Unraveling the interactions of 14-3-3 with the neuronal proteins L1 and alpha II spectrin

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Intracellular Amyloid β 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 β (Aβ) 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 Aβ precedes its extracellular deposition and is also associated with synaptic dysfunction in AD. However, the links between intracellular Aβ, 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 Aβ into extracellular space and the latter accumulates Aβ oligomers within cells. First we investigated the effects of intracellular Aβ oligomers on dendritic spines in primary neurons and their tau-dependency using tau knockout neurons. We found that intracellular Aβ oligomers caused a reduction in mushroom, or mature spines, independently of tau. We also found that intracellular Aβ oligomers significantly impaired the intracellular transport of BDNF, mitochondria, and recycling endosomes: cargoes essential for synaptic maintenance. A reduction in BDNF transport by intracellular Aβ oligomers was also observed in tau knockout neurons. Conclusions Our findings indicate that intracellular Aβ oligomers likely contribute to early synaptic pathology in AD and argue against the consensus that Aβ-induced spine loss and transport defects require tau

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

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

Ser¹¹⁸¹Ala substitution and RSLESD deletion disrupt binding of L1 to 14-3-3ζ.

<p><b>A.</b> Schematic of recombinant L1ICD constructs. The full-length L1ICD construct contains the RSLESD sequence, a potential 14-3-3 binding motif. L1ICD S1181A has a single amino acid substitution (S1181A) of a serine residue in this motif. Ser1181 can be phosphorylated by CKII. The RSLESD sequence, a potential 14-3-3-binding motif, is deleted in L1ICD ΔRSLESD. Wild-type and mutated L1ICD constructs were recombinantly expressed as 6xHis-tagged proteins, purified from bacterial lysates and used in pull-down experiments. <b>B.</b> Purified mutated L1ICD constructs were analyzed by Western blot using the 74-5H7 anti-L1 antibody. <b>C. </b><i>Upper panel</i>: 6xHis-tagged proteins, purified from bacterial lysates, were subjected to GST-14-3-3ζ pull-down assays after treatment with CKII. GST was used as a control. Pull-down eluates were analyzed by Western blot (WB) with the 74-5H7 anti-L1 antibody. <i>Lower panel</i>, GST and GST-14-3-3ζ were detected in pull-down eluates by Western blot (WB) with an anti-GST antibody, confirming that comparable amounts of GST or GST-14-3-3ζ were used in each pull-down.</p

14-3-3ζ is associated with L1 in endosomes.

<p><b>A. </b><i>Upper panel</i>, Immunoprecipitation (IP) of L1 from vesicle fractions was performed using the anti-L1 monoclonal antibody 557. Proteins were resolved by a SDS-PAGE gradient gel (4–20%) and analyzed by Western blotting (WB) with an anti-L1 antibody (74-5H7), which recognizes full-length L1 and L1 proteolytic fragments containing the L1ICD. Full-length L1 (∼200 kDa), indicated by an arrow, was successfully precipitated. <i>Lower panel</i>, Western blot analysis of the L1 immunoprecipitates was also performed with an isoform-specific anti-14-3-3ζ antibody, revealing the association of 14-3-3ζ with L1 in vesicle fractions. C: crude endosomal preparation; S: cytosolic compounds; P: crude membrane fraction; ctrl: control IgG used for immunoprecipitation. <b>B.</b> To confirm that the isolated fractions are enriched in endosomal markers, equal protein amounts from total brain homogenate and vesicle fractions were analyzed for expression of the early endosome marker Rab 4 (upper panel) and the late endosome marker Rab 9 (lower panel). H: homogenate. In <b>B</b>, the bands shown for the homogenate are from a different blot, but with the same protein amount loaded and the same film exposure time as for the fractions.</p

14-3-3ζ binds to CKII-phosphorylated L1ICD more strongly than to nonphosphorylated L1ICD.

<p><b>A. </b><i>Upper panel</i>: Equal amounts (15 µg each) of recombinant His-tagged L1ICD were incubated in the presence or absence of CKII. Where indicated, 5 µM TBB was included to specifically inhibit CKII. To exclude non-specific effects of DMSO in which TBB was dissolved, one reaction took place in the presence of this solvent. After treatment, a GST-14-3-3ζ pull-down was performed to investigate direct binding of L1ICD to 14-3-3. Pull-down eluates were analyzed by Western blotting (WB) with the 74-5H7 anti-L1 antibody. <i>Lower panel</i>, GST and GST-14-3-3ζ were detected in pull-down eluates by Western blotting (WB) with a GST antibody, confirming that comparable amounts of GST or GST-14-3-3ζ were used in each pull-down. <b>B.</b> CKII phosphorylation of L1ICD enhances its association with 14-3-3ζ. Quantification of the lower L1ICD-immunoreactive band in lane 1 (L1ICD only) and the upper L1ICD-immunoreactive band in lane 2 (L1ICD and CKII) was performed by densitometric analysis (<b>A</b>, <i>upper panel</i> shows a representative example). Data represent mean ±SEM of four independent experiments.</p