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The learning abilities of planarian worms (Dugesia tigrina) were assessed by using a number of Pavlovian conditioning paradigms. Experiment 1 showed that planaria were susceptible to basic conditioning in that they readily developed a conditioned response to a change in ambient luminance when it was consistently paired with an electric shock over a number of trials. In Experiment 2, the change in luminance was presented in a compound with a vibration stimulus during conditioning. Subsequent tests revealed poor conditioning of the elements compared with control groups in which the animals were conditioned in the presence of the elements alone, an instance of overshadowing. In Experiment 3, pre-training of one of the elements before compound conditioning resulted in blocking of learning about the other element. These results add to other studies that have reported cue competition effects in animal species belonging to different phyla (chordate, mollusk, arthropod), suggesting that learning in these phyla could be ruled by similar principles. The results are discussed adopting an evolutionary-comparative approach.

Evidence suggests that the acquisition of recognition memory depends upon CREB-dependent long-lasting changes in synaptic plasticity in the perirhinal cortex. The CREB-responsive microRNA miR-132 has been shown to regulate synaptic transmission and we set out to investigate a role for this microRNA in recognition memory and its underlying plasticity mechanisms. To this end we mediated the specific overexpression of miR-132 selectively in the rat perirhinal cortex and demonstrated impairment in short-term recognition memory. This functional deficit was associated with a reduction in both long-term depression and long-term potentiation. These results confirm that microRNAs are key coordinators of the intracellular pathways that mediate experience-dependent changes in the brain. In addition, these results demonstrate a role for miR-132 in the neuronal mechanisms underlying the formation of short-term recognition memory.

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

Heat shock proteins (HSPs) are associated with the proteinaceous inclusions that characterise many neurodegenerative diseases. This suggests they may be associated with disease aetiology and/or represents an attempt to remove abnormal protein aggregates. In this study the adenoviral mediated over-expression of HSP70 interacting protein (HIP) alone was shown to significantly reduce inclusion formation in both an in vitro model of Spinal Bulbar Muscular Atrophy and a primary neuronal model of polyglutamine disease. Experiments to determine the mechanism of action showed that: denatured luciferase activity (a measure of protein refolding) was not increased in the presence of HIP alone but was increased when HIP was co-expressed with HSP70 or Heat Shock cognate protein 70 (HSC70); the expression of polyglutamine inclusions in cortical neurons mediated an increase in the levels of HSC70 but not HSP70. Our data suggest that HIP may prevent inclusion formation by facilitating the constitutive HSC70 refolding cycle and possibly by preventing aggregation. HIP expression is not increased following stress and its over-expression may therefore reduce toxic polyglutamine aggregation events and contribute to an effective therapeutic strategy.

Background: “Mirrored” or complementary mammalian miRNAs have been predicted but none have yet been characterized. Results: miR-3120 and miR-214 are produced from the same intronic locus; miR-3120 regulates heat shock cognate protein 70 (Hsc70) and auxilin expression and vesicle uncoating. Conclusion: miR-3120 is a mirror miRNA regulating endocytic function. Significance: Mirror miRNAs are important mammalian gene regulatory units, and many more remain to be found and characterized., We show that a single gene locus gives rise to two fully processed and functional miRNAs, i.e. that due to imperfect base pairing, two distinct microRNAs (miRNAs) can be produced from the fully complementary DNA strands. The antisense strand encodes miR-214, which is transcribed by its own promoter, whereas a novel miRNA, miR-3120, is co-expressed with its host gene mRNA. We also found that miR-3120 regulates important aspects of cellular function that are similar to that of its host gene, dynamin-3. miR-3120 was found to be located in neuronal cell bodies and to target Hsc70 and auxilin, and its lentivirus-mediated expression inhibited the uncoating of clathrin-coated vesicles. Finally, mirror miRNAs are likely to represent a new group of miRNAs with complex roles in coordinating gene expression.

We describe the characteristics of neuron specific adenoviral (Ad) and lentiviral (LV) vectors that make them particularly suited to the study of (hsp70, hsp40 and hsj1a) gene function in neurons. Ad vectors are particularly well suited to in vitro studies and we demonstrate that the overexpression of Hsp70 and Hsp40 in primary hippocampal neurons increased luciferase activity following a denaturing stress. Furthermore, the Ad mediated expression of Hsp70 and Hsp40 was shown to protect hippocampal neurons from a denaturing excitotoxic stress. An example of how RNA interference (RNAi) technology can be combined with Ad vector delivery to mediate the effective knockdown of chaperone genes is also given. Lentiviral vectors are best suited to in vivo studies and we demonstrate they can be used to generate animal models of neurodegenerative disease. In this example LV vectors expressing EGFP-tagged polyglutamine constructs were injected into the striatum to generate a chronic model of polyglutamine disease. Furthermore, the lentiviral-mediated expression of Hsj1A was shown to decrease the number of neurons containing inclusions. These and other studies suggest that unlike other HSP40 members Hsj1A does not promote chaperone activity but instead ubiquityates proteins and hence targets them for degradation via the ubiquitin proteosome system

The anti-apoptotic effects of heat-shock protein (Hsp70) were assessed in SCG neurones following nerve growth factor (NGF) withdrawal. The results showed that the virally mediated expression of Hsp70 mirrored the effects of the c-Jun-N-terminal kinase (JNK) binding domain (JBD) of JNK interacting protein (an inhibitor of JNK and c-Jun activation) and suppressed the phosphorylation of c-Jun. Preventing c-Jun transcriptional activity subsequently led to reduced cytochrome c release and prevented caspase activation as indicated by a decrease in poly (ADP-ribose) polymerase-1 (PARP) cleavage. Together, these results show that Hsp70 is a highly effective inhibitor of apoptosis in sympathetic neurones and that it mediates this effect primarily by suppressing c-Jun transcriptional signalling.

We studied the ability of heat shock, DnaJ-like-1 (HSJ1) proteins (which contain DnaJ and ubiquitin-interacting motifs) to reduce polyglutamine-mediated inclusion formation. The experiments demonstrated that expression of heat shock protein 70 (hsp70), hsp40, HSJ1a, and HSJ1b significantly reduced protein inclusion formation in a model of spinal and bulbar muscular atrophy (SBMA). HSJ1a also mediated a significant decrease in the number of inclusions formed in a primary neuronal model of protein aggregation. Studies to elucidate the mechanisms underlying these reductions showed that hsp70 and hsp40 increased chaperone-mediated refolding. In contrast, expression of HSJ1 proteins did not promote chaperone activity but caused an increase in ubiquitylation. Furthermore, HSJ1a was associated with a ubiquitylated luciferase complex, and in the presence of HSJ1a but not an HSJ1a UIM mutant (HSJ1a-deltaUIM) there was a reduction in luciferase protein levels. Together these results show that HSJ1 proteins mediated an increase in target protein degradation via the ubiquitin-proteasome system (UPS). We also found that the expression of HSJ1a significantly decreased the number of neurons containing inclusions in an in vivo model of polyglutamine disease. These findings indicate that targeted modification of the UPS to facilitate degradation of misfolded proteins may represent a highly effective therapeutic avenue for the treatment of polyglutamine disease.

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described.