Distinct Transcriptomic Profiles in the Dorsal Hippocampus and Prelimbic Cortex Are Transiently Regulated following Episodic Learning.

A fundamental, evolutionarily conserved biological mechanism required for long-term memory formation is rapid induction of gene transcription upon learning in relevant brain areas. For episodic types of memories, two regions undergoing this transcription are the dorsal hippocampus (dHC) and prelimbic (PL) cortex. Whether and to what extent these regions regulate similar or distinct transcriptomic profiles upon learning remain to be understood. Here, we used RNA sequencing in the dHC and PL cortex of male rats to profile their transcriptomes in untrained conditions (baseline) and at 1 h and 6 d after inhibitory avoidance learning. We found that, of 33,713 transcripts, >14,000 were significantly expressed at baseline in both regions and ∼3000 were selectively enriched in each region. Gene Ontology biological pathway analyses indicated that commonly expressed pathways included synapse organization, regulation of membrane potential, and vesicle localization. The enriched pathways in the dHC were gliogenesis, axon development, and lipid modification, while in the PL cortex included vesicle localization and synaptic vesicle cycle. At 1 h after learning, 135 transcripts changed significantly in the dHC and 478 in the PL cortex; of these, only 34 were shared. Biological pathways most significantly regulated by learning in the dHC were protein dephosphorylation, glycogen and glucan metabolism, while in the PL cortex were axon development and axonogenesis. The transcriptome profiles returned to baseline by 6 d after training. Thus, a significant portion of dHC and PL cortex transcriptomic profiles is divergent, and their regulation upon learning is largely distinct and transient.SIGNIFICANCE STATEMENT Long-term episodic memory formation requires gene transcription in several brain regions, including the hippocampus and PFC. The comprehensive profiles of the dynamic mRNA changes that occur in these regions following learning are not well understood. Here, we performed RNA sequencing in the dorsal hippocampus and prelimbic cortex, a PFC subregion, at baseline, 1 h, and 6 d after episodic learning in rats. We found that, at baseline, dorsal hippocampus and prelimbic cortex differentially express a significant portion of mRNAs. Moreover, learning produces a transient regulation of region-specific profiles of mRNA, indicating that unique biological programs in different brain regions underlie memory formation.

CIM6P/IGF-2 Receptor Ligands Reverse Deficits in Angelman Syndrome Model Mice.

Angelman syndrome (AS), a genetic disorder that primarily affects the nervous system, is characterized by delayed development, intellectual disability, severe speech impairment, and problems with movement and balance (ataxia). Most affected children also have recurrent seizures (epilepsy). No existing therapies are capable of comprehensively treating the deficits in AS; hence, there is an urgent need to identify new treatments. Here we show that insulin-like growth factor 2 (IGF-2) and mannose-6-phosphate (M6P), ligands of two independent binding sites of the cation-independent M6P/IGF-2 receptor (CIM6P/IGF-2R), reverse most major deficits of AS modeled in mice. Subcutaneous injection of IGF-2 or M6P in mice modeling AS restored cognitive impairments as assessed by measurements of contextual and recognition memories, motor deficits assessed by rotarod and hindlimb clasping, and working memory/flexibility measured by Y-maze. IGF-2 also corrected deficits in marble burying and significantly attenuated acoustically induced seizures. An observational battery of tests confirmed that neither ligand changed basic functions including physical characteristics, general behavioral responses, and sensory reflexes, indicating that they are relatively safe. Our data provide strong preclinical evidence that targeting CIM6P/IGF-2R is a promising approach for developing novel therapeutics for AS. LAY SUMMARY: There is no effective treatment for the neurodevelopmental disorder Angelman syndrome (AS). Using a validated AS mouse model, the Ube3am-/p+ , in this study we show that systemic administration of ligands of the cation independent mannose-6-phosphate receptor, also known as insulin-like growth factor 2 receptor (CIM6P/IGF-2R) reverses cognitive impairment, motor deficits, as well as seizures associated with AS. Thus, ligands that activate the CIM6P/IGF-2R may represent novel, potential therapeutic targets for AS.

A role for CIM6P/IGF2 receptor in memory consolidation and enhancement.

Cation-independent mannose-6-phosphate receptor, also called insulin-like growth factor two receptor (CIM6P/IGF2R), plays important roles in growth and development, but is also extensively expressed in the mature nervous system, particularly in the hippocampus, where its functions are largely unknown. One of its major ligands, IGF2, is critical for long-term memory formation and strengthening. Using CIM6P/IGF2R inhibition in rats and neuron-specific knockdown in mice, here we show that hippocampal CIM6P/IGF2R is necessary for hippocampus-dependent memory consolidation, but dispensable for learning, memory retrieval, and reconsolidation. CIM6P/IGF2R controls the training-induced upregulation of de novo protein synthesis, including increase of Arc, Egr1, and c-Fos proteins, without affecting their mRNA induction. Hippocampal or systemic administration of mannose-6-phosphate, like IGF2, significantly enhances memory retention and persistence in a CIM6P/IGF2R-dependent manner. Thus, hippocampal CIM6P/IGF2R plays a critical role in memory consolidation by controlling the rate of training-regulated protein metabolism and is also a target mechanism for memory enhancement.

NLGN1 and NLGN2 in the prefrontal cortex: their role in memory consolidation and strengthening.

The prefrontal cortex (PFC) is critical for memory formation, but the underlying molecular mechanisms are poorly understood. Clinical and animal model studies have shown that changes in PFC excitation and inhibition are important for cognitive functions as well as related disorders. Here, we discuss recent findings revealing the roles of the excitatory and inhibitory synaptic proteins neuroligin 1 (NLGN1) and NLGN2 in the PFC in memory formation and modulation of memory strength. We propose that shifts in NLGN1 and NLGN2 expression in specific excitatory and inhibitory neuronal subpopulations in response to experience regulate the dynamic processes of memory consolidation and strengthening. Because excitatory/inhibitory imbalances accompany neuropsychiatric disorders in which strength and flexibility of representations play important roles, understanding these mechanisms may suggest novel therapies.

Neuronal PAS Domain Proteins 1 and 3 Are Master Regulators of Neuropsychiatric Risk Genes.

BACKGROUND: NPAS3 has been established as a robust genetic risk factor in major mental illness. In mice, loss of neuronal PAS domain protein 3 (NPAS3) impairs postnatal hippocampal neurogenesis, while loss of the related protein NPAS1 promotes it. These and other findings suggest a critical role for NPAS proteins in neuropsychiatric functioning, prompting interest in the molecular pathways under their control. METHODS: We used RNA sequencing coupled with chromatin immunoprecipitation sequencing to identify genes directly regulated by NPAS1 and NPAS3 in the hippocampus of wild-type, Npas1-/-, and Npas3-/- mice. Computational integration with human genetic and expression data revealed the disease relevance of NPAS-regulated genes and pathways. Specific findings were confirmed at the protein level by Western blot. RESULTS: This is the first in vivo, transcriptome-scale investigation of genes regulated by NPAS1 and NPAS3. These transcription factors control an ensemble of genes that are themselves also major regulators of neuropsychiatric function. Specifically, Fmr1 (fragile X syndrome) and Ube3a (Angelman syndrome) are transcriptionally regulated by NPAS3, as is the neurogenesis regulator Notch. Dysregulation of these pathways was confirmed at the protein level. Furthermore, NPAS1/3 targets show increased human genetic burden for schizophrenia and intellectual disability. CONCLUSIONS: Together, these data provide a clear, unbiased view of the full spectrum of genes regulated by NPAS1 and NPAS3 and show that these transcription factors are master regulators of neuropsychiatric function. These findings expose the molecular pathophysiology of NPAS1/3 mutations and provide a striking example of the shared, combinatorial nature of molecular pathways that underlie diagnostically distinct neuropsychiatric conditions.

P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

Effects of prenatal cocaine exposure on social development in mice.

Prenatal cocaine exposure (PCE) in humans and animals has been shown to impair social development. Molecules that mediate synaptic plasticity and learning in the medial prefrontal cortex (mPFC), specifically brain-derived neurotrophic factor (BDNF) and its downstream signaling molecule, early growth response protein 1 (egr1), have been shown to affect the regulation of social interactions (SI). In this study we determined the effects of PCE on SI and the corresponding ultrasonic vocalizations (USVs) in developing mice. Furthermore, we studied the PCE-induced changes in the constitutive expression of BDNF, egr1 and their transcriptional regulators in the mPFC as a possible molecular mechanism mediating the altered SI. In prenatal cocaine-exposed (PCOC) mice we identified increased SI and USV production at postnatal day (PD) 25, and increased SI but not USVs at PD35. By PD45 the expression of both social behaviors normalized in PCOC mice. At the molecular level, we found increased BDNF exon IV and egr1 mRNA in the mPFC of PCOC mice at PD30 that normalized by PD45. This was concurrent with increased EGR1 protein in the mPFC of PCOC mice at PD30, suggesting a role of egr1 in the enhanced SI observed in juvenile PCOC mice. Additionally, by measuring the association of acetylation of histone 3 at lysine residues 9 and 14 (acH3K9,14) and MeCP2 at the promoters of BDNF exons I and IV and egr1, our results provide evidence of promoter-specific alterations in the mPFC of PCOC juvenile mice, with increased association of acH3K9,14 only at the BDNF exon IV promoter. These results identify a potential PCE-induced molecular alteration as the underlying neurobiological mechanism mediating the altered social development in juvenile mice.

Effects of prolonged selective serotonin reuptake inhibition on the development and expression of L-DOPA-induced dyskinesia in hemi-parkinsonian rats.

Dopamine (DA) replacement therapy with l-DOPA is the standard treatment for Parkinson's disease (PD). Unfortunately chronic treatment often leads to the development of abnormal involuntary movements (AIMs) referred to as L-DOPA-induced dyskinesia (LID). Accumulating evidence has shown that compensatory plasticity in serotonin (5-HT) neurons contributes to LID and recent work has indicated that acute 5-HT transporter (SERT) blockade provides anti-dyskinetic protection. However neither the persistence nor the mechanism(s) of these effects have been investigated. Therefore the current endeavor sought to mimic a prolonged regimen of SERT inhibition in L-DOPA-primed and -naïve hemi-parkinsonian rats. Rats received 3 weeks of daily co-treatment of the selective 5-HT reuptake inhibitors (SSRIs) citalopram (0, 3, or 5 mg/kg) or paroxetine (0, 0.5, or 1.25 mg/kg) with L-DOPA (6 mg/kg) during which AIMs and motor performance were monitored. In order to investigate potential mechanisms of action, tissue levels of striatal monoamines were monitored and the 5-HT(1A) receptor antagonist WAY100635 (0.5 mg/kg) was used. Results revealed that prolonged SSRIs attenuated AIMs expression and development in L-DOPA-primed and -naïve subjects, respectively, without interfering with motor performance. Neurochemical analysis of striatal tissue indicated that a 3 week SERT blockade increased DA levels in L-DOPA-treated rats. Pharmacologically, anti-dyskinetic effects were partially reversed with WAY100635 signifying involvement of the 5-HT1A receptor. Collectively, these findings demonstrate that prolonged SERT inhibition provides enduring anti-dyskinetic effects in part via 5-HT(1A) receptors while maintaining L-DOPA's anti-parkinsonian efficacy by enhancing striatal DA levels.

Molecular mechanisms mediating a deficit in recall of fear extinction in adult mice exposed to cocaine in utero.

Prenatal cocaine exposure has been shown to alter cognitive processes of exposed individuals, presumed to be a result of long-lasting molecular alterations in the brain. In adult prenatal cocaine exposed (PCOC) mice we have identified a deficit in recall of fear extinction, a behavior that is dependent on the medial prefrontal cortex (mPFC) and the hippocampus. While we observed no change in the constitutive expression of brain derived neurotrophic factor (BDNF) protein and mRNA in the mPFC and hippocampus of adult PCOC mice, we observed blunted BDNF signaling in the mPFC of adult PCOC mice after fear extinction compared to the control animals. Specifically, during the consolidation phase of the extinction memory, we observed a decrease in BDNF protein and it's phospho-TrkB receptor expression. Interestingly, at this same time point there was a significant increase in total Bdnf mRNA levels in the mPFC of PCOC mice as compared with controls. In the Bdnf gene, we identified decreased constitutive binding of the transcription factors, MeCP2 and P-CREB at the promoters of Bdnf exons I and IV in the mPFC of PCOC mice, that unlike control mice remained unchanged when measured during the behavior. Finally, bilateral infusion of recombinant BDNF protein into the infralimbic subdivision of the mPFC during the consolidation phase of the extinction memory rescued the behavioral deficit in PCOC mice. In conclusion, these findings extend our knowledge of the neurobiologic impact of prenatal cocaine exposure on the mPFC of mice, which may lead to improved clinical recognition and treatment of exposed individuals.

Brain-derived neurotrophic factor genotype impacts the prenatal cocaine-induced mouse phenotype.

Prenatal cocaine exposure leads to persistent alterations in the growth factor brain-derived neurotrophic factor (BDNF), particularly in the medial prefrontal cortex (mPFC) and hippocampus, brain regions important in cognitive functioning. BDNF plays an important role in the strengthening of existing synaptic connections as well as in the formation of new contacts during learning. A single nucleotide polymorphism in the BDNF gene (Val66Met), leading to a Met substitution for Val at codon 66 in the prodomain, is common in human populations, with an allele frequency of 20-30% in Caucasians. To study the interaction between prenatal cocaine exposure and BDNF, we have utilized a line of BDNF Val66Met transgenic mice on a Swiss Webster background in which BDNF(Met) is endogenously expressed. Examination of baseline levels of mature BDNF protein in the mPFC of prenatally cocaine-treated wild-type (Val66Val) and Val66Met mice revealed significantly lower levels compared to prenatally saline-treated mice. In contrast, in the hippocampus of prenatally saline- and cocaine-treated adult Val66Met mice, there were significantly lower levels of mature BDNF protein compared to Val66Val mice. In extinction of a conditioned fear, we found that prenatally cocaine-treated Val66Met mice had a deficit in recall of extinction. Examination of mature BDNF protein levels immediately after the test for extinction recall revealed lower levels in the mPFC of prenatally cocaine-treated Val66Met mice compared to saline-treated mice. However, 2 h after the extinction test, there was increased BDNF exons I, IV, and IX mRNA expression in the prelimbic cortex of the mPFC in the prenatally cocaine-treated BDNF Val66Met mice compared to prenatally saline-treated mice. Taken together, our results suggest the possibility that prenatal cocaine-induced constitutive alterations in BDNF mRNA and protein expression in the mPFC differentially poises animals for alterations in behaviorally induced gene activation, which are interactive with BDNF genotype and differentially impact those behaviors. Such findings in our prenatal cocaine mouse model suggest a gene X environment interaction of potential clinical relevance.