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In vitro cultured cerebellar granule cells (cgc) undergo apoptotic death when the depolarising concentration of KCl 25 mM is adjusted to 5 mM. We investigated whether the apoptotic message can be transferred from a group of neurons to neighbouring but separated sister cultures by resorting to two different culture systems, one based on the use of concentric dishes, the other employing the transwells. The extent of transferred death was 38.6% in the two dishes system and 31.3% in the transwell system. The transfer of such death-inducing signal(s), accompanied by chromatin condensation, was inhibited by actinomicin D. Preliminary experiments aimed at identifying such apoptosis-inducing signal(s) suggest the involvement of beta-amyloid fragment(s).

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Background: Our laboratory has pioneered targeting pathological tau proteins for clearance with immunotherapies. We have shown that this approach leads to antibody uptake into neurons, clears tau aggregates from the brain and prevents functional impairments, such as cognitive decline, in mouse tauopathy models. Methods: To clarify the mechanism of action of tau antibodies, we are using na€ ıve and transfected (0N4R P301L tau) SHSY5Y human neuroblastoma cells. To mimic a pathological state, we have incubated these cells with paired helical filaments (PHF) enriched from Alzheimer's disease (AD) brain for 3 days, and subsequently incubated with tau antibody (Ser396/404 epitope) for 1, 4, or 6 days. Incubation was performed at 10% (normal) and 1% (compromising) FBS media conditions. In parallel, these cells were analyzed using our flow cytometry protocol and western blot analysis for antibody and PHF uptake as well as tau protein profile, respectively. Results: Transfected cells with 1% FBS had more PHF uptake than all other groups at 1, 4, and 6 days (p<0.0001, p¼0.0005, and p¼0.0003, respectively, n¼3). In both controls and sequentially (PHF and tau antibody) treated samples we observed a gradual increase in tau antibody uptake from 1 to 4 days in both cell lines and additional increase from 4 to 6 days in our transfected line. Only at 6 days of tau antibody incubation and in 1% FBS the transfected cells had significantly greater uptake of tau antibody compared to all other groups (26-39%, p¼ 0.0002, n¼3) and a decrease in total tau and phospho Ser199 tau (64%, p¼0.0013 and 49%, p¼0.0925, respectively, n¼3) levels compared to its PHF incubated control. Conclusions: The compromised transfected cells at 1% FBS and 6 days of antibody incubation show the most promising results as antibody uptake increases and tau levels decrease. Also, PHF seems to have limited effect on antibody uptake, but provides a condition for tau degradation. This neuroblastoma flow cytometry protocol may give us a new high throughput method to better understand the underlying cellular mechanisms and further development of this immunotherapy approach to treat AD and related tauopathies.Background: Structure/function disarrangement of mitochondria and disrupted ubiquitin homeostasis at synapses are early events in Alzheimer's Disease (AD) pathobiology. The coordinated interplay between the ubiquitin-proteasome system and the mitochondrial quality control is a critical process in maintaining a normal synaptic transmission in post-mitotic neurons which are strictly dependent on oxidative phosphorylation for their high energy demands. We reported that a 20-22 kDa NH 2 -tau fragment (aka NH 2 htau): (i) is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons (Amadoro et al., 2006(Amadoro et al., , 2008; (ii) compromises the mitochondrial biology thereby contributing to AD synaptic deficits either directly, by inhibiting the ANT-1-dependent ADP/ ATP exchange (Amadoro et al., 2010,2012) or indirectly, by impairing Parkin-...

mRNA; the axonal location of which is determined by a 91 base ciselement in the 3' untranslated region (3'UTR) known as the axonal targeting element (ATE). The ATE contains a binding site for HuD, a member of the embryonic lethal abnormal vision like family. HuD stabilises tau mRNA by binding a U-rich regulatory region controlling mRNA turnover. The full complement of trans-acting factors binding to the ATE responsible for the specific localisation of tau mRNA to axons is still to be identified. The objective of this study is to analyse the mechanisms regulating tau mRNA subcellular localisation by first identifying ATE-binding proteins using proteomic analysis. Methods: Trans-acting factors which bind the 3'UTR of tau mRNA were isolated using a novel RNA affinity purification procedure. We have designed the pOC vector, in a pBS backbone, containing a 19 bp tagging sequence adjacent to the BamHI site into which a DNA sequence encoding the ATE is subcloned. In-vitro transcription from the T3 promoter produces RNA tagged at the 3' end. Tagged RNA is annealed to a biotinylated oligoribonucleotide corresponding to the tag sequence in the antisense orientation. This oligoribonucleotide is synthesised with modified 2'-O-methyl bases increasing the stability of the RNA-RNA duplex and protecting it from RNase degradation. An immobilised tagged RNA matrix is prepared by binding the biotinylated RNA to magnetic streptavidin beads. Results: This novel protocol was validated by the specific isolation of HuD from cell extracts overexpressing recombinant HuD. Large scale studies were performed to isolate endogenous RNA-binding proteins from adult rat brain. We observe specific binding of endogenous trans-acting factors binding to the ATE of tau mRNA. Moreover, these proteins are isolated in amounts compatible with their identification by mass spectrometry; allowing global identification of tau ATE-binding proteins. Conclusions: Understanding the mechanisms by which RNA-binding proteins regulate tau mRNA processing will lead to increased understanding of the pathology of tauopathies and the development of novel therapeutic strategies.