The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased—rather than monoallelic—expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain.DOI: http://dx.doi.org/10.7554/eLife.07860.001
By using differential display PCR, we have identified 98 cDNA fragments from the rat dorsal hippocampus that are expressed differentially between the fast learners and slow learners in the water maze learning task. One of these cDNA fragments encodes the rat serum-and glucocorticoid-inducible kinase (sgk) gene. Northern blot analysis revealed that the sgk mRNA level was approximately 4-fold higher in the hippocampus of fast learners than slow learners. In situ hybridization results indicated that sgk mRNA level was increased markedly in CA1, CA3, and dentate gyrus of hippocampus in fast learners. Transient transfection of the sgk mutant DNA to the CA1 area impaired, whereas transfection of the sgk wild-type DNA facilitated water maze performance in rats. These results provide direct evidence that enhanced sgk expression facilitates memory consolidation of spatial learning in rats. These results also elucidate the molecular mechanism of glucocorticoidinduced memory facilitation in mammals. It is well accepted that long-term memory formation requires de novo RNA and protein synthesis. Evidence supporting this notion comes from the results that inhibition of mRNA and protein synthesis impairs long-term memory formation in various behavioral tasks in rats (1-3). This evidence suggests that neural activities associated with learning leads to the expression of various genes, and the protein products of these genes play important roles in the process of memory formation. Extensive efforts have been made to identify genes that are associated specifically with certain forms of learning and memory. For example, by using two-dimensional gel analysis, several candidate proteins have been identified that are related to long-term sensitization of the gill-withdrawal reflex in Aplysia (4). Screening in Drosophila mutants has yielded approximately 10 genes that are associated with the process of olfactory learning and memory (5). Further, by using a double-labeling method, proteins that show increased glycosylation as a result of training were identified in rats (6). The methods used in the above studies are effective; however, identifying and characterizing these genes takes a long time when using these methods. In addition, most of these studies were carried out in the invertebrate, in which the neuronal circuits and genes involved in memory formation probably are different from that in the vertebrate.In a more recent study, by using differential display-PCR (DD-PCR), we have successfully identified genes that are associated specifically with memory formation of one-way inhibitory avoidance learning in rats (7,8). Some of these results were confirmed further by a gene-knockout study (9). These results suggest that DD-PCR is an effective method in identifying genes that are involved in complex forms of learning and memory in the vertebrate. Therefore, we have adopted the same strategy in the present study to identify genes that are associated specifically with memory formation of spatial learning in rats. The Morris water maze...
Tyrosine kinase phosphorylation plays an important role in the induction of long-term potentiation (LTP). Focal adhesion kinase (FAK) is a 125 kDa nonreceptor tyrosine kinase that shows decreased phosphorylation in fyn mutant mice, and Fyn plays a critical role in LTP induction. By examining the role of FAK involved in LTP induction in dentate gyrus in vivo with medial perforant path stimulation, we found that both FAK and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation were increased significantly 5 and 10 min after LTP induction, whereas cAMP-responsive element binding protein (CREB) phosphorylation was increased 40 min later. Transfection of the dominant-negative FAK mutant construct HA-FAK(Y397F) impaired LTP, whereas transfection of the constitutively activated form HA-FAK(Delta1-100) reduced the threshold for LTP induction. Transfection of HA-FAK(Delta1-100) by itself did not induce long-lasting potentiation. Further, transfection of the HA-FAK(Y397F) construct decreased FAK, MAPK/ERK, and CREB phosphorylation, and the inhibition of MAPK/ERK decreased CREB phosphorylation. Moreover, blockade of NMDA receptor (NMDAR) did not decrease FAK, MAPK/ERK, and CREB phosphorylation although LTP induction was blunted by NMDAR antagonist. These biochemical changes were not associated with low-frequency stimulation either. Immunoprecipitation results revealed that tyrosine phosphorylation of NR2A and NR2B as well as the association of phosphorylated FAK with NR2A and NR2B was increased with LTP induction. These results together suggest that FAK is required, but not sufficient, for the induction of LTP in a NMDAR-independent manner and that MAPK/ERK and CREB are the downstream events of FAK activation. Further, FAK may interact with NR2A and NR2B to modulate LTP induction.
The process of long-term potentiation (LTP) consists of the early induction and late maintenance phases. Few studies have examined the cellular mechanisms underlying these two phases; their respective mRNA expression profiles have not yet been elucidated. Here we used the technique of PCR differential display to identify genes that are differentially expressed between the early and late phases of LTP in vivo. Our results indicated that the cDNA fragment corresponding to one mRNA with preferentially increased expression during the early, but not late, phase of LTP encodes the rat thyroid hormone-responsive protein (THRP) gene. In situ hybridization analysis confirmed the results obtained from the PCR differential display. Prior NMDA receptor blockade with MK801 prevented induction of LTP and decreased THRP mRNA expression in the dentate gyrus, as assayed by quantitative RT-PCR analysis. THRP antisense oligonucleotide treatment before tetanic stimulation also prevented induction of LTP. However, when THRP antisense oligonucleotide was administered after induction of LTP, it did not affect expression and maintenance of LTP. THRP is known to be responsive to thyroid hormone. Our results indicate that direct thyroid hormone (T3) injection into the dentate gyrus produces a long-lasting enhancement of synaptic efficacy of these neurons. T3 injection also markedly increased THRP mRNA expression in the dentate gyrus. Taken together, our results suggest that THRP mRNA expression plays an important role in the early phase, but not the late phase, of LTP and that both THRP and thyroid hormone are involved in synaptic plasticity in hippocampal neurons.
Systemic and intracellular metabolic states are critical factors affecting immune cell functions. The metabolic regulator AMP‐activated protein kinase (AMPK) senses AMP levels and mediates cellular responses to energy‐restrained conditions. The ubiquitously expressed AMPK participates in various biological functions in numerous cell types, including innate immune cell macrophages and osteoclasts, which are their specialized derivatives in bone tissues. Previous studies have demonstrated that the activation of AMPK promotes macrophage polarization toward anti‐inflammatory M2 status. Additionally, AMPK acts as a negative regulator of osteoclastogenesis, and upregulation of AMPK disrupts the differentiation of osteoclasts. However, the regulation and roles of AMPK in differentiated osteoclasts have not been characterized. Here, we report that inflammatory stimuli‐regulated‐AMPK activation of differentiated and undifferentiated osteoclasts in opposite ways. Lipopolysaccharide (LPS) inhibited the phosphorylation of AMPK in macrophages and undifferentiated osteoclasts, but it activated AMPK in differentiated osteoclasts. Inactivating AMPK decreased cellular responses against the activation of toll‐like receptor signaling, including the transcriptional activation of proinflammatory cytokines and the bone resorption genes TRAP, and MMP9. The elevation of bone resorption by LPS stimulation was disrupted by AMPK inhibitor, indicating the pivotal roles of AMPK in inflammation‐induced activities in differentiated osteoclasts. The AMPK activator metformin did not increase proinflammatory responses, possibly because other factors are also required for this regulation. Notably, changing the activation status of AMPK did not alter the expression levels of bone resorption genes in unstimulated osteoclasts, indicating the essential roles of AMPK in cellular responses to inflammatory stimuli but not in the maintenance of basal levels. Unlike its M2‐polarizing roles in macrophages, AMPK was not responsive to the M2 stimulus of interleukin‐4. Our observations revealed differences in the cellular properties of macrophages and osteoclasts as well as the complexity of regulatory mechanisms for osteoclast functions.
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