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Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impairments in social interaction, challenges with communication, and repetitive behaviors. Genetic mutations associated with ASD can either activate or inactivate the responsible proteins, affecting neuronal morphogenesis and contributing to the disorder’s hallmark features. However, the molecular mechanisms driving these changes remain incompletely understood. Here, we report for the first time that the small GTP/GDP-binding protein Arf6 and FE65, which act together with the genetically conserved engulfment and cell motility 2 (ELMO2) signalosome to control Rac1, underlie the excessive neuronal process elongation phenotype associated with the ASD-linked semaphorin-5 A (Sema5A) Arg676-to-Cys protein (p.Arg676Cys). Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13-mediated knockdown of Arf6 or FE65 reversed the excessively elongated processes in primary cortical neurons. Similar results were obtained in the N1E-115 cell line, a model capable of neuronal morphological differentiation. Moreover, expression of the ELMO2-binding domain of FE65 restored Rac1 activity required for process elongation, recapitulating the effects seen in the knockdown experiments. These findings suggest that signaling through FE65 specifically couples Sema5A p.Arg676Cy to the ELMO2 signalosome molecule, driving excessively elongated processes with elevated Rac1 activity. One or more of these molecules may provide possible therapeutic targets for correcting the cellular phenotypes associated with the Sema5A p.Arg676Cys mutation in ASD.

N‐methyl‐D‐aspartate receptors (NMDARs) are critical for the maturation and plasticity of glutamatergic synapses. In the hippocampus, NMDARs mainly contain GluN2A and/or GluN2B regulatory subunits. The amyloid precursor protein (APP) has emerged as a putative regulator of NMDARs, but the impact of this interaction to their function is largely unknown. By combining patch‐clamp electrophysiology and molecular approaches, we unravel a dual mechanism by which APP controls GluN2B‐NMDARs, depending on the life stage. We show that APP is highly abundant specifically at the postnatal postsynapse. It interacts with GluN2B‐NMDARs, controlling its synaptic content and mediated currents, both in infant mice and primary neuronal cultures. Upon aging, the APP amyloidogenic‐derived C‐terminal fragments, rather than APP full‐length, contribute to aberrant GluN2B‐NMDAR currents. Accordingly, we found that the APP processing is increased upon aging, both in mice and human brain. Interfering with stability or production of the APP intracellular domain normalized the GluN2B‐NMDARs currents. While the first mechanism might be essential for synaptic maturation during development, the latter could contribute to age‐related synaptic impairments. Rajão‐Saraiva et al. identified the amyloid precursor protein (APP) as an important regulator of NMDA receptors, acting through a dual age‐dependent mechanism. During postnatal development, APP controls GluN2B‐NMDAR synaptic content and currents, potentially contributing to synaptic maturation. Upon aging, the APP amyloidogenic intracellular fragments contribute to aberrant GluN2B‐NMDAR currents. This work highlights the importance of keeping APP processing under tight control, to ensure the normal functioning of glutamatergic synapses, being particularly relevant to understand age‐related synaptic impairments and Alzheimer's disease.

Autophagy is a vital cellular pathway in eukaryotic cells, including neurons, where it plays significant roles in neurodevelopment and maintenance. A crucial step in autophagy is the formation of the class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1), which is essential for initiating autophagosome biogenesis. Beclin 1 is the key component of PI3KC3-C1, and its interactors have been reported to affect autophagy. The brain-enriched adaptor protein FE65 has been shown to interact with Alzheimer’s disease amyloid precursor protein (APP) to alter the processing of APP. Additionally, FE65 has been implicated in various cellular pathways, including autophagy. We demonstrate here that FE65 positively regulates autophagy. FE65, through its C-terminus, has been shown to interact with Beclin 1. Notably, the overexpression of FE65 enhances Beclin 1-mediated autophagy, whereas this process is attenuated in FE65 knockout cells. Moreover, the stimulatory effect of FE65 on Beclin 1-mediated autophagy is diminished by an FE65 C-terminus deletion mutant that disrupts the FE65–Beclin 1 interaction. Lastly, we have found that the FE65-Beclin 1 interaction modulates the kinase activity of the PI3KC3-C1 complex. Together, we have identified FE65 as a novel Beclin 1 interactor, and this interaction potentiates autophagy.

A battery of genetically encoded calcium indicators (GECIs) with different binding kinetics and calcium affinities was developed over the recent years to permit long-term calcium imaging. GECIs are calcium buffers and therefore, expression of GECIs may interfere with calcium homeostasis and signaling pathways important for neuronal differentiation and survival. Our objective was to investigate if biolistically induced expression of five commonly used GECIs at two postnatal time points (days 14 and 22-25) could affect the morphological maturation of cortical neurons in organotypic slice cultures of rat visual cortex. Expression of GCaMP3 in both time windows, and of GCaMP5G and TN-XXL in the later time window impaired apical and /or basal dendrite growth of pyramidal neurons. With time, the proportion of GECI transfectants with nuclear filling increased, but only prolonged expression of TN-XXL caused higher levels of neurodegeneration. In multipolar interneurons, only GCaMP3 evoked a transient growth delay during the early time window. GCaMP6m and GCaMP6m-XC were quite “neuron-friendly”. Since growth-impaired neurons might not have the physiological responses typical of age-matched wildtype neurons the results obtained after prolonged developmental expression of certain GECIs might need to be interpreted with caution.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) leads to the formation of fragments, among which the intracellular domain of APP (AICD) was also identified to be a causative of early pathological events. AICD-counteracting proteins, such as Fe65, may serve as alternative therapeutic targets of Alzheimer’s disease (AD). The detection of elevated levels of Fe65 in the brains of both human patients and APP transgenic mice may further strengthen the hypothesis that influencing the interaction between Fe65 and APP may have a beneficial effect on the course of AD. Based on a PXP motif, proven to bind to the WW domain of Fe65, a new pentapeptide was designed and tested. The impedimental effect of P33 on the production of beta amyloid (Aβ) (soluble fraction and aggregated plaques) and on the typical features of the AD pathology (decreased dendritic spine density, synaptic markers, elevated inflammatory reactions) was also demonstrated. Significant enhancements of both learning ability and memory function were observed in a Morris water maze paradigm. The results led us to formulate the theory that P33 acts by altering the conformation of Fe65 via binding to its WW domain, consequently hindering any interactions between Fe65 and key members involved in APP processing.

Amyloid-β precursor protein (APP), a widely expressed cell-surface protein, is cleaved in the transmembrane region by γ-secretase. γ-Cleavage of APP produces the extracellular amyloid β-peptide of Alzheimer's disease and releases an intracellular tail fragment of unknown physiological function. We now demonstrate that the cytoplasmic tail of APP forms a multimeric complex with the nuclear adaptor protein Fe65 and the histone acetyltransferase Tip60. This complex potently stimulates transcription via heterologous Gal4- or LexA-DNA binding domains, suggesting that release of the cytoplasmic tail of APP by γ-cleavage may function in gene expression.

Summary Cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretase generates amyloid‐β (Aβ) and APP intracellular domain (AICD) peptides. Presenilin (PS) 1 or 2 is the catalytic component of the γ‐secretase complex. Mitochondrial dysfunction is an established phenomenon in Alzheimer's disease (AD), but the causes and role of PS1, APP, and APP's cleavage products in this process are largely unknown. We studied the effect of these AD‐associated molecules on mitochondrial features. Using cells deficient in PSs expression, expressing human wild‐type PS1, or PS1 familial AD (FAD) mutants, we found that PS1 affects mitochondrial energy metabolism (ATP levels and oxygen consumption) and expression of mitochondrial proteins. These effects were associated with enhanced expression of the mitochondrial master transcriptional coactivator PGC‐1α and its target genes. Importantly, PS1‐FAD mutations decreased PS1's ability to enhance PGC‐1α mRNA levels. Analyzing the effect of APP and its γ‐secretase‐derived cleavage products Aβ and AICD on PGC‐1α expression showed that APP and AICD increase PGC‐1α expression. Accordingly, PGC‐1α mRNA levels in cells deficient in APP/APLP2 or expressing APP lacking its last 15 amino acids were lower than in control cells, and treatment with AICD, but not with Aβ, enhanced PGC‐1α mRNA levels in these and PSs‐deficient cells. In addition, knockdown of the AICD‐binding partner Fe65 reduced PGC‐1α mRNA levels. Importantly, APP/AICD increases PGC‐1α expression also in the mice brain. Our results therefore suggest that APP processing regulates mitochondrial function and that impairments in the newly discovered PS1/APP/AICD/PGC‐1α pathway may lead to mitochondrial dysfunction and neurodegeneration.

Understanding the molecular mechanisms underlying amyloid precursor protein family (APP/APP-like proteins, APLP) function in the nervous system can be achieved by studying the APP/APLP interactome. In this review article, we focused on intracellular APP interacting proteins that bind the YENPTY internalization motif located in the last 15 amino acids of the C-terminal region. These proteins, which include X11/Munc-18-interacting proteins (Mints) and FE65/FE65Ls, represent APP cytosolic binding partners exhibiting different neuronal functions. A comparison of FE65 and APP family member mutant mice revealed a shared function for APP/FE65 protein family members in neurogenesis and neuronal positioning. Accumulating evidence also supports a role for membrane-associated APP/APLP proteins in synapse formation and function. Therefore, it is tempting to speculate that APP/APLP C-terminal interacting proteins transmit APP/APLP-dependent signals at the synapse. Herein, we compare our current knowledge of the synaptic phenotypes of APP/APLP mutant mice with those of mice lacking different APP/APLP interaction partners and discuss the possible downstream effects of APP-dependent FE65/FE65L or X11/Mint signaling on synaptic vesicle release, synaptic morphology and function. Given that the role of X11/Mint proteins at the synapse is well-established, we propose a model highlighting the role of FE65 protein family members for transduction of APP/APLP physiological function at the synapse.

Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid‐β peptide (Aβ) and liberates the carboxy‐terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Aβ generation. We determined the crystal structure of the AICD in complex with the C‐terminal phosphotyrosine‐binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB‐binding motif and two α helices. The amino‐terminal helix of the AICD is capped by threonine T 668 , an Alzheimer disease‐relevant phosphorylation site involved in Fe65‐binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T 668 phosphorylation‐dependent complex regulation at a molecular level. A molecular switch model is proposed.

Physiological function and pathology of the Alzheimer's disease causing amyloid precursor protein (APP) are correlated with its cytosolic adaptor Fe65 encompassing a WW and two phosphotyrosine-binding domains (PTBs). The C-terminal Fe65-PTB2 binds a large portion of the APP intracellular domain (AICD) including the GYENPTY internalization sequence fingerprint. AICD binding to Fe65-PTB2 opens an intra-molecular interaction causing a structural change and altering Fe65 activity. Here we show that in the absence of the AICD, Fe65-PTB2 forms a homodimer in solution and determine its crystal structure at 2.6 Å resolution. Dimerization involves the unwinding of a C-terminal α-helix that mimics binding of the AICD internalization sequence, thus shielding the hydrophobic binding pocket. Specific dimer formation is validated by nuclear magnetic resonance (NMR) techniques and cell-based analyses reveal that Fe65-PTB2 together with the WW domain are necessary and sufficient for dimerization. Together, our data demonstrate that Fe65 dimerizes via its APP interaction site, suggesting that besides intra- also intermolecular interactions between Fe65 molecules contribute to homeostatic regulation of APP mediated signaling.