Identification of a novel type of focal adhesion remodelling via FAK/FRNK replacement, and its contribution to cancer progression.

Numerous studies have investigated the various cellular responses against genotoxic stress, including those mediated by focal adhesions. We here identified a novel type of focal adhesion remodelling that occurs under genotoxic stress conditions, which involves the replacement of active focal adhesion kinase (FAK) with FAK-related non-kinase (FRNK). FRNK stabilized focal adhesions, leading to strong cell-matrix adhesion, and FRNK-depleted cells were easily detached from extracellular matrix upon genotoxic stress. This remodelling occurred in a wide variety of cells. In vivo, the stomachs of Frnk-knockout mice were severely damaged by genotoxic stress, highlighting the protective role of FRNK against genotoxic stress. FRNK was also found to play a vital role in cancer progression, because FRNK depletion significantly inhibited cancer dissemination and progression in a mouse cancer model. Furthermore, in human cancers, FRNK was predominantly expressed in metastatic tissues and not in primary tissues. We hence conclude that this novel type of focal adhesion remodelling reinforces cell adhesion and acts against genotoxic stress, which results in the protection of normal tissues, but in turn facilitates cancer progression.

Cell penetration efficiency analysis of different atomic force microscopy nanoneedles into living cells.

Over the last decade, nanoneedle-based systems have demonstrated to be extremely useful in cell biology. They can be used as nanotools for drug delivery, biosensing or biomolecular recognition inside cells; or they can be employed to select and sort in parallel a large number of living cells. When using these nanoprobes, the most important requirement is to minimize the cell damage, reducing the forces and indentation lengths needed to penetrate the cell membrane. This is normally achieved by reducing the diameter of the nanoneedles. However, several studies have shown that nanoneedles with a flat tip display lower penetration forces and indentation lengths. In this work, we have tested different nanoneedle shapes and diameters to reduce the force and the indentation length needed to penetrate the cell membrane, demonstrating that ultra-thin and sharp nanoprobes can further reduce them, consequently minimizing the cell damage.

Developmental trajectory of rule management system in children.

The ability to apply rules for environmental adaptation is crucial for human life. This capacity may require high-order cognitive control, such as when managing personal behavior by selecting among context-dependent internal rules. This process is poorly understood in children, especially in terms of the age at which multiple-rules processing becomes possible. We created a child-appropriate "rule management paradigm" to elucidate developmental changes in rule processing, and used it to investigate the trajectory of the rule management system in 322 children aged 4 to 6 years, with comparison to 57 adults. We found age-specific capacities in multiple-rules processing, with the majority of 4-year-olds failing at concurrent management of multiple-rules processing, a capacity that became well developed by age 6. Task performance in multiple-rules processing improved steeply with age and approached the adult level by late age 6. By contrast, single-rule processing on single-feature stimuli approached the adult level by age 5. Our main findings suggest that the critical period for the development of the multiple-rules processing system occurs before age 7, and is associated with the developmental period of the rule management system and other cognitive resources.

Shifting the balance of brain tryptophan metabolism elicited by isolation housing and systemic administration of lipopolysaccharide in mice.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase, is a key tryptophan (TRP) metabolic pathway. It shares TRP mainly with the serotonin (5-HT) pathway. Activation of the KYN pathway by stimulation of the inflammatory response system (IRS) is known to induce depressive symptoms. Thus, we considered that shifting the balance between the KYN and 5-HT systems in the brain to the KYN pathway closely relate to the etiology of depression. In the present study, we investigated the influence of environmental risk factors for depression, such as social isolation and activation of the IRS, on brain TRP metabolism. Male ICR mice (postnatal day 21) were divided into two housing conditions, isolation and group housing, reared for 4 weeks, and then given an intraperitoneal injection of lipopolysaccharide (LPS). We measured the TRP, KYN, and 5-HT levels in the prefrontal cortex, hippocampus, amygdala, and dorsal raphe nuclei. Isolation housing decreased the KYN/5-HT ratio in the amygdala and dorsal raphe nuclei. LPS increased the KYN/5-HT ratio in all regions except the dorsal raphe nuclei. Thus, isolation housing shifted the balance between the KYN and 5-HT pathways to the 5-HT pathway, whereas systemic administration of LPS shifted it to the KYN pathway.

Changes in brain tryptophan metabolism elicited by ageing, social environment, and psychological stress in mice.

The kynurenine (KYN) pathway, which is initiated by indoleamine 2,3-dioxygenase (IDO), is a tryptophan (TRP) metabolic pathway. It shares TRP with the serotonin (5-hydroxytryptamine, 5-HT) pathway. In major depression, activation of the KYN pathway may deplete 5-HT. In the present study we investigated the influence of various risk factors for depression, such as ageing, social isolation and psychological stress, on TRP metabolism. Male ICR mice (postnatal day, PND, 21) were divided into two housing conditions, isolation and group housing, reared for 4 weeks (young adult) or 5 months (adult) and exposed to novelty stress. We measured TRP, KYN and 5-HT contents in the prefrontal cortex, hippocampus, amygdala and dorsal raphe nuclei to investigate the balance between the KYN and 5-HT pathways. Ageing decreased TRP and KYN and increased 5-HT. Thus, ageing shifted the balance to the latter. In the younger group, social isolation decreased TRP and KYN and increased the 5-HT/TRP ratio, whereas novelty stress increased TRP and KYN and decreased the 5-HT/TRP ratio. Thus, social isolation shifted the balance to the latter, whereas novelty stress shifted it to the former. In the older group, these effects were restricted to specific brain regions. Ageing and social isolation counteracted novelty stress effects on TRP metabolism.

Age-dependent changes in dopaminergic projections from the substantia nigra pars compacta to the neostriatum.

Age-dependent changes in dopaminergic (DA) innervation of the neostriatum (Str) were studied in male F344/N rats. Projections from the substantia nigra pars compacta (SNc) to the neostriatum were quantified using electrophysiological methods at age points from 6 to 24 months. The percentage of DA neurons activated antidromically by electrical stimulation (P-index) of Str increased between 18 and 24 months. Additionally, the percentage of DA neurons showing multiple antidromic latencies from striatal stimulation (M-index), which suggests axonal branching of individual DA neurons, increased significantly between 6 and 12 months and 6 and 24 months. These results suggest that DA neurons exhibit increased axonal branching in the aged brain.

Involvement of neurotrophic factors in aging of noradrenergic innervations in hippocampus and frontal cortex.

In the present study, we investigated the age-dependent changes in the axon terminals of the locus coeruleus (LC) neurons in the frontal cortex and hippocampus, in which a high degree of axonal branching in the middle-aged brain was suggested to occur in our previous electrophysiological study. We used 6-, 13- and 25-month-old male F344/N rats, and performed Western blot analysis of the norepinephrine transporter (NET), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). NET expression level increased in the 13-month-old hippocampus, but was not altered by aging in the frontal cortex. BDNF expression level increased in the hippocampus, but did not change with age in the frontal cortex. On the other hand, GDNF expression level was increased with age in the frontal cortex, but was not in the hippocampus. These results suggest that the LC noradrenergic innervations may be locally regulated by different neurotrophic factors that exert their trophic actions at different target sites.

Effects of BDNF infusion on the axon terminals of locus coeruleus neurons of aging rats.

Using in vivo electrophysiological techniques and continuous local infusion methods, we examined the effects of brain-derived neurotrophic factor (BDNF) and its specific antibody (anti-BDNF) on the noradrenergic axon terminals of the locus coeruleus (LC) neurons in the frontal cortex of aging rats. Recently, we observed that LC neurons with multiple-threshold antidromic responses (multi-threshold LC neurons) increased critically between 15 and 17 months of age. To examine whether the BDNF is involved in this change occurred in the aging brain, we continuously infused BDNF into the frontal cortex for 14 days. Exogenous BDNF produced a marked increase in the multi-threshold LC neurons in the 13-month-old brain, accompanied with a decrease in threshold current. However, no morphological change in the noradrenergic axons was observed in the BDNF-infused cortex. In contrast, infusion of anti-BDNF led to a dose-dependent reduction of the multi-threshold LC neurons in the 19-month-old brain, accompanied with an increase in threshold current. These findings suggest that BDNF may contribute to functional changes in the presynaptic axon terminals of LC neurons in the aging brain.

Age-dependent interactive changes in serotonergic and noradrenergic cortical axon terminals in F344 rats.

In the frontal cortex of aging rats, we found an increase in sprouting of the noradrenergic (NA) axons originated from the locus coeruleus (LC). The serotonergic (5-HT) axons originating from the dorsal raphe (DR) share the same cortical area and their age-dependent changes and interactions with NA axons were still unclear. To compare quantitatively the extent of axonal sprouting of DR and LC neurons in the frontal cortex, we extracellularly recorded from both DR and LC neurons in the same animals and antidromically stimulated 32 cortical sites (a pair of stimulating electrodes was moved at 100-mum intervals from 500 to 2000 microm in depth). In addition, to examine the effects of degeneration of 5-HT axons on NA axons, and vice versa, we used specific neurotoxins for 5-HT (PCA) or NA (DSP-4) axons. We also used noradrenaline uptake inhibitor (maprotiline) to verify the effects of NA on degeneration of 5-HT axons. Results suggested that 5-HT axons sprouted between 15 and 17 months of age and noradrenaline accelerated the age-dependent change of 5-HT axons.

[Plasticity of central monoaminergic systems during aging].

The locus coeruleus (LC), located within the caudal pontine central gray, is composed of noradrenaline-containing neurons. The axons of these neurons form extensive collateral branches that project widely to many brain sites. The function of the LC is still unclear at present, however, LC neurons are known to exhibit marked axonal regeneration and sprouting in response to brain damage. We investigated the age-related changes in noradrenergic innervations of the frontal cortex, using in vivo electrophysiological techniques and immunohistochemistry. While noradrenergic innervations gradually decreased with age in the frontal cortex, a high degree of sprouting occurred in the LC axon terminals in middle age. Neither the electrophysiological properties of LC neurons nor NA levels in the frontal cortex changed with age. These findings suggested that the LC neurons preserve a strong capacity to remodel their axon terminals even in the aging brain. Exogenous brain-derived neurotrophic factor (BDNF) infusion caused a marked increase in the density of noradrenergic axon in the aged brain, but no trophic action of BDNF was observed in the young or middle-aged brain. The result suggests that BDNF is necessary for the maintenance of noradrenergic innervations in the aged brain.

BDNF is necessary for maintenance of noradrenergic innervations in the aged rat brain.

In the axon terminals of the locus coeruleus (LC) neurons, a high level of axonal branching was occurred in the middle-aged brain, and the increased branching was maintained in the aged brain. In the present study, we hypothesized that neurotrophic support is necessary for the morphological age-related changes seen in the noradrenergic innervations from the LC to frontal cortex. Through immunohistochemical and quantitative image analyses, we examined the age-dependent effects of brain-derived neurotrophic factor (BDNF) on the noradrenergic axon terminals in the frontal cortex of F344 rats. We continuously infused BDNF into the frontal cortex of young (6-months-old), middle-aged (13-months-old), or aged (25-months-old) rats. Exogenous BDNF infusion caused a marked increase in the density of noradrenergic axons in the aged brain, but no trophic action of BDNF was observed in the young and middle-aged brain. Neutralization of endogenous BDNF with a specific function-blocking antibody to BDNF led to a reduction in noradrenergic axons in the frontal cortex of 19-month-old rats. The present results suggest that BDNF is not involved in the augmentation of noradrenergic innervations in the aging brain, but it is necessary for the maintenance of noradrenergic innervations in the aged brain.

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus neurons.

Age-related changes in the release and uptake activity of presynaptic axon terminals of rat locus coeruleus (LC) noradrenergic neurons were studied in the frontal cortex using an extracellular single unit recording technique in vivo. Clonidine, a selective alpha(2) adrenergic agonist, and nisoxetine, a selective noradrenaline uptake inhibitor, were infused locally into the frontal cortex to examine the effects of these drugs on release and uptake activities of the axon terminals of LC neurons. Although the infusion of clonidine produced a marked suppression of release, the effect did not change with age. Infusion of nisoxetine caused an inhibition of uptake, but the effect was attenuated in aged rats. These results suggest that the release activity mediated by the presynaptic autoreceptor did not change with age, but the uptake activity mediated by the NA transporter declined with age in the axon terminals of LC neurons.

Specific uptake of Abeta1-40 in rat brain occurs in astrocyte, but not in microglia.

In the brain of a patient with Alzheimer's disease, beta amyloid peptide (Abeta) is thought to be taken up by glial cells such as astrocyte and microglia to be degraded. However, it is unclear whether the Abeta is absorbed by astrocyte or microglia. The purpose of our study is to determine which type of glial cell, astrocyte or microglia, can take up Abeta. Beta amyloid 1-40 (Abeta1-40) was directly infused into the frontal cortex or hippocampus for 14 days. Dual-labeling immunohistochemistry for Abeta1-40 with an astrocytic (GFAP) or microglial (CD11b) marker was performed to examine co-localization of Abeta1-40 and glial markers. In the Abeta1-40 infused site, immunoreactivity of Abeta1-40 was observed only in astrocytes, not in microglia. In addition, Abeta40-1, a reverse peptide of Abeta1-40, was not taken up by astrocytes. These results suggested that the astrocyte-specific uptake of Abeta occurred in the rat brain.