification of tryptophan, kynurenine and indole derivatives as well as four catecholamines, including dopamine, noradrenaline, homovanillic acid and 3,4-dihydroxyphenylacetic acid. The method utilises isocratic reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase and the detection potentials, was investigated. Separation of 13 compounds was achieved by a mobile phase consisting of 10% methanol in 50 mM sodium phosphate-acetate buffer, pH 4.10, containing 0.42 mM octanesulphonic acid. The calibration curve was linear over the range 12 pg to 300 ng on-column. The detection limits (SIN 3) depended on the working potential and were found to be between 10 and 100 pg injected. The method was reproducible with intra-day RSDs of 0.3 to 1.5% and inter-day RSDs of 0.5 to 4%.

Role of Mitochondrial Dynamics in Neuronal Development: Mechanism for Wolfram Syndrome.

Deficiency of the protein Wolfram syndrome 1 (WFS1) is associated with multiple neurological and psychiatric abnormalities similar to those observed in pathologies showing alterations in mitochondrial dynamics. The aim of this study was to examine the hypothesis that WFS1 deficiency affects neuronal function via mitochondrial abnormalities. We show that down-regulation of WFS1 in neurons leads to dramatic changes in mitochondrial dynamics (inhibited mitochondrial fusion, altered mitochondrial trafficking, and augmented mitophagy), delaying neuronal development. WFS1 deficiency induces endoplasmic reticulum (ER) stress, leading to inositol 1,4,5-trisphosphate receptor (IP3R) dysfunction and disturbed cytosolic Ca2+ homeostasis, which, in turn, alters mitochondrial dynamics. Importantly, ER stress, impaired Ca2+ homeostasis, altered mitochondrial dynamics, and delayed neuronal development are causatively related events because interventions at all these levels improved the downstream processes. Our data shed light on the mechanisms of neuronal abnormalities in Wolfram syndrome and point out potential therapeutic targets. This work may have broader implications for understanding the role of mitochondrial dynamics in neuropsychiatric diseases.

Mitochondrial biogenesis is required for axonal growth.

During early development, neurons undergo complex morphological rearrangements to assemble into neuronal circuits and propagate signals. Rapid growth requires a large quantity of building materials, efficient intracellular transport and also a considerable amount of energy. To produce this energy, the neuron should first generate new mitochondria because the pre-existing mitochondria are unlikely to provide a sufficient acceleration in ATP production. Here, we demonstrate that mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons. We also demonstrate that growth signals activating the CaMKKβ, LKB1-STRAD or TAK1 pathways also co-activate the AMPK–PGC-1α–NRF1 axis leading to the generation of new mitochondria to ensure energy for upcoming growth. In conclusion, our results suggest that neurons are capable of signalling for upcoming energy requirements. Earlier activation of mitochondrial biogenesis through these pathways will accelerate the generation of new mitochondria, thereby ensuring energy-producing capability for when other factors for axonal growth are synthesized. Summary: Mitochondrial biogenesis and ATP production are required for axonal growth and neuronal development in cultured rat cortical neurons.

Seizures, Ataxia, and Neuronal Loss in Cystatin B Heterozygous Mice

Summary:  Unverricht‐Lundborg disease (EPM1) has been considered to be an autosomal‐recessive disease related with loss of function mutations in the gene encoding cystatin B. Although heterozygous carriers are generally asymptomatic, earlier studies in Finnish EPM1 families have reported minor symptoms together with slight changes in the EEG recordings also in near relatives of patients. Here we tested the hypothesis that EPM1 phenotype is expressed also in heterozygous subjects using 17‐month‐old cystatin B deficient mice as a model of disease. Western blot analysis demonstrated a 50% decrease in cystatin B expression in the cerebellum of these animals. Heterozygous mice showed significantly impaired rotarod performance and were weaker in the grid test. Also the total seizure‐rating score of heterozygous animals was higher than in wild‐type mice. The stereological analysis revealed a significant decrease in the number of neurons in cerebral cortex and the granule cell layer of cerebellum. These results suggest that partial decrease in cystatin B expression in heterozygous mice could lead to the development of mild EPM1 phenotype.

Altered tryptophan metabolism in the brain of Cystatin B-deficient mice : A model system for progressive myoclonus epilepsy

Summary:  Purpose: Progressive myoclonus epilepsy of the Unverricht–Lundborg type (EPM1) is a rare neurologic disorder, associated with mutations in the Cystatin B (Cstb) gene. Mice lacking Cstb, a cysteine protease inhibitor of the cathepsine family of proteases, provide a mammalian model for EPM1 by displaying similarly progressive ataxia, myoclonic seizures, and neurodegeneration. However, the linkage of Cstb deficit on the molecular level to pathologic features like myoclonic jerks or tonic–clonic seizures has remained unclear. We examined the tryptophan (TRP) metabolism, along the serotonin (5HT) and kynurenine (KYN) pathway in the brain of Cstb‐deficient mice, in relation to their possible involvement in the seizure phenotype.

Reduced Serotonin and 3-Hydroxyanthranilic Acid Levels in Serum of Cystatin B-Deficient Mice, a Model System for Progressive Myoclonus Epilepsy

Summary:  Purpose: To evaluate the levels of tryptophan and its metabolites along serotonin (5‐HT) and kynurenine (KYN) pathways in serum of progressive myoclonus epilepsy (EPM1) patients and cystatin B (CSTB)‐deficient mice, a model system for EPM1.

Apomorphine-induced aggressive behaviour and post-mortem monoamine content in male Wistar rats.

The aim of this study was to investigate the monoamine content in post-mortem brain samples of control, apomorphine-aggressive, and apomorphine-non-aggressive adult male Wistar rats. The repeated apomorphine (1.0 mg/kg, (s.c.) once daily during 2 weeks) gradually induced aggressive behaviour in 18 animals out of 24. No unidirectional changes in the brain monoamine contents in four regions (frontal cortex, striatum, hippocampus, and hypothalamus) were detected as measured by high pressure liquid chromatography-electrochemical detection. In conclusion, our present experiment demonstrates that the development and intensity of apomorphine-induced aggressive behaviour do not correlate with the brain post-mortem monoamine content.

Role of 5-HT1A receptors in the mediation of acute citalopram effects: a 8-OH-DPAT challenge study.

(1) The study was aimed to investigate the effects of the minimal effective doses of acute citalopram (5 mg/kg), (+/-)-8-hydroxydipropylaminotetralin HBr (8-OH-DPAT; 0.1 mg/kg), and their combined treatment on the rat open field and forced swimming behaviour and post-mortem monoamine content. (2) The animals were prospectively divided into the vehicle- and para-chlorophenylalanine (p-CPA)-pretreated (350 mg/kg) groups. (3) Acute citalopram (5 mg/kg), 8-OH-DPAT (0.1 mg/kg), or their combined treatment had no major effect on the rat open field and forced swimming behaviour. (4) The post-mortem catecholamine content in four brain regions studied was unchanged in all treatment groups. (5) The combined 8-OH-DPAT (0.1 mg/kg) and citalopram (5 mg/kg) treatment partially reversed the p-CPA-induced decrease of serotonin (5-HT) and 5-hydroxy-indolacetic acid (5-HIAA) content. (6) The present experiments demonstrate that the 5-HT1A receptors mediate some of the selective serotonin reuptake inhibitor (SSRI)-induced biochemical phenomena.