Presenilin is necessary for the function of CBP in the adult Drosophila CNS

Dominant mutations in Presenilin (Psn) have been correlated with the formation of Aß- containing plaques in patients with inherited forms of Alzheimer\u27s disease (AD). However, a clear mechanism directly linking amyloid plaques to the pathology of familial or sporadic forms of AD has remained elusive. Thus, recent discoveries of several new substrates for Psn protease activity have sparked alternative hypotheses to explain the preclinical symptoms of AD. CBP (CREB-binding protein) is a haplo- insufficient transcriptional co-activator with histone acetyltransferase (HAT) activity that has been proposed to be a downstream target for Psn signaling. Individuals with reduced CBP levels have cognitive deficits that have been linked to several neurological disorders. However, there are contradictory reports in the vertebrate literature regarding the relationship between Psn activity and CBP levels. This dissertation using Drosophila melanogaster, provides evidence for the first time that Psn is required for normal CBP levels and for maintaining global acetylations of the central nervous system of the adult fly. This work also demonstrates that adult flies conditionally compromised for CBP display an altered geotaxis response to gravity that likely reflects a neurological defect. The association between Psn and CBP is most likely not direct, but through a signaling molecule released via Psn-mediated substrate processing. One possible candidate for this intermediate molecule is the transmembrane receptor Notch. Notch is attractive because it has been shown previously to be required for long-term memory, and I have provided evidence here that suggests Notch is required for neurite outgrowth through the culturing of primary neurons of the mushroom body (a region known to be important for learning and memory in flies). In addition I have located putative DNA binding sites for the Notch transcription factor Su(H) in the CBP enhancer, which suggests a regulatory role for Notch in CBP transcription. Although this is an attractive model, my data do not support it. Therefore it is likely that Notch and Psn/CBP control functions of the adult CNS through independent signaling molecules or pathways. If the model proves correct for vertebrates, it will have a significant impact on the development of new therapies and pharmaceuticals agents for the treatment of Alzheimer\u27s disease

Presenilin Controls CBP Levels in the Adult Drosophila Central Nervous System

Background: Dominant mutations in both human Presenilin (Psn) genes have been correlated with the formation of amyloid plaques and development of familial early-onset Alzheimer’s disease (AD). However, a definitive mechanism whereby plaque formation causes the pathology of familial and sporadic forms of AD has remained elusive. Recent discoveries of several substrates for Psn protease activity have sparked alternative hypotheses for the pathophysiology underlying AD. CBP (CREB-binding protein) is a haplo-insufficient transcriptional co-activator with histone acetly-transferase (HAT) activity that has been proposed to be a downstream target of Psn signaling. Individuals with altered CBP have cognitive deficits that have been linked to several neurological disorders. Methodology/Principal Findings: Using a transgenic RNA-interference strategy to selectively silence CBP, Psn, and Notch in adult Drosophila, we provide evidence for the first time that Psn is required for normal CBP levels and for maintaining specific global acetylations at lysine 8 of histone 4 (H4K8ac) in the central nervous system (CNS). In addition, flies conditionally compromised for the adult-expression of CBP display an altered geotaxis behavior that may reflect a neurological defect. Conclusions/Significance: Our data support a model in which Psn regulates CBP levels in the adult fly brain in a manner that is independent of Notch signaling. Although we do not understand the molecular mechanism underlying th

The HAT domain of CBP is required for H4K8ac in the adult <i>Drosophila</i> CNS.

<p>(A) depicts a typical roughened eye phenotype produced when <i>UAS-CBP-FLAD</i> is expressed with the <i>GMR-Gal4</i> driver during development. (B) displays a wing phenotype with a prominent notch (arrowhead) that is typically observed when <i>UAS-CBP-FLAD</i> is expressed with the <i>c96-Gal4</i> driver. Both phenotypes are similar to those detected when <i>UAS-CBPi</i> is expressed with the same drivers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014332#pone-0014332-g001" target="_blank">Fig. 1</a>). (C) is a control brain (no <i>Gal4</i>; <i>UAS-CBP-FLAD</i>; + heat shocks) stained for H4K8ac, and (D) is an experimental brain (<i>hsGal4; UAS-CBP-FLAD</i>; + heat shocks) stained with the same antibody under the same conditions. Note the dramatic reduction in staining intensity when the HAT-defective form of CBP is expressed.</p

Assaying geotaxis using a countercurrent device.

<p>Pictured is the countercurrent device we built for assaying geotaxis behavior. This apparatus was modeled directly from a similar design created by Seymour Benzer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014332#pone.0014332-Benzer1" target="_blank">[30]</a>. Each experiment starts as flies are placed in tube set 1 and given 3 sharp taps on a rubber mat to knock them to the bottom. Flies are then given 10 seconds to climb into the upper tube, after which time it is moved over to the bottom of set 2. They are again tapped to the bottom, allowed to climb for 10 seconds, and moved to the bottom of set 3. The procedure continues until set 6 is reached. At the end of the experiment, the percentage of the fly cohort in each tube is calculated. The experiment is calibrated in such a way that most of the wildtype flies (exhibiting normal negative geotaxis) accumulate in tube number six.</p

Psn affects H4K8ac levels in the adult brain.

<p>The above images are whole-mount adult fly brains stained for H4K8ac as an indicator of global acetylation levels. (A,D,G) represent one control group (<i>hsGal4; UAS-RNAi;</i> no heat shocks), (B,E,H) depict a second control group (no <i>Gal4</i>; <i>UAS-RNAi</i>; + heat shocks), and (C,F,I) represent the experimental group (hsGal4; <i>UAS-RNAi; +</i> heat shocks<i>).</i> Note that when <i>CBP</i> is silenced there is a dramatic effect on H4K8ac levels (compare C with the A and B controls); when <i>Psn</i> is silenced there is a significant but less dramatic effect (F); and when <i>Notch</i> is silenced (I) there is no noticeable effect. All brains were dissected, stained, and photographed using the same conditions and microscope settings.</p

Flies compromised for <i>CBP</i> exhibit poor geotaxis.

<p>(A) represents the mean distribution of flies within each tube after 6 successive trials. Tube distribution for each group is presented by a best-fit line, which is based on a linear regression. The slope of the experimental group (<i>hs-Gal4; UAS-CBPi</i>) indicates that the majority of these cohorts remained in the first sets of tubes, whereas the control groups accumulated in the last sets of tubes. (B) is the mean score for each group with a significant difference between the experimental group (<i>hsGal4</i>; <i>UAS-CBPi;</i> +heat shocks) and all three control groups (<i>F</i>_(3,53) = 11.8; <i>P</i><0.0001). There were no significant differences between genders or control groups. A (+) indicates a heat shock treatment and (-) indicates no heat shock.</p