Fe65 Is the Sole Member of Its Family That Mediates Transcription

RESEARCH ARTICLE Fe65 is the sole member of its family that mediates transcription regulated by the amyloid precursor protein Sabine Probst*, Maik Krüger, Larissa Kägi, Sarina Thöni, Daniel Schuppli, Roger M. Nitsch and Uwe Konietzko*,‡

ABSTRACT with extracellular matrix components or neighboring cells. A major The amyloid precursor protein (APP), a central molecule in determinant of amyloidogenic cleavage is the subcellular Alzheimer’s disease (AD), has physiological roles in cell adhesion localization of APP (Rajendran et al., 2008) and this is regulated and signaling, migration, neurite outgrowth and synaptogenesis. by proteins binding to the APP intracellular domain (AICD). Intracellular adapter proteins mediate the function of transmembrane Fe65 (also known as APBB1) is the most prominent AICD- proteins. Fe65 (also known as APBB1) is a major APP-binding binding protein, frequently found in yeast two hybrid screens using protein. Regulated intramembrane proteolysis (RIP) by γ-secretase AICD as bait (Bressler et al., 1996; Cao and Sudhof, 2001). As with releases the APP intracellular domain (AICD), together with the APP, Fe65 has two homologs, the Fe65-like proteins Fe65L1 and interacting proteins, from the membrane. We studied the impact of the Fe65L2 (also known as APBB2 and APBB3, respectively). The Fe65 family (Fe65, and its homologs Fe65L1 and Fe65L2, also importance of Fe65 in mediating APP function is revealed by the known as APBB2 and APBB3, respectively) on the nuclear signaling similar phenotypes of APP- and Fe65 family-knockout (KO) mice, function of the AICD. All Fe65 family members increased which show cortical dysplasia caused by altered neuroblast amyloidogenic processing of APP, generating higher levels of β- migration (Guénette et al., 2006; Herms et al., 2004; McLoughlin cleaved APP stubs and AICD. However, Fe65 was the only family and Miller, 2008) and deficits in formation of the neuromuscular member supporting AICD translocation to nuclear spots and its synapse (Strecker et al., 2016; Wang et al., 2005). In C. elegans, transcriptional activity. Using a recently established transcription knockdown of the orthologs of Fe65 or APP also reveals similar assay, we dissected the transcriptional activity of Fe65 and provide negative effects in the regulation of pharyngeal pumping (Zambrano strong evidence that Fe65 represents a transcription factor. We show et al., 2002). Fe65 function is thus strongly connected with APP/ that Fe65 relies on the lysine acetyltransferase Tip60 (also known as AICD signaling functions. Fe65 dominates AICD function even in KAT5) for nuclear translocation. Furthermore, inhibition of APP the presence of other AICD-binding proteins such as MINT1 (also α cleavage reduces nuclear Tip60 levels, but this does not occur in known as X11 and APBA1) or Jip1 (also known as MAPK8IP1), Fe65-knockout cells. The rate of APP cleavage therefore regulates as seen in pull-down assays or localization of AICD to different the nuclear translocation of AICD–Fe65–Tip60 (AFT) complexes, to nuclear compartments (Konietzko et al., 2010; Lau et al., 2000). The promote transcription by Fe65. crystal structure of AICD bound to the phosphotyrosine-binding domain 2 (PTB2) of Fe65 revealed an extraordinary extended KEY WORDS: APBB1, APP, Fe65L1, Fe65L2, Nuclear signaling, interaction interface, three times larger than the known peptide– Transcription assay PTB domain complexes (Radzimanowski et al., 2008), lending an explanation for the predominance of APP–Fe65 interaction among INTRODUCTION the plethora of interaction partners for AICD (Müller et al., 2008). Alterations in the complex metabolism of APP have been linked to Fe65 contains two PTB domains and a WW domain that can Alzheimer’s disease (AD). Proteolytic processing of APP generates simultaneously bind different proteins, and it can thus be viewed as several fragments, of which the Aβ peptide is currently the most a scaffolding protein (Chow et al., 2015a). Full-length APP can pursued therapeutic target – in line with the amyloid cascade anchor Fe65 at the membrane (Minopoli et al., 2001), forming a hypothesis (Hardy and Selkoe, 2002; Sevigny et al., 2016). In the scaffold, for instance with the ApoE receptor family (Herz and opposing non-amyloidogenic cleavage pathway, Aβ is destroyed Beffert, 2000), and γ-secretase-mediated cleavage of APP releases and a secreted APP fragment (sAPPα) is generated that has diverse the AICD–Fe65 complex, enabling it to translocate to the nucleus positive functions on neurons (Müller et al., 2017). The choice (Kimberly et al., 2001; Kinoshita et al., 2002). In the nucleus, between the two cleavage pathways determines the risk of AICD–Fe65 localizes together with the lysine acetyltransferase developing AD, as shown by genetic mutations in APP (Bertram Tip60 (also known as KAT5) in spherical AFT spots that represent and Tanzi, 2008). APP has properties of a cell adhesion molecule sites of transcription (Konietzko et al., 2010; von Rotz et al., 2004). (Soba et al., 2005), and its cleavage can be regulated by interaction Fusion of yeast Gal4 DNA-binding domains to APP or AICD has revealed that transactivation activity can be dramatically enhanced Institute for Regenerative Medicine – IREM, University of Zurich, Campus Schlieren, by co-expression of Fe65 (Cao and Sudhof, 2001). Further studies Wagistrasse 12, 8952 Schlieren – Zurich, Switzerland. have shown varying results for AICD–Fe65 signaling and reports on *These authors contributed equally to this work transcriptional activity of the other Fe65 family members are sparse. ‡Author for correspondence ([email protected]) We therefore studied the Fe65 family with respect to nuclear signaling and transcription, and performed detailed analysis of the U.K., 0000-0002-6897-232X role of different domains and motifs. We conclude that of the family,

Handling Editor: Maria Carmo-Fonseca only Fe65 is a transcription factor whose activity in the nucleus is

Received 14 December 2019; Accepted 11 July 2020 regulated by APP processing. Journal of Cell Science

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RESULTS Fe65 contains a unique stretch of 15 negatively charged amino The predicted NLS of Fe65 is not functional for nuclear import acids, with six more in close vicinity, that contains only one We aligned the three Fe65 family member amino acid sequences, positively charged lysine. We have added this motif (denoted A) in revealing the described conserved WW, PTB1 and PTB2 domains the Fe65 scheme. For a more accurate discussion we have termed the (see Fig. 7A). Fe65 and Fe65L1 are extended N-terminal to the WW N- and C-terminal parts bracketing these motifs and domains Nt and domain by around 250 and 290 amino acids, respectively. Only Ct, and the linkers in between from L1 to L3 (Fig. 1A, scaled to

Fig. 1. The predicted NLS of Fe65 is not functional in nuclear import. (A) Overview of the different Fe65 constructs used in this study. Fe65 is drawn scaled to amino acid numbering based on reported domains and alignments. The acidic region (A), the WW (W), the PTB1 and PTB2 domains are highlighted in grey. The N-terminal (Nt) and C-terminal (Ct) regions as well as the linker (L1, L2, 3) regions are shown in white. Internal deletions are drawn as a line, point mutations as a cross. (B) Mutation of the predicted NLS in Fe65 does not prevent nuclear AFT spot formation. The Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells, together with APP-Cit and CFP-Tip60, and imaged by confocal microscopy. Scale bar: 10 μm. Enlarged nucleus is magnified by a factor of 2. (C) SBP-myc-tagged Fe65 K701/703A mutant or wt Fe65 were transfected into HEK293 cells together with either myc-Tip60 or APP-3HA, followed by streptavidin-based purification. Blots show cell lysates (L) and eluates (E) after purification. Fe65 was detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies, and full-length APP (flAPP) and APP CTF with anti-HA antibodies. (D) The nuclear export import inhibitor leptomycin B (LMB) results in nuclear accumulation of Fe65 with mutations in the predicted NLS. Scale bar: 10 μm. (E) LMB analysis of all Fe65 mutants revealed that only simultaneous deletion of the WW domain and L2 prevents nuclear import of Fe65. Outline of DAPI-stained nuclei is overlaid in white. Scale bar: 5 μm. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242917. doi:10.1242/jcs.242917 amino acid numbering of Fe65). We made various Fe65 deletion or (Fig. 2D). We observed that Tip60 pulldown was strongly reduced mutation constructs as shown in Fig. 1A. The Fe65 mutants include by deletion of the Fe65 PTB1 domain as described previously (Cao progressive deletions of the N-terminus or C-terminus, deletion of and Sudhof, 2001). In addition, deletion of the N-terminal region of single domains or selected point mutations. All constructs were Fe65 as in the ΔN253 and ΔN357 constructs, also strongly reduced tagged at the N-terminus with a streptavidin-binding peptide (SBP) Tip60 pulldown. Thus, there seems to be a widespread interaction followed by a Myc tag. interface between Fe65 and Tip60, whereby both the Fe65 We wanted to verify the predicted C-terminal nuclear localization N-terminal region and the PTB1 domain are necessary for signal (NLS) in Fe65 and mutated two lysine residues to alanine. We enabling Tip60 pulldown. co-transfected fluorescently tagged APP-Citrine (Cit) and CFP- We used CRISPR-Cas9 editing with three different sgRNAs to Tip60 with wild-type (wt) Fe65 or the Fe65 K701/703A mutant into generate three independent HEK293 Fe65-KO cell lines. We did not HEK293 cells to observe the formation of nuclear AFT spots using identify an antibody that detects endogenous Tip60 and therefore confocal microscopy as described previously (Konietzko et al., transfected Tip60 into these cells. We performed inhibition of APP 2010; von Rotz et al., 2004). Although the mutation of the putative cleavage by γ-secretase and subcellular fractionation into cytosol/ NLS should prevent nuclear translocation, we clearly detected membrane and nuclear fractions. Inhibition of APP cleavage strongly nuclear AFT spots (Fig. 1B). We analyzed whether the mutant Fe65 attenuated nuclear Tip60 levels in wt HEK293 cells (Fig. 2E). In was still able to interact with APP and Tip60. HEK293 lysates co- contrast, the Fe65-KO cell lines had lower baseline nuclear Tip60 expressing SBP-tagged Fe65 K701/703A mutant or wt, together levels that were not affected by γ-secretase inhibition. Quantification with either Tip60 or APP, were subjected to streptavidin-based of nuclear Tip60 levels with normalization to histone 3 from all three purification. The Fe65 K701/703A mutant interacted with APP and Fe65 KO lines is shown in Fig. 2F. Tip60 levels in the cytosol/ Tip60 to similar degree to wt Fe65, in line with its capacity to form membrane fraction could not be determined, due to an unspecific band AFT spots (Fig. 1C). Fe65 undergoes nuclear export that can be detected by the Tip60 polyclonal antibody above the Tip60 signal. inhibited by leptomycin B (LMB), leading to nuclear accumulation Overall, these results show that processing of endogenous APP in due to ongoing nuclear import of Fe65 (von Rotz et al., 2004). LMB HEK293 cells controls the subcellular localization of expressed Tip60 treatment resulted in clear nuclear accumulation of the Fe65 K701/ via the endogenously expressed Fe65. These data identify Tip60 as a 703A mutant, further revealing that this putative NLS is not component that is bound to APP–Fe65 at the membrane and that co- necessary for nuclear import (Fig. 1D). We subjected all Fe65 translocates with AICD and Fe65 to the nucleus. constructs to LMB analysis (Fig. 1E). The N-terminal deletion series revealed lack of nuclear import starting with ΔN357, a Fe65 domains essential for AFT spot formation construct that still contains the predicted NLS, again showing its AICD–Fe65 complexes translocate to the nucleus where they, non-functionality. As the ΔN253 construct accumulated in the together with the lysine acetyl transferase Tip60, form AFT nucleus after LMB treatment, the NLS would be expected to reside complexes localized in spherical spots that represent transcription in the WW-L2 region of Fe65. Sole deletion of all motifs, including factories (Konietzko et al., 2010). Without Fe65, Tip60 localizes to the WW domain, or the L2 and Ct regions did not interfere with speckles, representing a different nuclear compartment that does not nuclear import of Fe65. Thus, we could not identify a single motif co-localize with AFT spots. Spots and speckles can be directing nuclear import of Fe65 in the WW domain or L2 – deletion unequivocally identified with confocal microscopy, and the of both is necessary to prevent nuclear import. occurrence of Tip60 in spots is a clear indication of the presence of Fe65 (Fig. S1). The Fe65 and Tip60 interaction promotes the nuclear The different Fe65 deletion constructs were co-transfected translocation of both proteins together with APP-Cit and CFP-Tip60 into HEK293 cells to The lack of a unique NLS in Fe65 prompted us to access the identify essential domains of Fe65 for AFT spot formation (Fig. 3). possibility that Fe65 uses the NLSs of Tip60 for nuclear Owing to the crowded nuclear environment, the antibody-mediated translocation. We transfected HEK293 cells with either APP, detection of Fe65 is not as clear, as fused fluorescent proteins and Fe65 or Tip60 alone, or in different combinations (Fig. 2A). Fe65 was spots cannot always be resolved. Nevertheless, the relocation of distributed throughout the cytosol and in the nucleus and also Tip60 Tip60 to spots clearly reveals the presence of Fe65. N-terminal Fe65 could be detected in both compartments. Co-expression of Fe65 and deletions until the beginning of the WW domain support AFT spot Tip60 resulted in enhanced nuclear localization of both proteins and formation. The ΔN357 and ΔN531 Fe65 constructs do not additional APP co-expression retained more Tip60 and Fe65 outside translocate with AICD to the nucleus and Tip60 resides in of the nucleus. We quantified the nuclear:cytoplasmic (nuc/cyt) ratio speckles, correlating with their lack of nuclear translocation. as described in the Materials and Methods (Fig. 2B). The nuc/cyt ratio Deletion of the WW domain or L2, together necessary for nuclear of both Fe65 and Tip60 was increased by ∼2-fold when the two were translocation, had differing effects. Fe65ΔWW localized together co-expressed as compared to single expression, suggesting that Fe65 with Tip60 in nuclear speckles devoid of AICD. Fe65ΔL2 bound to and Tip60 shuttle to the nucleus together in a complex. APP co- Tip60 and induced relocation to spots that also did not harbor expression reduced the nuclear signals of Tip60 and Fe65. At the AICD. Deletion of the Fe65 PTB1 domain, necessary for Tip60 same time, compared to expressing APP alone, the nuc/cyt ratio of binding in pulldowns, still enabled association of Fe65 with Tip60 APP increased when Fe65 and Tip60 were co-expressed. in speckles, but again no relocation to spots or nuclear co- To analyze the molecular interaction between Fe65 and Tip60 in localization of AICD could be detected. Removal of the PTB2 detail, we co-transfected Tip60 with SBP-tagged Fe65 deletion domain still resulted in Fe65–Tip60 localization to nuclear spots constructs and performed streptavidin purification. Western blot that were devoid of AICD. Finally, deletion of the C-terminal analysis revealed similar Tip60 expression levels in co-transfected residues after the PTB2 domain in Fe65ΔCt showed AFT spot cells and comparable expression of the Fe65 constructs in cell formation indistinguishable from that of wt Fe65. These data show lysates (Fig. 2C). Quantification of pulldowns were performed, that both the WW and PTB1 domains are necessary for spot normalizing Tip60 levels in the eluate to Fe65 levels in the eluate formation. Journal of Cell Science

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Fig. 2. Fe65 and Tip60 interact to promote the nuclear translocation of each other. (A) Confocal microscopy of HEK293 cells transfected with HA-Fe65, myc- Tip60 or APP-Cerulean alone, or in different combinations. Every row represents one experiment. Co-expression of Fe65 and Tip60 leads to less extranuclear staining. Scale bar: 100 μm. (B) Quantification of APP, Fe65 and Tip60 in nuclei demarcated by DAPI and calculation of cytosolic levels from total measurements. Nuclear/cytoplasmic (nuc/cyt) ratios are relative measures due to the different accessibility of nuclear and cytoplasmic antigens for antibodies (mean±s.d., n=14). *P<0.05, **P<0.01 (Kruskal–Wallis with Dunn’s test). (C) Identification of Fe65 domains necessary for interaction with Tip60. SBP-myc-Fe65 deletion constructs were co-expressed with CFP-Tip60. After streptavidin–dynabead isolation, proteins were eluted with biotin, and lysate and eluates were analyzed by western blotting with antibodies against the Myc tag and GFP. (D) Quantification of Tip60 in eluates, normalized to Fe65 eluate levels (mean±s.e.m., n=3). *P<0.05 (Mann–Whitney U-test). Tip60–Fe65 interaction is disrupted by deletion of the Fe65 N-terminal region (Nt-Ac-L1) or deletion of PTB1. (E) HEK293 wt and Fe65- KO cells were transfected with myc-Tip60 and treated with DAPT or control solution, followed by fractionation into cytosol/membrane (C/M) and nuclear (N) fractions. The Tip60 signal in the C/M fraction is occluded by an unspecific band (star). Fe65 is only detected in the C/M fraction of wt cells. (F) Quantification of Tip60, normalized to histone H3, in the N fraction of three independent KO cell lines versus n=3 wt cell lines (mean±s.d.). **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). Journal of Cell Science

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Transcriptional activity of Fe65 constructs possessed transcriptional activity, albeit far below wild-type levels We recently developed yeast Gal4-based cellular assays for nuclear (Fig. 5A). Additional inclusion of the WW domain (ΔC295) signaling and transcriptional activity (Konietzko et al., 2019). increased transcriptional activity over wild-type levels, yet Tip60 HEK293 cells were infected with lentiviral vectors to integrate resided in speckles, as for the Fe65ΔC254 construct (Fig. S3). 9×UAS-Citrine reporters into the genome (denoted HEK:UAS-Cit The ΔC516 construct, which only lacks the L3-PTB2-Ct part of cells). Expressing the fluorescent protein Citrine as a reporter Fe65, showed the strongest increase in reporter transcription. The circumvents enzymatic detection in cell lysates and enables ΔC516 construct includes all regions found to interact with Tip60 quantification by confocal microscopy in situ. We tagged Fe65 and leads to a dispersal of Tip60 speckles but failed to form with the Gal4 DNA-binding domain (Gal4-DBD) by replacing the spherical spots. SBP tag in all Fe65 constructs and used sole transfection of these Deletion of the PTB2 increased transcriptional activity, whereas constructs to determine their transcriptional activity via Citrine the C654F mutation did not affect it (Fig. 4). Both constructs are expression (Fig. 4A). Expression of wt Gal4-DBD-Fe65 in HEK: reported to lack APP binding (Borg et al., 1996; Cao and Sudhof, UAS-Cit cells induced the expression of Citrine, in contrast to 2001; Minopoli et al., 2001), and thus sequestration of Fe65 by APP lipofection alone, which only showed baseline leakage expression cannot explain this discrepancy. Besides APP binding, the PTB2 (Fig. 4B). Fiji software was used to measure total Citrine domain also mediates Fe65 dimer formation (Feilen et al., 2017). fluorescence and to outline and count DAPI-stained nuclei (zoom We hypothesized that Fe65 dimer formation could lead to Fe65 in Fig. 4B). We transfected the Fe65 constructs into different sequestration in the cytosol, akin to its sequestration at the passages of HEK:UAS-Cit cells to analyze levels of Citrine membrane by APP. We performed pulldown experiments with expression normalized to the number of nuclei (Fig. 4C). Deletion differently tagged Fe65 constructs and again saw the disruption of of the Fe65 Nt (ΔN144) resulted in a significant increase in Fe65 dimers by the PTB2 deletion, but the C654F mutation did not transcriptional activity that was returned to wild-type levels by interfere with dimer formation (Fig. 5B). additional deletion of the acidic motif and L1 (ΔN253). Further We directly compared the ΔC516 construct, which lacks both the deletion of the WW domain and L2 (ΔN357) reduced Citrine PTB2 domain and Ct, with the single deletions in HEK2:UAS-Cit- expression to baseline levels and the same was seen for Fe65 NLS cells. Whereas both single deletions showed an increase over ΔN531, which consists solely of PTB2 and Ct. Internal wild-type Fe65 activity as seen with the other reporter cells, the Fe65 deletions of the WW or PTB1 domains, or the L2 ΔC516 construct revealed even higher activity, demonstrating connecting them, caused a strong reduction in transcriptional independent inhibitory functions for the PTB2 domain and Ct activity of these constructs. By contrast, deletion of the PTB2 (Fig. 5C). We next deleted the inhibitory Nt region in addition to domain or Ct more than doubled the expression of Citrine. PTB2-Ct, but this construct showed no further increase in activity The C654F mutation in the PTB2 domain did not show a over the ΔC516 construct alone (Fig. 5D). These experiments reveal difference in transcriptional activity compared to wt Fe65. that the central part of Fe65, from the acidic motif to the PTB1 Mutation of the predicted NLS (K701/703A) significantly domain, contributes to the transcriptional activity and the bracketing increased Citrine expression over wt Fe65. Transfecting a full- Nt and PTB2-Ct have an inhibitory function. length Fe65 with an SBP tag replacing the Gal4-DBD showed no transcriptional activity as this construct does not bind the UAS Dissociation of AFT spot formation and Gal4/UAS-Cit assay elements preceding the Citrine reporter. We analyzed a DNA concentration series of Gal4-Fe65 revealing a Lentiviral reporter vectors were improved by fusing an NLS to sigmoidal response of Cit expression in two cell lines (Fig. 5E), as Citrine, which concentrates it in the nucleus (Konietzko et al., we reported for APP-Gal4 plus Fe65 (Konietzko et al., 2019). The 2019). This method is more accurate as it measures Citrine 1 µg DNA used in all experiments in this study is clearly in the fluorescence only in the DAPI-stained nuclei that are identified by saturated range of the assay. We reanalyzed selected Fe65 constructs Fiji software and counted for normalization. We infected mouse with increased or decreased transcriptional activity using only N2a neuroblastoma cells to derive a reporter cell line. As with the 150 ng DNA to target the linear range of the assay. The results HEK:UAS-Cit cell line, the N2a:UAS-Cit-NLS cell line showed mirrored those with 1 µg DNA with the same magnitude of change Citrine expression – now confined to the nucleus – after transfection of transcriptional activity (Fig. 5F). of Gal4-Fe65 (Fig. S2). We again analyzed all Gal4-Fe65 We designed a control construct with the Gal4-DBD fused to the constructs. Transcriptional activity of the different deletions fluorescent protein Cerulean (Cer; Gal4-myc-Cer-NLS) and closely replicated the results seen in HEK293 cells, with the confirmed that this construct showed no activity in the UAS-Cit exception of the ΔN253 construct that had reduced activity assay, in contrast to Gal4-Fe65 (Fig. 5G). Gal4-Fe65 transcriptional compared to wt Fe65 in N2a cells (Fig. 4D). activity is reduced upon co-expression of a control plasmid (Cer- We reevaluated some of the Fe65 constructs, including ΔN253, in NLS). We set this to 100% to judge the effect of the Gal4-DBD- HEK2:UAS-Cit-NLS cells (see Fig. 6M) to give a better signal-to- containing construct. Co-expression of Gal4-myc-Cer-NLS, as noise ratio than was seen in HEK:UAS-Cit cells (Konietzko et al., opposed to Cer-NLS, strongly reduced Gal4-Fe65-induced Cit 2019). Removal of the N-terminal 144 residues of Fe65 again expression due to the competition for UAS elements integrated in increased transcriptional activity, whereas further deletion of the the genome. We co-transfected Cit-AICD, HA-Fe65 and V5-Tip60, acidic motif with the adjacent L1 (ΔN253) reduced activity below together with the Gal4-myc-Cer-NLS construct. In contrast to the wild-type levels (Fig. 5A). We also reanalyzed the effect of competition seen in the UAS-Cit assay, imaging the Gal4-myc-Cer- mutating K701/703 in Fe65, which again led to a significant NLS construct via Myc staining or Cer fluorescence showed no increase in transcriptional activity. colocalization with AFT spots (Fig. 5H). These experiments show To complement the N-terminal deletion constructs of Fe65, we that the UAS-Cit assay merely measures transcriptional activity of constructed a set of C-terminal deletions (constructs depicted in Gal4-fused proteins, whereas AFT spot formation indicates the Fig. 1A). Fe65ΔC254, which lacks all C-terminal sequences property to localize to transcription factories at endogenous including the WW domain but retains the acidic motif, still promoters. Journal of Cell Science

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Fig. 3. Function of different Fe65 domains in AFT spot formation. Confocal microscopy of HEK293 cells transfected with full-length SBP-myc-Fe65 or deletion mutants, together with APP-Cit and CFP-Tip60. A representative image of each clone from a minimum of three different transfections is shown. Arrows denote a cell with nuclear AFT spots. Arrowheads denote a cell where Tip60 localizes to nuclear speckles that are devoid of Fe65 and AICD. A magnified view (factor of 2) of one cell is also shown. Nuclear signals from the fluorescent proteins are clearly resolved, whereas antibody staining of nuclear structures is restricted. Scale bar: 10 µm.

Fe65L1 and Fe65L2 do not promote transcription We transfected the constructs alone, or in different combinations We inserted the Fe65L1 and Fe65L2 sequences in the different with APP and/or Tip60 into HEK293 cells and analyzed their pUKBK vectors to label them with Gal4-myc- and SBP-myc-tags. localization by confocal microscopy. In contrast to Fe65, Fe65L1 is Journal of Cell Science

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Fig. 4. Transcriptional activity of Gal4-Fe65 constructs. (A) Schematic of the transactivation assay. HEK293 cells and N2a cells were infected with viral vectors to ensure nuclear localization of the reporter cassette that contains nine repeats of the Gal4 upstream activator sequence (UAS) driving Citrine (Cit) expression. The Gal4-DBD is fused N-terminally to the different Fe65 constructs. (B) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK:UAS-Cit cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (C) HEK:UAS-Cit cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. Total Cit intensity was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) N2a:UAS-Cit-NLS cells were transfected with various Gal4-Fe65 constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalized by the number of nuclei and the value for full-length Fe65 set to 100%. Bars depict mean±s.d. of n=21 images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction).

excluded from the nucleus, as seen in single confocal sections in line with the reported APP–Fe65L1 interaction (Fig. 6B). No (Fig. 6A). Co-expression of APP leads to relocation of diffuse interaction could be seen with Tip60, which located to nuclear

Fe65L1 cytoplasmic staining to cellular sites where APP is present, speckles, while Fe65L1 retained its diffuse cytoplasmic distribution Journal of Cell Science

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Fig. 5. See next page for legend.

(Fig. 6C). Co-expression of all three proteins again showed APP– Analysis of the third family member Fe65L2, revealed a major Fe65L1 colocalization throughout the cell, apart from in the localization to nuclear speckles when expressed alone (Fig. 6G). nucleus, where Tip60 is localized to speckles (Fig. 6D). To further Co-expression of APP completely relocated nuclear Fe65L2 to the show the lack of nuclear Fe65L1 translocation, we inhibited nuclear extranuclear locations of APP, confirming the known interaction export with LMB. In contrast to Fe65, where LMB treatment leads (Fig. 6H). Together with Tip60, nearly all Fe65L2 was seen to to accumulation in the nucleus (Fig. 6E), Fe65L1 could not be localize to the same nuclear speckle structures (Fig. 6I). With all detected in the nucleus (Fig. 6F). three proteins expressed, Fe65L2 localized with APP throughout the Journal of Cell Science

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Fig. 5. UAS-Cit assay refinements and dissociation from AFT spot in Fe65 are involved in Tip60 binding (Fig. 2D). We performed formation. HEK2:UAS-Cit-NLS cells were used for all transcription assays. pulldown experiments with the Fe65 family and the two fusion (A) Lipofection of Fe65 deletion constructs (schemes in Fig. 1A) was followed proteins to detect the interaction with Tip60. For Fe65L2 we used a by confocal microscopy analysis. Mean±s.d. of n=21 images from three replicates are shown. ***P<0.001 (one-way ANOVA with Bonferroni CFP-Tip60 construct as the Myc-Tip60 constructs runs at the same correction). (B) SBP-myc-Fe65 (SBP-Fe65) was co-transfected with mCherry- height in SDS-PAGE (Fig. 7D). Neither Fe65L1 or Fe65L2 show Fe65 (mChe-Fe65) wt, C654F or ΔPTB2 into HEK293 cells, followed by co-purification of Tip60, whereas both fusion constructs were able streptavidin-based purification. Myc and mCherry stainings were used to to pulldown Tip60. These data confirm the role of the Fe65 N- dissociate the different Fe65 species in lysates (L) and eluates (E). Bands terminus in binding Tip60 in addition to the PTB1 domain. marked with a star are leftover signals from the mChe-Fe65 staining. (C) UAS- We constructed fusion proteins of Fe65 and Fe65L1 to further Cit assay for comparison of Fe65 C-terminal deletions. Bars depict mean±s.d. delineate crucial motifs and domains (Fig. S4A). We replaced the Nt of n=21 confocal microscopy images from three replicates. *P<0.05; **P<0.01; – ***P<0.001; ns, not significant (Kruskal–Wallis with Dunn’s test). (D) UAS-Cit region of Fe65 preceding the acidic motif with the corresponding assay to compare combined N- and C-terminal deletions with sole deletion of 48 amino acids longer – sequence from Fe65L1 and saw no effect on the C-terminus. Mean±s.d. of n=21 images from three replicates are shown. transcriptional activity, but AFT spot formation was abolished ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (Fig. S4B,C). Additional exchange of the acidic motif and L1 in (E) Sigmoidal dose-response in two HEK:UAS-Cit-NLS cell lines infected once Fe65 with the Fe65L1 sequence resulted in a significant reduction of or twice with reporter viruses, and transfected with increasing amounts of Gal4- Citrine reporter transcription and also no spot formation. We Fe65 plasmids (n=6 images per concentration, mean±s.d.). (F) Transcriptional activity of Fe65 deletion constructs was analyzed with lipofection of 150 ng per expressed Fe65L1 lacking the N-terminal 290 residues. In contrast construct, in contrast to the 1 µg used in all other assays. Mean±s.d. of n=21 to full-length Fe65L1, this construct showed transcriptional activity, images from three replicates are shown. *P<0.05; ***P<0.001 (one-way albeit at quite low levels compared to Fe65, and no spot formation ANOVA with Bonferroni correction). (G) HEK2:UAS-Cit-NLS cells were occurred. Replacement of the L3-PTB2-Ct domains in the truncated transfected with Gal4-Fe65, alone or together with Cer-NLS or Gal4-myc-Cer- Fe65L1 with the Fe65 sequence further increased transcriptional NLS constructs and analyzed by confocal microscopy for Cit expression. Bars activity and enabled the formation of AFT spots. The mirror depict mean±s.d. of n=7 images. ***P<0.001 (one-way ANOVA with Bonferroni correction). (H) Cit-AICD, HA-Fe65, V5-Tip60 and the Gal4-myc-Cer-NLS construct, with the WW-L2-PTB1 sequence from Fe65 fused to the construct were co-transfected into HEK293 cells. Confocal microscopy L3-PTB2-Ct of Fe65L1 revealed even stronger transcriptional revealed the formation of AFT spots detected by the Cit signal. Gal4-myc-Cer- activity but no AFT spot formation. Finally, replacing the acidic NLS, detected by Cer fluorescence and myc staining, did not colocalize with motif plus L1 in Fe65 with the Fe65L1 sequence resulted in a small, AFT spots. Scale bar: 10 µm. but significant drop in transcriptional activity. These data show that several regions of Fe65 have evolved to better support transcription. cell and with Tip60 in nuclear speckles that revealed only a faint Activity is reduced by lack of the unique acidic region and exchange AICD signal (Fig. 6J). LMB treatment resulted in nuclear of the WW-L2-PTB1 central region by the Fe65L1 sequence. accumulation of Fe65L2 (Fig. 6K). Thus, all Fe65 family Furthermore, the L3-PTB2-Ct region of Fe65 is sufficient to enable members bind APP but differ strongly in their nuclear localization. spot formation when fused to a truncated Fe65L1. Gal4-DBD-fused Fe65L1 and Fe65L2 constructs were transfected into the HEK:UAS-Cit reporter line. Confocal The Fe65 family increases amyloidogenic APP processing microscopy analysis revealed that only Fe65 shows transcriptional and AICD levels activity. Expression of Fe65L1 or Fe65L2 did not raise Citrine To determine the impact of the Fe65 family members on APP expression above baseline (Fig. 6L). We further analyzed metabolism, we co-expressed them, or Cit-3HA as a control, transcriptional activity in HEK2:UAS-Cit-NLS cells (Konietzko together with a construct expressing 3myc-APP-3HA via a GAPDH et al., 2019). Expression of Gal4-Fe65 induced strong Citrine-NLS promoter (Gersbacher et al., 2013). We analyzed APP fragments expression localized to the nucleus with very low leakage using western blots – due to the approximately extra 5 kDa of the expression seen under control conditions (Fig. 6M). Comparison 3HA tag we were able to resolve overexpressed and endogenous of Fe65 family members in HEK2:UAS-Cit-NLS cells again APP fragments using an APP C-terminal antibody (Fig. S5A). showed transcriptional activity for Fe65, not for Fe65L1 and a DAPT incubation was performed to verify C-terminal fragments minor response for Fe65L2 (Fig. 6N). In conclusion, Fe65 is the sole (CTFs) and AICD bands by their increase or decrease, respectively. family member with transcriptional activity. We stained the different APP fragments using several antibodies and quantified n=3 experiments, normalizing the levels to GAPDH. The Dissection of transcriptional function with fusion proteins Fe65 family members partially increased the levels of endogenous The major difference in the protein sequence of the family members and expressed full-length APP (Fig. S5B). We did not detect a is in the 250 N-terminal residues of Fe65 and Fe65L1. Fe65L2 lacks change in the of levels of the α C-terminal fragment (α-CTF) of this region (Fig. 7A). To elucidate the function of the Fe65 APP, which were strongly enhanced by DAPT treatment (Fig. S5C, N-terminus, we fused the 250 residues to Fe65L2 or exchanged the S6C). All three Fe65 family members enhanced the levels of the 300 N-terminal residues of Fe65L1 with the Fe65 sequence β-CTF of both endogenous and overexpressed APP (Fig. S5D,F). (Fig. 7A). Constructs with N-terminal fusion of SBP were used Furthermore, AICD levels were also strongly increased for both by for confocal microscopy and N-terminal fusions of the Gal4-DBD all Fe65 family members (Fig. S5E,G). Identical results regarding were used to analyze transcriptional activity in HEK2:UAS-Cit- full-length APP, β-CTF, α-CTF and AICD were seen using an anti- NLS cells. Co-transfection of these constructs with APP and Tip60 HA antibody to detect overexpressed APP (Fig. S6A). Analysis of showed that neither was able to form AFT spots, with the Fe65L1 medium revealed no difference in sAPPα derived from both fusion still excluded from the nucleus and the Fe65L2 fusion overexpressed and endogenous APP, as well as no difference in residing in nuclear speckles (Fig. 7B). We analyzed the fusion 3myc-tagged sAPP levels (Fig. S6B). We measured the half-lives of proteins in the UAS-Cit assay and saw that neither acquired endogenous APP by performing a cycloheximide (CHX) time series transcriptional activity upon the fusion of the Fe65 N-terminal with concomitant expression of the Fe65 family members or a residues (Fig. 7C). We have shown that the 250 N-terminal residues control construct. Both Fe65 and Fe65L2 expression resulted in a Journal of Cell Science

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Fig. 6. Fe65L1 and Fe65L2 do not promote transcription. (A–J) HEK293 cells transfected with Fe65 family members, Tip60 and APP, were analyzed by confocal microscopy. Fe65L1 and Fe65L2 were transfected alone (A,G), together with APP (B,H), together with Tip60 (C,I) or with APP and Tip60 (D,J). (E,F,K) LMB treatment leads to nuclear accumulation of Fe65 and Fe65L2 but not Fe65L1. Scale bar: 10 µm. (L) Transcription assay with HEK:UAS-Cit cells imaged via confocal microscopy reveals lack of transcriptional activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). (M) Representative confocal microscopy of Gal4-Fe65-mediated Cit expression in HEK2:UAS-Cit-NLS cells, with DAPI-stained nuclei outlined by Fiji software. Scale bar: 200 µm. (N) Transcription assay with HEK2:UAS-Cit-NLS cells confirms lack of activity for Fe65L1 and Fe65L2. Bars depict mean±s.d. of n=21 images from three replicates. ***P<0.001 (one-way ANOVA with Bonferroni correction). stabilization of immature and mature APP (Fig. S7A). In Fe65L2 is the sole family member with high turnover conclusion, the common effect of the Fe65 family members on All Fe65 family members were expressed from the same APP metabolism is an increased amyloidogenic processing and promoter, yet Fe65L2 levels were much lower (Fig. S5A). increased AICD levels. We determined the half-life of Fe65 family members in the Journal of Cell Science

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Fig. 7. Fusion of the Fe65 N-terminus to Fe65L1 or Fe65L2 does not enable transcription. (A) Scheme of Fe65 family members and fusion proteins showing the conserved domains. Fe65L1 and Fe65L2 are adapted to Fe65 that is drawn scaled to amino acid numbering. Roughly, Fe65L1 has a 50 amino acids longer Nt and Fe65L2 starts only with the WW domain, has a L2 of half the size and a 23 residue extended Ct, all compared to Fe65. (B) Confocal microscopy of HEK293 cells transfected with the fusion constructs, together with APP-Cit and CFP-Tip60. Scale bar: 10 µm. (C) HEK2:UAS-Cit-NLS cells were transfected with the depicted constructs and analyzed by confocal microscopy for Cit expression. The sum of Cit intensity in the nuclei was normalizedby the number of nuclei and the value of full-length Fe65 set to 100%. Bars depict mean±s.d. (n=7). ***P<0.001; ns, not significant (one-way ANOVA with Bonferroni correction). (D) HEK293 cells were co-transfected with either myc-Tip60 or CFP-Tip60 and the SBP-myc-tagged Fe65 family and fusion constructs, followed by streptavidin-based pulldown. Blots show cell lysates (L) and eluates (E) after purification. Fe65 constructs were detected with anti-Myc antibodies, Tip60 with anti-Tip60 antibodies. GAPDH is only detected in lysates and not purified with the Fe65 family. above experiments that analyzed APP half-live by the CHX APP cleavage controls Fe65 nuclear signaling time series. Cell lysates were analyzed by SDS-PAGE and We co-transfected cells to generate AFT spots and analyzed the normalization was done with the highly stable GAPDH protein effects of inhibiting γ-secretase, which prevents the release of AICD (Fig. 8A; Fig. S7B). Quantification of protein levels showed from APP stubs. Whereas wild-type Fe65 supported AFT spot that Fe65 and Fe65L1 are quite stable with half-lives of formation under control conditions, incubation with the γ-secretase 24.4 and 35.6 h, respectively. Only Fe65L2 had a high inhibitor DAPT caused Fe65 to be retained at the membrane and turnover with a half-life of 1.5 h, in line with the lower levels Tip60 was not relocated from speckles to spots (Fig. 8B). The detected in Fig. S5A. ΔPTB2 Fe65 mutant cannot bind to APP, and DAPT was not able to Journal of Cell Science

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Fig. 8. Transcriptional activity of stable Fe65 is regulated by APP cleavage. (A) Half-life of Fe65 family members as determined by CHX-mediated inhibition of protein synthesis. Data points show mean±s.e.m. for n=5 experiments. Representative western blots are shown in Fig. S7B. (B) Confocal microscopy of HEK293 cells transfected with APP-Cit, CFP-Tip60 and either wt Fe65 or Fe65 mutants with disrupted APP binding. Treatment with DAPT revealed that APP retained only wt Fe65 outside of the nucleus with Tip60 located in speckles. Even with DAPT, both Fe65 mutants can still translocate to the nucleus and form FT spots that are devoid of AICD. Scale bar: 10 μm. (C) Transcription assay with HEK2:UAS-Cit-NLS cells. Gal4-Fe65 was co-transfected with APP, AICD or an empty vector and cells treated with DAPT or control solvent. Bars depict mean±s.d. of n=21 confocal microscopy images from three replicates. *P<0.05, ***P<0.001 (one-way ANOVA with Bonferroni correction). (D) Schematic overview of the role of Fe65 domains in transcription and spot formation. prevent its translocation to the nucleus and the formation of spots Inhibition of γ-secretase cleavage by DAPT slightly reduced Fe65 with Tip60, which were consequently devoid of AICD. The C654F activity under control conditions or with co-expression of AICD mutation in the PTB2 domain, which has been reported to inhibit when compared to the respective transfections without DAPT. APP binding (Minopoli et al., 2001), behaved in a similar manner to Inhibitor treatment resulted in a strong inhibition of Fe65 activity the ΔPTB2 mutant. when APP was co-expressed. These experiments show that the We further analyzed the influence of full-length APP and AICD turnover of APP determines the capacity of Fe65 to signal to the on the transactivation activity of Gal4-Fe65 using HEK2:UAS-Cit- nucleus and activate transcription. Fig. 8D shows a scheme of Fe65 NLS cells. APP and AICD had opposing effects, decreasing and depicting the regions regulating transcription, interacting with increasing Fe65 transactivation activity, respectively (Fig. 8C). Tip60 or APP and involved in AFT spot formation. Journal of Cell Science

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DISCUSSION Sudhof, 2001). Nevertheless, with immunocytochemistry, we saw We directly compared the three Fe65 family members with respect colocalization of the Tip60 NKSY to NASA mutant with Fe65 and to nuclear translocation, subnuclear location, transcriptional activity AICD in nuclear speckles (von Rotz et al., 2004). We now see a and influence on APP processing. We identified Fe65 as the sole similar effect with deletion of the PTB1 domain in Fe65 – mutant family member regulating transcription and see strong evidence that Fe65 localizes with Tip60 in nuclear speckles instead of spots and is Fe65 is a transcription factor regulated by APP processing. In not able to pulldown Tip60. Biochemical pull-down experiments addition, we show the role of extranuclear Tip60 in translocation of with several wash steps are harsher than fixing cells in situ. This AFT complexes to the nucleus. Interestingly, Fe65 was originally could explain why some interaction can still be detected via identified as a transcriptional activator (Duilio et al., 1991) and was colocalization using microscopy. Still, both NKSY mutation and only later shown to bind APP (Borg et al., 1996; Fiore et al., 1995; PTB1 deletion prevent the nuclear relocation of Fe65 and Tip60 Zambrano et al., 1997). In transcription assays with APP-Gal4, it from speckles to spots. was frequently reported that co-expression of Fe65 dramatically All Fe65 family members enhanced amyloidogenic processing increased transcriptional activity and deleting the APP-interacting with increases in β-CTF and AICD levels. Increased amyloidogenic PTB2 domain of Fe65 blocked this effect (Cao and Sudhof, 2001; processing has been described before for Fe65 (Chow et al., 2015b; Konietzko et al., 2019; Wiley et al., 2007; Zambrano et al., 2004). Lee et al., 2017; Sabo et al., 1999), but there are also studies Using a series of Fe65 deletion constructs, we showed that proposing the opposite (Hoe et al., 2006). For Fe65L1, increased Aβ constructs lacking K701/703 of the predicted NLS motif still secretion and AICD generation are reported (Chang et al., 2003), translocate to the nucleus and form AFT spots, whereas constructs and for Fe65L2 increased Aβ secretion (Tanahashi and Tabira, including the motif but lacking other domains do not accumulate in 2002). Taken together, the Fe65 family has an identical influence on the nucleus or form AFT spots. We conclude that the two lysine APP processing in vitro. This in line with Fe65/Fe65L1 double KO residues are not part of a NLS but nevertheless have a functional mice showing a reduction in Aβ peptides (Guénette et al., 2006). In role, as mutation to alanine increased the transcriptional activity of addition, knockin mice with the APP Y682G mutation that abolishes Fe65. In unpublished data we show that this relates to acetylation Fe65 binding show strongly increased sAPPα levels as well as of lysine residues by Tip60 (S.P., F. Riese, L.K., N. Russi, R.M.N. reduced sAPPβ (Barbagallo et al., 2010). The amyloidogenic and U.K., unpublished). Further experiments with Fe65 domain pathway mediates AICD and Fe65 nuclear signaling (Belyaev deletions or Fe65–Fe65L1 fusions did not reveal a confined motif et al., 2010; Flammang et al., 2012; Goodger et al., 2009). Thus, Fe65 for nuclear import in Fe65. The whole WW-L2-PTB1 region had to induces its own nuclear localization by promoting the amyloidogenic be deleted to disrupt nuclear localization. We conclude that Fe65 pathway. does not contain a classical NLS, but probably relies on a piggyback We detect some increase in full-length APP levels upon the mechanism as was previously proposed (Minopoli et al., 2001). expression of Fe65 family members, as well as stabilization of Tip60 mediates the accumulation of Fe65 and AICD in the mature and immature APP by Fe65 and Fe65L2. Stabilization of nucleus in AFT spots, and we had previously proposed that this is APP by Fe65 has been reported before, with stronger effects than due to sequestration of AICD–Fe65 complexes undergoing nucleo- reported here (Chow et al., 2015b). This difference might be cytoplasmic shuttling by nuclear Tip60 (von Rotz et al., 2004). explained by the different cells used for analysis. The HEK293 cells Nevertheless, we have also detected nucleo-cytoplasmic shuttling used in our analysis show endogenous Fe65 expression, whereas in of Tip60 and its colocalization with APP and Fe65 in neurites the CHO cells used in the cited study Fe65 is barely detectable (our (Konietzko et al., 2010). We now report that Fe65 also induces unpublished observations), leading to greater changes in APP nuclear localization of Tip60; thus, these two molecules interact to turnover when Fe65 is ectopically expressed. Fe65 is predominantly promote each other’s nuclear localization. Fe65, which lacks a expressed in neural tissue, including the adrenal gland (Sabo et al., unique NLS, therefore probably relies on the NLSs of Tip60 for 2003), and HEK293 cells are likely derived from the adrenal gland nuclear import. The nuclear localization of Tip60 is controlled by (Shaw et al., 2002). This might explain the high Fe65 expression in APP turnover, similar to Fe65. Inhibition of γ-secretase cleavage of HEK293 cells compared to non-neuronal cell lines such as CHO endogenous APP in HEK293 cells reduced nuclear Tip60 levels. and HeLa cells. This effect was abolished in Fe65-KO cells, showing that We directly compared all Fe65 family members with respect to endogenous levels of APP and Fe65 in HEK293 cells are transcriptional activity, using our recently developed Gal4/UAS-Cit sufficient to determine the nuclear localization of ectopically assay (Konietzko et al., 2019). Genomic integration of reporter expressed Tip60. sequences enables detection of bona fide nuclear signaling, as We showed that, for Fe65, the N-terminal 250 amino acids, as opposed to the transient transfections of reporter plasmids in the well as the reported PTB1 domain (Cao and Sudhof, 2001), are commonly used luciferase assays, which mainly reside in the necessary for Tip60 binding in pulldown experiments. Thus, the cytosol (discussed in Konietzko, 2012). We conclude that Tip60–Fe65 interaction involves widespread interaction surfaces. transcriptional activity is a unique property of Fe65. The lack of We found no indication of Fe65L1 interaction with Tip60, whereas transcriptional activity for Fe65L1 (Chang et al., 2003) and Fe65L2 Fe65L2 located to speckles together with Tip60, but also showed (Tanahashi and Tabira, 2002) have been noted before, using no interaction in pulldown experiments. Adding the Fe65 250 luciferase and chloramphenicol transferase assays. Analysis of N-terminal residues to FE65L2 or replacing the Fe65L1 N-terminal fusion proteins containing different parts of Fe65 and Fe65L1 residues with the Fe65 sequence was sufficient to enable the fusion revealed that various regions of Fe65 have evolved to better function proteins to pulldown Tip60. In conclusion, Fe65 shows stronger in transcription than the corresponding Fe65L1 sequences. This interaction with Tip60, which results in nuclear signaling properties concerns the central WW-L2-PTB1 region that together contributes not shared by the family members. to transcriptional activity. The WW domain of Fe65 has been Initially, yeast two-hybrid experiments had revealed that the previously shown to possess transcriptional activity (Cao and Fe65 L2-PTB1-L3 region binds Tip60 and mutation of the NKSY Sudhof, 2004; Telese et al., 2005). Furthermore, Fe65 contains a motif in the PTB1 domain disrupted pulldown of Fe65 (Cao and unique acidic region in the N-terminus. Both the Fe65 deletion Journal of Cell Science

13 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242917. doi:10.1242/jcs.242917 constructs and the Fe65/Fe65L1 fusion proteins revealed formation and presented an even higher transcriptional activity than transcriptional activity of this region. In conclusion, the central full-length Fe65. part of Fe65 from the acidic region to the PTB1 domain, contributes On first sight, this implies that AICD is not necessary for Fe65- to transcriptional activity. mediated transcription. However, transcriptional activity in the The Nt region preceding the acidic motif, as well as the C- assay relies on the Gal4-DBD that binds introduced UAS promoter terminal PTB2 domain and Ct, have an inhibitory effect on elements and the fused protein just needs to have transcriptional transcription. The PTB2 domain and Ct independently act to inhibit activity or attract transcription factors, not DNA-binding activity. transcription. Combined deletion further increases transcriptional We have shown that the Gal4-DBD does not colocalize with AFT activity above the similar increases seen with sole deletion of PTB2 spots but rather locates to the genomic integration sites of the UAS or Ct. Deletion of Nt alone increases transcription, but combined elements. The dissociation of transcriptional activity from the deletion with the Fe65 Ct region does not cause a further increase. capacity to form AFT spots relates to this. It is well possible that The inhibitory effect of the Ct region could be related to an only with bound AICD, is Fe65 is able to adopt a conformation to intramolecular interaction with the Nt region, shielding the central target endogenous promoters. We clearly see AICD localizing to transcriptionally active part of the molecule, similar to the model nuclear AFT complexes in our experiments and have shown that proposed by Cao and Sudhof (2004). The function of the PTB2 AFT spots localize to APP and KAI1 promoters (Konietzko et al., domain in suppressing transcription is discussed below. 2010), two proposed target genes of AICD and Fe65 (Baek et al., The UAS-Cit assay has a sigmoidal response curve with 2002; von Rotz et al., 2004). Furthermore, AICD has been shown to increasing amounts of Gal4-Fe65 lipofection, similar to the bind mediator subunits (Xu et al., 2011), which also form response we have reported recently for APP-Gal4/Fe65 transcription-dependent condensates that resemble AFT spots (Konietzko et al., 2019). There, we studied the effects of mutating (Cho et al., 2018). Dissection of Fe65 by deletion and fusion amino acids known to be targets of phosphorylation in the AICD of constructs revealed that, in addition to the WW and PTB1 domains, APP-Gal4 and could only detect changes of transcriptional activity the C-terminal L3-PTB2-Ct region is absolutely required for AFT in the linear range of the assay, not at saturating 1 µg DNA. The APP spot formation. It even enabled a N-terminally truncated Fe65L1 to G681A mutant that was shown to abolish Fe65 binding (Cao and localize to spots when replacing the Fe65L1 sequence. Besides Sudhof, 2004), disrupted APP-Gal4-induced transcriptional activity AICD, Fe65 and Tip60, more proteins are likely localized to spots both in the linear and the saturated range. Detecting changes that that could be scaffolded by the different Fe65 domains, such as SET only modulate transcriptional activity and are not on–off effects, and (Telese et al., 2005), akin to the cytosolic scaffolding function of is thus only possible in the linear range. Nearly all experiments Fe65 (Chow et al., 2015a). performed here were done with 1 µg Gal4-Fe65 constructs, clearly Fe65 is a long-lived protein with a half-life of ∼25 h, and control in the saturated range of the assay. Deletion of different domains in of its transcriptional activity is unlikely to be mediated by the F65 influenced transcriptional activity in both directions. This is a regulation of Fe65 levels. Although a cytosolic protein, in strong indication that Fe65 represents a transcription factor – sole fractionations it mostly distributes to the membrane fraction where manipulation of Fe65 is sufficient to bidirectionally change it binds to APP and other transmembrane proteins such as the ApoE transcriptional activity under saturating conditions. receptor family and calsyntenin/alcadein proteins (Araki et al., Spherical nuclear complexes are formed upon co-expression of 2004; Hoe et al., 2006; Kinoshita et al., 2001; Trommsdorff et al., APP, Fe65 and Tip60, designated as AFT spots (von Rotz et al., 1998). Nevertheless, in contrast to the APP family, which binds to 2004). These AFT spots localize to transcription factories the PTB2 domain, these transmembrane proteins bind the PTB1 (Konietzko et al., 2010) and their formation is a clear indication domain of Fe65 and it is not known whether that enables the of the capacity of nuclear signaling by APP and Fe65 (Gersbacher simultaneous binding of Tip60 as for APP–Fe65 complexes. All et al., 2013; Goodger et al., 2009). AFT spots are highly reminiscent currently described transmembrane interaction partners of Fe65 are of the recently described phase-separated condensates that contain substrates for γ-secretase-mediated RIP. We revealed that RNA polymerase II and other components of the transcriptional manipulation of γ-secretase cleavage of APP directly controlled machinery (Cho et al., 2018), with the same liquid-like properties, Fe65 nuclear translocation and transcriptional activity. Deletion of such as spherical shape, and the ability to flow and fuse (Konietzko the PTB2 domain prevented sequestration of Fe65 at the membrane et al., 2010). Acidic motifs in activation domains, such as the unique after inhibition of γ-secretase cleavage. Therefore, APP is a break region in Fe65 that is involved in transcription, have recently been for Fe65 signaling, and the rate of APP cleavage determines the connected to transcription and the formation of phase-separated strength of the transcriptional response as reported recently condensates (Boija et al., 2018). This further supports our findings (Konietzko et al., 2019). In contrast to Fe65, APP is turned over that AFT spots define nuclear loci of endogenous target gene rapidly with a half-life of less than an hour (Allinquant et al., 1995; transcription. Tip60 alone localizes to irregular speckles that Gersbacher et al., 2013; Weidemann et al., 1989). Proteomic represent a different nuclear compartment than the spots formed analysis of synaptosome protein turnover identified APP as a upon co-expression of Fe65. Deletion of either WW or PTB1 protein with one of the fastest turnover rates (Heo et al., 2018). In domains disrupted spot formation, with mutant Fe65 and Tip60 addition, the APP–Fe65 interaction is regulated by phosphorylation colocalizing in nuclear speckles. These constructs also showed of interacting residues in both proteins. APP Y682 phosphorylation strongly reduced transcriptional activity. inhibits Fe65 binding (Tamayev et al., 2009) and Fe65 S610 Although most constructs showed a correlation between phosphorylation similarly blocks APP binding (Chow et al., transcriptional activity and AFT spot formation, some revealed 2015b). Thus, the turnover of APP and modification of its a dissociation of the two phenomena. Deletion of L2 connecting binding to Fe65 determine transcriptional signaling by Fe65. WW and PTB1 domains did not disrupt spot formation of Fe65 The PTB2 deletion enhanced the transcriptional activity of Fe65, with Tip60, but prevented spot localization of AICD and but a C654F mutation that also disrupts APP binding (Borg et al., abolished transcriptional activity. Deleting the APP-binding PTB2 1996; Minopoli et al., 2001) had wild-type levels of activity. Fe65 domain inhibited AICD spot localization, but allowed FT spot has been shown to form dimers via the PTB2 domain (Feilen et al., Journal of Cell Science

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2017). We now show that the C654F mutant Fe65 can still form (mChe) via the AscI cleavage site. As a control construct we also fused the dimers. C654 is located in α-helix 3 of the Fe65 PTB2 in Fe65– Gal4-DBD via a Myc tag to Cerulean with a triple NLS sequence (Gal4- AICD complexes and its mutation creates a steric overlap with the myc-Cer-NLS). Table S1 provides an overview of the amino acid sequences YENPTY motif of AICD, thus disrupting APP binding of the different Fe65 family protein constructs. A CMV promoter was used (Radzimanowski et al., 2008). Fe65 dimerization occurs via β- to express all constructs, except for 3Myc-APP-3HA, which was expressed – β via a GAPDH promoter. The lentiviral reporter construct UAS-Cit-NLS has completion C654 is part of a -sheet that is far less vulnerable to been recently described (Konietzko et al., 2019). accommodating a phenylalanine (Feilen et al., 2017). On the other hand, the PTB2 domain is not necessary for spot localization, Cell culture, lipofection and drug treatment although it disrupts Fe65 dimers. We conclude that Fe65 dimers are Human embryonic kidney cells (HEK293; DSMZ, Braunschweig, located in the cytosol. Whereas PTB2 deletion disrupts both dimers Germany), the Lenti-X™ 293T cell line (#632180, Clonentech, Takara, and APP binding, leading to increased nuclear translocation and Berkely, USA) and N2a (DSMZ, Braunschweig, Germany) cells were transcriptional activity of Fe65, the C654F mutation keeps Fe65 cultured in Dulbecco’s modified Eagle’s medium (DMEM, #41965039, dimers intact and thus some Fe65 is retained in the cytosol. ThermoFisher Scientific) containing 10% fetal bovine serum (FBS; Stimulation of amyloidogenic APP cleavage by Fe65 has previously #10270106, ThermoFisher Scientific) using standard conditions (37°C, been shown to be regulated by T579 phosphorylation in the PTB2 5% CO2, humified atmosphere). Cells were free of mycoplasma contamination. For microscopy experiments, cells were grown on four- domain. This suppressed Fe65 PTB2 intermolecular dimerization – well-chamber glass slides (Falcon), successively coated with poly-L- and enhanced FE65 APP complex formation (Lee et al., 2017). The ornithine (50 μg/ml; Sigma-Aldrich) and fibronectin (5 μg/ml; Sigma- picture that emerges is a reservoir of cytosolic Fe65 dimers, whose Aldrich). Cells were transfected with expression constructs using dissociation and APP association is regulated, whereby the binding Lipofectamine™ 2000 Transfection Reagent (#11668019, ThermoFisher of Fe65 PTB2 to AICD opens up Fe65 for additional binding of Scientific) according to the manufacturer’s instructions, and fixed or lysed Tip60. Upon γ-secretase-mediated cleavage of APP, the AFT after 20 to 26 h. Plasmid DNA concentration was measured on a Nanodrop complex can then translocate to the nucleus with the help of the 2000 (ThermoScientific) and 500 ng of undigested DNA was routinely NLSs from Tip60, to form AFT spots. analyzed on agarose gels to ensure identical quality (i.e. supercoiled status, Fe65/Fe65L1 double KO mice show a much more severe which affects lipofection efficiency). Drugs were added with medium phenotype than the single KOs (Guénette et al., 2006). Because replacement 2.5 h after transfection. The nuclear export inhibitor leptomycin B (LMB, #431050, Calbiochem, in ethanol) was used at only Fe65 has transcriptional activity, the severe phenotype cannot 10 ng/ml for 18 h and the γ-secretase inhibitor DAPT (#D5942, Sigma, in be explained by the loss of transcriptional function of Fe65, but DMSO) was used at 1 or 3 µM for 20 h. To inhibit protein synthesis, cells probably the role of the Fe65 family as cytosolic scaffold (Chow were treated with 100 μg/ml cycloheximide (CHX, #66819, Sigma) 22 h et al., 2015a). Nevertheless, in contrast to Fe65L1 single KO, Fe65- after transfection for 0.5 h to 4 h. KO animals have reduced early long-term potentiation, pointing towards defects in synaptic plasticity (Strecker et al., 2016). In Generation of Fe65-KO cells support of this, AICD has been shown to regulate transcription of HEK293 Fe65-KO cells were generated using Alt-R® CRISPR-Cas9 (IDT, NMDAR subunits and modify electrical properties of neuronal Coralville, Iowa, US). The crRNA/tracrRNA/Cas9 complexes were created signaling (Pousinha et al., 2019, 2017). Furthermore, APP also using 0.2 nmol Alt-R® CRISPR-Cas9 crRNA against Fe65 (HEK293 Fe65ko clone 1: #Hs.Cas9.APBB1.1.AA, 5′-ATTGCGATTCTGGTCAC- regulates activity-dependent genes to influence inhibitory ′ neurotransmission (Opsomer et al., 2020). APP turnover is GGT-3 , starting at amino acid 84; HEK293 Fe65ko clone 3: #CD.Cas9.- TPCK3618.AB, 5′-CCCCACGGAATACCAACCCA-3′, starting at amino regulated by synaptic activity (Cirrito et al., 2005; Kamenetz acid 359; HEK293 Fe65ko clone 4: #CD.Cas9.ZGJY0202.AH, 5′-ACCC- et al., 2003). Thus, activity-dependent synapse to nuclear signaling AGTGATGAGGCCCCAA-3′, starting at amino acid 320), 0.5 nmol Alt- by AICD–Fe65–Tip60 complexes might be involved in regulating R® CRISPR-Cas9 tracrRNA (#1072532) and 61 pmol Alt-R® S.p. Cas9 synapse function. nuclease V3 (#1081058). The ribonucleoprotein complexes were electro- porated into 1.5×106 HEK293 cells using the Neon™ Transfection System MATERIALS AND METHODS 100 µl Kit (#MPK10096, ThermoFisher Scientific), and applying one 30 DNA constructs ms pulse of 1150 V (Yu et al., 2016). Single clones were generated using The CFP-Tip60 (human, isoform 2), Citrine-AICD (Cit-AICD), human limited dilution on 96-well plates. Deletion of Fe65 was verified by APP695-Citrine (APP-Cit) and human Fe65 expression constructs were immunoblotting. previously described (von Rotz et al., 2004). APP695-Cerulean (APP-Cer) (Kohli et al., 2012) and Myc-Tip60 (Konietzko et al., 2010) are as described Virus production and cell line generation previously. The Myc tag in Tip60 was further exchanged with a V5 tag. The Lentiviral vector production and generation of HEK:UAS-Cit, HEK1:UAS- 3myc-APP695-3HA construct was previously described (Gersbacher et al., Cit-NLS and HEK2:UAS-Cit-NLS cells was as recently described 2013) and AICD-3HA was derived from this construct by PCR-based (Konietzko et al., 2019). To generate the N2a:UAS-Cit-NLS reporter cell cloning. Mouse Fe65L1 (courtesy of Suzanne Guénette and Stefan Kins, TU line, 1.25×105 N2a cells were plated and infected the next day with Kaiserslautern, Germany) and human Fe65L2 (variant 4, OHu05198, concentrated UAS-Cit-NLS reporter virus (5×108 particles). Cell lines were Genescript) were cloned into the pUKBK vector system (Kohli et al., 2012). isolated by limited dilution on 96-well plates. We used PCR-based cloning methods to extend the pUKBK vectors with N- terminal tags – streptavidin-binding protein (SBP) was derived from a Streptavidin purification construct described previously (Kohli et al., 2012), and the Gal4 DNA- Pulldown assays were performed to purify the SBP-tagged constructs and to binding domain (DBD) was derived from APP-Gal4 (courtesy of Thomas co-precipitate bound proteins. At ∼22 h following transfection, HEK293 Südhof, Department of Molecular and Cellular Physiology, Stanford cells were lysed in homogenization buffer [HB; 140 mM KCl, 20 mM University School of Medicine, USA). SBP and Gal4-DBD were HEPES pH 7.2, 10 mM NaCl, 5% (v/v) glycerol, 2 mM MgSO4, 1% (v/v) followed by a Myc tag and a unique AscI cleavage site designed to code Triton-X100, 2 mM DTT, EDTA-free Protease-Inhibitor Cocktail for glycine-alanine-proline. Fe65 family proteins and mutants thereof were (#11873580001, Roche) and 2 mM Phenantrolen]. Lysates were pushed inserted after the AscI site. Deletion, point mutation, and fusion protein 10× through a 26 G needle, incubated on a rotating wheel for 15 min at 4°C, constructs of Fe65 family proteins were created using standard PCR-based and cleared by centrifugation at 800 g for 10 min at 4°C. 100 μl of the cloning methods. The SBP-myc tag was further exchanged with mCherry supernatant was used as lysate sample; the remaining 700 μl was incubated Journal of Cell Science

15 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242917. doi:10.1242/jcs.242917 with Dynabeads® M-280 Streptavidin (#11205D, ThermoFisher Scientific) DAPI, and cells were mounted with Mowiol containing 2.5% 1,4- for 4 h at 4°C. Next, the beads were washed, and the bound proteins were diazabicyclo(2.2.2)octane (DABCO) (Valnes and Brandtzaeg, 1985). eluted with 14 mM biotin. Confocal microscopy Subcellular fractionation Images were acquired on a TCS/SP8 confocal microscope (Leica) with a Cells were lysed with the same buffer as for streptavidin purification and 63× glycerol objective and a pinhole of 0.5 airy. Hybrid detectors (HyDs) transferred to Eppendorf tubes. Tubes were vortexed with maximum speed were operated in standard mode with 8-bit intensity resolution and detection for 20 s, with the finger placed on top of the tube to achieve reproducible windows adjusted to fluorophore emission spectra. DAPI was excited with a conditions. After 15 min on a turning wheel at 4°C, cell were vortexed again 405 nm diode laser and detected with a HyD window of 407–507 nm. Cit for 20 s and centrifuged for 15 min at 800 g. The supernatant was kept as was excited with the 514 nm line of the argon laser and detected with a HyD cytosol/membrane (C/M) fraction and the pellet resuspended in streptavidin window of 516–560 nm with Cy3 co-staining or 516–610 nm when no Cy3 purification buffer and centrifuged for 15 min at 800 g. The nuclear pellet was present. CFP was excited with the 458 nm line of the argon laser and was resuspended in RIPA buffer, 125 Units benzonase added, incubated at detected with a HyD window of 460–512 nm. In this case, the HyD window 37°C for 5 min, and resuspended by vortexing and trituration with 18G and for DAPI was set from 407–456 nm to prevent signal pickup from Cer. Cy3 22G canula. was excited with a 561 nm laser and detected with a HyD window of 563– 631 nm. Alexa Fluor 647 was excited with a 633 nm helium-neon laser and – Western blotting detected with a HyD window of 635 750 nm. Images shown are mostly – Medium was collected, and cells were lysed using RIPA buffer [50 mM maximum projections of 5 8 z-stacks encompassing the nucleus (unless Tris-HCl pH 7.6, 150 mM NaCl, 1% NP40, 0.1% SDS, 0.5% sodium noted). Single sections imaged with a 20× glycerol objective and a pinhole deoxycholate, 2 mM EDTA, 1 tablet EDTA-free protease inhibitor cocktail/ of 1 airy, were used for UAS-Cit assay measurements and determination of 50 ml (#04693159001, Sigma)]. After centrifugation for 5 min at 20,817 g, the nuc/cyt ratio. For quantification of the nuc/cyt ratio, we measured the 4°C, equal amounts of lysate supernatant and collected medium were total fluorescence and subtracted the fluorescence signal in the DAPI- separated on Novex™ 10%–20% Tricine Protein Gels (#EC6625BOX, demarcated nuclei to derive the cytoplasmic signal. Background ThermoFisher Scientific), and transferred on a 0.1 µm Amersham™ fluorescence was determined with non-transfected and stained cells to Protran™ nitrocellulose blotting membrane (#GE10600000, GE calculate the Limit of Blank (Armbruster and Pry, 2008), which was Healthcare). Blots were blocked with PBS, 0.05% Tween-20, 5% milk subtracted from fluorescence measurements. All fluorophores used are and incubated in blocking solution with following primary antibodies: HA consistently color-coded in all images: CFP and Cer (cyan), Cit (yellow), high affinity (rat IgG1, 3F10, monoclonal, 1:1000, #11867431001, lot# Cy3 (red), Alexa Fluor 647 (blue) and DAPI (white). 15645900, Roche), APP C-term (rabbit IgG, Y188, monoclonal, 1:250– 1:1000, #ab32136, lot# GR211501-24, Abcam), β-amyloid 1-16 (mouse UAS-Cit transactivation assay IgG1, 6E10, monoclonal, 1:1000, #SIG-39320, lot# B228658, Biolegend), Image acquisition and quantification for UAS-Cit assay experiments was c-myc (mouse IgG1K, 9E10, monoclonal, 1:1000, #11667149001, lot# performed as recently described (Konietzko et al., 2019). Briefly, we used a 11776800, Roche), mCherry (rat IgG2a, 16D7, monoclonal, 1:2000, 20× glycerol objective and HyDs were operated in count mode with 12-bit #M11217, lot# UD284615, ThermoFisher), Tip60 (rabbit, polyclonal, intensity resolution to image Cit, DAPI and Alexa Fluor 647. After adjusting 1:500, #ab137518, lot# GR315199-11, Abcam), acetylated histone H3 laser intensity and accumulation number to not saturate the HyDs, we (rabbit, polyclonal, 1:4000, #06-599, lot# DAM1588236, Millipore), Fe65 selected frames to image via the DAPI signal to select areas of similar cell (mouse, OTI3H9 monoclonal, 1:1000, #MA5-27408, lot# UE2761282, density, adjusted the z-position to achieve the brightest DAPI signal and ThermoFisher) and GAPDH (mouse IgG1, monoclonal, 1:5000, acquired 7 images per condition and passage (frame 775×775 µm, ∼1000– #H86504M, lot# H86504M, Meridian). Secondary antibodies were 2000 cells per frame). This way, the Cit channel is imaged for the first time peroxidase-linked goat anti-mouse-IgG (1:2500, #115-035-146, lot# during actual acquisition, preventing any bias or bleaching. All image 142637, Jackson ImmunoResearch), peroxidase-linked donkey anti-rabbit- analysis was performed using Fiji/ImageJ and described in detail in IgG (1:2000, #NA9340, lot# 9672941, GE Healthcare) and peroxidase- Konietzko et al. (2019). We measured total Cit fluorescence in the case of linked goat anti-rat-IgG (1:2500, #NA935, lot# 354130, GE Healthcare). UAS-Cit reporters or nuclear Cit fluorescence in the case of UAS-Cit-NLS For improved APP and AICD detection, the membranes were air-dried and reporters. DAPI was used to determine the nuclear outline and cell number. incubated with boiling PBS after transfer (Pimplikar and Suryanarayana, Data from Fiji were transferred to Excel or Prism for further analysis. 2011), followed by blocking with Tris-buffered saline (TBS) with 0.1% Tween-20 and 10% FBS. Protein bands were detected with the ImageQuant Statistical analysis LAS 4000 (GE Healthcare Life Sciences), using Pierce™ ECL Western Statistics were calculated with Prism 8 (GraphPad). Data were controlled for Blotting Substrate (#32106, ThermoFisher Scientific) or with ECL Prime normal distribution by Shapiro–Wilk testing. Normal distributed data were Western Blotting Detection Reagent (#RPN2232, GE Healthcare). analyzed by Student’s t-test or one-way ANOVA with multiple comparisons The ImageQuant TL software was used to quantify the latest exposure against control or wild type. Non-normal distributed data were analyzed by before the brightest band was saturated on the blot. APP, Fe65, Fe65L1 and Kruskal–Wallis or Mann–Whitney U-test. Asterisks above the horizontal Fe65L2 protein levels were normalized to GAPDH. For the CHX chase significance line refer to all bars with tick marks except when the ticks are experiments, the normalized protein levels were fitted to the one-term marked individually (*P<0.05, **P<0.01 and ***P<0.001. Bars not exponential model f(x)=a×e(bx) using MATLAB R2014b. The obtained indicated by a tick mark are not significantly different. Data are presented coefficient b was further used to calculate the half-life using the formula: as mean±s.d. for UAS-Cit assays and determination of nuc/cyt ratios. Data ln(2)/−b. are presented with mean±s.e.m. for western blots.

Immunocytochemistry Competing interests HEK293 cells were fixed with 4% (w/v) paraformaldehyde, blocked with The authors declare no competing or financial interests. TBS supplemented with 0.2% Triton X-100, 5% goat serum, 5% horse serum, and incubated with primary antibodies (same as for western blots, Author contributions 5-fold less diluted) in blocking solution. Highly cross-purified secondary Conceptualization: S.P., U.K.; Methodology: S.P., M.K., L.K., S.T., D.S., U.K.; ™ Software: M.K.; Validation: L.K., S.T.; Formal analysis: S.P., M.K., U.K.; antibodies from Jackson ImmunoResearch were Cy 3-conjugated donkey Investigation: S.P., M.K., L.K., U.K.; Resources: M.K., L.K., S.T., D.S.; Writing - ® anti-mouse-IgG (#715-165-151), Alexa Fluor 647-conjugated donkey original draft: U.K.; Writing - review & editing: S.P., R.M.N., U.K.; Visualization: S.P., ® anti-mouse-IgG (#715-606-151) and Alexa Fluor 647-conjugated U.K.; Supervision: R.M.N., U.K.; Project administration: R.M.N., U.K.; Funding donkey anti-rat-IgG (#712-605-153). Cell nuclei were stained with acquisition: U.K. Journal of Cell Science

16 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs242917. doi:10.1242/jcs.242917

Funding Synaptic activity regulates interstitial fluid amyloid-β levels in vivo. Neuron 48, This work was supported by the Swiss National Science Foundation SNF 913-922. doi:10.1016/j.neuron.2005.10.028 (Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung) Duilio, A., Zambrano, N., Mogavero, A. R., Ammendola, R., Cimino, F. and 31003A_166177 and the resources of the IREM. Russo, T. (1991). A rat brain mRNA encoding a transcriptional activator homologous to the DNA binding domain of retroviral integrases. Nucleic Acids Supplementary information Res. 19, 5269-5274. doi:10.1093/nar/19.19.5269 Feilen, L. P., Haubrich, K., Strecker, P., Probst, S., Eggert, S., Stier, G., Sinning, Supplementary information available online at I., Konietzko, U., Kins, S., Simon, B. et al. (2017). Fe65-PTB2 Dimerization https://jcs.biologists.org/lookup/doi/10.1242/jcs.242917.supplemental Mimics Fe65-APP Interaction. Front. Mol. 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