Tina Wahle

PTEN recruitment controls synaptic and cognitive function in Alzheimer's models

Dyshomeostasis of amyloid-β peptide (Aβ) is responsible for synaptic malfunctions leading to cognitive deficits ranging from mild impairment to full-blown dementia in Alzheimers disease. Aβ appears to skew synaptic plasticity events toward depression. We found that inhibition of PTEN, a lipid phosphatase that is essential to long-term depression, rescued normal synaptic function and cognition in cellular and animal models of Alzheimers disease. Conversely, transgenic mice that overexpressed PTEN displayed synaptic depression that mimicked and occluded Aβ-induced depression. Mechanistically, Aβ triggers a PDZ-dependent recruitment of PTEN into the postsynaptic compartment. Using a PTEN knock-in mouse lacking the PDZ motif, and a cell-permeable interfering peptide, we found that this mechanism is crucial for Aβ-induced synaptic toxicity and cognitive dysfunction. Our results provide fundamental information on the molecular mechanisms of Aβ-induced synaptic malfunction and may offer new mechanism-based therapeutic targets to counteract downstream Aβ signaling.

Modification of γ-secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease

Inherited familial Alzheimers disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta‐peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ‐secretase complex formation. Moreover we found that peroxynitrite (ONOO−), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ‐secretase. This was associated with an increased association of the two PS1 fragments, PS1‐CTF and PS1‐NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ42 and increased the Aβ42/Aβ40 ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.

Dysregulated ADAM10-Mediated Processing of APP during a Critical Time Window Leads to Synaptic Deficits in Fragile X Syndrome.

The Fragile X mental retardation protein (FMRP) regulates neuronal RNA metabolism, and its absence or mutations leads to the Fragile X syndrome (FXS). The β-amyloid precursor protein (APP) is involved in Alzheimers disease, plays a role in synapse formation, and is upregulated in intellectual disabilities. Here, we show that during mouse synaptogenesis and in human FXS fibroblasts, a dual dysregulation of APP and the α-secretase ADAM10 leads to the production of an excess of soluble APPα (sAPPα). In FXS, sAPPα signals through the metabotropic receptor that, activating the MAP kinase pathway, leads to synaptic and behavioral deficits. Modulation of ADAM10 activity in FXS reduces sAPPα levels, restoring translational control, synaptic morphology, and behavioral plasticity. Thus, proper control of ADAM10-mediated APP processing during a specific developmental postnatal stage is crucial for healthy spine formation and function(s). Downregulation of ADAM10 activity at synapses may be an effective strategy for ameliorating FXS phenotypes.

Down-regulation of the ATP-binding Cassette Transporter 2 (Abca2) Reduces Amyloid-β Production by Altering Nicastrin Maturation and Intracellular Localization

Background: The intracellular sterol transporter Abca2 has been genetically linked to Alzheimer disease. Results: Abca2 depletion reduces γ-secretase cleavage of APP without affecting γ-cleavage of Notch and alters maturation and intracellular localization of Nicastrin, a member of the γ-secretase complex. Conclusion: Abca2 depletion affects γ-secretase cleavage in a substrate-distinctive manner. Significance: Abca2 is an important regulator of γ-secretase-mediated APP proteolysis and therefore Aβ generation. Clinical, pharmacological, biochemical, and genetic evidence support the notion that alteration of cholesterol homeostasis strongly predisposes to Alzheimer disease (AD). The ATP-binding cassette transporter-2 (Abca2), which plays a role in intracellular sterol trafficking, has been genetically linked to AD. It is unclear how these two processes are related. Here we demonstrate that down-regulation of Abca2 in mammalian cells leads to decreased amyloid-β (Aβ) generation. In vitro studies revealed altered γ-secretase complex formation in Abca2 knock-out cells due to the altered levels, post-translational modification, and subcellular localization of Nicastrin. Reduced Abca2 levels in mammalian cells in vitro, in Drosophila melanogaster and in mice resulted in altered γ-secretase processing of APP, and thus Aβ generation, without affecting Notch cleavage.

The role of CNI-1493 in the function of primary microglia with respect to amyloid-β.

Amyloid-β (Aβ) oligomer toxicity is a crucial factor in the development of Alzheimers disease. Therefore, the aim of therapeutic research is to target the modification of secretase activity, increase Aβ degradation, reduce Aβ formation, and modulate Aβ-induced neuroinflammation. Recently, the p38 MAP kinase inhibitor CNI-1493 has been shown to reduce plaque load and has led to an improvement in memory performance in a transgenic mouse model. We examined the role of CNI-1493 in the microglial inflammatory response to Aβ using both a microglia cell line as well as primary microglia isolated from mesocortices. MTT assays were performed to quantify cell viability. FACS analysis was used to measure phagocytosis. We used ELISA to analyse cytokine concentrations in response to CNI-1493 treatment. Western-blot/Dot-blot techniques were used to show the interaction of CNI-1493 with Aβ-oligomers as well as to measure apoptosis in microglia cells. RT-PCR was used to analyze secretase expression, and secretase function was determined using fluorimetric assays. CNI-1493 is able to prevent oligomer formation as well as apoptosis in microglia. A significant reduction was found in the Aβ-induced release of IL-6 and TNF-α in the presence of CNI-1493. Phagocytosis is an essential Aβ removal mechanism and was enhanced by CNI-1493 in primary microglia. CNI-1493 also influenced the α-secretase product C83 with an increase in the treated cells, while a simultaneous reduction in Aβ secretion was also observed. We hypothesize that CNI-1493 not only reduces neuroinflammation and consequent neurodegeneration, but also leads to a shift in AβPP-processing towards the non-amyloidogenic pathway. Therefore, CNI-1493 is a promising candidate for the treatment of AD.

Alterations in phosphatidylethanolamine levels affect the generation of Aβ

Several studies suggest that the generation of Aβ is highly dependent on the levels of cholesterol within membranes’ detergent‐resistant microdomains (DRM). Indeed, the β‐amyloid precursor protein (APP) cleaving machinery, namely β‐ and γ‐secretases, has been shown to be present in DRM and its activity depends on membrane cholesterol levels. Counterintuitive to the localization of the cleavage machinery, the substrate, APP, localizes to membranes’ detergent‐soluble microdomains enriched in phospholipids (PL), indicating that Aβ generation is highly dependent on the capacity of enzyme and substrate to diffuse along the lateral plane of the membrane and therefore on the internal equilibrium of the different lipids of DRM and non‐DRM domains. Here, we studied to which extent changes in the content of a main non‐DRM lipid might affect the proteolytic processing of APP. As phosphatidylethanolamine (PE) accounts for the majority of PL, we focused on its impact on the regulation of APP proteolysis. In mammalian cells, siRNA‐mediated knock‐down of PE synthesis resulted in decreased Aβ owing to a dual effect: promoted α‐secretase cleavage and decreased γ‐secretase processing of APP. In vivo, in Drosophila melanogaster, genetic reduction in PL synthesis results in decreased γ‐secretase‐dependent cleavage of APP. These results suggest that modulation of the membrane‐soluble domains could be a valuable alternative to reduce excessive Aβ generation.

E3 ligase mahogunin (MGRN1) influences amyloid precursor protein maturation and secretion

Altered processing of the Amyloid Precursor Protein (APP) is a well-recognized central pathogenic mechanism in Alzheimers Disease (AD), and regulation of APP processing is a major focus of research in the AD field. However, how age-associated cellular and molecular changes contribute to changes in the amyloidogenic processing of APP have not been extensively clarified so far. We here provide evidence that the processing of APP is influenced by the e3 ubiquitin ligase Mahogunin (MGRN1), a neuroprotective molecule whose levels decrease with aging. Specifically, the expression of MGRN1 inhibits the maturation of APP by sequestering it in the secretory pathway. This sequestration significantly delayed the proteolytic processing of APP, resulting in a reduced β-amyloid (Aβ) peptide release into the extracellular environment. Accordingly, a reduction of MGRN1 levels in hippocampal neurons, as it occurs during physiological aging, leads to an increased Aβ40 and Aβ42 release. We therefore propose that age contributes to the amyloidogenic processing of APP by altering its intracellular trafficking along the secretory pathway due in part to the down-regulation of MGRN1.

P2-181: Amino acid sequence of BACE1 and BACE2 determine different subcellular transport and localisation

memory formation--in PrP-MAPT mice that over-express normal human fetal tau (3R/0N isoform). Methods: Field potential recordings were made from the lateral perforant path of the dentate gyrus of either wild-type or heterozygous mice aged 2 and 4 months, respectively, and include assessments of basal transmission, paired-pulse facilitation (PPF) and long-term potentiation (LTP). Results: At 2 months, no difference in the magnitude of LTP between genotypes was observed (Het: 176.4 10.2%, n 6; WT: 171.3 9.1, n 6) but a subtle impairment in basal transmission was detected in the Het’s even at this early age. By 4 months of age, differences in basal transmission, PPF, and LTP (WT: 178.2 6.4, n 6; Het: 148.1 11.9, n 9) were observed. Interestingly, 4M Het LTP data revealed that some slices had normal, WT LTP magnitudes while others were impaired. This duality suggests that at 4 months, Het mice may reside on a threshold between normal and abnormal and that other factors influence the progression of symptoms. Further studies will include assessments in 8-10M old Tau transgenic mice. Conclusions: These data suggest that overexpression of normal tau protein influences hippocampal synaptic properties, similar to deficits found in Tg2576 mice, early in development and thus may be important in the disease progression.