Multiple forms of activity-dependent competition refine hippocampal circuits in vivo

Efficient memory formation relies on the establishment of functional hippocampal circuits. It has been proposed that synaptic connections are refined by neural activity to form functional brain circuitry. However, it is not known whether and how hippocampal connections are refined by neural activity in vivo. Using a mouse genetic system in which restricted populations of neurons in the hippocampal circuit are inactivated, we show that inactive axons are eliminated after they develop through a competition with active axons. Remarkably, in the dentate gyrus, which undergoes neurogenesis throughout life, axon refinement is achieved by a competition between mature and young neurons. These results demonstrate that activity-dependent competition plays multiple roles in the establishment of functional memory circuits in vivo.     

The identification of cDNA clones that include the 3′ end of mRNA

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3′ end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.     

Identification of thyroid regulatory elements in the Na-K-ATPase α3 gene promoter

A –1027 bp to +108 bp region of Na-K-ATPase ₃ gene promoter has been searched for the presence of thyroid response elements (TRE). Computer analysis of this sequence using a consensus TRE sequence revealed the presence of four putative TRE rich regions referred to as regions I (–636 to –457 bp), II (–218 to –106 bp), III (–106 to –6 bp) and IV (–6 to +108 bp). Cotransfection of the luciferase linked full length construct as well as constructs progressively devoid of the TRE rich regions in Cos1 cells revealed that regions I and III are positively regulated by T ₃ whereas there are some sequences in region II which can suppress the positive regulatory effect of region III but not of region I, TRE IV seems to have no functional role. EMSA of the three functional TRE rich regions (I, II and III) showed strong and specific interaction with thyroid hormone receptor (TR) cloned and expressed in baculovirus. The overall results suggest the regulation of Na-K-ATPase ₃ gene by T ₃ is complex involving several thyroidal regulatory elements.     

Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene

Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the -galactosidase expression pattern in transformed lines carrying different lengths of 5 flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5 flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5 flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5 flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5 flanking DNA is not necessary for embryonic survival and development to adult flies. Correspondence to: P.M. Salvaterra     

3′-UTR polymorphism of dopamine transporter gene in Hadza and Datoga males

A study of VNTR polymorphism and molecular structure of 3′-UTR of the dopamine transporter gene (DAT1/SLC6A3) was performed in the Hadza and Datoga males. It was shown that Hadza and Datoga differed in allele and genotype frequencies. Allele with 9 repeats in 3′-UTR, as well as the DAT1 homozygous genotype 9/9, is more common in the Hadza. The allele with ten repeats, as well as the homozygous phenotype 10/10, is more common in the Datoga. Molecular structure of DAT1 alleles with 3, 8, and 12 repeats was determined for the first time. In addition, it was found that the DAT1 allele with 11 repeats in the Datoga significantly differed in the type and arrangement of repeats from those previously described in other populations. We suggest that variations in the repeats number and type in the 3′-UTR of allelic variants may affect the dopamine transporter gene function.     

β-amyloid-induced changes in calcium homeostasis in cultured hippocampal neurons of the rat

A two-wave technique of calciometry with the use of a fluorescence dye, fura-2/AM, was applied for examination of the effect of a protein, β-amyloid (the main component of senile plaques in Alzheimer’s disease), on calcium homeostasis in cultured neurons of the rat hippocampus; β-amyloid was added to the culture medium. In most neurons, the effect of β-amyloid appeared as a more than twofold increase in the basic calcium concentration, as compared with the control (153.4 ± 11.5 and 71.7 ± 5.4 nM, respectively; P < 0.05). The characteristics of calcium transients induced by application of hyperpotassium solution also changed; the amplitude of these transients decreased, and the duration of a part corresponding to calcium release from the cell (rundown of the transient) increased. The mean amplitude of calcium transients under control conditions was 447.5 ± 20.1 nM, while after incubation in the presence of β-amyloid this index dropped to 278.4 ± 22.6 nM. Under control conditions, the decline phase of calcium transients lasted, on average, 100 ± 6 sec, while after incubation of hippocampal cell cultures in the presence of β-amyloid this phase lasted 250 ± 10 sec. Therefore, an excess of β-amyloid influences significantly calcium homeostasis in the nerve cells by disturbing functions of the calcium-controlling systems, such as voltage-operated calcium channels of the plasma membrane and calcium stores of the mitochondria and endoplasmic reticulum. Neirofiziologiya/Neurophysiology, Vol. 40, No. 1, pp. 9–12, January–February, 2008.     

Estrogen-induced upregulation and 3′-UTR shortening of CDC6

3′-Untranslated region (UTR) shortening of mRNAs via alternative polyadenylation (APA) has important ramifications for gene expression. By using proximal APA sites and switching to shorter 3′-UTRs, proliferating cells avoid miRNA-mediated repression. Such APA and 3′-UTR shortening events may explain the basis of some of the proto-oncogene activation cases observed in cancer cells. In this study, we investigated whether 17 β-estradiol (E2), a potent proliferation signal, induces APA and 3′-UTR shortening to activate proto-oncogenes in estrogen receptor positive (ER+) breast cancers. Our initial probe based screen of independent expression arrays suggested upregulation and 3′-UTR shortening of an essential regulator of DNA replication, CDC6 (cell division cycle 6), upon E2 treatment. We further confirmed the E2- and ER-dependent upregulation and 3′UTR shortening of CDC6, which lead to increased CDC6 protein levels and higher BrdU incorporation. Consequently, miRNA binding predictions and dual luciferase assays suggested that 3′-UTR shortening of CDC6 was a mechanism to avoid 3′-UTR-dependent negative regulations. Hence, we demonstrated CDC6 APA induction by the proliferative effect of E2 in ER+ cells and provided new insights into the complex regulation of APA. E2-induced APA is likely to be an important but previously overlooked mechanism of E2-responsive gene expression.     

Neuroprotective effect of ketamine against TNF-α-induced necroptosis in hippocampal neurons

Tumour necrosis factor-α (TNF-α), a crucial cytokine, has various homeostatic and pathogenic bioactivities. The aim of this study was to assess the neuroprotective effect of ketamine against TNF-α-induced motor dysfunction and neuronal necroptosis in male C57BL/6J mice in vivo and HT-22 cell lines in vitro. The behavioural testing results of the present study indicate that ketamine ameliorated TNF-α-induced neurological dysfunction. Moreover, immunohistochemical staining results showed that TNF-α-induced brain dysfunction was caused by necroptosis and microglial activation, which could be attenuated by ketamine pre-treatment inhibiting reactive oxygen species production and mixed lineage kinase domain-like phosphorylation in hippocampal neurons. Therefore, we concluded that ketamine may have neuroprotective effects as a potent inhibitor of necroptosis, which provides a new theoretical and experimental basis for the application of ketamine in TNF-α-induced necroptosis-associated diseases.     

Conserved elements in the 3′ untranslated regions of c-fos and actin mRNAs

In this study, the nucleotide sequences of the 3 untranslated regions (UTR) of the mouse and human c-fos genes, and the rat and human -actin genes were examined. It is shown (i) that the 3 UTR of c-fos is highly conserved between mouse and man, (ii) that multiple copies of a 12 bp element occur, in clusters, in the 3 UTR both of c-fos and of -actin. This conserved 12 bp element is analogous to the putative repressor binding site previously identified (Renan,Bioscience Reports,5 (1985), 739–753). These findings provide additional support for the proposal that regulatory signals are located in the 3 UTR's of certain genes.     

Identification of elements in human long 3′ UTRs that inhibit nonsense-mediated decay

The nonsense-mediated mRNA decay (NMD) pathway serves an important role in gene expression by targeting aberrant mRNAs that have acquired premature termination codons (PTCs) as well as a subset of normally processed endogenous mRNAs. One determinant for the targeting of mRNAs by NMD is the occurrence of translation termination distal to the poly(A) tail. Yet, a large subset of naturally occurring mRNAs contain long 3′ UTRs, many of which, according to global studies, are insensitive to NMD. This raises the possibility that such mRNAs have evolved mechanisms for NMD evasion. Here, we analyzed a set of human long 3′ UTR mRNAs and found that many are indeed resistant to NMD. By dissecting the 3′ UTR of one such mRNA, TRAM1 mRNA, we identified a cis element located within the first 200 nt that inhibits NMD when positioned in downstream proximity of the translation termination codon and is sufficient for repressing NMD of a heterologous reporter mRNA. Investigation of other NMD-evading long 3′ UTR mRNAs revealed a subset that, similar to TRAM1 mRNA, contains NMD-inhibiting cis elements in the first 200 nt. A smaller subset of long 3′ UTR mRNAs evades NMD by a different mechanism that appears to be independent of a termination-proximal cis element. Our study suggests that different mechanisms have evolved to ensure NMD evasion of human mRNAs with long 3′ UTRs.