Intracellular amyloid ß oligomers impair organelle transport and induce dendritic spine loss in primary neurons.

INTRODUCTION: Synaptic dysfunction and intracellular transport defects are early events in Alzheimer's disease (AD). Extracellular amyloid ß (Aß) oligomers cause spine alterations and impede the transport of proteins and organelles such as brain-derived neurotrophic factor (BDNF) and mitochondria that are required for synaptic function. Meanwhile, intraneuronal accumulation of Aß precedes its extracellular deposition and is also associated with synaptic dysfunction in AD. However, the links between intracellular Aß, spine alteration, and mechanisms that support synaptic maintenance such as organelle trafficking are poorly understood. RESULTS: We compared the effects of wild-type and Osaka (E693Δ)-mutant amyloid precursor proteins: the former secretes Aß into extracellular space and the latter accumulates Aß oligomers within cells. First we investigated the effects of intracellular Aß oligomers on dendritic spines in primary neurons and their tau-dependency using tau knockout neurons. We found that intracellular Aß oligomers caused a reduction in mushroom, or mature spines, independently of tau. We also found that intracellular Aß oligomers significantly impaired the intracellular transport of BDNF, mitochondria, and recycling endosomes: cargoes essential for synaptic maintenance. A reduction in BDNF transport by intracellular Aß oligomers was also observed in tau knockout neurons. CONCLUSIONS: Our findings indicate that intracellular Aß oligomers likely contribute to early synaptic pathology in AD and argue against the consensus that Aß-induced spine loss and transport defects require tau.

Amyloid-ß oligomers induce tau-independent disruption of BDNF axonal transport via calcineurin activation in cultured hippocampal neurons.

Disruption of fast axonal transport (FAT) is an early pathological event in Alzheimer's disease (AD). Soluble amyloid-ß oligomers (AßOs), increasingly recognized as proximal neurotoxins in AD, impair organelle transport in cultured neurons and transgenic mouse models. AßOs also stimulate hyperphosphorylation of the axonal microtubule-associated protein, tau. However, the role of tau in FAT disruption is controversial. Here we show that AßOs reduce vesicular transport of brain-derived neurotrophic factor (BDNF) in hippocampal neurons from both wild-type and tau-knockout mice, indicating that tau is not required for transport disruption. FAT inhibition is not accompanied by microtubule destabilization or neuronal death. Significantly, inhibition of calcineurin (CaN), a calcium-dependent phosphatase implicated in AD pathogenesis, rescues BDNF transport. Moreover, inhibition of protein phosphatase 1 and glycogen synthase kinase 3ß, downstream targets of CaN, prevents BDNF transport defects induced by AßOs. We further show that AßOs induce CaN activation through nonexcitotoxic calcium signaling. Results implicate CaN in FAT regulation and demonstrate that tau is not required for AßO-induced BDNF transport disruption.

The 14-3-3ζ protein binds to the cell adhesion molecule L1, promotes L1 phosphorylation by CKII and influences L1-dependent neurite outgrowth.

BACKGROUND: The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1. METHODOLOGY/PRINCIPAL FINDINGS: By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3ζ). Interestingly, in an in vitro phosphorylation assay, 14-3-3ζ promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3ζ on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3ζ, which impairs interactions of 14-3-3ζ with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3ζ. CONCLUSIONS/SIGNIFICANCE: Our results suggest that 14-3-3ζ, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1.

Binding of alphaII spectrin to 14-3-3beta is involved in NCAM-dependent neurite outgrowth.

Members of the 14-3-3 protein family have been implicated in neuronal migration, synaptic plasticity and learning. Using affinity chromatography followed by mass spectrometry analysis, we show here that the cytoskeletal protein alphaII spectrin is a novel ligand of 14-3-3beta. We found that 14-3-3beta interacts with alphaII spectrin via the mode 2 14-3-3 binding motif RLIQS(1302)HP. Binding required phosphorylation of Ser(1302) by casein kinase II and was enhanced in the presence of calmodulin. Co-immunoprecipitation of alphaII spectrin and 14-3-3beta with the neural cell adhesion molecule NCAM suggested that the 14-3-3-spectrin-interaction affects NCAM function. Indeed, disruption of the 14-3-3beta/alphaII spectrin interaction by mutating Ser(1302) to Ala enhanced NCAM-dependent neurite outgrowth. Our results indicate that the phosphorylation-dependent interaction between 14-3-3beta and alphaII spectrin acts as a switch between positive and negative regulation of neurite outgrowth stimulated by NCAM, representing a novel and acute mechanism preventing uncontrolled elongation of neuronal processes.