CW-Tau fights proteinopathy by improving autophagy and diminishing Tau seeding activity

CW-Tau fights proteinopathy by improving autophagy and diminishing Tau seeding activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CW-Tau fights proteinopathy by improving autophagy and diminishing Tau seeding activity Francisco Vallejo-Bedia, Indalo Domene-Serrano, Anastasia Stoliarov, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6725217/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract CW-Tau, a novel MAPT transcript variant generated by intron 12 retention, has been identified. Compared with classical Tau isoforms, this isoform has greater microtubule-binding properties, a reduced aggregation propensity, and faster turnover. Notably, CW-Tau RNA and protein levels are decreased in Alzheimer’s disease (AD) brains. To investigate its molecular interactions, we performed affinity chromatography using the W-Tau peptide, which is encoded by intron 12, and identified key binding partners involved in microtubule dynamics, vesicle trafficking, and autophagy. The W-Tau peptide interacts with tubulin, suggesting the presence of an additional microtubule-binding domain, and binds lactate dehydrogenase, preventing its aggregation. Furthermore, the W-Tau peptide is associated with vesicle trafficking regulators, including Rab proteins and Rab-GDP dissociation inhibitors, as well as autophagy-related proteins such as MAP1BLC1 and PARK7. Unlike classical Tau isoforms, which disrupt these pathways, CW-Tau functionally preserves vesicle trafficking and autophagic flux. Importantly, while classical Tau isoforms promote pathological transmission of Tau either nakedly or via exosome-mediated export, CW-Tau counteracts this process, demonstrating potent antiseeding activity. By preventing Tau aggregation and propagation, CW-Tau may play a protective role against proteinopathy-related neurodegeneration. These findings suggest that CW-Tau is a promising therapeutic tool for AD, offering potential strategies to restore cellular proteostasis and inhibit Tau pathology. Tau MAPT aggregation vesicle trafficking autophagy exovesicles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Tau is a microtubule-associated protein predominantly expressed in the central nervous system (CNS). Its principal function is to stabilize microtubules by regulating their dynamic equilibrium, thereby influencing vesicle trafficking. In the CNS, Tau plays a key role in establishing and maintaining neuronal polarity and promoting neurite outgrowth( 1 ). It also regulates the axonal transport of endocytic and exocytic vesicles ( 2 ), as well as organelles such as mitochondria ( 3 ), endosomes and lysosomes ( 4 ), peroxisomes and the endoplasmic reticulum. Under pathological conditions, such as Tau overexpression, kinesin-dependent organelle trafficking is impaired, resulting in neurodegeneration ( 5 , 6 ). These trafficking defects correlate with altered membrane trafficking pathways observed in Alzheimer’s disease, including synaptic vesicle disruption and increased levels of extracellular microvesicle markers ( 7 , 8 ). Tau is encoded by the MAPT gene located on chromosome 17 and undergoes extensive alternative splicing ( 9 , 10 ). This process gives rise to a family of Tau isoforms defined by specific exon combinations, yielding six distinct isoforms in the CNS ( 11 , 12 ). Recently, we identified a novel Tau isoform generated by intron 12 retention ( 13 ). This isoform lacks exon 13 but includes a unique human-specific 16-amino acid peptide encoded by intron 12, leading to a truncated C-terminal end ( 13 ). Notably, this intron-derived peptide introduces two tryptophans—absent in the classical Tau protein—prompting us to name the isoform CW-Tau. CW-Tau has distinct properties, including (a) greater microtubule binding, (b) greater solubility than other Tau isoforms, and (c) lower RNA and protein levels in the brains of AD patients than in those of nondemented individuals ( 13 – 15 ). Additionally, its specific peptide, W-Tau, has chaperone-like activity ( 15 ). The discovery of other Tau isoforms arising from intron 11 retention ( 16 ) highlights an overlooked mechanism for Tau diversity. To explore the function of CW-Tau, we identified human brain proteins that bind to the W-Tau peptide, particularly those involved in microtubule dynamics, membrane trafficking, chaperone activity, and protein clearance. Tubulin and lactate dehydrogenase (LDH), which are highly abundant in the brain, interact with the W-Tau peptide, as do lower-abundance proteins linked to autophagy (LC1-MAP1B) and vesicle trafficking/secretion (Rab-GDIs and several RABs). Consistently, CW-Tau expression enhances autophagy flux and vesicle trafficking, suggesting more efficient protein clearance and faster turnover than classical Tau isoforms do. Moreover, CW-Tau effectively inhibited Tau seeding activity. Materials and methods 3.1 Proteomic analysis The W-Tau peptide (sequence: KKVKGVGWVGCCPWVYGH), derived from Tau intron 12, was purchased from Abyntek Biopharma S.L. ( 13 ). W-Tau peptide interactions with human brain extracts from cortical samples from patients with sporadic Alzheimer's disease and control individuals (HUB-IDIBELL Brain Bank, Spain) were analyzed via CNBr-activated Sepharose 4B affinity chromatography ( 17 ). Proteins were concentrated at the stacking/resolving gel interface of SDS‒PAGE, digested with trypsin, and desalted via OMIX Pipette C18 tips (Agilent Technologies) ( 18 , 19 ). Protein identification was performed via reverse-phase liquid chromatography coupled with tandem mass spectrometry (RP-LC‒MS/MS) via an LTQ-Orbitrap-Velos-Pro (Thermo Scientific) under a dynamic exclusion setting ( 20 , 21 ). Peptide identification was conducted via PEAKS Studio XPro, which searches against the UniProt database. Functional network analysis was performed via STRING ( 22 – 24 ). 3.2 Microtubule binding assay Microtubules were purified from C57 mouse brains, polymerized, incubated with W-Tau, and analyzed via dot blotting via anti-CW-Tau antibodies and electron microscopy ( 25 ). 3.3 Lactate Dehydrogenase Aggregation Lactate dehydrogenase (LDH) was incubated with W-Tau at 80°C, and aggregation was visualized via transmission electron microscopy (JEM1400 Flash Transmission Electron Microscope, Jeol). 3.4 Immunofluorescence analysis SH-SY5Y cells were transduced with pWPI vectors expressing the T30 and CW-T30 isoforms ( 26 ) and then fixed and stained with antibodies against β-tubulin (SIGMA, T4026), GFP (ABCAM, 13970), Tau (NUVUSBIO, NB100-822247), LC3 (SIGMA, L7543), EEA1 (BD Biosciences, 610457), Rab11A1 (Invitrogen, 71–5300), CD63 (DSHB, H5C6), and LAMP1 (DSHB, H4A3) and with DAPI (4',6-diamidino-2-phenylindole, Merck, 268298). When indicated, the cells were treated with LysoTracker Red DND-99 (Invitrogen, L7528) before fixation. Confocal microscopy analysis was performed via Fiji software, and the vesicles were segmented via CellPose Plug-in ( 27 ). 3.5 Study of Autophagy Modulation HEK293T cells (CRL-11268, ATCC) were transfected with Tau constructs (pSG5, Agilent, 216201) ( 28 ) and treated with CCCP and bafilomycin A1 to assess autophagic responses via Western blot analysis as previously described ( 29 ). 3.6 Protein aggregation assays HEK293T cells were treated with bafilomycin A1 and MG-132 to induce protein aggregation, which was analyzed via a Proteostat Aggresome Detection Kit (Enzo Life Sciences, 51023) followed by confocal microscopy analysis. 3.7 Determination of Tau export in exovesicles HEK293T cells transfected with pSG5 encoding T30 or CW-T30 ( 28 ) were cultured, and their extracellular medium was fractionated by sequential centrifugation as previously described ( 30 ). Exosome presence was confirmed via nanoparticle tracking analysis, and Tau in different fractions was detected by Western blotting. 3.8 Tau aggregation assay HEK293T Tau RD P301S FRET biosensor cells (CRL-3275, ATTC) were transfected with Alzheimer’s-derived Tau seeds as previously described ( 31 ) and with Tau-expressing vectors. Alternatively, cells were treated with different Tau fractions ( 30 ). The FRET signal was analyzed via confocal microscopy and Fiji software ( 27 ). 3.9 Statistical analysis The experimental data were analyzed via GraphPad Prism 9.0 and Excel. The results are expressed as the mean ± SEM, and statistical comparisons were conducted via one-way or two-way ANOVA. Significance levels are represented as follows: ns (not significant), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Additional methodological details are provided in the Supplementary Materials and Methods section. Results 4.1 Identification of abundant human brain proteins that bind to the intron 12 peptide of the novel Tau isoform A peptide sequence (KKVKGVGWVGCCPWVYGH), comprising two lysines from exon 12 and a 16-amino acid region from intron 12, was conjugated to a Sepharose column. Protein extracts from nondemented (C) and Alzheimer’s disease (AD) brains were chromatographed, retaining ~ 20% of the total brain proteins in both cases. SDS‒PAGE analysis revealed two predominant retained proteins, with electrophoretic mobilities consistent with those of tubulin and lactate dehydrogenase (LDH). Mass spectrometry analysis revealed a significant interaction of the W-Tau peptide with tubulin alpha chain 1B (TBA1B) ( supplementary Fig. 1A ), which was specifically enriched in the AD brain (Table 1 ). Table 1 Proteomic Profiling of Tubulin Isotypes and Lactate Dehydrogenase Interacting with W-Tau in Human Brains. Proteins binding to W-Tau were identified via reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for both control and AD samples. Enrichment ratios were calculated to assess protein binding. Proteins identified include tubulin alpha chain 1B (TBA1B), lactate dehydrogenase A (LDHA), and B (LDHB). CONTROL ALZHEIMER DISEASE Accession Pass-through Eluted Ratio Pass-through Eluted Ratio Tubulin subtype P68363|TBA1B_HUMAN 2834300 891220 0.314 312840 3576400 11.432 Lactate dehydrogenase P00338|LDHA_HUMAN 43397000 26541000 0.611 44151000 13896000 0.314 P07195|LDHB_HUMAN 129050000 122800000 0.951 133290000 112410000 0.843 Given that Tau isoforms bind tubulin via microtubule-binding repeats ( 11 , 12 ), the interaction between W-Tau and tubulin suggests a novel tubulin-binding region within the CW-Tau isoform. Polymerization assays with mouse brain microtubules confirmed W-Tau’s association with polymerized microtubules, enhancing polymerization, as observed by electron microscopy (Fig. 1A-C). Additional interactions were identified with MAP1B, MAP2, and Tau itself (Table 2 , Supplementary Fig. 2A-C ), reinforcing its role in microtubule dynamics. To examine the impact of CW-Tau on microtubule stability, HEK293T cells were transfected with expression vectors encoding two Tau isoforms: the classical T30 isoform (with three microtubule-binding repeats and no N-terminal inserts) and the CW-Tau isoform (generated by intron 12 retention) ( 28 ), which are thought to be the most abundant isoform in the human brain. While T30 disrupted microtubule morphology, CW-Tau preserved cytoskeletal integrity, suggesting its stabilizing role (Fig. 1D). Additionally, W-Tau potentially interacts with tubulin polymerization-promoting protein (TPPP), which is known to regulate microtubule dynamics, particularly in AD brains (Table 2 , Supplementary Fig. 2D ( 32 , 33 )). Table 2 Proteomic Analysis of MAPs and Related Proteins Interacting with W-Tau Peptide in Human Brains. Proteins binding to W-Tau were identified via reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for both control and AD samples. Enrichment ratios were calculated to assess protein binding. Identified proteins include microtubule-associated proteins (MAPs) 1B (MAP1B), light-chain MAP1B (LC1-MAP1B), MAP2 (MTAP2), Tau, and Tubulin polymerization-promoter protein (TPPP). CONTROL ALZHEIMER DISEASE Accession Pass-through Eluted Ratio Pass-through Eluted Ratio MAPs and related proteins P46821|MAP1B_HUMAN 2261300 225860 0.099 3548200 2008400 0.566 LC1-MAP1B_HUMAN 1599214 184824 0.115 1105500 885294 0.800 P11137|MTAP2_HUMAN 2420300 1047700 0.432 894180 537010 0.600 P10636|TAU_HUMAN 10759000 1593900 0.148 3925400 2631000 0.670 O94811|TPPP_HUMAN 4604800 320230 0.069 238300 1110000 4.657 In addition to microtubules, the W-Tau peptide exhibited affinity for LDH1 and LDH2 (Table 1 , Supplementary Fig. 1B-C ), indicating a potential additional LDH-binding site in CW-Tau. Since Tau isoforms can act as chaperones to prevent LDH aggregation ( 34 ), we tested W-Tau’s chaperone-like function following Tian et al.’s protocol ( 34 ). Electron microscopy revealed that W-Tau significantly reduced LDH aggregation, similar to Tau 42 (Fig. 1E). These results support W-Tau’s chaperone role, which aligns with previous findings that it prevents Tau and β-amyloid aggregation in vitro ( 15 ). 4.2 CW-Tau maintains autophagic flux in HEK293T cells Detailed analysis of tryptic peptides from MAP1B that interact with the W-Tau peptide revealed enrichment within the light chain 1 (LC1-MAP1B) region of the precursor MAP1B protein, which comprises 2,459 amino acids (Table 2 and Supplementary Fig. 2A ). The heavy chain (HC) spans residues 1–2,185, whereas the light chain (LC) includes residues 2,210–2,459 ( 35 ), containing an acidic region (amino acids 2,337–2,459) likely responsible for W-Tau binding. LC1-MAP1B is also involved in autophagy regulation through its interaction with syntaxin 17 (Stx17), which responds to nutrient availability ( 36 ). Additionally, the W-Tau peptide potentially interacted with PARK7/DJ-1 in both control and AD brain samples (Table 3 , Supplementary Fig. 3K ). DJ-1 is a critical component of the PINK1/parkin-mediated mitophagy pathway, facilitating the recruitment of optineurin to depolarized mitochondria ( 37 ). Given the relevance of autophagy in Alzheimer's disease pathology ( 38 ) involving W-Tau with LC1-MAP1B and PARK7—key regulators of autophagy and mitophagy, respectively ( 36 , 37 )—we investigated the role of CW-Tau in mitophagy in comparison with the classical Tau isoform. To this end, HEK293T cells expressing various Tau isoforms were treated with CCCP, a mitochondrial uncoupler that induces mitophagy, either alone or in combination with bafilomycin A1. Bafilomycin inhibits lysosomal acidification by targeting the vacuolar H⁺-ATPase, thereby preventing autophagosome–lysosome fusion and degradation of vesicular contents ( 26 ) (Fig. 2A). In these experiments, we compared classical Tau 3R (T30) with its corresponding CW-Tau isoform. Table 3 Proteomic Analysis of Vesicle Trafficking and Neurodegeneration Biomarkers Interacting with W-Tau Peptide in Human Brains. W-Tau-binding proteins were identified by reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for control and AD samples, with enrichment ratios calculated. Identified proteins include RAB GDP dissociation inhibitors (GDIA and GDIB), Rab proteins (Rab5C, Rab6B, Rab7A, Rab11B), Fatty acid binding protein 7 (FABP7), serine-threonine phosphatase 1 (CPPED), tyrosine-protein phosphatase SHPS1, apolipoprotein E (APOE), Parkinson disease protein 7 (PARK7), and ubiquitin-conjugating enzyme E2N (UBE2N). CONTROL ALZHEIMER DISEASE Accession Pass-through Eluted Ratio Pass-through Eluted Ratio Vesicle trafficking related P31150|GDIA_HUMAN 60749000 46603000 0.767 37907000 142050000 3.747 P50395|GDIB_HUMAN 7489500 6400000 0.854 5303900 11555000 2.178 P51148|RAB5C_HUMAN 507410 75972 0.149 502810 840520 1.671 Q9NRW1|RAB6B_HUMAN 2318900 3975300 1.714 1658600 8083200 4.873 P51149|RAB7A_HUMAN 842110 859590 1.020 1313300 2331800 1.775 Q15907|RB11B_HUMAN 298330 407600 1.366 892820 547980 0.613 O15540|FABP7_HUMAN 1008200 2339300 2.320 1961500 2460400 1.254 Q9BRF8|CPPED_HUMAN 457490 827020 1.807 247760 1404300 5.667 P78324|SHPS1_HUMAN 472120 532770 1.128 363650 195090 0.536 Neurodegeneration biomarkers P02649 | APOE_HUMAN 741100 1633100 2.203 1307300 749110 0.573 Q99497|PARK7_HUMAN 16604000 19949000 1.201 12080000 22930000 1.898 P61088|UBE2N_HUMAN 1776800 2392600 1.346 1272800 2826900 2.221 We first examined the levels of LC3, a marker of autophagic vesicle dynamics. The overexpression of T30 modestly increased LC3 synthesis, suggesting the induction of autophagosomes (Fig. 2B and C ), which is consistent with the finding that autophagy is activated in response to Tau aggregation. In contrast, CW-Tau maintained LC3 levels comparable to those in control cells, indicating no autophagy induction—likely due to its lower aggregation propensity ( 14 ). Analysis of the LC3 degradation ratio revealed a marked impairment in autophagic flux with T30, whereas CW-Tau preserved normal degradation (Fig. 2B and D ). Similarly, p62, an autophagy adaptor that links ubiquitinated cargo to LC3, accumulated in T30-expressing cells but remained unaltered under CW-Tau conditions (Fig. 2B and E) . The degradation ratio of p62 also decreased significantly with T30 but not with CW-Tau (Fig. 2B and F ). Immunofluorescence analysis supported these findings. In SH-SY5Y neuroepithelioma cells transduced with lentiviral vectors expressing GFP alone, GFP-T30, or GFP–CW-Tau, the number of LC3 puncta (representing autophagic vesicles) increased modestly with T30 but remained unchanged with CW-Tau (Fig. 2G-I), indicating preservation of autophagic flux in the latter. Collectively, these results indicate that overexpression of the aggregation-prone T30 Tau isoform impairs autophagic flux by hindering the degradative phase, leading to vesicle accumulation. In contrast, CW-Tau preserves both the synthesis and degradation phases, maintaining autophagic homeostasis. The observed autophagy induction in T30-expressing cells may reflect a compensatory response to Tau aggregation, which fails to proceed to completion due to degradation blockade. CW-Tau, which is less prone to aggregation, does not trigger such a response. Interestingly, when early endosomal markers were analyzed, both T30 and CW-Tau expression induced early endosome clustering, with a more pronounced effect on CW-Tau-expressing cells (Fig. 2H-J). This may reflect an increase in endosome size with a concomitant decrease in number, potentially indicating altered function ( 39 ) or increased endocytic activity. 4.3 Identification of vesicle trafficking-related proteins that bind to the Intron 12 peptide Given the essential role of vesicle trafficking and Rab GTPases in neurodegenerative processes ( 40 ), we explored potential interactions between the W-Tau peptide and proteins involved in these pathways (Table 3 ). Affinity chromatography using the W-Tau peptide and brain extracts from AD patients revealed retention of the Rab-GDP dissociation inhibitors GDIA and GDIB (Table 3 , Supplementary Fig. 3A-B ). These proteins modulate intracellular membrane trafficking by inhibiting GDP-to-GTP exchange with Rab GTPases, facilitating their extraction from membranes and maintaining them in the inactive GDP-bound state ( 41 , 42 ). GDIA is expressed primarily in neural and sensory tissues, whereas GDIB is ubiquitously expressed ( 43 ). In addition, we detected putative interactions of the W-Tau peptide with several Rab family members, including Rab5C, Rab6B, Rab7A, and Rab11B (Table 3 , Supplementary Fig. 3C-F ). Rab5C is critical for early endosome formation and is implicated in Parkin-mediated delivery of damaged mitochondria to Rab5-positive endosomes via the ESCRT machinery, supporting lysosomal degradation ( 44 ). Rab6B is associated with retrograde transport in neurons ( 45 ), whereas Rab7A governs endolysosomal trafficking, orchestrating early-to-late endosome maturation, microtubule-mediated transport, and endosome positioning ( 46 ). Rab7A is also essential for phagosome fusion with late endosomes and lysosomes, a key step in autophagic degradation ( 47 ). Rab11B participates in both constitutive and regulated secretion ( 48 ). Proteomic analysis also confirmed W-Tau peptide interactions with UBE2N, FABP7, CPPED, and SHPS1 (Table 3 , Supplementary Fig. 3G-I ). Although these proteins play secondary roles in vesicle trafficking, the results of the STRING network analysis support their involvement in related pathways. Considering these interactions, particularly with Rab proteins and GDIs, and given the central role of Rab GTPases in vesicular transport and secretion, we propose that the CW-Tau isoform may influence these processes. Notably, several of the identified proteins have established links to neurodegenerative diseases. Among the W-Tau interactors, PARK7/DJ-1 is associated with Parkinson’s disease ( 49 ) and Tau inclusions ( 50 ). Abnormal Rab5 activation has been reported in AD brains, where it contributes to endocytic dysfunction ( 51 ). Furthermore, UBE2N has been proposed as an AD biomarker ( 52 ). In support of these disease connections, the W-Tau peptide also binds two proteins strongly associated with AD pathology—apolipoprotein E (ApoE) (Table 3 , Supplementary Fig. 3J ) and Tau itself (Table 2 , Supplementary Fig. 2C )—suggesting a potential role in the disease. 4.4 CW-Tau preserves LAMP1 vesicle size, number and acidification. Given the potential interactions between the CW-Tau and Rab proteins and GDIs, we next examined the effects of different Tau isoforms on vesicle trafficking, with a focus on LAMP1-positive compartments. We also assessed whether the impaired autophagic degradation phase previously observed with classical Tau is absent in CW-Tau-expressing cells by analyzing lysosomal distribution and maturation status. To this end, SH-SY5Y cells were infected with lentivectors encoding either GFP alone, GFP + classical Tau 3R (T30), or GFP + CW-Tau. The cells were then labeled with LysoTracker Red DND-99, an acidotropic fluorescent probe that targets acidic organelles, to assess lysosomal dynamics. LAMP1, a membrane protein predominantly associated with lysosomes but also present in early/late endosomes and multivesicular bodies (MVBs), was used to identify vesicular compartments (Fig. 3A). To characterize these vesicle populations, segmentation analysis classified them into three size categories: ≤2 µm² (primary and mature lysosomes), 2–5 µm² (amphisomes, autophagolysosomes, MVBs), and ≥ 5 µm² (early and late endosomes) ( 53 , 54 ) (Fig. 3B). Confocal imaging and quantification revealed a significant increase in the number of LAMP1-positive vesicles across all size ranges in T30-overexpressing cells, whereas CW-Tau cells presented vesicle counts comparable to those of the GFP control (Fig. 3A –C ). In T30 cells, vesicles ≤ 2 µm² (primary/mature lysosomes) were notably enlarged—a feature not observed in CW-Tau or control cells (Fig. 3D). The enlargement of these lysosomes is consistent with previous findings in neurodegenerative diseases such as AD ( 55 , 56 ). The size increase trend extended, albeit less markedly, to the 2–5 µm² category. Vesicles ≥ 5 µm² also increased in size, suggesting endosomal compartment accumulation in T30 cells. To assess compartment acidity, LysoTracker Red DND-99 staining was performed. T30 overexpression significantly increased the number of acidified vesicles, particularly those in the ≤ 2 µm² category, whereas the number of CW-Tau cells remained similar to that of the control (Fig. 3A –E ). The colocalization of LysoTracker and LAMP1 confirmed this increase in acidified vesicles in T30 cells, especially within the ≤ 2 µm² and 2–5 µm² compartments, which was consistent with mature lysosomes, amphisomes, autophagosomes, and MVBs (Fig. 3F). For vesicles ≥ 5 µm², the trend persisted but was less defined. Overall, T30 expression led to greater numbers of acidified vesicles per cell (Fig. 3G), with increased acidity in LAMP1 + compartments (Fig. 3H). However, this effect was attenuated when LAMP1 + LysoTracker + double-positive vesicles were considered (Fig. 3I), suggesting an accumulation of mature lysosomes without significant pH alterations compared with the control. This finding aligns with elevated cathepsin D maturation in T30 cells (Fig. 3J). In contrast, CW-Tau-expressing cells preserved a greater proportion of primary lysosomes (Fig. 3F, area ≤ 2 µm² LysoTracker-negative vesicles), indicating intact lysosomal biogenesis. Together, these results demonstrate that the overexpression of classical Tau 3R (T30) leads to the accumulation and enlargement of LAMP1-positive vesicles, particularly acidified mature lysosomes, which is indicative of impaired autophagic flux—likely due to defective fusion with autophagosomes or endosomes. Conversely, CW-Tau maintains lysosomal number, size, acidity, and biogenesis at levels comparable to those of the control, supporting functional lysosomal trafficking and degradation. 4.5 CW-Tau prevents protein aggregation On the basis of previous findings that classical Tau impairs autophagy and disrupts vesicular trafficking—effects not observed with CW-Tau—we investigated whether these differences influence protein aggregation via the Proteostat™ assay. HEK293T cells transfected with either classical Tau 3R (T30) or the corresponding CW-Tau isoform were treated with bafilomycin A1, an inhibitor of lysosomal acidification and autophagy, or MG-132, a proteasome inhibitor that impairs protein degradation via the ubiquitin‒proteasome system. Immunofluorescence imaging and quantification revealed that, compared with no treatment, overexpression of classical Tau led to a significant increase in protein accumulation and aggregation in control cells (Fig. 4A –B ). In contrast, CW-Tau overexpression did not induce notable aggregation, with levels comparable to those of the control. These findings align with prior reports describing the antiaggregation properties of the W-Tau peptide ( 15 ). Upon treatment, cells transfected with the empty vector showed significant aggregate accumulation in response to both bafilomycin A1 and MG-132, particularly the latter, which was consistent with the expected outcomes of autophagy or proteasome inhibition (Fig. 4C). In contrast, in T30-expressing cells, bafilomycin A1 did not further increase aggregation, suggesting that autophagy was impaired by Tau expression (Fig. 4D). Moreover, the increase in aggregation in response to MG-132 treatment was markedly attenuated in T30 cells compared with that in control cells, where MG-132 induced a fivefold increase in aggregation. Conversely, in CW-Tau-expressing cells, both bafilomycin A1 and MG-132 significantly increased protein aggregation to levels comparable to those of the control vector, indicating that CW-Tau preserves the functionality of both the autophagic and proteasomal degradation pathways (Fig. 4E). Collectively, these results indicate that CW-Tau prevents protein aggregation by maintaining proteostasis, preserving both autophagy and proteasome activity. In contrast, the overexpression of classical Tau promotes protein aggregation, likely through the intrinsic inhibition of autophagy. 4.6 CW-Tau is efficiently exported to the extracellular medium via exosomes Tau is known to be secreted into the extracellular space via multiple pathways, including direct release, tunneling nanotubes, and extracellular vesicles such as exosomes, particularly under pathological conditions. Considering the observed interaction between the W-Tau peptide and Rab proteins—especially Rab11A, a key regulator of both constitutive and regulated secretion ( 48 )—we investigated whether CW-Tau is differentially exported from cells compared with classical Tau. HEK293T cells were transfected with either the classical Tau 3R isoform lacking inserts (T30) or the corresponding CW-Tau isoform generated by intron 12 retention. We analyzed the Tau distribution in both the intracellular compartment and the extracellular medium, separating the latter into a soluble ("naked") fraction and an exosome-enriched fraction (Fig. 5A). Despite differences in their intracellular expression levels, both isoforms were detected in the extracellular space. However, normalization to intracellular expression revealed that CW-Tau was exported less efficiently than T30 overall (Fig. 5A –B ). Notably, when the naked Tau fraction was analyzed, both isoforms were exported with comparable efficiency (Fig. 5A –C ). In contrast, the exosome-enriched fraction presented significantly lower CW-Tau levels than did the T30 fraction (Fig. 5A –D ), suggesting isoform-specific differences in exosome-mediated secretion. These findings imply that Tau may influence neighboring cells through both naked-eye export and exosome-mediated pathways. Exosomes are released via the fusion of multivesicular bodies (MVBs) with the plasma membrane ( 57 ), a process regulated by Rab family members such as Rab11a. Rab11a is implicated in both constitutive and regulated secretion, facilitating MVB docking and fusion via interactions with the exocyst complex ( 58 , 59 ). It is also involved in the formation of recycling endosomes that contribute to vesicle recycling and secretion. To assess whether Tau isoforms affect the distribution of Rab11a, we analyzed SH-SY5Y cells expressing either T30 or CW-Tau via lentiviral vectors (GFP-tagged). Rab11a localization was evaluated via kurtosis analysis, a statistical measure of data clustering indicative of protein aggregation versus normal vesicular distribution. T30 expression significantly increased Rab11a kurtosis, suggesting abnormal protein aggregation rather than physiological vesicular distribution. This effect was not detected in CW-Tau-expressing cells (Fig. 5E –F ). Rab11a clustering may indicate impaired vesicle trafficking, potentially linked to microtubule destabilization induced by T30 overexpression (Fig. 1D). We further examined the expression of CD63, a tetraspanin enriched in late endosomes and MVBs that plays a key role in exosome biogenesis and cargo selection. CD63 levels per cell were markedly reduced in T30-expressing cells but remained unchanged in CW-Tau-expressing cells (Fig. 5E –G ). This decrease in MVBs contrasts with the observed accumulation of small- and medium-sized LAMP1 + /Lysotracker + vesicles, corresponding to mature lysosomes and autophagolysosomes (Fig. 3F). The reduction in the intracellular CD63 signal may result from impaired fusion of MVBs with lysosomes or autophagolysosomes, favoring MVB fusion with the plasma membrane and subsequent exosomal release of T30 (Fig. 5C). Together, these data suggest that classical Tau (T30) disrupts normal vesicle trafficking and secretion pathways, altering Rab11a distribution and reducing intracellular CD63 levels, which may promote excessive exosomal Tau release. In contrast, CW-Tau maintains Rab11a localization and MVB integrity, resulting in reduced Tau secretion via exosomes and overall preservation of vesicle trafficking homeostasis. 4.7 CW-Tau prevents Tau seeding activity and the propagation of Tauopathy Building on the previously demonstrated antiaggregation effects of CW-Tau and the W-Tau peptide in vitro ( 28 ), we evaluated whether CW-Tau could inhibit Tau seeding activity in vivo via a cellular biosensor model. For this purpose, we employed HEK293T TAU RD P301S FRET reporter cells, which generate a fluorescence resonance energy transfer (FRET) signal upon Tau aggregation (Fig. 6A). As a positive control, the treatment of these cells with Tau seeds derived from Alzheimer’s disease (AD) patient brain extracts induced a robust increase in the FRET signal (Fig. 6B). Cotransfection of these cells with both Tau seeds and a plasmid encoding the classical T30 (Tau 3R) isoform led to a dose-dependent increase in FRET activity, indicating that T30 promotes Tau aggregation (Fig. 6D, top row; quantification in Fig. 6C ). Conversely, coexpression of CW-Tau significantly reduced Tau aggregation induced by brain-derived seeds in a dose-dependent manner (Fig. 6D, bottom row; quantification in Fig. 6C ). This antiaggregation effect may be attributed to the previously observed binding affinity of the W-Tau peptide for Tau (Table 2 , Supplementary Fig. 2C ), suggesting that CW-Tau interferes with Tau oligomerization by disrupting interactions within the microtubule-binding domain. To investigate whether the antiaggregation effects of CW-Tau could be transferred between cells, we treated TAU FRET cells with Tau seeds in combination with increasing volumes of extracellular medium collected from cells overexpressing either T30 or CW-Tau. While the addition of extracellular medium from T30-expressing cells significantly increased Tau seeding, the addition of medium from CW-Tau-expressing cells reduced aggregation in a dose-dependent manner (Fig. 6E and H ). Since Tau can be secreted as a free protein or within extracellular vesicles, we separated the extracellular medium into vesicle-free and exosome-enriched fractions via ultracentrifugation. These fractions were then applied to the reporter cells at equal concentrations. Vesicle-free T30 was efficiently internalized and led to a dose-dependent increase in the FRET signal, which was consistent with enhanced seeding activity (Fig. 6F and I ). Although CW-Tau was present at lower levels in the vesicle-free fraction (Fig. 5A), it retained the ability to suppress Tau aggregation upon transfer, confirming that its antiseeding activity is preserved during intercellular transmission. Further analysis revealed that the exosome-enriched fraction from T30-expressing cells also induced robust Tau aggregation in a dose-dependent manner (Fig. 6G –J ). Notably, despite the reduced export of CW-Tau via exosomes (Fig. 5A and D ), the exosomal fraction derived from CW-Tau-expressing cells was able to transmit antiaggregation effects. Treatment with the highest concentration of this fraction significantly decreased the FRET signal, confirming that the protective properties of CW-Tau are also conveyed through exosomes (Fig. 6G –J ). These findings demonstrate that CW-Tau effectively inhibits pathological Tau seeding and aggregation in a cellular context. Moreover, its antiaggregation activity can be transmitted to recipient cells via both vesicle-free and exosome-mediated mechanisms. Together, these properties underscore the therapeutic potential of CW-Tau to counteract Tau pathology and limit the intercellular propagation of Tau aggregates. Discussion In a previous study, we identified a novel Tau isoform, CW-Tau, characterized by the retention of intron 12, which encodes a 16-amino-acid sequence containing two tryptophans (W), and by the absence of the C-terminal region, including exon 13 ( 13 ). This isoform has high microtubule-binding affinity, increased solubility, and lower RNA and protein levels in AD brains than nondemented controls do ( 13 , 14 ). Additionally, a peptide encoded by intron 12 exhibits chaperone-like activity, effectively inhibiting Tau and amyloid-β aggregation in vitro ( 15 ). In this study, we provide mechanistic insight into the distinctive cellular properties of CW-Tau and its potential therapeutic relevance. We show that the W-Tau peptide directly interacts with tubulin, functioning as an auxiliary microtubule-binding domain that promotes microtubule polymerization in vitro. Unlike canonical Tau isoforms, whose overexpression disrupts microtubule architecture, CW-Tau preserves cytoskeletal integrity. Interestingly, the binding of CW-Tau to tubulin is increased in AD samples, potentially due to pathological posttranslational modifications, such as tyrosination and detyrosination ( 60 ). These modifications have been associated with increased Tau phosphorylation and synaptotoxicity ( 61 ). Future studies should assess whether the affinity of CW-Tau for tubulin is modulated by these modifications and whether this affects Tau phosphorylation and neurotoxicity. Exon 13 of MAPT is constitutively expressed across all canonical Tau isoforms and encodes phosphorylation sites such as those recognized by PHF-1 antibodies (S396 and S404), which modulate microtubule binding and have been implicated in Tau aggregation and Tauopathy pathology ( 62 , 63 ). These residues also play key roles in regulating synaptic plasticity via long-term potentiation ( 64 ). Research has demonstrated that the W-Tau peptide prevents Tau and amyloid-β aggregation ( 15 ). In addition to microtubule interactions, the W-Tau peptide binds lactate dehydrogenase (LDH), a known Tau interactor, and prevents its self-aggregation via a chaperone-like mechanism ( 34 ). This may involve interaction motifs such as EVHHQ (in amyloid-β) and EVHKQ (in LDH), although interactions with acidic residues in LDH (e.g., 101-DLDED-105 or 360-EEE-362) remain to be confirmed ( 65 ). We also identified interactions between W-Tau and other brain proteins, including LC1-MAP1B, several Rab proteins and GDIs (primarily in AD brain extracts). MAP1B-LC1 is a cytoskeletal protein that inhibits autophagosome formation by linking syntaxin 17 to microtubules and preventing the Drp1-mediated mitochondrial fission necessary for recycling through autophagy ( 66 ). Classic Tau overexpression impairs autophagic flux, increasing vesicle accumulation while reducing the degradation of damaged mitochondria. CW-Tau’s interaction with MAP1B-LC1 may alleviate this inhibition, restoring mitochondrial recycling and protein turnover. Additionally, CW-Tau associates with PARK7, a key regulator of mitophagy ( 37 ), suggesting a broader role in maintaining mitochondrial homeostasis. W-Tau also binds GDIs A and B, which regulate Rab cycling and autophagy by mediating GTPase membrane association ( 41 , 67 – 69 ). This interaction could facilitate Rab activation, promoting vesicular trafficking and autophagic flux. In contrast, classical Tau overexpression disrupts Rab11 dynamics and leads to the accumulation of LAMP1 + vesicles—particularly mature lysosomes—indicating the defective fusion of autophagosomes and endosomes, leading to severe autophagy impairment. Exosomes, which are secreted from multivesicular bodies (MVBs), are key mediators of proteostasis, balancing degradation via lysosomal fusion or the secretion of aggregated proteins. In autophagy-deficient contexts, exosome release is upregulated, promoting the secretion of pathological aggregates such as Tau ( 70 ). W-Tau’s interaction with Rab11B—a known regulator of constitutive and regulated secretion ( 48 )—is particularly relevant, as classical Tau expression induces Rab11 clustering and enhances exosomal Tau release. Rab11 also facilitates MVB fusion with the plasma membrane in a Ca²⁺-dependent manner ( 71 , 72 ) and mediates the secretion of α-synuclein inclusions ( 73 ). Although CW-Tau is secreted in exosomes, its expression does not compromise vesicular trafficking or autophagy, thereby limiting exosomal export. This is supported by preserved CD63 labeling in CW-Tau-expressing cells, in contrast to classical Tau, which markedly reduces CD63 levels, likely due to excessive MVB depletion via exosome release. Importantly, the W-Tau peptide displays differential protein-binding affinities between healthy and AD brains. Tubulin, MAPs, Rabs, GDIs, LC1-MAP1B, and PARK7 all exhibit enhanced interactions in AD tissue, suggesting that the protective functions of CW-Tau may be potentiated under pathological conditions. Conversely, in nonpathological brains, W-Tau preferentially binds Rab11 and lipid-related proteins such as APOE and FABP7, suggesting a physiological role in secretion and lipid metabolism. The biological implications of these distinct binding patterns require further investigation. Finally, we show that both CW-Tau and classical Tau isoforms are secreted into the extracellular space, either freely or within exosomes. This allows their aggregation-modifying properties to influence neighboring cells. CW-Tau-containing extracellular media or exosome fractions exhibit antiaggregation activity, highlighting their therapeutic potential for the protective effects of cell-to-cell transmission. Conclusions In summary, CW-Tau promotes microtubule polymerization, preserves vesicle trafficking, enhances autophagic flux, and reduces protein aggregation. Furthermore, its protective properties are transferable to neighboring cells via secretory pathways. These characteristics position CW-Tau as a promising candidate for therapeutic intervention in Tauopathies. Declarations Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness: PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by the CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). Schemes in Figures 1, 2, 3 and 6 were created with BioRender.com. The Centro de Biología Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S). Funding for publication is provided by the Open Access Publishing Support Program of the CSIC. Competing interest: The authors declare that they have no competing interests. Availability of data and materials: The datasets supporting the conclusions of this article are available in the Figshare repository: Raw proteomic data and analysis https://doi.org/10.6084/m9.figshare.28770797.v1 Microtubule and LDH binding https://doi.org/10.6084/m9.figshare.28771124.v1. Autophagy analysis https://doi.org/10.6084/m9.figshare.28771076.v1. Vesicle trafficking analysis https://doi.org/10.6084/m9.figshare.28771091.v2. FRET Tau https://doi.org/10.6084/m9.figshare.28771049.v1. Ethics approval: The study received approval from The Ethical Committee of Universidad Autónoma de Madrid (CEI-98-1831). The biobank HUB-IDIBELL is authorized by the Directorate General for Health Planning and Research of the Department of Health of the Generalitat of Catalunya and is registered in the National Register of Biobanks of the Instituto de Salud Carlos III in accordance with current Spanish legislation (Law 14/2007, of July 3, about Biomedical Research, and RD 1716/2011, of November 18). Author contributions: Conceptualization and designing of the work, V.G-E, and J.A.; acquisition, F.V.-B., I.D.-S., A.S., R.C., M.P., E.M., N.S., D.R.-G., and V.G.-E.; analysis or interpretation of data, F.V.-B., I.D.-S., A.S., M.P., E.M., D.R.-G., F.H., V.G.-E., and J.A.; Writing – Original Draft, F.V.-B., I.D.-S., A.S., E.M., V.G.-E., and J.A.; Writing – Review & Editing and final approval of the version F.V.-B., I.D.-S., A.S., R.C., M.P., E.M., N.S., D.R.-G., F.H., V.G.-E., and J.A. References Drubin DG, Nelson WJ. Origins of cell polarity. Cell. 1996;84(3):335–44. 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García-Escudero","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIiWNgGAWjYPACCSjNBsTsDSRr4TlAkm0gLRIJ+NUYHGC/+LmixkKegb394WeeMpt8fsk3hp9uMNjY49bCUyx55piEYQPPGWNpnnNpljNn5xhL5zCkJTbg0CLZwJMg2dggwbj/Rg4bM2/bYQOD2zkGQC2HcToPqCX5J1CLfYP882dALf8NDG6eMf6dw/Afp8P4GdiPgWxJbJBgMANqOWBgcIPHDGjLAUZcDuNn5mGzbDgmkdzAk2MsOedcsoFkT1qZdY5BMk6/sLG3P77ZUFNn28B+/OGHN2V2Bvzshzffzqmww+kwBmYeA2zCWAVhgP0BPtlRMApGwSgYBQwMAH7aS4a/Vq3QAAAAAElFTkSuQmCC","orcid":"","institution":"Universidad Autónoma de Madrid (UAM)","correspondingAuthor":true,"prefix":"","firstName":"Vega","middleName":"","lastName":"García-Escudero","suffix":""},{"id":463228685,"identity":"e2e1f9ae-7fb7-476f-a4de-a11acf0c4aaa","order_by":10,"name":"Jesús Avila","email":"","orcid":"","institution":"Centro de Biología Molecular Severo Ochoa (UAM-CSIC)","correspondingAuthor":false,"prefix":"","firstName":"Jesús","middleName":"","lastName":"Avila","suffix":""}],"badges":[],"createdAt":"2025-05-22 12:53:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6725217/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6725217/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83614480,"identity":"1cd42748-9fd9-4808-82ff-2d59d2a43c0a","added_by":"auto","created_at":"2025-05-29 13:17:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2482180,"visible":true,"origin":"","legend":"\u003cp\u003eThe \u003cem\u003eW-Tau peptide binds to microtubules and lactate dehydrogenase.\u003c/em\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA\u003c/strong\u003e. Schematic of the microtubule binding assay (created with BioRender.com). \u003cstrong\u003eB.\u003c/strong\u003eDot blot showing W-Tau binding to tubulin at various tubulin:W-Tau ratios. \u003cstrong\u003eC.\u003c/strong\u003eElectron microscopy images of tubulin polymerization ± W-Tau, with quantification.\u003cstrong\u003e D. \u003c/strong\u003eConfocal images of SH-SY5Y cells expressing classical Tau (T30) or CW-Tau via lentiviral infection, showing GFP, Tau, and β-tubulin staining; the microtubule surface area was quantified in nm²/cell via FIJI. Scale bar: 50 µm. \u003cstrong\u003eE.\u003c/strong\u003e Electron microscopy of LDH aggregation after thermal denaturation (80 °C, 100 s), with or without Tau42 or W-Tau peptide at varying LDH ratios; aggregation was quantified via FIJI. Scale bar: 100 nm. All the data are presented as the means ± SEMs (one-way ANOVA: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns: not significant).\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/5722ec60443470863aa379f0.jpg"},{"id":83614482,"identity":"e9ba0eea-ca53-4bac-a1e9-74df348d0bc1","added_by":"auto","created_at":"2025-05-29 13:17:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1811770,"visible":true,"origin":"","legend":"\u003cp\u003eModulation of autophagic flux by Tau isoforms. \u003cstrong\u003eA. \u003c/strong\u003eSchematic of macroautophagy showing CCCP as a mitophagy inducer and bafilomycin A1 (Baf A1) as a lysosomal acidification inhibitor that blocks autophagosome‒lysosome fusion (created with BioRender.com) \u003cstrong\u003eB‒F. \u003c/strong\u003eHEK293T cells were transfected with pSG5 vectors encoding Tau isoforms (T30 or CW‒T30) or empty vector and then treated for 24 h with 5 μM CCCP ± 200 nM Baf A1 for the final 6 h. \u003cstrong\u003eB.\u003c/strong\u003eRepresentative Western blots for total Tau (Tau5), LC3, p62, and β-actin. \u003cstrong\u003eC-F.\u003c/strong\u003eQuantification of the ratios of LC3-II synthesis (\u003cstrong\u003eC\u003c/strong\u003e), LC3-II degradation (\u003cstrong\u003eD\u003c/strong\u003e), p62 accumulation (\u003cstrong\u003eE\u003c/strong\u003e), and p62 degradation (\u003cstrong\u003eF\u003c/strong\u003e). \u003cstrong\u003eG-H.\u003c/strong\u003eConfocal microscopy of SH-SY5Y cells expressing GFP alone, T30, or CW-Tau, which were stained for GFP (green), Tau (red), and LC3 (white, \u003cstrong\u003eG\u003c/strong\u003e) or EEA1 (white, \u003cstrong\u003eH\u003c/strong\u003e).\u003cstrong\u003e I‒J.\u003c/strong\u003e Quantification of LC3 puncta (\u003cstrong\u003eI\u003c/strong\u003e) and EEA1 puncta (\u003cstrong\u003eJ\u003c/strong\u003e) per cell. The graphs present the means ± SEMs (one-way ANOVA: *p ≤ 0.05, **p ≤ 0.01, ns: not significant).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/0b0c5cf5f695352d244e9a7b.jpg"},{"id":83615194,"identity":"9b714de9-1068-494c-91f0-02be8c0be607","added_by":"auto","created_at":"2025-05-29 13:25:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2216284,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of Tau isoform overexpression on LAMP1 vesicle number, size, and acidification. \u003cstrong\u003eA. \u003c/strong\u003eConfocal images of SH-SY5Y cells expressing GFP alone as control (C), Tau 3R (T30, T), or CW-Tau (CW) immunolabeled for GFP (green), LysoTracker Red DND-99 (red), LAMP1 (white), and merge of the last two. Scale bar: 50 µm. \u003cstrong\u003eB\u003c/strong\u003e. Schematic classification of LAMP1\u003csup\u003e+\u003c/sup\u003e vesicles by size and acidity (created with BioRender.com). \u003cstrong\u003eC‒D.\u003c/strong\u003e Quantification of LAMP1\u003csup\u003e+\u003c/sup\u003e vesicles per cell (\u003cstrong\u003eC\u003c/strong\u003e) and vesicle size (\u003cstrong\u003eD\u003c/strong\u003e), categorized by area: ≤2 μm² (primary/mature lysosomes), 2‒5 μm² (amphisomes, autolysosomes, MVBs), and ≥5 μm² (early/late endosomes). \u003cstrong\u003eE.\u003c/strong\u003e Quantification of LysoTracker+ vesicles per cell by area. \u003cstrong\u003eF.\u003c/strong\u003e Colocalization ratio of LAMP1\u003csup\u003e+\u003c/sup\u003e and LysoTracker+ vesicles by area. \u003cstrong\u003eG\u003c/strong\u003e. Cell-wide acidity based on total LysoTracker intensity. \u003cstrong\u003eH\u003c/strong\u003e. Acidity of LAMP1+ vesicles (LysoTracker intensity per LAMP1\u003csup\u003e+\u003c/sup\u003e vesicle). \u003cstrong\u003eI. \u003c/strong\u003eLysosomal acidity, calculated as the ratio of LysoTracker intensity to the number of LAMP1\u003csup\u003e+\u003c/sup\u003e and LysoTracker+ vesicles. \u003cstrong\u003eJ.\u003c/strong\u003e Western blot and quantification of cathepsin B maturation, shown as the cathepsin D/procathepsin D ratio. \u003cstrong\u003eJ.\u003c/strong\u003e All the data are presented as the means ± SEMs (one-way ANOVA: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant).\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/dba19f3de4f23566e770f557.jpg"},{"id":83614483,"identity":"0119b1cb-6e62-436a-aa54-f49090d3417d","added_by":"auto","created_at":"2025-05-29 13:17:23","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1138705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCW-Tau does not induce protein aggregation\u003c/em\u003e. \u003cstrong\u003eA. \u003c/strong\u003eConfocal images of HEK293T cells transfected with empty pSG5, Tau 3R (T30), or CW-Tau plasmids. The cells were treated with 200 nM bafilomycin A1 or 5 µM MG-132 for 6 h and stained with a Proteostat Aggresome Detection Kit. Scale bar: 50 µm. \u003cstrong\u003eB. \u003c/strong\u003eQuantification of protein aggregation under control conditions, expressed as puncta per cell (FIJI). \u003cstrong\u003eC-E\u003c/strong\u003e. Quantification of protein aggregation in cells transfected with empty vector (C), T30 (D), or CW-Tau (E) with or without bafilomycin A1 or MG-132 treatment. The data are shown as the means ± SEMs (one-way ANOVA: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: not significant).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/87d6eab8649af851c0b9eec0.jpg"},{"id":83614487,"identity":"7c9e32f8-41a6-4e8c-93c8-ca3a080fc629","added_by":"auto","created_at":"2025-05-29 13:17:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1962501,"visible":true,"origin":"","legend":"\u003cp\u003eSecretion of different Tau isoforms\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003eA. \u003c/strong\u003eWestern blot analysis of HEK293T cells transfected with empty vector (C), Tau 3R (T30), or CW-Tau. Intracellular Tau was detected in whole-cell lysates via an anti-T12 antibody; β-actin served as a loading control. Equal volumes of extracellular medium were analyzed postclarification. Media were ultracentrifuged following the methods of Simon et al. (30) to separate naked and exosome-associated Tau fractions. \u003cstrong\u003eB-D.\u003c/strong\u003e Quantification of (\u003cstrong\u003eB\u003c/strong\u003e) total extracellular Tau relative to intracellular Tau (normalized to β-actin), (\u003cstrong\u003eC\u003c/strong\u003e) the naked Tau fraction, and (\u003cstrong\u003eD\u003c/strong\u003e) the exosomal Tau fraction, each relative to the intracellular level. \u003cstrong\u003eE.\u003c/strong\u003e Confocal images of SH-SY5Y cells transduced with GFP, GFP-T30, or GFP-CW-Tau lentivectors. Immunostaining for GFP (green), Tau (red), and Rab11 or CD63 (white) is shown. Scale bar: 50 µm.\u003cstrong\u003e F-G.\u003c/strong\u003e Quantification of Rab11 kurtosis (\u003cstrong\u003eF\u003c/strong\u003e) and CD63 fluorescence intensity (\u003cstrong\u003eG\u003c/strong\u003e) per cell. The data are shown as the means ± SEMs (n = 3 for Western blots, Student’s t test; n = 4 for immunofluorescence, 10 images/experiment, one-way ANOVA; *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001; ns: not significant).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/abca62be467e22ff1841c0d9.jpg"},{"id":83615196,"identity":"1a051c60-dfbc-4926-909c-9ae365c71e6b","added_by":"auto","created_at":"2025-05-29 13:25:23","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1156349,"visible":true,"origin":"","legend":"\u003cp\u003eCW-Tau inhibits Tau seeding activity and propagation. \u003cstrong\u003eA.\u003c/strong\u003eExperimental design for Tau aggregation assessment via TAU FRET cells transfected with AD brain-derived Tau seeds alone or with pSG5 plasmids encoding Tau isoforms, or with extracellular medium/exosome fractions from HEK293T cells expressing Tau isoforms (created with BioRender.com). \u003cstrong\u003eB.\u003c/strong\u003eConfocal images of untreated or AD seed-treated TAU FRET cells. \u003cstrong\u003eC.\u003c/strong\u003e Quantification of the FRET ratio (fluorescence intensity of each condition normalized to that of the AD-seed-only control) from the images shown in \u003cstrong\u003eD\u003c/strong\u003e. \u003cstrong\u003eD. \u003c/strong\u003eRepresentative images of TAU FRET cells cotransfected with AD seeds and increasing amounts of pSG5 encoding T30 or CW-Tau. \u003cstrong\u003eE\u003c/strong\u003e. Similar setup using increasing amounts of extracellular medium from cells expressing T30 or CW-Tau. \u003cstrong\u003eF. \u003c/strong\u003eTreatment with the naked fraction of extracellular medium (ultracentrifuged per Simon et al. (30)). \u003cstrong\u003eG.\u003c/strong\u003e Treatment with exosome-enriched fractions from cells expressing T30 or CW-Tau.\u003cstrong\u003e H-J\u003c/strong\u003e Graphs show the FRET quantification or images shown in \u003cstrong\u003eE\u003c/strong\u003e, \u003cstrong\u003eF\u003c/strong\u003e and\u003cstrong\u003e G,\u003c/strong\u003erespectively. The data are presented as the means ± SEMs (n = 3, 10 images per replicate). One-way ANOVA: *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/70a95415b5bdec09442d3f27.jpg"},{"id":94672122,"identity":"8d8188ed-86b9-4989-b7eb-459755d34752","added_by":"auto","created_at":"2025-10-29 13:39:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12175775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/1d7b5c4e-e4b8-4417-8c65-5d0123c02221.pdf"},{"id":83614479,"identity":"b9610e31-5579-426f-8d2b-5ff5d985f814","added_by":"auto","created_at":"2025-05-29 13:17:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":47362,"visible":true,"origin":"","legend":"","description":"","filename":"extendedmaterialsandmethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/33d787fff4f6a67550ccc261.docx"},{"id":83615195,"identity":"49b49861-8476-4155-b044-20f59d7984b6","added_by":"auto","created_at":"2025-05-29 13:25:23","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":240732,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/5829dee6a589ea8394f66753.pdf"},{"id":83614485,"identity":"271d6043-d53c-41a5-9831-e15e1ab69577","added_by":"auto","created_at":"2025-05-29 13:17:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":418700,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/41701c3cd2dc2487bfd22d16.pdf"},{"id":83614489,"identity":"beda7491-c727-4c74-bd8a-d51323bd7c88","added_by":"auto","created_at":"2025-05-29 13:17:23","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":460586,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6725217/v1/9fce6ba7872ea09835079a87.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CW-Tau fights proteinopathy by improving autophagy and diminishing Tau seeding activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTau is a microtubule-associated protein predominantly expressed in the central nervous system (CNS). Its principal function is to stabilize microtubules by regulating their dynamic equilibrium, thereby influencing vesicle trafficking. In the CNS, Tau plays a key role in establishing and maintaining neuronal polarity and promoting neurite outgrowth(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). It also regulates the axonal transport of endocytic and exocytic vesicles (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), as well as organelles such as mitochondria (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), endosomes and lysosomes (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e), peroxisomes and the endoplasmic reticulum. Under pathological conditions, such as Tau overexpression, kinesin-dependent organelle trafficking is impaired, resulting in neurodegeneration (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). These trafficking defects correlate with altered membrane trafficking pathways observed in Alzheimer\u0026rsquo;s disease, including synaptic vesicle disruption and increased levels of extracellular microvesicle markers (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTau is encoded by the MAPT gene located on chromosome 17 and undergoes extensive alternative splicing (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). This process gives rise to a family of Tau isoforms defined by specific exon combinations, yielding six distinct isoforms in the CNS (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Recently, we identified a novel Tau isoform generated by intron 12 retention (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This isoform lacks exon 13 but includes a unique human-specific 16-amino acid peptide encoded by intron 12, leading to a truncated C-terminal end (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Notably, this intron-derived peptide introduces two tryptophans\u0026mdash;absent in the classical Tau protein\u0026mdash;prompting us to name the isoform CW-Tau.\u003c/p\u003e \u003cp\u003eCW-Tau has distinct properties, including (a) greater microtubule binding, (b) greater solubility than other Tau isoforms, and (c) lower RNA and protein levels in the brains of AD patients than in those of nondemented individuals (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Additionally, its specific peptide, W-Tau, has chaperone-like activity (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). The discovery of other Tau isoforms arising from intron 11 retention (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e) highlights an overlooked mechanism for Tau diversity.\u003c/p\u003e \u003cp\u003eTo explore the function of CW-Tau, we identified human brain proteins that bind to the W-Tau peptide, particularly those involved in microtubule dynamics, membrane trafficking, chaperone activity, and protein clearance. Tubulin and lactate dehydrogenase (LDH), which are highly abundant in the brain, interact with the W-Tau peptide, as do lower-abundance proteins linked to autophagy (LC1-MAP1B) and vesicle trafficking/secretion (Rab-GDIs and several RABs).\u003c/p\u003e \u003cp\u003eConsistently, CW-Tau expression enhances autophagy flux and vesicle trafficking, suggesting more efficient protein clearance and faster turnover than classical Tau isoforms do. Moreover, CW-Tau effectively inhibited Tau seeding activity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Proteomic analysis\u003c/h2\u003e \u003cp\u003eThe W-Tau peptide (sequence: KKVKGVGWVGCCPWVYGH), derived from Tau intron 12, was purchased from Abyntek Biopharma S.L. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). W-Tau peptide interactions with human brain extracts from cortical samples from patients with sporadic Alzheimer's disease and control individuals (HUB-IDIBELL Brain Bank, Spain) were analyzed via CNBr-activated Sepharose 4B affinity chromatography (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Proteins were concentrated at the stacking/resolving gel interface of SDS‒PAGE, digested with trypsin, and desalted via OMIX Pipette C18 tips (Agilent Technologies) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Protein identification was performed via reverse-phase liquid chromatography coupled with tandem mass spectrometry (RP-LC‒MS/MS) via an LTQ-Orbitrap-Velos-Pro (Thermo Scientific) under a dynamic exclusion setting (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Peptide identification was conducted via PEAKS Studio XPro, which searches against the UniProt database. Functional network analysis was performed via STRING (\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Microtubule binding assay\u003c/h2\u003e \u003cp\u003eMicrotubules were purified from C57 mouse brains, polymerized, incubated with W-Tau, and analyzed via dot blotting via anti-CW-Tau antibodies and electron microscopy (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Lactate Dehydrogenase Aggregation\u003c/h2\u003e \u003cp\u003eLactate dehydrogenase (LDH) was incubated with W-Tau at 80\u0026deg;C, and aggregation was visualized via transmission electron microscopy (JEM1400 Flash Transmission Electron Microscope, Jeol).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Immunofluorescence analysis\u003c/h2\u003e \u003cp\u003eSH-SY5Y cells were transduced with pWPI vectors expressing the T30 and CW-T30 isoforms (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and then fixed and stained with antibodies against β-tubulin (SIGMA, T4026), GFP (ABCAM, 13970), Tau (NUVUSBIO, NB100-822247), LC3 (SIGMA, L7543), EEA1 (BD Biosciences, 610457), Rab11A1 (Invitrogen, 71\u0026ndash;5300), CD63 (DSHB, H5C6), and LAMP1 (DSHB, H4A3) and with DAPI (4',6-diamidino-2-phenylindole, Merck, 268298). When indicated, the cells were treated with LysoTracker Red DND-99 (Invitrogen, L7528) before fixation. Confocal microscopy analysis was performed via Fiji software, and the vesicles were segmented via CellPose Plug-in (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Study of Autophagy Modulation\u003c/h2\u003e \u003cp\u003eHEK293T cells (CRL-11268, ATCC) were transfected with Tau constructs (pSG5, Agilent, 216201) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) and treated with CCCP and bafilomycin A1 to assess autophagic responses via Western blot analysis as previously described (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Protein aggregation assays\u003c/h2\u003e \u003cp\u003eHEK293T cells were treated with bafilomycin A1 and MG-132 to induce protein aggregation, which was analyzed via a Proteostat Aggresome Detection Kit (Enzo Life Sciences, 51023) followed by confocal microscopy analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Determination of Tau export in exovesicles\u003c/h2\u003e \u003cp\u003eHEK293T cells transfected with pSG5 encoding T30 or CW-T30 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e) were cultured, and their extracellular medium was fractionated by sequential centrifugation as previously described (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Exosome presence was confirmed via nanoparticle tracking analysis, and Tau in different fractions was detected by Western blotting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Tau aggregation assay\u003c/h2\u003e \u003cp\u003eHEK293T Tau RD P301S FRET biosensor cells (CRL-3275, ATTC) were transfected with Alzheimer\u0026rsquo;s-derived Tau seeds as previously described (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and with Tau-expressing vectors. Alternatively, cells were treated with different Tau fractions (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The FRET signal was analyzed via confocal microscopy and Fiji software (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were analyzed via GraphPad Prism 9.0 and Excel. The results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, and statistical comparisons were conducted via one-way or two-way ANOVA. Significance levels are represented as follows: ns (not significant), * p\u0026thinsp;\u0026le;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026le;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026le;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026le;\u0026thinsp;0.0001.\u003c/p\u003e \u003cp\u003eAdditional methodological details are provided in the \u003cb\u003eSupplementary Materials and Methods\u003c/b\u003e section.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e4.1 Identification of abundant human brain proteins that bind to the intron 12 peptide of the novel Tau isoform\u003c/p\u003e \u003cp\u003eA peptide sequence (KKVKGVGWVGCCPWVYGH), comprising two lysines from exon 12 and a 16-amino acid region from intron 12, was conjugated to a Sepharose column. Protein extracts from nondemented (C) and Alzheimer\u0026rsquo;s disease (AD) brains were chromatographed, retaining\u0026thinsp;~\u0026thinsp;20% of the total brain proteins in both cases. SDS‒PAGE analysis revealed two predominant retained proteins, with electrophoretic mobilities consistent with those of tubulin and lactate dehydrogenase (LDH). Mass spectrometry analysis revealed a significant interaction of the W-Tau peptide with tubulin alpha chain 1B (TBA1B) (\u003cb\u003esupplementary Fig.\u0026nbsp;1A\u003c/b\u003e), which was specifically enriched in the AD brain (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eProteomic Profiling of Tubulin Isotypes and Lactate Dehydrogenase Interacting with W-Tau in Human Brains.\u003c/em\u003e Proteins binding to W-Tau were identified via reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for both control and AD samples. Enrichment ratios were calculated to assess protein binding. Proteins identified include tubulin alpha chain 1B (TBA1B), lactate dehydrogenase A (LDHA), and B (LDHB).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eALZHEIMER DISEASE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTubulin subtype\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP68363|TBA1B_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2834300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e891220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e312840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3576400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e11.432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eLactate dehydrogenase\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP00338|LDHA_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e43397000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26541000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.611\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44151000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e13896000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.314\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP07195|LDHB_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e129050000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e122800000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.951\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e133290000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e112410000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.843\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGiven that Tau isoforms bind tubulin via microtubule-binding repeats (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), the interaction between W-Tau and tubulin suggests a novel tubulin-binding region within the CW-Tau isoform. Polymerization assays with mouse brain microtubules confirmed W-Tau\u0026rsquo;s association with polymerized microtubules, enhancing polymerization, as observed by electron microscopy (Fig.\u0026nbsp;1A-C). Additional interactions were identified with MAP1B, MAP2, and Tau itself (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2A-C\u003c/b\u003e), reinforcing its role in microtubule dynamics.\u003c/p\u003e \u003cp\u003eTo examine the impact of CW-Tau on microtubule stability, HEK293T cells were transfected with expression vectors encoding two Tau isoforms: the classical T30 isoform (with three microtubule-binding repeats and no N-terminal inserts) and the CW-Tau isoform (generated by intron 12 retention) (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), which are thought to be the most abundant isoform in the human brain. While T30 disrupted microtubule morphology, CW-Tau preserved cytoskeletal integrity, suggesting its stabilizing role (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eAdditionally, W-Tau potentially interacts with tubulin polymerization-promoting protein (TPPP), which is known to regulate microtubule dynamics, particularly in AD brains (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2D\u003c/b\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eProteomic Analysis of MAPs and Related Proteins Interacting with W-Tau Peptide in Human Brains.\u003c/em\u003e Proteins binding to W-Tau were identified via reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for both control and AD samples. Enrichment ratios were calculated to assess protein binding. Identified proteins include microtubule-associated proteins (MAPs) 1B (MAP1B), light-chain MAP1B (LC1-MAP1B), MAP2 (MTAP2), Tau, and Tubulin polymerization-promoter protein (TPPP).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eALZHEIMER DISEASE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e\u003cb\u003eMAPs and related proteins\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP46821|MAP1B_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2261300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e225860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3548200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2008400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.566\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLC1-MAP1B_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1599214\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e184824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1105500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e885294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.800\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP11137|MTAP2_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2420300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1047700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e894180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e537010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.600\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP10636|TAU_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10759000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1593900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3925400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2631000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.670\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO94811|TPPP_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4604800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e320230\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.069\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e238300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1110000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.657\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn addition to microtubules, the W-Tau peptide exhibited affinity for LDH1 and LDH2 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;1B-C\u003c/b\u003e), indicating a potential additional LDH-binding site in CW-Tau. Since Tau isoforms can act as chaperones to prevent LDH aggregation (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), we tested W-Tau\u0026rsquo;s chaperone-like function following Tian et al.\u0026rsquo;s protocol (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Electron microscopy revealed that W-Tau significantly reduced LDH aggregation, similar to Tau 42 (Fig.\u0026nbsp;1E). These results support W-Tau\u0026rsquo;s chaperone role, which aligns with previous findings that it prevents Tau and β-amyloid aggregation in vitro (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.2 CW-Tau maintains autophagic flux in HEK293T cells\u003c/h2\u003e \u003cp\u003eDetailed analysis of tryptic peptides from MAP1B that interact with the W-Tau peptide revealed enrichment within the light chain 1 (LC1-MAP1B) region of the precursor MAP1B protein, which comprises 2,459 amino acids (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e \u003cb\u003eand Supplementary Fig.\u0026nbsp;2A\u003c/b\u003e). The heavy chain (HC) spans residues 1\u0026ndash;2,185, whereas the light chain (LC) includes residues 2,210\u0026ndash;2,459 (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e), containing an acidic region (amino acids 2,337\u0026ndash;2,459) likely responsible for W-Tau binding. LC1-MAP1B is also involved in autophagy regulation through its interaction with syntaxin 17 (Stx17), which responds to nutrient availability (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdditionally, the W-Tau peptide potentially interacted with PARK7/DJ-1 in both control and AD brain samples (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;3K\u003c/b\u003e). DJ-1 is a critical component of the PINK1/parkin-mediated mitophagy pathway, facilitating the recruitment of optineurin to depolarized mitochondria (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the relevance of autophagy in Alzheimer's disease pathology (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) involving W-Tau with LC1-MAP1B and PARK7\u0026mdash;key regulators of autophagy and mitophagy, respectively (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e)\u0026mdash;we investigated the role of CW-Tau in mitophagy in comparison with the classical Tau isoform.\u003c/p\u003e \u003cp\u003eTo this end, HEK293T cells expressing various Tau isoforms were treated with CCCP, a mitochondrial uncoupler that induces mitophagy, either alone or in combination with bafilomycin A1. Bafilomycin inhibits lysosomal acidification by targeting the vacuolar H⁺-ATPase, thereby preventing autophagosome\u0026ndash;lysosome fusion and degradation of vesicular contents (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) (Fig.\u0026nbsp;2A). In these experiments, we compared classical Tau 3R (T30) with its corresponding CW-Tau isoform.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eProteomic Analysis of Vesicle Trafficking and Neurodegeneration Biomarkers Interacting with W-Tau Peptide in Human Brains.\u003c/em\u003e W-Tau-binding proteins were identified by reverse-phase liquid chromatography (RP-LC-MS/MS) and bioinformatics analysis with PEAKS Studio XPro. Binding areas of CW-Tau for each protein are shown for control and AD samples, with enrichment ratios calculated. Identified proteins include RAB GDP dissociation inhibitors (GDIA and GDIB), Rab proteins (Rab5C, Rab6B, Rab7A, Rab11B), Fatty acid binding protein 7 (FABP7), serine-threonine phosphatase 1 (CPPED), tyrosine-protein phosphatase SHPS1, apolipoprotein E (APOE), Parkinson disease protein 7 (PARK7), and ubiquitin-conjugating enzyme E2N (UBE2N).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eALZHEIMER DISEASE\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePass-through\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eEluted\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRatio\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003e\u003cb\u003eVesicle trafficking related\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP31150|GDIA_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60749000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46603000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37907000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e142050000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.747\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP50395|GDIB_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7489500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6400000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.854\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5303900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e11555000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP51148|RAB5C_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e507410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e502810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e840520\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.671\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ9NRW1|RAB6B_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2318900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e3975300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1658600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8083200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.873\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP51149|RAB7A_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e842110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e859590\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1313300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2331800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.775\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ15907|RB11B_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e298330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e407600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.366\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e892820\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e547980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.613\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO15540|FABP7_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1008200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2339300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.320\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1961500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2460400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.254\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ9BRF8|CPPED_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e457490\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e827020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.807\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e247760\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1404300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5.667\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP78324|SHPS1_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e472120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e532770\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e363650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e195090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.536\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cb\u003eNeurodegeneration biomarkers\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP02649 |\u0026nbsp;APOE_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e741100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1633100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2.203\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1307300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e749110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.573\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQ99497|PARK7_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16604000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19949000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12080000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e22930000\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.898\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP61088|UBE2N_HUMAN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1776800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2392600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.346\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1272800\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2826900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.221\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe first examined the levels of LC3, a marker of autophagic vesicle dynamics. The overexpression of T30 modestly increased LC3 synthesis, suggesting the induction of autophagosomes (Fig.\u0026nbsp;2B \u003cb\u003eand C\u003c/b\u003e), which is consistent with the finding that autophagy is activated in response to Tau aggregation. In contrast, CW-Tau maintained LC3 levels comparable to those in control cells, indicating no autophagy induction\u0026mdash;likely due to its lower aggregation propensity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnalysis of the LC3 degradation ratio revealed a marked impairment in autophagic flux with T30, whereas CW-Tau preserved normal degradation (Fig.\u0026nbsp;2B \u003cb\u003eand D\u003c/b\u003e). Similarly, p62, an autophagy adaptor that links ubiquitinated cargo to LC3, accumulated in T30-expressing cells but remained unaltered under CW-Tau conditions (Fig.\u0026nbsp;2B \u003cb\u003eand E)\u003c/b\u003e. The degradation ratio of p62 also decreased significantly with T30 but not with CW-Tau (Fig.\u0026nbsp;2B \u003cb\u003eand F\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eImmunofluorescence analysis supported these findings. In SH-SY5Y neuroepithelioma cells transduced with lentiviral vectors expressing GFP alone, GFP-T30, or GFP\u0026ndash;CW-Tau, the number of LC3 puncta (representing autophagic vesicles) increased modestly with T30 but remained unchanged with CW-Tau (Fig.\u0026nbsp;2G-I), indicating preservation of autophagic flux in the latter.\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that overexpression of the aggregation-prone T30 Tau isoform impairs autophagic flux by hindering the degradative phase, leading to vesicle accumulation. In contrast, CW-Tau preserves both the synthesis and degradation phases, maintaining autophagic homeostasis. The observed autophagy induction in T30-expressing cells may reflect a compensatory response to Tau aggregation, which fails to proceed to completion due to degradation blockade. CW-Tau, which is less prone to aggregation, does not trigger such a response.\u003c/p\u003e \u003cp\u003eInterestingly, when early endosomal markers were analyzed, both T30 and CW-Tau expression induced early endosome clustering, with a more pronounced effect on CW-Tau-expressing cells (Fig.\u0026nbsp;2H-J). This may reflect an increase in endosome size with a concomitant decrease in number, potentially indicating altered function (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e) or increased endocytic activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Identification of vesicle trafficking-related proteins that bind to the Intron 12 peptide\u003c/h2\u003e \u003cp\u003eGiven the essential role of vesicle trafficking and Rab GTPases in neurodegenerative processes (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), we explored potential interactions between the W-Tau peptide and proteins involved in these pathways (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAffinity chromatography using the W-Tau peptide and brain extracts from AD patients revealed retention of the Rab-GDP dissociation inhibitors GDIA and GDIB (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;3A-B\u003c/b\u003e). These proteins modulate intracellular membrane trafficking by inhibiting GDP-to-GTP exchange with Rab GTPases, facilitating their extraction from membranes and maintaining them in the inactive GDP-bound state (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). GDIA is expressed primarily in neural and sensory tissues, whereas GDIB is ubiquitously expressed (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, we detected putative interactions of the W-Tau peptide with several Rab family members, including Rab5C, Rab6B, Rab7A, and Rab11B (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;3C-F\u003c/b\u003e). Rab5C is critical for early endosome formation and is implicated in Parkin-mediated delivery of damaged mitochondria to Rab5-positive endosomes via the ESCRT machinery, supporting lysosomal degradation (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Rab6B is associated with retrograde transport in neurons (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), whereas Rab7A governs endolysosomal trafficking, orchestrating early-to-late endosome maturation, microtubule-mediated transport, and endosome positioning (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Rab7A is also essential for phagosome fusion with late endosomes and lysosomes, a key step in autophagic degradation (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Rab11B participates in both constitutive and regulated secretion (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProteomic analysis also confirmed W-Tau peptide interactions with UBE2N, FABP7, CPPED, and SHPS1 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;3G-I\u003c/b\u003e). Although these proteins play secondary roles in vesicle trafficking, the results of the STRING network analysis support their involvement in related pathways.\u003c/p\u003e \u003cp\u003eConsidering these interactions, particularly with Rab proteins and GDIs, and given the central role of Rab GTPases in vesicular transport and secretion, we propose that the CW-Tau isoform may influence these processes.\u003c/p\u003e \u003cp\u003eNotably, several of the identified proteins have established links to neurodegenerative diseases. Among the W-Tau interactors, PARK7/DJ-1 is associated with Parkinson\u0026rsquo;s disease (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e) and Tau inclusions (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Abnormal Rab5 activation has been reported in AD brains, where it contributes to endocytic dysfunction (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Furthermore, UBE2N has been proposed as an AD biomarker (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In support of these disease connections, the W-Tau peptide also binds two proteins strongly associated with AD pathology\u0026mdash;apolipoprotein E (ApoE) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;3J\u003c/b\u003e) and Tau itself (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e)\u0026mdash;suggesting a potential role in the disease.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e4.4 CW-Tau preserves LAMP1 vesicle size, number and acidification.\u003c/h2\u003e \u003cp\u003eGiven the potential interactions between the CW-Tau and Rab proteins and GDIs, we next examined the effects of different Tau isoforms on vesicle trafficking, with a focus on LAMP1-positive compartments. We also assessed whether the impaired autophagic degradation phase previously observed with classical Tau is absent in CW-Tau-expressing cells by analyzing lysosomal distribution and maturation status.\u003c/p\u003e \u003cp\u003eTo this end, SH-SY5Y cells were infected with lentivectors encoding either GFP alone, GFP\u0026thinsp;+\u0026thinsp;classical Tau 3R (T30), or GFP\u0026thinsp;+\u0026thinsp;CW-Tau. The cells were then labeled with LysoTracker Red DND-99, an acidotropic fluorescent probe that targets acidic organelles, to assess lysosomal dynamics. LAMP1, a membrane protein predominantly associated with lysosomes but also present in early/late endosomes and multivesicular bodies (MVBs), was used to identify vesicular compartments (Fig.\u0026nbsp;3A).\u003c/p\u003e \u003cp\u003eTo characterize these vesicle populations, segmentation analysis classified them into three size categories: \u0026le;2 \u0026micro;m\u0026sup2; (primary and mature lysosomes), 2\u0026ndash;5 \u0026micro;m\u0026sup2; (amphisomes, autophagolysosomes, MVBs), and \u0026ge;\u0026thinsp;5 \u0026micro;m\u0026sup2; (early and late endosomes) (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e) (Fig.\u0026nbsp;3B).\u003c/p\u003e \u003cp\u003eConfocal imaging and quantification revealed a significant increase in the number of LAMP1-positive vesicles across all size ranges in T30-overexpressing cells, whereas CW-Tau cells presented vesicle counts comparable to those of the GFP control (Fig.\u0026nbsp;3A\u003cb\u003e\u0026ndash;C\u003c/b\u003e). In T30 cells, vesicles\u0026thinsp;\u0026le;\u0026thinsp;2 \u0026micro;m\u0026sup2; (primary/mature lysosomes) were notably enlarged\u0026mdash;a feature not observed in CW-Tau or control cells (Fig.\u0026nbsp;3D). The enlargement of these lysosomes is consistent with previous findings in neurodegenerative diseases such as AD (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). The size increase trend extended, albeit less markedly, to the 2\u0026ndash;5 \u0026micro;m\u0026sup2; category. Vesicles\u0026thinsp;\u0026ge;\u0026thinsp;5 \u0026micro;m\u0026sup2; also increased in size, suggesting endosomal compartment accumulation in T30 cells.\u003c/p\u003e \u003cp\u003eTo assess compartment acidity, LysoTracker Red DND-99 staining was performed. T30 overexpression significantly increased the number of acidified vesicles, particularly those in the \u0026le;\u0026thinsp;2 \u0026micro;m\u0026sup2; category, whereas the number of CW-Tau cells remained similar to that of the control (Fig.\u0026nbsp;3A\u003cb\u003e\u0026ndash;E\u003c/b\u003e). The colocalization of LysoTracker and LAMP1 confirmed this increase in acidified vesicles in T30 cells, especially within the \u0026le;\u0026thinsp;2 \u0026micro;m\u0026sup2; and 2\u0026ndash;5 \u0026micro;m\u0026sup2; compartments, which was consistent with mature lysosomes, amphisomes, autophagosomes, and MVBs (Fig.\u0026nbsp;3F). For vesicles\u0026thinsp;\u0026ge;\u0026thinsp;5 \u0026micro;m\u0026sup2;, the trend persisted but was less defined. Overall, T30 expression led to greater numbers of acidified vesicles per cell (Fig.\u0026nbsp;3G), with increased acidity in LAMP1\u003csup\u003e+\u003c/sup\u003e compartments (Fig.\u0026nbsp;3H). However, this effect was attenuated when LAMP1\u003csup\u003e+\u003c/sup\u003eLysoTracker\u003csup\u003e+\u003c/sup\u003e double-positive vesicles were considered (Fig.\u0026nbsp;3I), suggesting an accumulation of mature lysosomes without significant pH alterations compared with the control. This finding aligns with elevated cathepsin D maturation in T30 cells (Fig.\u0026nbsp;3J).\u003c/p\u003e \u003cp\u003eIn contrast, CW-Tau-expressing cells preserved a greater proportion of primary lysosomes (Fig.\u0026nbsp;3F, area\u0026thinsp;\u0026le;\u0026thinsp;2 \u0026micro;m\u0026sup2; LysoTracker-negative vesicles), indicating intact lysosomal biogenesis.\u003c/p\u003e \u003cp\u003eTogether, these results demonstrate that the overexpression of classical Tau 3R (T30) leads to the accumulation and enlargement of LAMP1-positive vesicles, particularly acidified mature lysosomes, which is indicative of impaired autophagic flux\u0026mdash;likely due to defective fusion with autophagosomes or endosomes. Conversely, CW-Tau maintains lysosomal number, size, acidity, and biogenesis at levels comparable to those of the control, supporting functional lysosomal trafficking and degradation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e4.5 CW-Tau prevents protein aggregation\u003c/h2\u003e \u003cp\u003eOn the basis of previous findings that classical Tau impairs autophagy and disrupts vesicular trafficking\u0026mdash;effects not observed with CW-Tau\u0026mdash;we investigated whether these differences influence protein aggregation via the Proteostat\u0026trade; assay. HEK293T cells transfected with either classical Tau 3R (T30) or the corresponding CW-Tau isoform were treated with bafilomycin A1, an inhibitor of lysosomal acidification and autophagy, or MG-132, a proteasome inhibitor that impairs protein degradation via the ubiquitin‒proteasome system.\u003c/p\u003e \u003cp\u003eImmunofluorescence imaging and quantification revealed that, compared with no treatment, overexpression of classical Tau led to a significant increase in protein accumulation and aggregation in control cells (Fig.\u0026nbsp;4A\u003cb\u003e\u0026ndash;B\u003c/b\u003e). In contrast, CW-Tau overexpression did not induce notable aggregation, with levels comparable to those of the control. These findings align with prior reports describing the antiaggregation properties of the W-Tau peptide (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUpon treatment, cells transfected with the empty vector showed significant aggregate accumulation in response to both bafilomycin A1 and MG-132, particularly the latter, which was consistent with the expected outcomes of autophagy or proteasome inhibition (Fig.\u0026nbsp;4C). In contrast, in T30-expressing cells, bafilomycin A1 did not further increase aggregation, suggesting that autophagy was impaired by Tau expression (Fig.\u0026nbsp;4D). Moreover, the increase in aggregation in response to MG-132 treatment was markedly attenuated in T30 cells compared with that in control cells, where MG-132 induced a fivefold increase in aggregation.\u003c/p\u003e \u003cp\u003eConversely, in CW-Tau-expressing cells, both bafilomycin A1 and MG-132 significantly increased protein aggregation to levels comparable to those of the control vector, indicating that CW-Tau preserves the functionality of both the autophagic and proteasomal degradation pathways (Fig.\u0026nbsp;4E).\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that CW-Tau prevents protein aggregation by maintaining proteostasis, preserving both autophagy and proteasome activity. In contrast, the overexpression of classical Tau promotes protein aggregation, likely through the intrinsic inhibition of autophagy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e4.6 CW-Tau is efficiently exported to the extracellular medium via exosomes\u003c/h2\u003e \u003cp\u003eTau is known to be secreted into the extracellular space via multiple pathways, including direct release, tunneling nanotubes, and extracellular vesicles such as exosomes, particularly under pathological conditions. Considering the observed interaction between the W-Tau peptide and Rab proteins\u0026mdash;especially Rab11A, a key regulator of both constitutive and regulated secretion (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u0026mdash;we investigated whether CW-Tau is differentially exported from cells compared with classical Tau.\u003c/p\u003e \u003cp\u003eHEK293T cells were transfected with either the classical Tau 3R isoform lacking inserts (T30) or the corresponding CW-Tau isoform generated by intron 12 retention. We analyzed the Tau distribution in both the intracellular compartment and the extracellular medium, separating the latter into a soluble (\"naked\") fraction and an exosome-enriched fraction (Fig.\u0026nbsp;5A).\u003c/p\u003e \u003cp\u003eDespite differences in their intracellular expression levels, both isoforms were detected in the extracellular space. However, normalization to intracellular expression revealed that CW-Tau was exported less efficiently than T30 overall (Fig.\u0026nbsp;5A\u003cb\u003e\u0026ndash;B\u003c/b\u003e). Notably, when the naked Tau fraction was analyzed, both isoforms were exported with comparable efficiency (Fig.\u0026nbsp;5A\u003cb\u003e\u0026ndash;C\u003c/b\u003e). In contrast, the exosome-enriched fraction presented significantly lower CW-Tau levels than did the T30 fraction (Fig.\u0026nbsp;5A\u003cb\u003e\u0026ndash;D\u003c/b\u003e), suggesting isoform-specific differences in exosome-mediated secretion. These findings imply that Tau may influence neighboring cells through both naked-eye export and exosome-mediated pathways.\u003c/p\u003e \u003cp\u003eExosomes are released via the fusion of multivesicular bodies (MVBs) with the plasma membrane (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), a process regulated by Rab family members such as Rab11a. Rab11a is implicated in both constitutive and regulated secretion, facilitating MVB docking and fusion via interactions with the exocyst complex (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). It is also involved in the formation of recycling endosomes that contribute to vesicle recycling and secretion.\u003c/p\u003e \u003cp\u003eTo assess whether Tau isoforms affect the distribution of Rab11a, we analyzed SH-SY5Y cells expressing either T30 or CW-Tau via lentiviral vectors (GFP-tagged). Rab11a localization was evaluated via kurtosis analysis, a statistical measure of data clustering indicative of protein aggregation versus normal vesicular distribution. T30 expression significantly increased Rab11a kurtosis, suggesting abnormal protein aggregation rather than physiological vesicular distribution. This effect was not detected in CW-Tau-expressing cells (Fig.\u0026nbsp;5E\u003cb\u003e\u0026ndash;F\u003c/b\u003e). Rab11a clustering may indicate impaired vesicle trafficking, potentially linked to microtubule destabilization induced by T30 overexpression (Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eWe further examined the expression of CD63, a tetraspanin enriched in late endosomes and MVBs that plays a key role in exosome biogenesis and cargo selection. CD63 levels per cell were markedly reduced in T30-expressing cells but remained unchanged in CW-Tau-expressing cells (Fig.\u0026nbsp;5E\u003cb\u003e\u0026ndash;G\u003c/b\u003e). This decrease in MVBs contrasts with the observed accumulation of small- and medium-sized LAMP1\u003csup\u003e+\u003c/sup\u003e/Lysotracker\u003csup\u003e+\u003c/sup\u003e vesicles, corresponding to mature lysosomes and autophagolysosomes (Fig.\u0026nbsp;3F). The reduction in the intracellular CD63 signal may result from impaired fusion of MVBs with lysosomes or autophagolysosomes, favoring MVB fusion with the plasma membrane and subsequent exosomal release of T30 (Fig.\u0026nbsp;5C).\u003c/p\u003e \u003cp\u003eTogether, these data suggest that classical Tau (T30) disrupts normal vesicle trafficking and secretion pathways, altering Rab11a distribution and reducing intracellular CD63 levels, which may promote excessive exosomal Tau release. In contrast, CW-Tau maintains Rab11a localization and MVB integrity, resulting in reduced Tau secretion via exosomes and overall preservation of vesicle trafficking homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e4.7 CW-Tau prevents Tau seeding activity and the propagation of Tauopathy\u003c/h2\u003e \u003cp\u003eBuilding on the previously demonstrated antiaggregation effects of CW-Tau and the W-Tau peptide in vitro (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), we evaluated whether CW-Tau could inhibit Tau seeding activity in vivo via a cellular biosensor model. For this purpose, we employed HEK293T TAU RD P301S FRET reporter cells, which generate a fluorescence resonance energy transfer (FRET) signal upon Tau aggregation (Fig.\u0026nbsp;6A).\u003c/p\u003e \u003cp\u003eAs a positive control, the treatment of these cells with Tau seeds derived from Alzheimer\u0026rsquo;s disease (AD) patient brain extracts induced a robust increase in the FRET signal (Fig.\u0026nbsp;6B). Cotransfection of these cells with both Tau seeds and a plasmid encoding the classical T30 (Tau 3R) isoform led to a dose-dependent increase in FRET activity, indicating that T30 promotes Tau aggregation (Fig.\u0026nbsp;6D, top row; quantification in \u003cb\u003eFig.\u0026nbsp;6C\u003c/b\u003e). Conversely, coexpression of CW-Tau significantly reduced Tau aggregation induced by brain-derived seeds in a dose-dependent manner (Fig.\u0026nbsp;6D, bottom row; quantification in \u003cb\u003eFig.\u0026nbsp;6C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThis antiaggregation effect may be attributed to the previously observed binding affinity of the W-Tau peptide for Tau (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e), suggesting that CW-Tau interferes with Tau oligomerization by disrupting interactions within the microtubule-binding domain.\u003c/p\u003e \u003cp\u003eTo investigate whether the antiaggregation effects of CW-Tau could be transferred between cells, we treated TAU FRET cells with Tau seeds in combination with increasing volumes of extracellular medium collected from cells overexpressing either T30 or CW-Tau. While the addition of extracellular medium from T30-expressing cells significantly increased Tau seeding, the addition of medium from CW-Tau-expressing cells reduced aggregation in a dose-dependent manner (Fig.\u0026nbsp;6E \u003cb\u003eand H\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eSince Tau can be secreted as a free protein or within extracellular vesicles, we separated the extracellular medium into vesicle-free and exosome-enriched fractions via ultracentrifugation. These fractions were then applied to the reporter cells at equal concentrations. Vesicle-free T30 was efficiently internalized and led to a dose-dependent increase in the FRET signal, which was consistent with enhanced seeding activity (Fig.\u0026nbsp;6F \u003cb\u003eand I\u003c/b\u003e). Although CW-Tau was present at lower levels in the vesicle-free fraction (Fig.\u0026nbsp;5A), it retained the ability to suppress Tau aggregation upon transfer, confirming that its antiseeding activity is preserved during intercellular transmission.\u003c/p\u003e \u003cp\u003eFurther analysis revealed that the exosome-enriched fraction from T30-expressing cells also induced robust Tau aggregation in a dose-dependent manner (Fig.\u0026nbsp;6G\u003cb\u003e\u0026ndash;J\u003c/b\u003e). Notably, despite the reduced export of CW-Tau via exosomes (Fig.\u0026nbsp;5A \u003cb\u003eand D\u003c/b\u003e), the exosomal fraction derived from CW-Tau-expressing cells was able to transmit antiaggregation effects. Treatment with the highest concentration of this fraction significantly decreased the FRET signal, confirming that the protective properties of CW-Tau are also conveyed through exosomes (Fig.\u0026nbsp;6G\u003cb\u003e\u0026ndash;J\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThese findings demonstrate that CW-Tau effectively inhibits pathological Tau seeding and aggregation in a cellular context. Moreover, its antiaggregation activity can be transmitted to recipient cells via both vesicle-free and exosome-mediated mechanisms. Together, these properties underscore the therapeutic potential of CW-Tau to counteract Tau pathology and limit the intercellular propagation of Tau aggregates.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn a previous study, we identified a novel Tau isoform, CW-Tau, characterized by the retention of intron 12, which encodes a 16-amino-acid sequence containing two tryptophans (W), and by the absence of the C-terminal region, including exon 13 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This isoform has high microtubule-binding affinity, increased solubility, and lower RNA and protein levels in AD brains than nondemented controls do (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Additionally, a peptide encoded by intron 12 exhibits chaperone-like activity, effectively inhibiting Tau and amyloid-β aggregation in vitro (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In this study, we provide mechanistic insight into the distinctive cellular properties of CW-Tau and its potential therapeutic relevance.\u003c/p\u003e \u003cp\u003eWe show that the W-Tau peptide directly interacts with tubulin, functioning as an auxiliary microtubule-binding domain that promotes microtubule polymerization in vitro. Unlike canonical Tau isoforms, whose overexpression disrupts microtubule architecture, CW-Tau preserves cytoskeletal integrity. Interestingly, the binding of CW-Tau to tubulin is increased in AD samples, potentially due to pathological posttranslational modifications, such as tyrosination and detyrosination (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). These modifications have been associated with increased Tau phosphorylation and synaptotoxicity (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). Future studies should assess whether the affinity of CW-Tau for tubulin is modulated by these modifications and whether this affects Tau phosphorylation and neurotoxicity.\u003c/p\u003e \u003cp\u003eExon 13 of MAPT is constitutively expressed across all canonical Tau isoforms and encodes phosphorylation sites such as those recognized by PHF-1 antibodies (S396 and S404), which modulate microtubule binding and have been implicated in Tau aggregation and Tauopathy pathology (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). These residues also play key roles in regulating synaptic plasticity via long-term potentiation (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearch has demonstrated that the W-Tau peptide prevents Tau and amyloid-β aggregation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to microtubule interactions, the W-Tau peptide binds lactate dehydrogenase (LDH), a known Tau interactor, and prevents its self-aggregation via a chaperone-like mechanism (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). This may involve interaction motifs such as EVHHQ (in amyloid-β) and EVHKQ (in LDH), although interactions with acidic residues in LDH (e.g., 101-DLDED-105 or 360-EEE-362) remain to be confirmed (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe also identified interactions between W-Tau and other brain proteins, including LC1-MAP1B, several Rab proteins and GDIs (primarily in AD brain extracts). MAP1B-LC1 is a cytoskeletal protein that inhibits autophagosome formation by linking syntaxin 17 to microtubules and preventing the Drp1-mediated mitochondrial fission necessary for recycling through autophagy (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Classic Tau overexpression impairs autophagic flux, increasing vesicle accumulation while reducing the degradation of damaged mitochondria. CW-Tau\u0026rsquo;s interaction with MAP1B-LC1 may alleviate this inhibition, restoring mitochondrial recycling and protein turnover. Additionally, CW-Tau associates with PARK7, a key regulator of mitophagy (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), suggesting a broader role in maintaining mitochondrial homeostasis.\u003c/p\u003e \u003cp\u003eW-Tau also binds GDIs A and B, which regulate Rab cycling and autophagy by mediating GTPase membrane association (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan additionalcitationids=\"CR68\" citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). This interaction could facilitate Rab activation, promoting vesicular trafficking and autophagic flux. In contrast, classical Tau overexpression disrupts Rab11 dynamics and leads to the accumulation of LAMP1\u0026thinsp;+\u0026thinsp;vesicles\u0026mdash;particularly mature lysosomes\u0026mdash;indicating the defective fusion of autophagosomes and endosomes, leading to severe autophagy impairment.\u003c/p\u003e \u003cp\u003eExosomes, which are secreted from multivesicular bodies (MVBs), are key mediators of proteostasis, balancing degradation via lysosomal fusion or the secretion of aggregated proteins. In autophagy-deficient contexts, exosome release is upregulated, promoting the secretion of pathological aggregates such as Tau (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e). W-Tau\u0026rsquo;s interaction with Rab11B\u0026mdash;a known regulator of constitutive and regulated secretion (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e)\u0026mdash;is particularly relevant, as classical Tau expression induces Rab11 clustering and enhances exosomal Tau release. Rab11 also facilitates MVB fusion with the plasma membrane in a Ca\u0026sup2;⁺-dependent manner (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e) and mediates the secretion of α-synuclein inclusions (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough CW-Tau is secreted in exosomes, its expression does not compromise vesicular trafficking or autophagy, thereby limiting exosomal export. This is supported by preserved CD63 labeling in CW-Tau-expressing cells, in contrast to classical Tau, which markedly reduces CD63 levels, likely due to excessive MVB depletion via exosome release.\u003c/p\u003e \u003cp\u003eImportantly, the W-Tau peptide displays differential protein-binding affinities between healthy and AD brains. Tubulin, MAPs, Rabs, GDIs, LC1-MAP1B, and PARK7 all exhibit enhanced interactions in AD tissue, suggesting that the protective functions of CW-Tau may be potentiated under pathological conditions. Conversely, in nonpathological brains, W-Tau preferentially binds Rab11 and lipid-related proteins such as APOE and FABP7, suggesting a physiological role in secretion and lipid metabolism. The biological implications of these distinct binding patterns require further investigation.\u003c/p\u003e \u003cp\u003eFinally, we show that both CW-Tau and classical Tau isoforms are secreted into the extracellular space, either freely or within exosomes. This allows their aggregation-modifying properties to influence neighboring cells. CW-Tau-containing extracellular media or exosome fractions exhibit antiaggregation activity, highlighting their therapeutic potential for the protective effects of cell-to-cell transmission.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, CW-Tau promotes microtubule polymerization, preserves vesicle trafficking, enhances autophagic flux, and reduces protein aggregation. Furthermore, its protective properties are transferable to neighboring cells via secretory pathways. These characteristics position CW-Tau as a promising candidate for therapeutic intervention in Tauopathies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was funded by the Spanish Ministry of Economy and Competitiveness: PID2020-113204GB-I00 (F.H.) and PID2021-123859OB-100 from MCIN/AEI/10.13039/501100011033/FEDER, UE (J.A.). It was also supported by the CSIC through an intramural grant (201920E104) (J.A.) and the Centre for Networked Biomedical Research on Neurodegenerative Diseases (J.A.). Schemes in Figures 1, 2, 3 and 6 were created with BioRender.com. The Centro de Biolog\u0026iacute;a Molecular Severo Ochoa (CBMSO) is a Severo Ochoa Center of Excellence (MICIN, award CEX2021-001154-S). Funding for publication is provided by the Open Access Publishing Support Program of the CSIC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe datasets supporting the conclusions of this article are available in the\u0026nbsp;Figshare\u0026nbsp;repository:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003eRaw proteomic data and analysis https://doi.org/10.6084/m9.figshare.28770797.v1\u003c/li\u003e\n \u003cli\u003eMicrotubule and LDH binding https://doi.org/10.6084/m9.figshare.28771124.v1.\u003c/li\u003e\n \u003cli\u003eAutophagy analysis https://doi.org/10.6084/m9.figshare.28771076.v1.\u003c/li\u003e\n \u003cli\u003eVesicle trafficking analysis https://doi.org/10.6084/m9.figshare.28771091.v2.\u003c/li\u003e\n \u003cli\u003eFRET Tau https://doi.org/10.6084/m9.figshare.28771049.v1.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThe study received approval from The Ethical Committee of Universidad Aut\u0026oacute;noma de Madrid (CEI-98-1831). The biobank HUB-IDIBELL is authorized by the Directorate General for Health Planning and Research of the Department of Health of the Generalitat of Catalunya and is registered in the National Register of Biobanks of the Instituto de Salud Carlos III in accordance with current Spanish legislation (Law 14/2007, of July 3, about Biomedical Research, and RD 1716/2011, of November 18).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003eConceptualization and designing of the work, V.G-E, and J.A.; acquisition, F.V.-B., I.D.-S., A.S., R.C., M.P., E.M., N.S., D.R.-G., and V.G.-E.; analysis or interpretation of data, F.V.-B., I.D.-S., A.S., M.P., E.M., D.R.-G., F.H., V.G.-E., and J.A.; Writing \u0026ndash; Original Draft, F.V.-B., I.D.-S., A.S., E.M., V.G.-E., and J.A.; Writing \u0026ndash; Review \u0026amp; Editing and final approval of the version F.V.-B., I.D.-S., A.S., R.C., M.P., E.M., N.S., D.R.-G., F.H., V.G.-E., and J.A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDrubin DG, Nelson WJ. 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Hum Mol Genet. 2014;23(25):6732\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Tau, MAPT, aggregation, vesicle trafficking, autophagy, exovesicles","lastPublishedDoi":"10.21203/rs.3.rs-6725217/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6725217/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCW-Tau, a novel MAPT transcript variant generated by intron 12 retention, has been identified. Compared with classical Tau isoforms, this isoform has greater microtubule-binding properties, a reduced aggregation propensity, and faster turnover. Notably, CW-Tau RNA and protein levels are decreased in Alzheimer\u0026rsquo;s disease (AD) brains. To investigate its molecular interactions, we performed affinity chromatography using the W-Tau peptide, which is encoded by intron 12, and identified key binding partners involved in microtubule dynamics, vesicle trafficking, and autophagy. The W-Tau peptide interacts with tubulin, suggesting the presence of an additional microtubule-binding domain, and binds lactate dehydrogenase, preventing its aggregation. Furthermore, the W-Tau peptide is associated with vesicle trafficking regulators, including Rab proteins and Rab-GDP dissociation inhibitors, as well as autophagy-related proteins such as MAP1BLC1 and PARK7.\u003c/p\u003e \u003cp\u003eUnlike classical Tau isoforms, which disrupt these pathways, CW-Tau functionally preserves vesicle trafficking and autophagic flux. Importantly, while classical Tau isoforms promote pathological transmission of Tau either nakedly or via exosome-mediated export, CW-Tau counteracts this process, demonstrating potent antiseeding activity. By preventing Tau aggregation and propagation, CW-Tau may play a protective role against proteinopathy-related neurodegeneration.\u003c/p\u003e \u003cp\u003eThese findings suggest that CW-Tau is a promising therapeutic tool for AD, offering potential strategies to restore cellular proteostasis and inhibit Tau pathology.\u003c/p\u003e","manuscriptTitle":"CW-Tau fights proteinopathy by improving autophagy and diminishing Tau seeding activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-29 13:17:18","doi":"10.21203/rs.3.rs-6725217/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e612578-c1d2-4ff4-8fe9-711dddc7cff5","owner":[],"postedDate":"May 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-29T08:08:57+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-29 13:17:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6725217","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6725217","identity":"rs-6725217","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}