Search

RNA-binding proteins (RBPs) can undergo phase separation and form condensates, processes that, in turn, can be critical for their functionality. In a recent study, Huang, Ellis, and colleagues show that cellular stress can trigger transient alterations in nuclear TAR DNA-binding protein 43 (TDP-43), leading to changes crucial for proper neuronal function. These findings have implications for understanding neurological TDP-43 proteinopathies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Protein synthesis is essential for cells to perform life metabolic processes. Pathological alterations of protein content can lead to particular diseases. Cells have an intrinsic array of mechanisms and pathways that are activated when protein misfolding, accumulation, aggregation or mislocalization occur. Some of them (like the unfolded protein response) represent complex interactions between endoplasmic reticulum sensors and elongation factors that tend to increase expression of chaperone proteins and/or repress translation in order to restore protein homeostasis (also known as proteostasis). This is even more important in neurons, as they are very susceptible to harmful effects associated with protein overload and proteostatic mechanisms are less effective with age. Several neurodegenerative pathologies such as Alzheimer's, Parkinson's, and Huntington's diseases, amyotrophic lateral sclerosis and frontotemporal dementia exhibit a particular molecular signature of distinct, unbalanced protein overload. In amyotrophic lateral sclerosis and frontotemporal dementia, the majority of cases present intracellular inclusions of ubiquitinated transactive response DNA-binding protein of 43 kDa (TDP-43). TDP-43 is an RNA binding protein that participates in RNA metabolism, among other functions. Dysregulation of TDP-43 (e.g. aggregation and mislocalization) can dramatically affect neurons, and this has been linked to disease development. Expression of amyotrophic lateral sclerosis/frontotemporal dementia TDP-43-related mutations in cellular and animal models has been shown to recapitulate key features of the amyotrophic lateral sclerosis/frontotemporal dementia disease spectrum. These variants can be causative of degeneration onset and progression. Most neurodegenerative diseases (including amyotrophic lateral sclerosis and frontotemporal dementia) have no cure at the moment; however, modulating translation has recently emerged as an attractive approach that can be performed at several steps (i.e. regulating activation of initiation and elongation factors, inhibiting unfolded protein response activation or inducing chaperone expression and activity). This review focuses on the features of protein imbalance in neurodegenerative disorders and the relevance of developing therapeutical compounds aiming at restoring proteostasis. We strive to highlight the importance of research on drugs that, not only restore protein imbalance without compromising translational activity of cells, but are also as safe as possible for the patients., Competing Interests: None

Multiple sclerosis (MS), especially in its progressive phase, involves early axonal and neuronal damage resulting from a combination of inflammatory mediators, demyelination, and loss of trophic support. During progressive disease stages, a microenvironment is created within the central nervous system (CNS) favoring the arrival and retention of inflammatory cells. Active demyelination and neurodegeneration have also been linked to microglia (MG) and astrocyte (AST)-activation in early lesions. While reactive MG can damage tissue, exacerbate deleterious effects, and contribute to neurodegeneration, it should be noted that activated MG possess neuroprotective functions as well, including debris phagocytosis and growth factor secretion. The progressive form of MS can be modeled by the prolonged administration to cuprizone (CPZ) in adult mice, as CPZ induces highly reproducible demyelination of different brain regions through oligodendrocyte (OLG) apoptosis, accompanied by MG and AST activation and axonal damage. Therefore, our goal was to evaluate the effects of a reduction in microglial activation through orally administered brain-penetrant colony-stimulating factor-1 receptor (CSF-1R) inhibitor BLZ945 (BLZ) on neurodegeneration and its correlation with demyelination, astroglial activation, and behavior in a chronic CPZ-induced demyelination model. Our results show that BLZ treatment successfully reduced the microglial population and myelin loss. However, no correlation was found between myelin preservation and neurodegeneration, as axonal degeneration was more prominent upon BLZ treatment. Concomitantly, BLZ failed to significantly offset CPZ-induced astroglial activation and behavioral alterations. These results should be taken into account when proposing the modulation of microglial activation in the design of therapies relevant for demyelinating diseases. Cover Image for this issue: https://doi.org/10.1111/jnc.15394., (© 2021 International Society for Neurochemistry.)

Objectives: The impact of chronic exposure to environmental adversities on brain regions involved in cognition and mental health depends on whether it occurs during the perinatal period, childhood, adolescence or adulthood. The effects of these adversities on the brain and behavior arise as a function of the timing of the exposure and their co-occurrence with the development of specific regions. Here we aimed to explore the behavioral phenotypes derived from two nutritional stress paradigms which differed in the timing of exposure: a low-protein perinatal diet during gestation and lactation and a low-protein diet during adolescence. Methods: Locomotor and exploratory activity, recognition memory and aversive memory were measured in CF-1 8-week-old male mice subjected to perinatal malnutrition (LP-P) or adolescent malnutrition (LP-A), and their respective controls with normal protein diet (NP-P and NP-A). Results: By using the open field test, we found that LP-P and LP-A mice showed reduced exploratory activity compared to controls, but no alterations in their locomotor activity. Recognition memory was impaired only in LP-P mice. Interestingly, aversive memory was not altered in LP-P mice but was enhanced in LP-A mice. Considering the stress-inoculation theory, we hypothesized that protein malnutrition during adolescence represents a challenging but still moderate stressful environment, which promotes active coping in face of later adversity. Conclusion: Our results indicate that while perinatal malnutrition impairs recognition memory, adolescent malnutrition enhances aversive memory, showing dissimilar adaptive responses.

TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo . Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Charif, Luchelli, Vila, Blaustein and Igaz.)

Dysregulation of TAR DNA-binding protein 43 (TDP-43) is a hallmark feature of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two fatal neurodegenerative diseases. TDP-43 is a ubiquitously expressed RNA-binding protein with many physiological functions, playing a role in multiple aspects of RNA metabolism. We developed transgenic mice conditionally overexpressing human wild-type TDP-43 protein (hTDP-43-WT) in forebrain neurons, a model that recapitulates several key features of FTD. After post-weaning transgene (TG) induction during 1 month, these mice display an early behavioral phenotype, including impaired cognitive and social function with no substantial motor abnormalities. In order to expand the analysis of this model, we took advantage of the temporal and regional control of TG expression possible in these mice. We behaviorally evaluated mice at two different times: after 2 weeks of post-weaning TG induction (0.5 month group) and after subsequent TG suppression for 2 weeks following that time point [1 month (sup) group]. We found no cognitive abnormalities after 0.5 month of hTDP-43 expression, evaluated with a spatial working memory task (Y-maze test). Suppression of TG expression with doxycycline (Dox) at this time point prevented the development of cognitive deficits previously observed at 1 month post-induction, as revealed by the performance of the 1 month (sup) group. On the other hand, sociability deficits (assessed through the social interaction test) appeared very rapidly after Dox removal (0.5 month) and TG suppression was not sufficient to reverse this phenotype, indicating differential vulnerability to hTDP-43 expression and suppression. Animals evaluated at the early time point (0.5 month) post-induction do not display a motor phenotype, in agreement with the results obtained after 1 month of TG expression. Moreover, all motor tests (open field, accelerated rotarod, limb clasping, hanging wire grip) showed identical responses in both control and bigenic animals in the suppressed group, demonstrating that this protocol and treatment do not cause non-specific effects in motor behavior, which could potentially mask the phenotypes in other domains. Our results show that TDP-43-WT mice have a phenotype that qualifies them as a useful model of FTD and provide valuable information for susceptibility windows in therapeutic strategies for TDP-43 proteinopathies.

Frontotemporal Dementia (FTD) and amyotrophic lateral sclerosis (ALS) are two neurodegenerative diseases associated to mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43). To investigate in depth the behavioral phenotype associated with this proteinopathy, we used as a model transgenic (Tg) mice conditionally overexpressing human wild-type TDP 43 protein (hTDP-43-WT) in forebrain neurons. We previously characterized these mice at the neuropathological level and found progressive neurodegeneration and other features that evoke human TDP-43 proteinopathies of the FTD/ALS spectrum. In the present study we analyzed the behavior of mice at multiple domains, including motor, social and cognitive performance. Our results indicate that young hTDP-43-WT Tg mice (1 month after post-weaning transgene induction) present a normal motor phenotype compared to control littermates, as assessed by accelerated rotarod performance, spontaneous locomotor activity in the open field test and a mild degree of spasticity shown by a clasping phenotype. Analysis of social and cognitive behavior showed a rapid installment of deficits in social interaction, working memory (Y-maze test) and recognition memory (novel object recognition test) in the absence of overt motor abnormalities. To investigate if the motor phenotype worsen with age, we analyzed the behavior of mice after long-term (up to 12 months) transgene induction. Our results reveal a decreased performance on the rotarod test and in the hanging wire test, indicating a motor phenotype that was absent in younger mice. In addition, long-term hTDP-43-WT expression led to hyperlocomotion in the open field test. In sum, these results demonstrate a time-dependent emergence of a motor phenotype in older hTDP-43-WT Tg mice, recapitulating aspects of clinical FTD presentations with motor involvement in human patients, and providing a complementary animal model for studying TDP-43 proteinopathies.

The medial prefrontal cortex (mPFC) is known for its role in decision making and memory processing, including the participation in the formation of extinction memories. However, little is known regarding its contribution to aversive memory consolidation. Here we demonstrate that neural activity and protein synthesis are required in the dorsal mPFC for memory formation of a conditioned taste aversion (CTA) task and that this region is involved in the retrieval of recent and remote long-term CTA memory. In addition, both NMDA receptor and CaMKII activity in dorsal mPFC are needed for CTA memory consolidation, highlighting the complexity of mPFC functions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Transactive response DNA-binding protein 43 (TDP-43) mislocalization and aggregation are hallmark features of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). We have previously shown in mice that inducible overexpression of a cytoplasmically localized form of TDP-43 (TDP-43-ΔNLS) in forebrain neurons evokes neuropathological changes that recapitulate several features of TDP-43 proteinopathies. Detailed behavioral phenotyping could provide further validation for its usage as a model for FTD. In the present study, we performed a battery of behavioral tests to evaluate motor, cognitive, and social phenotypes in this model. We found that transgene (Tg) induction by doxycycline removal at weaning led to motor abnormalities including hyperlocomotion in the open field test, impaired coordination and balance in the rotarod test, and increased spasticity as shown by a clasping phenotype. Cognitive assessment demonstrated impaired recognition and spatial memory, measured by novel object recognition and Y-maze tests. Remarkably, TDP-43-ΔNLS mice displayed deficits in social behavior, mimicking a key aspect of FTD. To determine whether these symptoms were reversible, we suppressed Tg expression for 14 d in 1.5-month-old mice showing an established behavioral phenotype but modest neurodegeneration and found that motor and cognitive deficits were ameliorated; however, social performance remained altered. When Tg expression was suppressed in 6.5-month-old mice showing overt neurodegeneration, motor deficits were irreversible. These results indicate that TDP-43-ΔNLS mice display several core behavioral features of FTD with motor neuron disease, possibly due to functional changes in surviving neurons, and might serve as a valuable tool to unveil the underlying mechanisms of this and other TDP-43 proteinopathies., (Copyright © 2014 the authors 0270-6474/14/3415244-16$15.00/0.)

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by cytoplasmic protein aggregates in the brain and spinal cord that include TAR-DNA binding protein 43 (TDP-43). TDP-43 is normally localized in the nucleus with roles in the regulation of gene expression, and pathological cytoplasmic aggregates are associated with depletion of nuclear protein. Here, we generated transgenic mice expressing human TDP-43 with a defective nuclear localization signal in the forebrain (hTDP-43-ΔNLS), and compared them with mice expressing WT hTDP-43 (hTDP-43-WT) to determine the effects of mislocalized cytoplasmic TDP-43 on neuronal viability. Expression of either hTDP-43-ΔNLS or hTDP-43-WT led to neuron loss in selectively vulnerable forebrain regions, corticospinal tract degeneration, and motor spasticity recapitulating key aspects of FTLD and primary lateral sclerosis. Only rare cytoplasmic phosphorylated and ubiquitinated TDP-43 inclusions were seen in hTDP-43-ΔNLS mice, suggesting that cytoplasmic inclusions were not required to induce neuronal death. Instead, neurodegeneration in hTDP-43 and hTDP-43-ΔNLS-expressing neurons was accompanied by a dramatic downregulation of the endogenous mouse TDP-43. Moreover, mice expressing hTDP-43-ΔNLS exhibited profound changes in gene expression in cortical neurons. Our data suggest that perturbation of endogenous nuclear TDP-43 results in loss of normal TDP-43 function(s) and gene regulatory pathways, culminating in degeneration of selectively vulnerable affected neurons.

The disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS) was identified recently as the TDP-43 (TAR DNA-binding protein 43), thereby providing a molecular link between these two disorders. In FTLD-U and ALS, TDP-43 is redistributed from its normal nuclear localization to form cytoplasmic insoluble aggregates. Moreover, pathological TDP-43 is abnormally ubiquitinated, hyperphosphorylated, and N-terminally cleaved to generate C-terminal fragments (CTFs). However, the specific cleavage site(s) and the biochemical properties as well as the functional consequences of pathological TDP-43 CTFs remained unknown. Here we have identified the specific cleavage site, Arg(208), of a pathological TDP-43 CTF purified from FTLD-U brains and show that the expression of this and other TDP-43 CTFs in cultured cells recapitulates key features of TDP-43 proteinopathy. These include the formation of cytoplasmic aggregates that are ubiquitinated and abnormally phosphorylated at sites found in FTLD-U and ALS brain and spinal cord samples. Furthermore, we observed splicing abnormalities in a cell culture system expressing TDP-43 CTFs, and this is significant because the regulation of exon splicing is a known function of TDP-43. Thus, our results show that TDP-43 CTF expression recapitulates key biochemical features of pathological TDP-43 and support the hypothesis that the generation of TDP-43 CTFs is an important step in the pathogenesis of FTLD-U and ALS.

TAR DNA-binding protein (TDP-43) has been recently described as a major pathological protein in both frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. However, little is known about the relative abundance and distribution of different pathological TDP-43 species, which include hyperphosphorylated, ubiquitinated, and N-terminally cleaved TDP-43. Here, we developed novel N-terminal (N-t) and C-terminal (C-t)-specific TDP-43 antibodies and performed biochemical and immunohistochemical studies to analyze cortical, hippocampal, and spinal cord tissue from frontotemporal dementia with ubiquitin-positive inclusions and amyotrophic lateral sclerosis cases. C-t-specific TDP-43 antibodies revealed similar abundance, morphology, and distribution of dystrophic neurites and neuronal cytoplasmic inclusions in cortex and hippocampus compared with previously described pan-TDP-43 antibodies. By contrast, N-t-specific TDP-43 antibodies only detected a small subset of these lesions. Biochemical studies confirmed the presence of C-t TDP-43 fragments but not extreme N-t fragments. Surprisingly, immunohistochemical analysis of inclusions in spinal cord motor neurons in both diseases showed that they are N-t and C-t positive. TDP-43 inclusions in Alzheimer's disease brains also were examined, and similar enrichment in C-t TDP-43 fragments was observed in cortex and hippocampus. These results show that the composition of the inclusions in brain versus spinal cord tissues differ, with an increased representation of C-t TDP-43 fragments in cortical and hippocampal regions. Therefore, regionally different pathogenic processes may underlie the development of abnormal TDP-43 proteinopathies.

TAR DNA-binding protein 43 (TDP-43) is the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Although normal TDP-43 is a nuclear protein, pathological TDP-43 is redistributed and sequestered as insoluble aggregates in neuronal nuclei, perikarya, and neurites. Here we recapitulate these pathological phenotypes in cultured cells by altering endogenous TDP-43 nuclear trafficking and by expressing mutants with defective nuclear localization (TDP-43-DeltaNLS) or nuclear export signals (TDP-43-DeltaNES). Restricting endogenous cytoplasmic TDP-43 from entering the nucleus or preventing its exit out of the nucleus resulted in TDP-43 aggregate formation. TDP-43-DeltaNLS accumulates as insoluble cytoplasmic aggregates and sequesters endogenous TDP-43, thereby depleting normal nuclear TDP-43, whereas TDP-43-DeltaNES forms insoluble nuclear aggregates with endogenous TDP-43. Mutant forms of TDP-43 also replicate the biochemical profile of pathological TDP-43 in FTLD-U/ALS. Thus, FTLD-U/ALS pathogenesis may be linked mechanistically to deleterious perturbations of nuclear trafficking and solubility of TDP-43.

Pathological TDP-43 is the major disease protein in frontotemporal lobar degeneration characterized by ubiquitin inclusions (FTLD-U) with/without motor neuron disease (MND) and in amyotrophic lateral sclerosis (ALS). As Guamanian parkinsonism-dementia complex (PDC) or Guamanian ALS (G-PDC or G-ALS) of the Chamorro population may present clinically similar to FTLD-U and ALS, TDP-43 pathology may be present in the G-PDC and G-ALS. Thus, we examined cortical or spinal cord samples from 54 Guamanian subjects for evidence of TDP-43 pathology. In addition to cortical neurofibrillary and glial tau pathology, G-PDC was associated with cortical TDP-43 positive dystrophic neurites and neuronal and glial inclusions in gray and/or white matter. Biochemical analyses showed the presence of FTLD-U-like insoluble TDP-43 in G-PDC, but not in Guam controls (G-C). Spinal cord pathology of G-PDC or G-ALS was characterized by tau positive tangles as well as TDP-43 positive inclusions in lower motor neurons and glial cells. G-C had variable tau and negligible TDP-43 pathology. These results indicate that G-PDC and G-ALS are associated with pathological TDP-43 similar to FTLD-U with/without MND as well as ALS, and that neocortical or hippocampal TDP-43 pathology distinguishes controls from disease subjects better than tau pathology. Finally, we conclude that the spectrum of TDP-43 proteinopathies should be expanded to include neurodegenerative cognitive and motor diseases, affecting the Chamorro population of Guam.

TDP-43 was recently identified as the major disease protein in neuronal inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). TDP-43 becomes redistributed from the nucleus to the cytoplasm, ubiquitinated, hyperphosphorylated and cleaved to generate C-terminal fragments, thereby linking mismetabolism of TDP-43 to the pathogenesis of FTLD-U. The function of TDP-43 is unclear, however it has been shown that TDP-43 might act as transcription repressor and activator of exon skipping through interaction with proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family as well as a scaffold for nuclear bodies through interactions with survival motor neuron protein. To investigate whether these binding partners might be associated with TDP-43 pathology, we studied the expression and localization of proteins of the hnRNP family (hnRNP A1, A2/B1, C1/C2) and SMN protein in affected brain regions in patients with sporadic and familial FTLD-U and normal control brains by immunohistochemistry and biochemical analysis. In contrast to TDP-43, no changes in subcellular distribution, no labeling of pathologic inclusions and no biochemical alterations were detectable for the tested hnRNPs and SMN in FTLD-U brains compared to controls. These results argue against a role of these binding partners in the pathogenesis of FTLD-U and emphasize the specificity of TDP-43 as marker for FTLD-U pathology.

It is widely accepted that the formation of long-term memory (LTM) requires mRNA translation, but little is known about the cellular mechanisms in the brain that regulate this process. Mammalian target of rapamycin (mTOR) is a key regulator of translational efficacy and capacity. Here, we show that LTM formation of one-trial inhibitory avoidance (IA) in rats, a hippocampus-dependent fear-motivated learning task, requires mTOR activation. IA training is specifically associated with a rapid increase in the phosphorylation state of mTOR and its substrate ribosomal S6 kinase (p70S6K). Bilateral intra-CA1 infusion of rapamycin, a selective mTOR inhibitor, 15 min before, but not immediately after training completely hinders IA LTM without affecting short-term memory (STM) retention. Therefore, our findings indicate that the regulation of hippocampal mRNA translation is a major control step in memory consolidation.

Persistence is the most characteristic attribute of long-term memory (LTM). To understand LTM, we must understand how memory traces persist over time despite the short-lived nature and rapid turnover of their molecular substrates. It is widely accepted that LTM formation is dependent upon hippocampal de novo protein synthesis and Brain-Derived Neurotrophic Factor (BDNF) signaling during or early after acquisition. Here we show that 12 hr after acquisition of a one-trial associative learning task, there is a novel protein synthesis and BDNF-dependent phase in the rat hippocampus that is critical for the persistence of LTM storage. Our findings indicate that a delayed stabilization phase is specifically required for maintenance, but not formation, of the memory trace. We propose that memory formation and memory persistence share some of the same molecular mechanisms and that recurrent rounds of consolidation-like events take place in the hippocampus for maintenance of the memory trace.

1. According to its duration there are, at least, two major forms of memory in mammals: short term memory (STM) which develops in a few seconds and lasts several hours and long-term memory (LTM) lasting days, weeks and even a lifetime. In contrast to LTM, very little is known about the neural, cellular and molecular requirements for mammalian STM formation. 2. Here we show that early activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in the hippocampus is required for the establishment of STM for a one-trial inhibitory avoidance task in the rat. Immediate posttraining infusion of U0126 (a selective inhibitor of ERK kinase) into the CA1 region of the dorsal hippocampus blocked STM formation. 3. Reversible inactivation of the entorhinal cortex through muscimol infusion produced deficits in STM and a selective and rapid decrease in hippocampal ERK2 activation.4. Together with our previous findings showing a rapid decrease in ERK2 activation and impaired STM after blocking BDNF function, the present results strongly suggest that ERK2 signaling in the hippocampus is a critical step in STM processing.

Most studies regarding altered gene expression after learning are performed using multi-trial tasks, which do not allow a clear discrimination of memory acquisition, consolidation and retrieval. We screened for candidate memory-modulated genes in the hippocampus at 3 and 24 h after one-trial inhibitory avoidance (IA) training, using a cDNA array containing 1176 genes. While 33 genes were modulated by training (respect to shocked-only animals), most of them were upregulated (27 genes) and only 6 were downregulated. To confirm and extend these findings, we performed RT-PCRs and analyzed differences in protein levels in rat hippocampus using immunoblot assays. We found several proteins upregulated 24 h after training: extracellular signal-regulated kinase ERK2, Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha), Syntaxin 1a, c-fos and Homer 1a. The total level of none of these proteins were found to be altered when measured 3-h post-training. Several of the mRNAs corresponding to the upregulated proteins were changed at 3 h but not 24 h. Additionally, a number of other candidates were identified for the first time as modulated by learning. The results presented here suggest that single-trial tasks can expose previously unseen differences in dynamic regulation of gene expression after behavioral manipulations, both at the transcriptional and translational levels, and reveal a diversity of gene products modulated by this task, allowing deeper understanding of the molecular basis of memory formation.

For several decades, neuroscientists have provided many clues that point out the involvement of de novo gene expression during the formation of long-lasting forms of memory. However, information regarding the transcriptional response networks involved in memory formation has been scarce and fragmented. With the advent of genome-based technologies, combined with more classical approaches (i.e., pharmacology and biochemistry), it is now feasible to address those relevant questions--which gene products are modulated, and when that processes are necessary for the proper storage of memories--with unprecedented resolution and scale. Using one-trial inhibitory (passive) avoidance training of rats, one of the most studied tasks so far, we found two time windows of sensitivity to transcriptional and translational inhibitors infused into the hippocampus: around the time of training and 3-6 h after training. Remarkably, these periods perfectly overlap with the involvement of hippocampal cAMP/PKA (protein kinase A) signaling pathways in memory consolidation. Given the complexity of transcriptional responses in the brain, particularly those related to processing of behavioral information, it was clearly necessary to address this issue with a multi-variable, parallel-oriented approach. We used cDNA arrays to screen for candidate inhibitory avoidance learning-related genes and analyze the dynamic pattern of gene expression that emerges during memory consolidation. These include genes involved in intracellular kinase networks, synaptic function, DNA-binding and chromatin modification, transcriptional activation and repression, translation, membrane receptors, and oncogenes, among others. Our findings suggest that differential and orchestrated hippocampal gene expression is necessary in both early and late periods of long-term memory consolidation. Additionally, this kind of studies may lead to the identification and characterization of genes that are relevant for the pathogenesis of complex psychiatric disorders involving learning and memory impairments, and may allow the development of new methods for the diagnosis and treatment of these diseases.

Rats with cannulae in the dorsal CA1 region of the hippocampus were trained in one-trial step-down inhibitory avoidance, and submitted to four consecutive daily test sessions without the footshock. This produced extinction of the conditioned response in control animals. The bilateral infusion into the CA1 region of the dorsal hippocampus of two different inhibitors of gene transcription, DRB (80 microg/side) or alpha-amanitin (25 pg/side), or of the protein synthesis inhibitor, anisomycin (80 microg/side) blocked extinction of the CR. The treatments were effective when given 15 min before, but not 1 or 3h after the first test session. Retrieval itself was not affected by the drugs. The treatments did not affect general activity in an open field or anxiety levels measured in an elevated plus maze. The data indicate that gene transcription and protein synthesis are necessary at the time of the first test session in order to generate extinction. These requirements are to be expected from learning that involves new synaptic associations.

Information storage in the brain is a temporally graded process involving different memory types or phases. It has been assumed for over a century that one or more short-term memory (STM) processes are involved in processing new information while long-term memory (LTM) is being formed. It has been repeatedly reported that LTM requires de novo RNA synthesis around the time of training. Here we show that LTM formation of a one-trial inhibitory avoidance training in rats, a hippocampal-dependent form of contextual fear conditioning, depends on two consolidation periods requiring synthesis of new mRNAs. By injecting the RNA polymerase II inhibitors 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole or alpha-amanitin into the CA1 region of the dorsal hippocampus at various times before and after training, we found that hippocampal gene expression is critical in two time windows: around the time of training and 3-6 hr after training. Interestingly, these two periods of sensitivity to transcriptional inhibitors are similar to those observed using the protein synthesis inhibitor anisomycin. These findings underscore the parallel dependence of LTM formation of contextual fear on mRNA and protein synthesis in the hippocampus and suggest that the two time periods of anisomycin-induced amnesia depend at least in part on new mRNA synthesis.

The main communicators between the neuroendocrine and immune systems are cytokines and hormones. We studied the molecular interaction between immune activators (cytokines and T-cell receptors [TCRs]) and the glucocorticoid receptor (GR) in cells in which glucocorticoids play a key regulatory function: (1) cellular targets of TNF-induced cytotoxicity; (2) the pituitary gland; and (3) thymic cells. Cytokines (TNF-alpha and IL-1) increase glucocorticoid-induced transcriptional activity of the GR via the DNA-glucocorticoid response elements (GREs) in cells transfected with a glucocorticoid-inducible reporter plasmid. As a functional physiological correlate, priming of fibroblastic cells with a low dose of TNF significantly increases the sensitivity to glucocorticoid inhibition of TNF-induced apoptosis (without involving NF-kappa B). Priming of AtT-20 mouse corticotrophs and Cushing pituitary cells with IL-1 increases the sensitivity to glucocorticoid inhibition of CRH-induced ACTH/POMC expression. In thymocytes, activation of the T-cell receptor counteracts the glucocorticoid-induced thymic apoptosis by downregulating the glucocorticoid action on GRE-driven apoptotic genes. Thus, cytokines and immune mediators prevent their own deleterious effects not only by stimulating glucocorticoid production, but also by modifying the sensitivity of the target cells for the glucocorticoid counter-regulatory action. The functional cross-talk at the molecular level between immune signals and glucocorticoids is essential to determine the biological response to both mediators and constitutes the ultimate level of interaction between the immune and neuroendocrine mediators.