An open-label pilot trial of minocycline in children as a treatment for Angelman syndrome.

BACKGROUND: Minocycline, a member of the tetracycline family, has a low risk of adverse effects and an ability to improve behavioral performance in humans with cognitive disruption. We performed a single-arm open-label trial in which 25 children diagnosed with Angelman syndrome (AS) were administered minocycline to assess the safety and tolerability of minocycline in this patient population and determine the drug's effect on the cognitive and behavioral manifestations of the disorder. METHODS: Participants, age 4-12 years old, were randomly selected from a pool of previously screened children for participation in this study. Each child received 3 milligrams of minocycline per kilogram of body weight per day for 8 weeks. Participants were assessed during 3 study visits: baseline, after 8-weeks of minocycline treatment and after an 8-week wash out period. The primary outcome measure was the Bayley Scales of Infant and Toddler Development 3rd Edition (BSID-III). Secondary outcome measures included the Clinical Global Impressions Scale (CGI), Vineland Adaptive Behavior Scales 2nd Edition (VABS-II), Preschool Language Scale 4th Edition (PLS-IV) and EEG scores. Observations were considered statistically significant if p < 0.05 using ANOVA and partial eta squared (η(2)) was calculated to show effect size. Multiple comparisons testing between time points were carried out using Dunnett's post hoc testing. RESULTS: Significant improvement in the mean raw scores of the BSID-III subdomains communication and fine motor ability as well as the subdomains auditory comprehension and total language ability of the PLS-IV when baseline scores were compared to scores after the washout period. Further, improvements were observed in the receptive communication subdomain of the VABS-II after treatment with minocycline. Finally, mean scores of the BSID-III self-direction subdomain and CGI scale score were significantly improved both after minocycline treatment and after the wash out period. CONCLUSION: The clinical and neuropsychological measures suggest minocycline was well tolerated and causes improvements in the adaptive behaviors of this sample of children with Angelman syndrome. While the optimal dosage and the effects of long-term use still need to be determined, these findings suggest further investigation into the effect minocycline has on patients with Angelman syndrome is warranted. TRIAL REGISTRATION: NCT01531582 - clinicaltrials.gov.

Activity-dependent changes in MAPK activation in the Angelman Syndrome mouse model.

Angelman Syndrome (AS) is a devastating neurological disorder caused by disruption of the maternal UBE3A gene. Ube3a protein is identified as an E3 ubiquitin ligase that shows neuron-specific imprinting. Despite extensive research evaluating the localization and basal expression profiles of Ube3a in mouse models, the molecular mechanisms whereby Ube3a deficiency results in AS are enigmatic. Using in vitro and in vivo systems we show dramatic changes in the expression of Ube3a following synaptic activation. In primary neuronal culture, neuronal depolarization was found to increase both nuclear and cytoplasmic Ube3a levels. Analogous up-regulation in maternal and paternal Ube3a expression was observed in Ube3a-YFP reporter mice following fear conditioning. Absence of Ube3a led to deficits in the activity-dependent increases in ERK1/2 phosphorylation, which may contribute to reported deficits in synaptic plasticity and cognitive function in AS mice. Taken together, our findings provide novel insight into the regulation of Ube3a by synaptic activity and its potential role in kinase regulation.

Reelin supplementation recovers sensorimotor gating, synaptic plasticity and associative learning deficits in the heterozygous reeler mouse.

The lipoprotein receptor ligand Reelin is important for the processes of normal synaptic plasticity, dendritic morphogenesis, and learning and memory. Heterozygous reeler mice (HRM) show many neuroanatomical, biochemical, and behavioral features that are associated with schizophrenia. HRM show subtle morphological defects including reductions in dendritic spine density, altered synaptic plasticity and behavioral deficits in associative learning and memory and pre-pulse inhibition. The present studies test the hypothesis that in vivo elevation of Reelin levels can rescue synaptic and behavioral phenotypes associated with HRM. We demonstrate that a single in vivo injection of Reelin increases GAD67 expression and alters dendritic spine morphology. In parallel we observed enhancement of hippocampal synaptic function and associative learning and memory. Reelin supplementation also increases pre-pulse inhibition. These results suggest that characteristics of HRM, similar to those observed in schizophrenia, are sensitive to Reelin levels and can be modified with Reelin supplementation in male and female adults.

Adeno-associated virus-mediated rescue of the cognitive defects in a mouse model for Angelman syndrome.

Angelman syndrome (AS), a genetic disorder occurring in approximately one in every 15,000 births, is characterized by severe mental retardation, seizures, difficulty speaking and ataxia. The gene responsible for AS was discovered to be UBE3A and encodes for E6-AP, an ubiquitin ligase. A unique feature of this gene is that it undergoes maternal imprinting in a neuron-specific manner. In the majority of AS cases, there is a mutation or deletion in the maternally inherited UBE3A gene, although other cases are the result of uniparental disomy or mismethylation of the maternal gene. While most human disorders characterized by severe mental retardation involve abnormalities in brain structure, no gross anatomical changes are associated with AS. However, we have determined that abnormal calcium/calmodulin-dependent protein kinase II (CaMKII) regulation is seen in the maternal UBE3A deletion AS mouse model and is responsible for the major phenotypes. Specifically, there is an increased αCaMKII phosphorylation at the autophosphorylation sites Thr(286) and Thr(305/306), resulting in an overall decrease in CaMKII activity. CaMKII is not produced until after birth, indicating that the deficits associated with AS are not the result of developmental abnormalities. The present studies are focused on exploring the potential to rescue the learning and memory deficits in the adult AS mouse model through the use of an adeno-associated virus (AAV) vector to increase neuronal UBE3A expression. These studies show that increasing the levels of E6-AP in the brain using an exogenous vector can improve the cognitive deficits associated with AS. Specifically, the associative learning deficit was ameliorated in the treated AS mice compared to the control AS mice, indicating that therapeutic intervention may be possible in older AS patients.

Reelin supplementation enhances cognitive ability, synaptic plasticity, and dendritic spine density.

Apolipoprotein receptors belong to an evolutionarily conserved surface receptor family that has intimate roles in the modulation of synaptic plasticity and is necessary for proper hippocampal-dependent memory formation. The known lipoprotein receptor ligand Reelin is important for normal synaptic plasticity, dendritic morphology, and cognitive function; however, the in vivo effect of enhanced Reelin signaling on cognitive function and synaptic plasticity in wild-type mice is unknown. The present studies test the hypothesis that in vivo enhancement of Reelin signaling can alter synaptic plasticity and ultimately influence processes of learning and memory. Purified recombinant Reelin was injected bilaterally into the ventricles of wild-type mice. We demonstrate that a single in vivo injection of Reelin increased activation of adaptor protein Disabled-1 and cAMP-response element binding protein after 15 min. These changes correlated with increased dendritic spine density, increased hippocampal CA1 long-term potentiation (LTP), and enhanced performance in associative and spatial learning and memory. The present study suggests that an acute elevation of in vivo Reelin can have long-term effects on synaptic function and cognitive ability in wild-type mice.

Inhibition of the interactions between eukaryotic initiation factors 4E and 4G impairs long-term associative memory consolidation but not reconsolidation.

Considerable evidence indicates that the general blockade of protein synthesis prevents both the initial consolidation and the postretrieval reconsolidation of long-term memories. These findings come largely from studies of drugs that block ribosomal function, so as to globally interfere with both cap-dependent and -independent forms of translation. Here we show that intra-amygdala microinfusions of 4EGI-1, a small molecule inhibitor of cap-dependent translation that selectively disrupts the interaction between eukaryotic initiation factors (eIF) 4E and 4G, attenuates fear memory consolidation but not reconsolidation. Using a combination of behavioral and biochemical techniques, we provide both in vitro and in vivo evidence that the eIF4E-eIF4G complex is more stringently required for plasticity induced by initial learning than for that triggered by reactivation of an existing memory.

Insights into synaptic function from mouse models of human cognitive disorders.

Modern approaches to the investigation of the molecular mechanisms underlying human cognitive disease often include multidisciplinary examination of animal models engineered with specific mutations that spatially and temporally restrict expression of a gene of interest. This approach not only makes possible the development of animal models that demonstrate phenotypic similarities to their respective human disorders, but has also allowed for significant progress towards understanding the processes that mediate synaptic function and memory formation in the nondiseased state. Examples of successful mouse models where genetic manipulation of the mouse resulted in recapitulation of the symptomatology of the human disorder and was used to significantly expand our understanding of the molecular mechanisms underlying normal synaptic plasticity and memory formation are discussed in this article. These studies have broadened our knowledge of several signal transduction cascades that function throughout life to mediate synaptic physiology. Defining these events is key for developing therapies to address disorders of cognitive ability.

Defining mild cognitive impairment: impact of varying decision criteria on neuropsychological diagnostic frequencies and correlates.

OBJECTIVE: To examine the impact of varying decision criteria on neuropsychological diagnostic frequencies and on their correlates. DESIGN: Descriptive and correlational study. SETTING: Florida Alzheimer's Disease Research Center. PARTICIPANTS: A sample of 373 individuals with comprehensive baseline analyses participating in a longitudinal study of cognitive decline and early Alzheimer disease. MEASUREMENTS: Mild cognitive impairment (MCI) diagnoses were made on the basis of four sets of decision criteria created by crossing two approaches: varying the number of impaired test results required for a diagnosis within any domain (1 test versus 2) and varying the performance level required to determine impairment (1.5 or 2 standard deviations [SDs] below the normative mean) for any test. RESULTS: Under each criteria set, single-domain amnestic MCI was the most frequent MCI diagnosis. MCI global and subtype diagnosis frequencies were inversely related to the stringency of the criteria. The single test-1.5 SD criterion identified the largest number of cases as qualifying for an MCI diagnosis, and the two test-2.0 SD cutoff identified the fewest. Across all sets of criteria, the authors found significant positive associations between neuropsychological diagnoses and Clinical Dementia Rating score categories. Significant relationships between diagnoses and both apolipoprotein E (APOE) genotype and magnetic resonance imaging ratings of medial temporal atrophy (MTA) application were found only for the two test-1.5 SD and two test-2.0 SD cutoffs. CONCLUSION: MCI diagnosis frequencies are substantively affected by the stringency of the criteria, but the relative rankings of MCI subtype diagnoses are fairly consistent regardless of the stringency of the criteria. Significant associations of neuropsychological diagnoses with independent markers such as APOE genotype and MTA are only found with more stringent criteria, suggesting that a coherent network of associations reflecting cognitive decline occurs with more restrictive definitions for impairment.

mGluR5 positive allosteric modulators facilitate both hippocampal LTP and LTD and enhance spatial learning.

Highly selective positive allosteric modulators (PAMs) of metabotropic glutamate receptor subtype 5 (mGluR5) have emerged as a potential approach to treat positive symptoms associated with schizophrenia. mGluR5 plays an important role in both long-term potentiation (LTP) and long-term depression (LTD), suggesting that mGluR5 PAMs may also have utility in improving impaired cognitive function. However, if mGluR5 PAMs shift the balance of LTP and LTD or induce a state in which afferent activity induces lasting changes in synaptic function that are not appropriate for a given pattern of activity, this could disrupt rather than enhance cognitive function. We determined the effect of selective mGluR5 PAMs on the induction of LTP and LTD at the Schaffer collateral-CA1 synapse in the hippocampus. mGluR5-selective PAMs significantly enhanced threshold theta-burst stimulation (TBS)-induced LTP. In addition, mGluR5 PAMs enhanced both DHPG-induced LTD and LTD induced by the delivery of paired-pulse low-frequency stimulation. Selective potentiation of mGluR5 had no effect on LTP induced by suprathreshold TBS or saturated LTP. The finding that potentiation of mGluR5-mediated responses to stimulation of glutamatergic afferents enhances both LTP and LTD and supports the hypothesis that the activation of mGluR5 by endogenous glutamate contributes to both forms of plasticity. Furthermore, two systemically active mGluR5 PAMs enhanced performance in the Morris water maze, a measure of hippocampus-dependent spatial learning. Discovery of small molecules that enhance both LTP and LTD in an activity-appropriate manner shows a unique action on synaptic plasticity that may provide a novel approach for the treatment of impaired cognitive function.

Activation of exchange protein activated by cyclic-AMP enhances long-lasting synaptic potentiation in the hippocampus.

cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term potentiation (LTP) of synaptic strength. However, cAMP may also initiate signaling via a guanine nucleotide exchange protein directly activated by cAMP (Epac). The role of Epac in hippocampal synaptic plasticity is unknown. We found that in area CA1 of mouse hippocampal slices, activation of Epac enhances maintenance of LTP without affecting basal synaptic transmission. The persistence of this form of LTP requires extracellular signal-regulated protein kinase (ERK) and new protein synthesis, but not transcription. Because ERK is involved in translational control of long-lasting plasticity and memory, our data suggest that Epac is a crucial link between cAMP and ERK during some forms of protein synthesis-dependent LTP. Activation of Epac represents a novel signaling pathway for rapid regulation of the stability of enduring forms of LTP and, perhaps, of hippocampus- dependent long-term memories.

Cap-dependent translation initiation and memory.

It is widely accepted that changes in gene expression contribute to enduring modifications of synaptic strength and are required for long-term memory. This is an exciting, wide-open area of research at this moment, one of those areas where it is clear that important work is underway but where the surface has just been scratched in terms of our understanding. Much attention has been given to the mechanisms of gene transcription; however, the regulation of transcription is only one route of manipulating gene expression. Regulation of mRNA translation is another route, and is the ultimate step in the control of gene expression, enabling cells to regulate protein production without altering mRNA synthesis or transport. One of the main advantages of this mechanism over transcriptional control in the nucleus lies in the fact that it endows local sites with independent decision-making authority, a consideration that is of particular relevance in neurons with complex synapto-dendritic architecture. There are a growing number of groups that are taking on the challenge of identifying the mechanisms responsible for regulating the process of mRNA translation during synaptic plasticity and memory formation. In this chapter we will discuss what has been discovered with regard to the localization and regulation of mRNA translation during specific types of neuronal activity in the mammalian central nervous system. The data are most complete for cap-dependent translation; therefore, particular attention will be paid to the machinery that initiates cap-dependent translation and its regulation during synaptic plasticity as well as the behavioral phenotypes consequent to its dysregulation.

Group III mGluR regulation of synaptic transmission at the SC-CA1 synapse is developmentally regulated.

Group III metabotropic glutamate receptors (mGluRs) reduce synaptic transmission at the Schaffer collateral-CA1 (SC-CA1) synapse in rats by a presynaptic mechanism. Previous studies show that low concentrations of the group III-selective agonist, L-AP4, reduce synaptic transmission in slices from neonatal but not adult rats, whereas high micromolar concentrations reduce transmission in both age groups. L-AP4 activates mGluRs 4 and 8 at much lower concentrations than those required to activate mGluR7, suggesting that the group III mGluR subtype modulating transmission is a high affinity receptor in neonates and a low affinity receptor in adults. The previous lack of subtype selective ligands has made it difficult to test this hypothesis. We have measured fEPSPs in the presence of novel subtype selective agents to address this question. We show that the effects of L-AP4 can be blocked by LY341495 in both neonates and adults, verifying that these effects are mediated by mGluRs. In addition, the selective mGluR8 agonist, DCPG, has a significant effect in slices from neonatal rats but does not reduce synaptic transmission in adult slices. The mGluR4 selective allosteric potentiator, PHCCC, is unable to potentiate the L-AP4-induced effects at either age. Taken together, our data suggest that group III mGluRs regulate transmission at the SC-CA1 synapse throughout development but there is a developmental regulation of the subtypes involved so that both mGluR7 and mGluR8 serve this role in neonates whereas mGluR7 is involved in regulating transmission at this synapse throughout postnatal development.

ERK and mTOR signaling couple beta-adrenergic receptors to translation initiation machinery to gate induction of protein synthesis-dependent long-term potentiation.

beta-Adrenergic receptors critically modulate long-lasting synaptic plasticity and long-term memory in the mammalian hippocampus. Persistent long-term potentiation of synaptic strength requires protein synthesis and has been correlated with some forms of hippocampal long-term memory. However, the intracellular processes that initiate protein synthesis downstream of the beta-adrenergic receptor are unidentified. Here we report that activation of beta-adrenergic receptors recruits ERK and mammalian target of rapamycin signaling to facilitate long-term potentiation maintenance at the level of translation initiation. Treatment of mouse hippocampal slices with a beta-adrenergic receptor agonist results in activation of eukaryotic initiation factor 4E and the eukaryotic initiation factor 4E kinase Mnk1, along with inhibition of the translation repressor 4E-BP. This coordinated activation of translation machinery requires concomitant ERK and mammalian target of rapamycin signaling. Taken together, our data identify distinct signaling pathways that converge to regulate beta-adrenergic receptor-dependent protein synthesis during long-term synaptic potentiation in the hippocampus. We suggest that beta-adrenergic receptors play a crucial role in gating the induction of long-lasting synaptic plasticity at the level of translation initiation, a mechanism that may underlie the ability of these receptors to influence the formation of long-lasting memories.

Behavioral alterations in mice lacking the translation repressor 4E-BP2.

The requirement for de novo protein synthesis during multiple forms of learning, memory and behavior is well-established; however, we are only beginning to uncover the regulatory mechanisms that govern this process. In order to determine how translation initiation is regulated during neuroplasticity we engineered mutant C57Bl/6J mice that lack the translation repressor eukaryotic initiation factor 4E-binding protein 2 (4E-BP2) and have previously demonstrated that 4E-BP2 plays a critical role in hippocampus-dependent synaptic plasticity and memory. Herein, we examined the 4E-BP2 knockout mice in a battery of paradigms to address motor activity and motor skill learning, anxiety and social dominance behaviors, working memory and conditioned taste aversion. We found that the 4E-BP2 knockout mice demonstrated altered activity in the rotating rod test, light/dark exploration test, spontaneous alternation T-maze and conditioned taste aversion test. The information gained from these studies builds a solid foundation for future studies on the specific role of 4E-BP2 in various types of behavior, and for a broader, more detailed examination of the mechanisms of translational control in the brain.

Regulation of eukaryotic initiation factor 4E by converging signaling pathways during metabotropic glutamate receptor-dependent long-term depression.

Long-term depression (LTD) is an activity-dependent decrease in synaptic efficacy that can be induced in hippocampal area CA1 by pharmacological application of the selective group I metabotropic glutamate receptor (mGluR) agonist 3,5-diyhroxyphenylglycine (DHPG). Recent work has demonstrated that DHPG-induced LTD recruits at least two signal transduction pathways known to couple to translation, the mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling pathway and the phosphoinositide 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway. However, it remains unclear which translation factors are engaged by these two signaling pathways during mGluR-LTD. In this study, we investigated whether the group I mGluRs couple to the cap-dependent translation proteins: Mnk1, eIF4E, and 4E-BP. We found that both the MEK-ERK and PI3K-mTOR signaling pathways are critical for the DHPG-induced regulation of these translation factors. Furthermore, we demonstrate that increasing eIF4F complex availability via the genetic elimination of 4E-BP2 can enhance the degree of LTD achieved by DHPG application in an ERK-dependent manner. Our results provide direct evidence that cap-dependent translation is engaged during mGluR-LTD and demonstrate that the MEK-ERK and PI3K-mTOR signaling pathways converge to regulate eIF4E activity after induction of DHPG-LTD.

The translation repressor 4E-BP2 is critical for eIF4F complex formation, synaptic plasticity, and memory in the hippocampus.

Long-lasting synaptic plasticity and memory requires mRNA translation, yet little is known as to how this process is regulated. To explore the role that the translation repressor 4E-BP2 plays in hippocampal long-term potentiation (LTP) and learning and memory, we examined 4E-BP2 knock-out mice. Interestingly, genetic elimination of 4E-BP2 converted early-phase LTP to late-phase LTP (L-LTP) in the Schaffer collateral pathway, likely as a result of increased eIF4F complex formation and translation initiation. A critical limit for activity-induced translation was revealed in the 4E-BP2 knock-out mice because L-LTP elicited by traditional stimulation paradigms was obstructed. Moreover, the 4E-BP2 knock-out mice also exhibited impaired spatial learning and memory and conditioned fear-associative memory deficits. These results suggest a crucial role for proper regulation of the eIF4F complex by 4E-BP2 during LTP and learning and memory in the mouse hippocampus.

NMDA receptor activation results in PKA- and ERK-dependent Mnk1 activation and increased eIF4E phosphorylation in hippocampal area CA1.

Protein synthesis is essential for the stabilization of glutamate receptor-dependent forms of long-lasting hippocampal synaptic plasticity and for the consolidation of memory, but the signal transduction mechanisms that regulate translation factors during these processes are not well understood. As a first step towards understanding how translation is activated during synaptic plasticity, we investigated how the eukaryotic initiation factor 4E (eIF4E), a rate-limiting mRNA cap-binding protein, and its kinase, Mnk1, are regulated by protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and N-methyl-D-aspartate (NMDA) receptor activation in hippocampal area CA1. We found that treatment of mouse hippocampal slices with either phorbol ester, to activate PKC, or forskolin, to activate PKA, resulted in activation of Mnk1 and increased eIF4E phosphorylation that was dependent on extracellular signal-regulated kinase (ERK). Similarly, brief treatment of hippocampal slices with NMDA resulted in activation of Mnk1 and increased phosphorylation of eIF4E. The NMDA-induced activation of Mnk1 and increased phosphorylation of eIF4E were dependent on PKA and ERK, but not PKC, and were present in synaptoneurosome preparations. Immunohistochemical analysis revealed that the PKA- and ERK-dependent increases in Mnk1 activation induced by NMDA also occurred in dendrites. These findings identify a specific regulatory pathway that can couple NMDA receptor activation to translation initiation factors in the hippocampus, and may represent a mechanism for triggering dendritic protein synthesis during long-term potentiation and long-term memory formation.

Synaptic plasticity and translation initiation.

It is widely accepted that protein synthesis, including local protein synthesis at synapses, is required for several forms of synaptic plasticity. Local protein synthesis enables synapses to control synaptic strength independent of the cell body via rapid protein production from pre-existing mRNA. Therefore, regulation of translation initiation is likely to be intimately involved in modulating synaptic strength. Our understanding of the translation-initiation process has expanded greatly in recent years. In this review, we discuss various aspects of translation initiation, as well as signaling pathways that might be involved in coupling neurotransmitter and neurotrophin receptors to the translation machinery during various forms of synaptic plasticity.