Essential role of ROS - 8-Nitro-cGMP signaling in long-term memory of motor learning and cerebellar synaptic plasticity.

Although reactive oxygen species (ROS) are known to have harmful effects in organisms, recent studies have demonstrated expression of ROS synthases at various parts of the organisms and the controlled ROS generation, suggesting possible involvement of ROS signaling in physiological events of individuals. However, physiological roles of ROS in the CNS, including functional roles in higher brain functions or neuronal activity-dependent ROS production, remain to be elucidated. Here, we demonstrated involvement of ROS - 8-NO2-cGMP signaling in motor learning and synaptic plasticity in the cerebellum. In the presence of inhibitors of ROS signal or ROS synthases, cerebellar motor learning was impaired, and the stimulus inducing long-term depression (LTD), cellular basis for the motor learning, failed to induce LTD but induced long-term potentiation (LTP)-like change at cerebellar synapses. Furthermore, ROS was produced by LTD-inducing stimulus in enzyme-dependent manner, and excess administration of the antioxidant vitamin E impaired cerebellar motor learning, suggesting beneficial roles of endogenous ROS in the learning. As a downstream signal, involvement of 8-NO2-cGMP in motor learning and cerebellar LTD were also revealed. These findings indicate that ROS - 8-NO2-cGMP signal is activated by neuronal activity and is essential for cerebellum-dependent motor learning and synaptic plasticity, demonstrating involvement of the signal in physiological function of brain systems.

Reversal of spatial memory impairment by phosphodiesterase 3 inhibitor cilostazol is associated with reduced neuroinflammation and increased cerebral glucose uptake in aged male mice.

The nucleotide second messenger 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP) mediate fundamental functions of the brain, including learning and memory. Phosphodiesterase 3 (PDE3) can hydrolyze both cAMP and cGMP and appears to be involved in the regulation of their contents in cells. We previously demonstrated that long-term administration of cilostazol, a PDE3 inhibitor, maintained good memory performance in aging mice. Here, we report on studies aimed at determining whether cilostazol also reverses already-impaired memory in aged male mice. One month of oral 1.5% cilostazol administration in 22-month-old mice reversed age-related declines in hippocampus-dependent memory tasks, including the object recognition and the Morris water maze. Furthermore, cilostazol reduced neuroinflammation, as evidenced by immunohistochemical staining, and increased glucose uptake in the brain, as evidence by positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). These results suggest that already-expressed memory impairment in aged male mice that depend on cyclic nucleotide signaling can be reversed by inhibition of PDE3. The reversal of age-related memory impairments may occur in the central nervous system, either through cilostazol-enhanced recall or strengthening of weak memories that otherwise may be resistant to recall.

The AP-1 transcription factor JunB is required for Th17 cell differentiation.

Interleukin (IL)-17-producing T helper (Th17) cells are crucial for host defense against extracellular microbes and pathogenesis of autoimmune diseases. Here we show that the AP-1 transcription factor JunB is required for Th17 cell development. Junb-deficient CD4+ T cells are able to develop in vitro into various helper T subsets except Th17. The RNA-seq transcriptome analysis reveals that JunB is crucial for the Th17-specific gene expression program. Junb-deficient mice are completely resistant to experimental autoimmune encephalomyelitis, a Th17-mediated inflammatory disease, and naive T helper cells from such mice fail to differentiate into Th17 cells. JunB appears to activate Th17 signature genes by forming a heterodimer with BATF, another AP-1 factor essential for Th17 differentiation. The mechanism whereby JunB controls Th17 cell development likely involves activation of the genes for the Th17 lineage-specifying orphan receptors RORγt and RORα and reduced expression of Foxp3, a transcription factor known to antagonize RORγt function.

Deficits in memory-guided limb movements impair obstacle avoidance locomotion in Alzheimer's disease mouse model.

Memory function deficits induced by Alzheimer's disease (AD) are believed to be one of the causes of an increased risk of tripping in patients. Working memory contributes to accurate stepping over obstacles during locomotion, and AD-induced deficits of this memory function may lead to an increased risk of contact with obstacles. We used the triple transgenic (3xTg) mice to examine the effects of memory deficits in terms of tripping and contact with obstacles. We found that the frequency of contact of the hindlimbs during an obstacle avoidance task increased significantly in 10-13 month-old 3xTg (Old-3xTg) mice compared with control mice. However, no changes in limb kinematics during unobstructed locomotion or successful obstacle avoidance locomotion were observed in the Old-3xTg mice. Furthermore, we found that memory-based movements in stepping over an obstacle were impaired in these mice. Our findings suggest that working memory deficits as a result of AD are associated with an increased risk of tripping during locomotion.

The kick-in system: a novel rapid knock-in strategy.

Knock-in mouse models have contributed tremendously to our understanding of human disorders. However, generation of knock-in animals requires a significant investment of time and effort. We addressed this problem by developing a novel knock-in system that circumvents several traditional challenges by establishing stem cells with acceptor elements enveloping a particular genomic target. Once established, these acceptor embryonic stem (ES) cells are efficient at directionally incorporating mutated target DNA using modified Cre/lox technology. This is advantageous, because knock-ins are not restricted to one a priori selected variation. Rather, it is possible to generate several mutant animal lines harboring desired alterations in the targeted area. Acceptor ES cell generation is the rate-limiting step, lasting approximately 2 months. Subsequent manipulations toward animal production require an additional 8 weeks, but this delimits the full period from conception of the genetic alteration to its animal incorporation. We call this system a "kick-in" to emphasize its unique characteristics of speed and convenience. To demonstrate the functionality of the kick-in methodology, we generated two mouse lines with separate mutant versions of the voltage-dependent potassium channel Kv7.2 (Kcnq2): p.Tyr284Cys (Y284C) and p.Ala306Thr (A306T); both variations have been associated with benign familial neonatal epilepsy. Adult mice homozygous for Y284C, heretofore unexamined in animals, presented with spontaneous seizures, whereas A306T homozygotes died early. Heterozygous mice of both lines showed increased sensitivity to pentylenetetrazole, possibly due to a reduction in M-current in CA1 hippocampal pyramidal neurons. Our observations for the A306T animals match those obtained with traditional knock-in technology, demonstrating that the kick-in system can readily generate mice bearing various mutations, making it a suitable feeder technology toward streamlined phenotyping.