USP8 prevents aberrant NF-κB and Nrf2 activation by counteracting ubiquitin signals from endosomes

K63-linked ubiquitin chains attached to plasma membrane proteins serve as tags for endocytosis and endosome-to-lysosome sorting. USP8 is an essential deubiquitinase for the maintenance of endosomal functions. Prolonged depletion of USP8 leads to cell death, but the major effects on cellular signaling pathways are poorly understood. Here, we show that USP8 depletion causes aberrant accumulation of K63-linked ubiquitin chains on endosomes and induces immune and stress responses. Upon USP8 depletion, two different decoders for K63-linked ubiquitin chains, TAB2/3 and p62, were recruited to endosomes and activated the TAK1-NF-κB and Keap1-Nrf2 pathways, respectively. Oxidative stress, an environmental stimulus that potentially suppresses USP8 activity, induced accumulation of K63-linked ubiquitin chains on endosomes, recruitment of TAB2, and expression of the inflammatory cytokine. The results demonstrate that USP8 is a gatekeeper of misdirected ubiquitin signals and inhibits immune and stress response pathways by removing K63-linked ubiquitin chains from endosomes.</p

A Novel Form of Memory for Auditory Fear Conditioning at a Low-Intensity Unconditioned Stimulus

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short-tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus

Consolidation of auditory fear memories formed by weak unconditioned stimuli requires NMDA receptor activation and de novo

BACKGROUND: Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. We previously showed that the selective ablation of striatal neurons in the adult brain impairs the long-term, but not short-term, memory for auditory fear conditioning with a lower-intensity footshock. This finding raises an intriguing possibility that long-term auditory fear memory may be consolidated in the striatum. RESULTS: There was a significant difference in the freezing responses between two groups of mice subjected to paired and unpaired conditioning, indicating that the auditory fear conditioning with a lower-intensity footshock is an associative learning. Post-conditioning infusion of NMDA receptor inhibitors into the striatum suppressed the consolidation of auditory fear memory when mice were conditioned with a low-intensity footshock. Furthermore, intra-striatum infusion of protein synthesis blocker anisomycin immediately or 1 h after the conditioning prevented the formation of auditory fear memory. On the other hand, the infusion of anisomycin 3 h after conditioning exerted little effect on the auditory fear conditioning, consistent with the presence of a critical time window of protein synthesis for memory consolidation. CONCLUSIONS: These results suggest that NMDA receptors and de novo protein synthesis in the striatum are crucial for the consolidation of auditory fear memory formed with a low-intensity unconditioned stimulus

Ablation of NMDA receptors enhances the excitability of hippocampal CA3 neurons.

Synchronized discharges in the hippocampal CA3 recurrent network are supposed to underlie network oscillations, memory formation and seizure generation. In the hippocampal CA3 network, NMDA receptors are abundant at the recurrent synapses but scarce at the mossy fiber synapses. We generated mutant mice in which NMDA receptors were abolished in hippocampal CA3 pyramidal neurons by postnatal day 14. The histological and cytological organizations of the hippocampal CA3 region were indistinguishable between control and mutant mice. We found that mutant mice lacking NMDA receptors selectively in CA3 pyramidal neurons became more susceptible to kainate-induced seizures. Consistently, mutant mice showed characteristic large EEG spikes associated with multiple unit activities (MUA), suggesting enhanced synchronous firing of CA3 neurons. The electrophysiological balance between fast excitatory and inhibitory synaptic transmission was comparable between control and mutant pyramidal neurons in the hippocampal CA3 region, while the NMDA receptor-slow AHP coupling was diminished in the mutant neurons. In the adult brain, inducible ablation of NMDA receptors in the hippocampal CA3 region by the viral expression vector for Cre recombinase also induced similar large EEG spikes. Furthermore, pharmacological blockade of CA3 NMDA receptors enhanced the susceptibility to kainate-induced seizures. These results raise an intriguing possibility that hippocampal CA3 NMDA receptors may suppress the excitability of the recurrent network as a whole in vivo by restricting synchronous firing of CA3 neurons

Ablation of medium-spiny projection neurons in the striatum of mutant mice.

<p>A, Immunoreactivity for calbindin in the dorsal striatum of control (upper) and mutant (lower) mice. B, Immunoreactivity for tyrosine hydroxylase and substance P in substantia nigra of control and mutant mice. C, Immunoreactivity for GAD and enkephalin in GP of control and mutant mice. Abbreviations: CP, caudate putamen; GP, globus pallidus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Scale bars, 1 mm.</p

NeuN-immunohistochemstry.

<p>A, Immunohistochemical analysis for neuronal marker NeuN in control (left) and mutant (right) mice 13 days after mock and RU-486 administration, respectively. Scale bar, 1 mm. B, Higher magnification of NeuN-immunohistochemistry in various brain regions. Scale bars, 0.1 mm. C, NeuN immunoreactive (NeuN⁺)-cell density in the CP after drug administration. <i>n</i> = 8–9 each. D, Densities of NeuN-positive cells in the NAc core (NAcC, open circles) and the NAc shell (NAcS, filled circles) after RU-486 treatment of <i>Gng7^+/mCrePR</i>; <i>+/Eno2-STOP-DTA</i> mice (<i>n</i> = 8–9 each). E, Densities of NeuN-positive cells in the lateral amygdala (LA) of control and mutant mice 22 days after mock and RU-486 treatment, respectively (<i>n</i> = 15 each, <i>F</i>_1,28 = 0.23, <i>P</i> = 0.64, one-way ANOVA). Abbreviations: Au, auditory cortex; CA1, hippocampal CA1 region; CP, caudate putamen; Cx, cortex; GP, globus pallidus; MGN, medial geniculate nucleus of thalamus; NAc, nucleus accumbens; OT, olfactory tubercle; PAG, periaqueductal gray; Sp, septum.</p

Impairment of long-term fear memory.

<p>A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses to tone were measured 1 or 3 h and 24 h after conditioning. B, Freezing responses of control (open circles, <i>n</i> = 8) and mutant (filled circles, <i>n</i> = 5) mice 1 h (left) and 24 h (right) after conditioning. C, Freezing responses of control (open circles, <i>n</i> = 6) and mutant (filled circles, <i>n</i> = 4) 3 h (left) and 24 h (right) after conditioning. D, Experimental design. Mice were subjected to auditory fear conditioning with a footshock at 0.3 mA or 0.5 mA. One day after conditioning, the conditioned mice were injected with RU-486 or vehicle. Their freezing responses were measured 14 days after drug treatment. E, Mice were subjected to auditory fear conditioning with a weak footshock at 0.3 mA. Freezing responses of mock-injected (open circles, <i>n</i> = 7) and RU-486-injected (filled circles, <i>n</i> = 8) mice on the conditioning (left) and test (right) days. F, Mice were subjected to auditory fear conditioning with the standard footshock at 0.5 mA. Freezing responses of mock-injected (open circles, <i>n</i> = 6) and RU-486-injected (filled circles, <i>n</i> = 7) mice on the conditioning (left) and test (right) days.</p

Impaired freezing responses of mutant mice after auditory fear conditioning with a low-intensity footshock.

<p>A, Experimental design. Mice were injected with RU-486 or vehicle. Fourteen days after treatment, the animals were subjected to auditory fear conditioning. B, Freezing responses of control (open circles, <i>n</i> = 9) and mutant (filled circles, <i>n</i> = 8) mice on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with the standard intensity of footshock (0.5 mA, an arrow). Solid lines represent tone. C, Freezing responses of control (open circles, <i>n</i> = 8) and mutant (filled circles, <i>n</i> = 11) mice and RU-486-treated Gγ7-mCrePR mice (RU-486 control) (shaded triangles, <i>n</i> = 7) on the conditioning (left) and test (right) days. Auditory fear conditioning was carried out with a low intensity of footshock (0.3 mA, an arrow). Solid lines represent tone. D, Current thresholds of control (open bar), RU-486-control (shaded bar) and mutant (filled bar) mice for flinch and jump reactions (<i>n</i> = 6 each).</p

Inducible ablation of striatal neurons.

<p>A, Schema for striatal neuron ablation induced by RU-486 administration. B, TUNEL staining (green) counterstained with DAPI (blue) in brain sections of control (left) and mutant (right) mice 10 days after mock and RU-486 administration, respectively. Scale bars, 1 mm. Abbreviations: Ce, cerebellum; Cx, cortex; Hi, hippocampus; Po, pons; St, striatum; Th, thalamus.</p