Impact of JNK and Its Substrates on Dendritic Spine Morphology

The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates

Impact of JNK and Its Substrates on Dendritic Spine Morphology

The protein kinase JNK1 exhibits high activity in the developing brain, where it regulates dendrite morphology through the phosphorylation of cytoskeletal regulatory proteins. JNK1 also phosphorylates dendritic spine proteins, and Jnk1-/- mice display a long-term depression deficit. Whether JNK1 or other JNKs regulate spine morphology is thus of interest. Here, we characterize dendritic spine morphology in hippocampus of mice lacking Jnk1-/- using Lucifer yellow labelling. We find that mushroom spines decrease and thin spines increase in apical dendrites of CA3 pyramidal neurons with no spine changes in basal dendrites or in CA1. Consistent with this spine deficit, Jnk1-/- mice display impaired acquisition learning in the Morris water maze. In hippocampal cultures, we show that cytosolic but not nuclear JNK, regulates spine morphology and expression of phosphomimicry variants of JNK substrates doublecortin (DCX) or myristoylated alanine-rich C kinase substrate-like protein-1 (MARCKSL1), rescue mushroom, thin, and stubby spines differentially. These data suggest that physiologically active JNK controls the equilibrium between mushroom, thin, and stubby spines via phosphorylation of distinct substrates

Parp1 hyperactivity couples DNA breaks to aberrant neuronal calcium signalling and lethal seizures

Defects in DNA single-strand break repair (SSBR) are linked with neurological dysfunction but the underlying mechanisms remain poorly understood. Here, we show that hyperactivity of the DNA strand break sensor protein Parp1 in mice in which the central SSBR protein Xrcc1 is conditionally deleted (Xrcc1Nes-Cre) results in lethal seizures and shortened lifespan. Using electrophysiological recording and synaptic imaging approaches, we demonstrate that aberrant Parp1 activation triggers seizure-like activity in Xrcc1-defective hippocampus ex vivo and deregulated presynaptic calcium signalling in isolated hippocampal neurons in vitro. Moreover, we show that these defects are prevented by Parp1 inhibition or deletion and, in the case of Parp1 deletion, that the lifespan of Xrcc1Nes-Cre mice is greatly extended. This is the first demonstration that lethal seizures can be triggered by aberrant Parp1 activity at unrepaired SSBs, highlighting PARP inhibition as a possible therapeutic approach in hereditary neurological disease

JNK1 controls dendritic field size in L2/3 and L5 of the motor cortex, constrains soma size, and influences fine motor coordination

Peer reviewe

Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

TDP2 is a 5’-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II. TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for HTS-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanised’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2

XRCC1 mutation is associated with PARP1 hyperactivation and cerebellar ataxia

XRCC1 is a molecular scaffold protein that assembles multi-protein complexes involved in DNA single-strand break repair1,2. Here we show that biallelic mutations in the human XRCC1 gene are associated with ocular motor apraxia, axonal neuropathy, and progressive cerebellar ataxia. Cells from a patient with mutations in XRCC1 exhibited not only reduced rates of single-strand break repair but also elevated levels of protein ADP-ribosylation. This latter phenotype is recapitulated in a related syndrome caused by mutations in the XRCC1 partner protein PNKP3,4,5 and implicates hyperactivation of poly(ADP-ribose) polymerase/s as a cause of cerebellar ataxia. Indeed, remarkably, genetic deletion of Parp1 rescued normal cerebellar ADP-ribose levels and reduced the loss of cerebellar neurons and ataxia in Xrcc1-defective mice, identifying a molecular mechanism by which endogenous single-strand breaks trigger neuropathology. Collectively, these data establish the importance of XRCC1 protein complexes for normal neurological function and identify PARP1 as a therapeutic target in DNA strand break repair-defective disease

JNK regulates dendrite and spine architecture in the central nervous system influencing spatial learning and motor tasks

JNK1 is a MAP-kinase that has proven a significant player in the central nervous system. It regulates brain development and the maintenance of dendrites and axons. Several novel phosphorylation targets of JNK1 were identified in a screen performed in the Coffey lab. These proteins were mainly involved in the regulation of neuronal cytoskeleton, influencing the dynamics and stability of microtubules and actin. These structural proteins form the dynamic backbone for the elaborate architecture of the dendritic tree of a neuron. The initiation and branching of the dendrites requires a dynamic interplay between the cytoskeletal building blocks. Both microtubules and actin are decorated by associated proteins which regulate their dynamics. The dendrite-specific, high molecular weight microtubule associated protein 2 (MAP2) is an abundant protein in the brain, the binding of which stabilizes microtubules and influences their bundling. Its expression in non-neuronal cells induces the formation of neurite-like processes from the cell body, and its function is highly regulated by phosphorylation. JNK1 was shown to phosphorylate the proline-rich domain of MAP2 in vivo in a previous study performed in the group. Here we verify three threonine residues (T1619, T1622 and T1625) as JNK1 targets, the phosphorylation of which increases the binding of MAP2 to microtubules. This binding stabilizes the microtubules and increases process formation in non-neuronal cells. Phosphorylation-site mutants were engineered in the lab. The non-phosphorylatable mutant of MAP2 (MAP2- T1619A, T1622A, T1625A) in these residues fails to bind microtubules, while the pseudo-phosphorylated form, MAP2- T1619D, T1622D, Thr1625D, efficiently binds and induces process formation even without the presence of active JNK1. Ectopic expression of the MAP2- T1619D, T1622D, Thr1625D in vivo in mouse brain led to a striking increase in the branching of cortical layer 2/3 (L2/3) pyramidal neurons, compared to MAP2-WT. The dendritic complexity defines the receptive field of a neuron and dictates the output to the postsynaptic cells. Previous studies in the group indicated altered dendrite architecture of the pyramidal neurons in the Jnk1-/- mouse motor cortex. Here, we used Lucifer Yellow loading and Sholl analysis of neurons in order to study the dendritic branching in more detail. We report a striking, opposing effect in the absence of Jnk1 in the cortical layers 2/3 and 5 of the primary motor cortex. The basal dendrites of pyramidal neurons close to the pial surface at L2/3 show a reduced complexity. In contrast, the L5 neurons, which receive massive input from the L2/3 neurons, show greatly increased branching. Another novel substrate identified for JNK1 was MARCKSL1, a protein that regulates actin dynamics. It is highly expressed in neurons, but also in various cancer tissues. Three phosphorylation target residues for JNK1 were identified, and it was demonstrated that their phosphorylation reduces actin turnover and retards migration of these cells. Actin is the main cytoskeletal component in dendritic spines, the site of most excitatory synapses in pyramidal neurons. The density and gross morphology of the Lucifer Yellow filled dendrites were characterized and we show reduced density and altered morphology of spines in the motor cortex and in the hippocampal area CA3. The dynamic dendritic spines are widely considered to function as the cellular correlate during learning. We used a Morris water maze to test spatial memory. Here, the wild-type mice outperformed the knock-out mice during the acquisition phase of the experiment indicating impaired special memory. The L5 pyramidal neurons of the motor cortex project to the spinal cord and regulate the movement of distinct muscle groups. Thus the altered dendrite morphology in the motor cortex was expected to have an effect on the input-output balance in the signaling from the cortex to the lower motor circuits. A battery of behavioral tests were conducted for the wild-type and Jnk1-/- mice, and the knock-outs performed poorly compared to wild-type mice in tests assessing balance and fine motor movements. This study expands our knowledge of JNK1 as an important regulator of the dendritic fields of neurons and their manifestations in behavior

Mindfulnessin hyödyntäminen ahdistuneisuuden hoidossa : opas ahdistuneisuuden hallintaan

Tässä opinnäytetyössä käsitellään tietoisuustaitoharjoitusten vaikutuksia ahdistuneisuuden hoidossa. Ahdistuneisuus on hyvin yleinen vaiva, johon liittyy niin psyykkisiä kuin fyysisiäkin oireita. Ahdistuksen psyykkisiä oireita ovat esimerkiksi huoli ja murehtiminen, pelokkuus ja levottomuus. Ahdistuksen fyysisiä oireita ovat muun muassa sydämen tykytykset, hikoilu, vapina ja vatsaoireet. Ahdistus on yleistä ja lievä ahdistuneisuus on jokaiselle tuttu, tilapäisenä täysin normaali tunne. Ahdistus voi vaihdella lievästä jännityksestä paniikkikohtauksiin. Jos ahdistus ei poistu, on pitkittynyttä ja se haittaa normaalia elämää on ahdistuneisuuden hoitoon monia erilaisia keinoja, niin lääkkeettömiä kuin lääkkeellisiäkin. Ahdistuneisuushäiriössä yleisinä hoitomuotoina ovat psykoterapia ja lääkehoito. Mindfulness on yksi lääkkeetön hoitomenetelmä, johon tässä opinnäytetyössä keskityttiin. Mindfulness-harjoitusten avulla helpotetaan näitä ahdistuksen oireita, rauhoitetaan kehoa ja mieltä. Opinnäytetyössä käsiteltiin mindfulnessia ahdistuksen hoitomuotona ja erilaisia mindfulness-harjoitteita kuten rentoutusharjoituksia ja hengitysharjoituksia. Opinnäytetyössä käytetty menetelmä on kehittämistyö. Opinnäytetyö sisältää kirjallisen tuotoksen sekä sähköisessä muodossa olevan oppaan. Tässä työssä kehitettiin opas mindfulness-pohjaisista tietoisuustaitoharjoituksista ahdistuksen hallintaan psykoterapiayritys TeraPiialle. Opas sisältää tietoa mindfulnessista ja ahdistuksesta sekä tietoisuustaitoharjoituksia. Kehittämistyö lisää terapia-asiakkaiden tietoa mindfulnessista ja ahdistuksesta sekä antaa keinoja ahdistuksen hallintaan. Opas toimii myös terapeuttien työn tukena. Jatkokehitysideana kehittämistyöllemme voisi olla oppaan harjoitteiden tekeminen äänite- tai videomuotoon. Toinen jatkokehitysidea voisi olla samankaltaisen oppaan tekeminen mindfulnessin hyödyntämisestä esimerkiksi masennuksen tai unettomuuden hallinnassa.This thesis discusses the benefits of mindfulness-exercises as a part of treating anxiety symptoms. Anxiety is a very common ailment with both mental and physical symptoms. Mental symptoms of anxiety include worry and anxiety, fear, and restlessness. Physical symptoms of anxiety include palpitations, sweating, tremors, and abdominal symptoms. Anxiety is common and mild, temporary anxiety is a familiar feeling for most of us. Anxiety can range from mild tension to panic attacks. If the anxiety does not go away, if it becomes prolonged and interferes with normal life, there are many different ways to treat anxiety, both non-medical and medical. Common treatments for anxiety disorder include psychotherapy and medication. Mindfulness is one drug-free method of treatment that this thesis focuses on. Mindfulness-exercises help relieve these anxiety symptoms, calm the body and mind. The thesis deals with mindfulness as a treatment for anxiety and various mindfulness exercises, such as relaxation and breathing exercises. This thesis was conducted as a development work. The thesis includes a written output and a guide in electric format. The purpose of this thesis was to produce a guide on mindfulness based conscious presence as a way of coping with anxiety for a psychotherapy company TeraPiia. The guide includes theory on anxiety and also contains mindfulness-exercises to prevent anxiety or reduce the anxious feeling in case it is already present. The goal of the development work is to increase therapy clients’ knowledge of mindfulness and provide concrete ways to manage anxiety. The guide can also be used by therapists as a part of their job. An idea for further development is to produce a recording or a video of the development work. Another idea is to produce a guide to utilizing mindfulness in the treatment of for example depression or insomnia

Deazaflavin inhibitors of TDP2 with cellular activity can affect etoposide influx and/or efflux

Tyrosyl DNA phosphodiesterase 2 (TDP2) facilitates the repair of topoisomerase II (TOP2)-linked DNA double-strand breaks and, as a consequence, is required for cellular resistance to TOP2 “poisons”. Recently, a deazaflavin series of compounds were identified as potent inhibitors of TDP2, in vitro. Here, however, we show that while some deazaflavins can induce cellular sensitivity to the TOP2 poison etoposide, they do so independently of TDP2 status. Consistent with this, both the cellular level of etoposide-induced TOP2 cleavage complexes and the intracellular concentration of etoposide was increased by incubation with deazaflavin, suggesting an impact of these compounds on etoposide uptake/efflux. In addition, deazaflavin failed to increase the level of TOP2 cleavage complexes or sensitivity induced by m-AMSA, which is a different class of TOP2 poison to which TDP2-defective cells are also sensitive. In conclusion, while deazaflavins are potent inhibitors of TDP2 in vitro, their limited cell permeability and likely interference with etoposide influx/efflux limits their utility in cells