The role of MeCP2 in activity-dependent brain processes

Rett syndrome (RTT) is a neurodevelopmental disorder that is caused by mutations in the X-linked gene MECP2 and results in cognitive impairment, epilepsy and motor dysfunction. Deletion or silencing of Mecp2 in the brain of mice recapitulates many of the main phenotypes of the disorder and has been fundamental in understanding the actions of MeCP2 although the precise molecular function remains unknown. MeCP2 is classically thought to have a role in repressing gene transcription through recruitment of histone deacetylase proteins. Studies have also shown that Mecp2 can be modified posttranslationally in response to neuronal activity. The aim of this thesis was to investigate the activity-dependent alterations in MeCP2 and the association this has with levels of acetylated histone proteins, a marker of active gene transcription thus gaining a better insight into how neuronal activity affects the function of MeCP2. Furthermore at the network level, a second objective was to characterise the effects loss of functional MeCP2 has on the regulation of network circuitry in the brain and in particular the development of epileptiform activities. To study this I focussed on the hippocampus in wild-type and Mecp2 mutant mice and in vivo administered the convulsant drug kainic acid (25mg/kg; IP) followed by seizure scoring and in vitro by application of various epileptogenic agents (kainic acid, bicuculline and 4-aminopyridine) to acute hippocampal slices. Having characterised the network properties, I then quantified protein alterations in phosphorylation of Mecp2, acetylated histone H3 and H4 and immediate early genes, c-Fos and Egr-1 in the hippocampus using western blot and quantitative immunohistochemistry techniques. Based on a modified seizure scale (eight stages with lower being less severe), administration of kainic acid in vivo to Mecp2-deficient male mice resulted in a higher seizure score (mean = 6 ± 0.7 vs. 4 ± 0.2 units in wild-type) and more rapid onset (77% of mice show seizures after 10 minutes compared to 5% of wild-type mice). Field recording data collected in vitro following application of kainic acid to hippocampal slice from Mecp2-deficient mice show a significant increase in gamma power oscillation (1059 ± 379µV2) compared to slices from WT mice (287 ± 178µV2) which had a lower mean power. Application of bicuculline revealed hippocampal slices from Mecp2-deficient mice had increased frequency of spontaneous epileptiform field events (1µM and 3µM bicuculline) and elevated duration of spontaneous and evoked epileptiform field events (10µM bicuculline). Similarly, 4-aminopyridine (4-AP) administration to hippocampal slices resulted in Mecp2-deficient mice displaying increased frequency of spontaneous field events (50µM 4-AP) and epileptic ictal-like events (88% of slices from Mecp2stop/y mice displayed these events compared to 43% of slices from WT mice). Furthermore there was an increase in spontaneous and evoked field events following application of 30µM and 10µM 4-AP. Western blot experiments using hippocampal extracts from WT and Mecp2-deficient male mice treated with the convulsant drug kainic acid or saline (vehicle control) revealed Mecp2 is highly phosphorylated at serine 421 (3.4 ± 0.5 fold, p< 0.01) upon induction of neuronal activity compared to saline controls but there was no change in histone H3 or H4 acetylated proteins. A complementary quantitative immunohistochemistry approach was used to assess variations in histone H3 or H4 acetylated proteins at the single cell level from heterozygous female mice (displaying a mosaic expression of Mecp2) treated with kainic acid or saline. These results revealed there was no difference in the levels of either acetylated histone H3 of H4 protein between Mecp2 positive and negative nuclei. However there was a clear cell-autonomous effect in terms of a 5% reduction in the nuclear volume of Mecp2-deficient cells. Quantification of immediate early gene signal in Mecp2+/- heterozygous female mice treated with kainic acid or saline using the same immunohistochemistry method showed there was no difference in the distribution of c-Fos intensities between Mecp2 positive and negative nuclei following any treatment. However there was a greater proportion of Mecp2-deficient nuclei (~20%) expressing c-Fos under saline control conditions and following neuronal activity. Furthermore there was a reduction in the percentage of Mecp2 negative nuclei in the top 25% of Egr-1 intensities in the CA1 following three hours of neuronal activity (33.5 ± 2.3% for Mecp2 negative nuclei and 19.1 ± 1.7% for Mecp2 positive nuclei). In summary my results show at the network level there is a reduction in seizure threshold and increase power of gamma oscillations in the hippocampus of Mecp2-deficient mice which could lead to a state of network hyperexcitability and switch activities from physiological oscillatory rhythms to more pathological ones. An imbalance in inhibitory neuron regulation could partially contribute to alterations in network excitability to overall promote epileptogenesis. At the cellular level I report that Mecp2 can become phosphorylated following induction of neuronal activity but this is not associated with alterations in global histone acetylation. Nonetheless Mecp2-deficient cells display a reduction in nuclear volume and have alterations in c-Fos and Egr-1 levels following induction of neuronal activity. Overall my data contribute to the understanding of how the presence or absence of MeCP2 affects network excitability and how in turn neuronal excitability affects MeCP2 phosphorylation, histone acetylation and the activation of immediate early genes

Wnt signalling tunes neurotransmitter release by directly targeting Synaptotagmin-1

The functional assembly of the synaptic release machinery is well understood; however, how signalling factors modulate this process remains unknown. Recent studies suggest that Wnts play a role in presynaptic function. To examine the mechanisms involved, we investigated the interaction of release machinery proteins with Dishevelled-1 (Dvl1), a scaffold protein that determines the cellular locale of Wnt action. Here we show that Dvl1 directly interacts with Synaptotagmin-1 (Syt-1) and indirectly with the SNARE proteins SNAP25 and Syntaxin (Stx-1). Importantly, the interaction of Dvl1 with Syt-1, which is regulated by Wnts, modulates neurotransmitter release. Moreover, presynaptic terminals from Wnt signalling-deficient mice exhibit reduced release probability and are unable to sustain high-frequency release. Consistently, the readily releasable pool size and formation of SNARE complexes are reduced. Our studies demonstrate that Wnt signalling tunes neurotransmitter release and identify Syt-1 as a target for modulation by secreted signalling proteins.Fil: Ciani, Lorenza. University College London; Estados UnidosFil: Marzo, Aude. University College London; Estados UnidosFil: Boyle, Kieran. University College London; Estados UnidosFil: Stamatakou, Eleanna. University College London; Estados UnidosFil: Lopes, Douglas M.. University College London; Estados UnidosFil: Anane, Derek. University College London; Estados UnidosFil: McLeod, Faye. University College London; Estados UnidosFil: Rosso, Silvana Beatriz. University College London; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gibb, Alasdair. University College London; Estados UnidosFil: Salinas, Patricia C.. University College London; Estados Unido

Restoring synapse integrity and memory in Alzheimer’s disease by downregulation of the Wnt antagonist Dickkopf-3

Increasing evidence supports a role of deficient Wnt signaling in Alzheimer’s disease (AD). Recent studies reveal that the secreted Wnt antagonist Dickkopf-3 (DKK3) is elevated in the human AD brain. Here, we investigate the contribution of DKK3 to synapse integrity in the healthy and AD brain. We uncover a novel genetic link between DKK3 gene variants and AD risk. Our findings show that DKK3 protein is increased in different human brain fractions consistent with disease progression. In the hAPP-J20 and hAPPNL-G-F/NL-G-F AD models, DKK3 accumulates at plaques in the brain. Oligomers of amyloid-β enhance the secretion of DKK3 from cultured neurons and DKK3 secretion is also increased in hippocampal slices of hAPP-J20 mice. In addition, gain-of-function experiments revealed that DKK3 decreases the density of excitatory synapses through inhibition of the canonical Wnt/GSK3β pathway but increases inhibitory synapse density through activation of the Wnt/JNK pathway. Our studies demonstrate that in vivo DKK3 downregulation restores synapse number in hAPP-J20 mice. Importantly, DKK3 knockdown improves memory in this AD model. Collectively, our findings identify DKK3 as a novel driver of synapse defects and memory impairment in AD

Physics and Chemistry of Planet-Forming Disks in Extreme Radiation Environments

Our knowledge about the formation history of planetary systems is obtained by comparing the demographics of proto-planetary disks with the exoplanetary system population. Most of the disks that we have been able to characterize to date are located in nearby low-mass star forming regions. However, it is well known that most stars form in denser environments and therefore, it is questionable that the well studied population of planet forming disks is representative of those in which most exoplanets were assembled. Due to their large distances and high densities, so far it has been impossible to study the physical and chemical properties of proto-planetary disks in massive star-forming regions. We will exploit the unique resolution and sensitivity of JWST/MIRI to explore for the first time the impact of disk evaporation on the disk structure, warm disk chemistry, and dust mineralogy, all of which are important for planet formation models and exoplanet atmosphere composition. The derived physical and chemical properties will be compared to similar data of low-mass star forming regions of JWST GTO programmes

Reversal of Synapse Degeneration by Restoring Wnt Signaling in the Adult Hippocampus

Synapse degeneration occurs early in neurodegenerative diseases and correlates strongly with cognitive decline in Alzheimer's disease (AD). The molecular mechanisms that trigger synapse vulnerability and those that promote synapse regeneration after substantial synaptic failure remain poorly understood. Increasing evidence suggests a link between a deficiency in Wnt signaling and AD. The secreted Wnt antagonist Dickkopf-1 (Dkk1), which is elevated in AD, contributes to amyloid-β-mediated synaptic failure. However, the impact of Dkk1 at the circuit level and the mechanism by which synapses disassemble have not yet been explored. Using a transgenic mouse model that inducibly expresses Dkk1 in the hippocampus, we demonstrate that Dkk1 triggers synapse loss, impairs long-term potentiation, enhances long-term depression, and induces learning and memory deficits. We decipher the mechanism involved in synapse loss induced by Dkk1 as it can be prevented by combined inhibition of the Gsk3 and RhoA-Rock pathways. Notably, after loss of synaptic connectivity, reactivation of the Wnt pathway by cessation of Dkk1 expression completely restores synapse number, synaptic plasticity, and long-term memory. These findings demonstrate the remarkable capacity of adult neurons to regenerate functional circuits and highlight Wnt signaling as a targetable pathway for neuronal circuit recovery after synapse degeneration

Polysome Profiling of mAb Producing CHO Cell Lines Links Translational Control of Cell Proliferation and Recombinant mRNA Loading onto Ribosomes with Global and Recombinant Protein Synthesis

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilise available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localised to the translationally active heavy polysomes. The highest titre cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5? untranslated regions that might be utilised to enhance recombinant transcript translation