JIP1-Mediated JNK Activation Negatively Regulates Synaptic Plasticity and Spatial Memory.

The c-Jun N-terminal kinase (JNK) signal transduction pathway is implicated in learning and memory. Here, we examined the role of JNK activation mediated by the JNK-interacting protein 1 (JIP1) scaffold protein. We compared male wild-type mice with a mouse model harboring a point mutation in the Jip1 gene that selectively blocks JIP1-mediated JNK activation. These male mutant mice exhibited increased NMDAR currents, increased NMDAR-mediated gene expression, and a lower threshold for induction of hippocampal long-term potentiation. The JIP1 mutant mice also displayed improved hippocampus-dependent spatial memory and enhanced associative fear conditioning. These results were confirmed using a second JIP1 mutant mouse model that suppresses JNK activity. Together, these observations establish that JIP1-mediated JNK activation contributes to the regulation of hippocampus-dependent, NMDAR-mediated synaptic plasticity and learning.SIGNIFICANCE STATEMENT The results of this study demonstrate that c-Jun N-terminal kinase (JNK) activation induced by the JNK-interacting protein 1 (JIP1) scaffold protein negatively regulates the threshold for induction of long-term synaptic plasticity through the NMDA-type glutamate receptor. This change in plasticity threshold influences learning. Indeed, mice with defects in JIP1-mediated JNK activation display enhanced memory in hippocampus-dependent tasks, such as contextual fear conditioning and Morris water maze, indicating that JIP1-JNK constrains spatial memory. This study identifies JIP1-mediated JNK activation as a novel molecular pathway that negatively regulates NMDAR-dependent synaptic plasticity and memory.

Protection against ß-amyloid neurotoxicity by a non-toxic endogenous N-terminal ß-amyloid fragment and its active hexapeptide core sequence.

High levels (µM) of beta amyloid (Aß) oligomers are known to trigger neurotoxic effects, leading to synaptic impairment, behavioral deficits, and apoptotic cell death. The hydrophobic C-terminal domain of Aß, together with sequences critical for oligomer formation, is essential for this neurotoxicity. However, Aß at low levels (pM-nM) has been shown to function as a positive neuromodulator and this activity resides in the hydrophilic N-terminal domain of Aß. An N-terminal Aß fragment (1-15/16), found in cerebrospinal fluid, was also shown to be a highly active neuromodulator and to reverse Aß-induced impairments of long-term potentiation. Here, we show the impact of this N-terminal Aß fragment and a shorter hexapeptide core sequence in the Aß fragment (Aßcore: 10-15) to protect or reverse Aß-induced neuronal toxicity, fear memory deficits and apoptotic death. The neuroprotective effects of the N-terminal Aß fragment and Aßcore on Aß-induced changes in mitochondrial function, oxidative stress, and apoptotic neuronal death were demonstrated via mitochondrial membrane potential, live reactive oxygen species, DNA fragmentation and cell survival assays using a model neuroblastoma cell line (differentiated NG108-15) and mouse hippocampal neuron cultures. The protective action of the N-terminal Aß fragment and Aßcore against spatial memory processing deficits in amyloid precursor protein/PSEN1 (5XFAD) mice was demonstrated in contextual fear conditioning. Stabilized derivatives of the N-terminal Aßcore were also shown to be fully protective against Aß-triggered oxidative stress. Together, these findings indicate an endogenous neuroprotective role for the N-terminal Aß fragment, while active stabilized N-terminal Aßcore derivatives offer the potential for therapeutic application.

Regulation of presynaptic Ca2+, synaptic plasticity and contextual fear conditioning by a N-terminal ß-amyloid fragment.

Soluble ß-amyloid has been shown to regulate presynaptic Ca(2+) and synaptic plasticity. In particular, picomolar ß-amyloid was found to have an agonist-like action on presynaptic nicotinic receptors and to augment long-term potentiation (LTP) in a manner dependent upon nicotinic receptors. Here, we report that a functional N-terminal domain exists within ß-amyloid for its agonist-like activity. This sequence corresponds to a N-terminal fragment generated by the combined action of α- and ß-secretases, and resident carboxypeptidase. The N-terminal ß-amyloid fragment is present in the brains and CSF of healthy adults as well as in Alzheimer's patients. Unlike full-length ß-amyloid, the N-terminal ß-amyloid fragment is monomeric and nontoxic. In Ca(2+) imaging studies using a model reconstituted rodent neuroblastoma cell line and isolated mouse nerve terminals, the N-terminal ß-amyloid fragment proved to be highly potent and more effective than full-length ß-amyloid in its agonist-like action on nicotinic receptors. In addition, the N-terminal ß-amyloid fragment augmented theta burst-induced post-tetanic potentiation and LTP in mouse hippocampal slices. The N-terminal fragment also rescued LTP inhibited by elevated levels of full-length ß-amyloid. Contextual fear conditioning was also strongly augmented following bilateral injection of N-terminal ß-amyloid fragment into the dorsal hippocampi of intact mice. The fragment-induced augmentation of fear conditioning was attenuated by coadministration of nicotinic antagonist. The activity of the N-terminal ß-amyloid fragment appears to reside largely in a sequence surrounding a putative metal binding site, YEVHHQ. These findings suggest that the N-terminal ß-amyloid fragment may serve as a potent and effective endogenous neuromodulator.

Hippocampal c-Jun-N-terminal kinases serve as negative regulators of associative learning.

In the adult mouse, signaling through c-Jun N-terminal kinases (JNKs) links exposure to acute stress to various physiological responses. Inflammatory cytokines, brain injury and ischemic insult, or exposure to psychological acute stressors induce activation of hippocampal JNKs. Here we report that exposure to acute stress caused activation of JNKs in the hippocampal CA1 and CA3 subfields, and impaired contextual fear conditioning. Conversely, intrahippocampal injection of JNKs inhibitors sp600125 (30 µm) or D-JNKI1 (8 µm) reduced activity of hippocampal JNKs and rescued stress-induced deficits in contextual fear. In addition, intrahippocampal administration of anisomycin (100 µg/µl), a potent JNKs activator, mimicked memory-impairing effects of stress on contextual fear. This anisomycin-induced amnesia was abolished after cotreatment with JNKs selective inhibitor sp600125 without affecting anisomycin's ability to effectively inhibit protein synthesis as measured by c-Fos immunoreactivity. We also demonstrated milder and transient activation of the JNKs pathway in the CA1 subfield of the hippocampus during contextual fear conditioning and an enhancement of contextual fear after pharmacological inhibition of JNKs under baseline conditions. Finally, using combined biochemical and transgenic approaches with mutant mice lacking different members of the JNK family (Jnk1, Jnk2, and Jnk3), we provided evidence that JNK2 and JNK3 are critically involved in stress-induced deficit of contextual fear, while JNK1 mainly regulates baseline learning in this behavioral task. Together, these results support the possibility that hippocampal JNKs serve as a critical molecular regulator in the formation of contextual fear.

c-Jun N-terminal kinases in memory and synaptic plasticity.

The c-Jun N-terminal kinases (JNK) belong to the subfamily of mitogen-activated protein kinases (MAPK). JNK is an important signaling enzyme that is involved in many facets of cellular regulation including gene expression, cell proliferation and programmed cell death. Activation of JNK isoforms (JNK1, 2, and 3) is regarded as a molecular switch in stress signal transduction. The activation of JNK pathways is also critical for pathological death associated with neurodegenerative diseases. Considering that a variety of stressors activate JNK, it is surprising that the role of hippocampal JNK in memory and synaptic plasticity has not yet been systematically investigated. Here we summarize the emerging evidence for the functions of hippocampal JNK in memory and synaptic plasticity, including our recent demon-stration that JNK isoforms play critical roles in regulation of contextual fear conditioning under stressful and baseline conditions. We postulate that sustained activation of the hippocampal JNK2 and JNK3 pathways is involved in the initial stress response that ultimately leads to deficits in memory and long-term potentiation, whereas transient JNK1 activation regulates baseline contextual fear conditioning. Results obtained within the framework of our recent findings will be used for future work, which will differentiate mechanisms underlying beneficial short-term JNK action from prolonged JNK activation that may lead to memory deficits and neurodegeneration.

Small-conductance Ca2+-activated potassium type 2 channels regulate the formation of contextual fear memory.

Small-conductance, Ca2+ activated K+ channels (SK channels) are expressed at high levels in brain regions responsible for learning and memory. In the current study we characterized the contribution of SK2 channels to synaptic plasticity and to different phases of hippocampal memory formation. Selective SK2 antisense-treatment facilitated basal synaptic transmission and theta-burst induced LTP in hippocampal brain slices. Using the selective SK2 antagonist Lei-Dab7 or SK2 antisense probes, we found that hippocampal SK2 channels are critical during two different time windows: 1) blockade of SK2 channels before the training impaired fear memory, whereas, 2) blockade of SK2 channels immediately after the training enhanced contextual fear memory. We provided the evidence that the post-training cleavage of the SK2 channels was responsible for the observed bidirectional effect of SK2 channel blockade on memory consolidation. Thus, Lei-Dab7-injection before training impaired the C-terminal cleavage of SK2 channels, while Lei-Dab7 given immediately after training facilitated the C-terminal cleavage. Application of the synthetic peptide comprising a leucine-zipper domain of the C-terminal fragment to Jurkat cells impaired SK2 channel-mediated currents, indicating that the endogenously cleaved fragment might exert its effects on memory formation by blocking SK2 channel-mediated currents. Our present findings suggest that SK2 channel proteins contribute to synaptic plasticity and memory not only as ion channels but also by additionally generating a SK2 C-terminal fragment, involved in both processes. The modulation of fear memory by down-regulating SK2 C-terminal cleavage might have applicability in the treatment of anxiety disorders in which fear conditioning is enhanced.

Cholecystokinin-B receptor gene expression in cerebellum, pre-frontal cortex and cingulate gyrus and its association with suicide.

Suicide is a complex behaviour. Genetic and environmental factors are implicated in suicide. Both factors require genes to exert their effects. One gene hypothesized to be involved in the pathophysiology of suicide is cholecystokinin. Alterations in cholecystokinin receptor binding have been reported to be significant in young suicide victims as compared to matched controls in the frontal and cingulate cortex. In this study we report the Cholecystokinin-B gene expression using RT-PCR, between suicide completers [(N = 10); mean age 37.2+/-12 years] and control subjects [(N = 10); mean age 37.6+/-11.9 years]. Cholecystokinin-B gene expression was significantly higher in the cerebellum (P = 0.006), cingulate gyrus (P = 0.024) and pre-frontal cortex (P = 0.017) of suicide completers when compared to their age and sex-matched controls.

Polymorphisms at the DRD2 locus in early-onset alcohol dependence in the Indian population.

The susceptibility to alcohol dependence is probably of polygenic origin. Association studies have attempted to identify possible candidate genes that may contribute to the risk to developing dependence. Severe forms of the alcoholism phenotype have been associated with an increased frequency of the Taq A1 allele at the DRD2 locus. Ethnic stratification and non-comparable phenotype may have contributed to the contradictory results in previous studies. We identified probands, using the Schedules of Assessment of Neuropsychiatry (SCAN) schedule, who had onset of alcohol dependence (ICD-10) before 25 years of age. Family members were interviewed using the Family Interview for Genetic Studies (FIGS) schedule to identify patients who had two first-degree relatives with alcohol dependence. Fifty subjects who fulfilled the criteria were selected for the study. These were compared to a normal population from a similar background. The allele frequencies did not differ between the two groups. The Taq1a polymorphism does not seem to be associated with alcoholism in this group of severely affected, young age of onset probands in the southern Indian population.

Suppression of the MEK/ERK signaling pathway reverses depression-like behaviors of CRF2-deficient mice.

The neuropeptide corticotropin-releasing factor (CRF) plays a critical role in the proper functioning of the stress response system through its actions on its receptors, CRF receptor 1 (CRF1) and CRF receptor 2 (CRF2), located at multiple anatomical sites. Clinical data indicate that stress response dysfunctions, such as excessive CRF activity and hyperstimulation of CRF1, are present in a range of stress-related disorders, including depression and anxiety disorders. Our previous work along with that of other laboratories has demonstrated that mice deficient in CRF2 (CRF2-/-) display increased anxiety and depression-like behaviors. In this study, we found CRF2-/- mice display increased hippocampal levels of activated (phosphorylated) mitogen-activated protein kinase (MAP kinase)/ERK kinase (MEK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ribosomal protein S6 kinases 1 (RSK1). These changes can be explained by overactive hippocampal CRF1, in view of the finding that the application of the nonselective CRF receptor antagonist [Glu(11,16)] astressin ([Glu(11,16)]Ast) into the dorsal hippocampus of mutant mice returned the levels of the phosphorylated proteins to baseline. Moreover, inhibition of the hippocampal MEK/ERK pathway with the specific MEK inhibitor U0126, decreased depression-like behaviors in the forced swim test and tail suspension test of CRF2-/- mice. Similarly, treatment with [Glu(11,16)]Ast reversed depression phenotype of CRF2-/- mice without affecting the phenotype of wild-type littermates. Our results support an involvement of CRF receptors in the development of depression, such that elevated hippocampal CRF1 activity, in the absence of CRF2, produces a depression-dominated phenotype through the activation of the MEK/ERK pathway.

Differential activation of CRF receptor subtypes removes stress-induced memory deficit and anxiety.

The objective of this study was to investigate the role of corticotropin-releasing factor receptors 1 (CRF(1)) and 2 (CRF(2)) in anxiety-like behavior and learning of C57BL/6J mice after exposure to a stressful stimulus. When C57BL/6J mice were exposed to immobilization (1 h) serving as stressful stimulus, context- and tone-dependent fear conditioning were impaired if the training followed immediately after immobilization. The stress-induced impairment of context-dependent fear conditioning was prevented by specific blockade of CRF(2) of the lateral septum (LS) with anti-sauvagine-30. Immobilization did not only affect conditioned fear, but also enhanced, through CRF(2) of the LS, anxiety-like behavior determined with the elevated plus maze. Recovery from stress-induced anxiety and impairment of context-dependent fear conditioning was observed after 1 h delay of training and required hippocampal CRF(1), as indicated by the finding that this recovery was prevented by blockade of intrahippocampal CRF(1). It was concluded that exposure to a stressor initially affected both anxiety-like behavior and contextual conditioned fear through septal CRF(2), while the later activation of hippocampal CRF(1) resulted in the return to baseline levels of both processes. Intraventricular injection of mouse urocortin 2, a CRF(2)-selective agonist, removed the stress-induced anxiety and learning impairment, but did not reduce the activation of the hypothalamic pituitary adrenal axis indicative of the hormonal stress response. We propose that the enhanced anxiety is the component of the stress response responsible for the memory deficit.