# A primary cilia-autophagy axis in hippocampal neurons is essential to maintain cognitive resilience

Manon Rivagorda, David Romeo-Guitart, Victoria Blanchet, François Mailliet, Valérie Boitez, Natalie Barry, Dimitrije Milunov, Eleni Siopi, Nicolas Goudin, Stéphanie Moriceau, et al.

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## To cite this version:

Manon Rivagorda, David Romeo-Guitart, Victoria Blanchet, François Mailliet, Valérie Boitez, et al. A primary cilia-autophagy axis in hippocampal neurons is essential to maintain cognitive resilience. Nature Aging, 2025, 5 (3), pp.450-467. 10.1038/s43587-024-00791-0. hal-05138018

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# A primary cilia-autophagy axis in hippocampal neurons is essential to maintain cognitive resilience

Received: 30 May 2023

Accepted: 6 December 2024

Published online: 21 February 2025

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Manon Rivagorda¹,⁸,⁹, David Romeo-Guitart Ⓓ ¹,⁹, Victoria Blanchet¹, François Mailliet¹, Valérie Boitez¹, Natalie Barry¹, Dimitrije Milunov², Eleni Siopi¹, Nicolas Goudin³, Stéphanie Moriceau Ⓓ ¹,⁴, Chiara Guerrera Ⓓ ⁵, Michel Leibovici¹, Soham Saha Ⓓ ², Patrice Codogno⁶, Eugenia Morselli⁷, Etienne Morel Ⓓ ⁶, Anne-Sophie Armand¹,¹⁰ & Franck Oury Ⓓ ¹,¹⁰ ☒

Blood-borne factors are essential to maintain neuronal synaptic plasticity and cognitive resilience throughout life. One such factor is osteocalcin (OCN), a hormone produced by osteoblasts that influences multiple physiological processes, including hippocampal neuronal homeostasis. However, the mechanism through which this blood-borne factor communicates with neurons remains unclear. Here we show the importance of a core primary cilium (PC) protein-autophagy axis in mediating the effects of OCN. We found that the OCN receptor GPR158 is present at the PC of hippocampal neurons and mediates the regulation of autophagy machinery by OCN. During aging, autophagy and PC core proteins are reduced in neurons, and restoring their levels is sufficient to improve cognitive impairments in aged mice. Mechanistically, the induction of this axis by OCN is dependent on the PC-dependent cAMP response element-binding protein signaling pathway. Altogether, this study demonstrates that the PC-autophagy axis is a gateway to mediate communication between blood-borne factors and neurons, and it advances understanding of the mechanisms involved in age-related cognitive decline.

During aging, organisms undergo a decline in tissue function, leading to reduced systemic, cellular and organ performance, with several functional consequences, such as decreased cognitive capacity¹⁻³. Among the different brain regions affected by aging, the hippocampus is particularly susceptible³⁻⁵. Central hallmarks of hippocampal aging include alterations in neuronal homeostasis and adaptive functional properties, leading to deficits in synaptic plasticity and memory⁴,⁶⁻⁹. Many efforts are now focused on the identification of molecular pathways involved in these processes to develop effective preventive or therapeutic strategies to treat age-related cognitive deficits.

Growing evidence has shown that administration of plasma derived from young animals is sufficient to improve hippocampal-dependent

cognitive impairments in aged mice, underlying the importance of blood-circulating factors in brain aging⁹⁻¹⁶. Leveraging technological advances in omics analyses led to the identification of blood-borne factors that can foster neuronal homeostasis and cognitive resilience. Among them, we and others have identified the bone-derived hormone osteocalcin (OCN), which crosses the blood-brain barrier and impacts neuronal homeostasis and cognitive fitness via activation of its receptor GPR158, a G-protein-coupled receptor (GPCR) expressed in hippocampal neurons¹⁷⁻¹⁹. Circulating OCN levels are reduced in aged mice, whereas their restoration is sufficient to improve age-related cognitive deficits by the induction of autophagy machinery¹⁸⁻²⁰.
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**Fig. 1** | GPR158 localized in PC of hippocampal neurons. **a**, Volcano plot showing protein fold change between vehicle-treated and OCN-treated hippocampi. **b**, Protein ontology network showing enrichment for PC core proteins in the hippocampus treated with OCN versus vehicle. **c**, 2D image (left) and 3D rendering (right) of GPR158 (green) localization in PC from primary hippocampal neurons treated with vehicle or OCN. Scale bar, 1 µm. Graph bar represents the quantification of GPR158 spots in PC (vehicle *n* = 12, OCN *n* = 10, from two independent experiments). **d**, Co-immunoprecipitation of TULP3 with GPR158 or IFT20 in N2a cells and representative image of co-immunofluorescence

of GPR158 (green) and TULP3 (white) in the PC stained with ACIII (orange) of hippocampal neurons. Scale bar, 1 µm. **e**, 2D image (top) and 3D rendering (bottom) of GPR158 (green) localization in PC from *Gpr158*⁻/⁻ primary hippocampal neurons infected with WT or mutated *Gpr158* and treated or not with OCN. Scale bar, 1 µm. Quantification of GPR158 spots found in each neuronal PC from three independent cultures (in 10-25 neurons per dot). PC was stained by ACIII in each corresponding panel. Data are represented as mean ± s.e.m. The *P* value was determined by two-tailed Student's *t*-test compared to control group (c) or by one-way ANOVA followed by a post hoc Sidak correction (e).
Autophagy is a catabolic cellular process, whereby proteins and organelles are engulfed in autophagosomes (APs), formed by the activity of different autophagy proteins, and then transported to lysosomes for degradation, contributing directly to cell metabolism and energy production<sup>21-23</sup>. In terminally differentiated cells such as neurons, autophagy-mediated degradation of intracellular material is key to maintain cellular homeostasis and promotes cell survival<sup>24-26</sup>. However, beyond its basal functions, autophagy machinery could also be induced by various stimuli (stress, hormones and nutrients), which mediate the adaptive cellular responses to changes in environmental conditions<sup>27-29</sup>.

In the brain, stimulated autophagy promotes activity-dependent synaptic plasticity in hippocampal neurons and memory formation<sup>20,30-33</sup>. During aging, autophagy levels are reduced in the hippocampus, leading to a decline in the capacity of neurons to conserve their ability to integrate novel stimuli, and, thereby, in memory functions<sup>20,33,34</sup>. Importantly, administration of young blood is sufficient to restore the level of essential autophagy-related proteins in hippocampal neurons of old mice, which is a prerequisite to foster cognitive resilience<sup>20</sup>. Among these cognitive resilience factors, we and others identified OCN or growth differentiation factor 11 (GDF11)<sup>20,34,35</sup>. However, the regulatory mechanisms mediating the induction of neuronal autophagy by these blood-borne factors remain largely elusive.

Recent work in the kidney has shown that autophagic machinery can be mobilized in response to systemic stimuli by direct or indirect connection with core primary cilia (PC) proteins, namely intraflagellar transports (IFTs) (IFT20 and IFT88) or kinesin family member 3A (KIF3A)<sup>36-38</sup>. In neurons, the link between PC and autophagy machinery has not yet been characterized.

PC are non-motile single organelles, acting as cellular 'antenna' sensing and transmitting changes in the systemic milieu<sup>39,40</sup>. Importantly, mutations in ciliary proteins are implicated in a wide variety of genetic disorders, known as ciliopathies, which, among other phenotypes, lead to neurological defects<sup>41</sup>. Previous research indicates that depletion of core cilia proteins in different brain regions results in altered cognitive function<sup>42-44</sup>. Numerous studies also indicate that PC are hubs for various GPCRs, the largest families of membrane protein sensors, involved in many physiological processes, including hormonal systems<sup>45-49</sup>. Knowing that GPR158 is the receptor of OCN in hippocampal neurons led us to hypothesize that PC and/or core PC proteins constitute a gateway through which the blood factor OCN modulates neuronal autophagy machinery and, therefore, cognitive fitness.

In the present study, we confirmed that GPR158 could be localized at the PC and is required for OCN-dependent autophagy induction in hippocampal neurons. Consistently, we show here that core PC proteins are required for the mobilization of key autophagy players by OCN in hippocampal neurons. In aged hippocampi, neuronal PC present an abnormal morphology, correlated with a reduction of the major core ciliary protein levels. Conversely, restoration of their levels in old hippocampi is sufficient to improve age-related neuronal autophagy and cognitive deficits. Lastly, we show that core PC proteins are required to integrate the rejuvenating effects of OCN in the hippocampus, thereby ameliorating age-related cognitive deficits. Altogether, these findings identify a paradigm for the neuron/systemic milieu communication and advance understanding of the regulatory mechanism mediating the rejuvenating effects of the blood-borne factors, such as OCN, in hippocampal neurons.

## Results

### OCN triggers a modulation in PC-related protein expression

We previously showed that OCN can influence neuronal homeostasis and cognitive fitness by mobilizing autophagic machinery in hippocampal neurons<sup>20</sup>. Exploring the mechanism by which this regulation occurs, we performed comparative proteomic analysis in hippocampi from mice injected with vehicle or OCN. As a result, we found that OCN treatment in the hippocampus enhanced the levels of PC-related

proteins and synaptic plasticity (Fig. 1a,b and Extended Data Fig. 1a-c). Interestingly, recent studies proposed that autophagic machinery can be triggered in response to various stimuli, such as serum deprivation and shear stress, by direct or indirect connection with core PC proteins, namely IFTs (such as IFT20 and IFT88) and kinesin (including KIF3A, a kinesin-2 family motor protein)<sup>36-40</sup>. These proteins allow the transportation of ciliary cargoes and are essential for the formation, maintenance and signaling activity of the PC<sup>50,51</sup>.

### GPR158 is present at the PC of hippocampal neurons

The PC was described as a signaling hub for various GPCRs that sense and transduce changes of the systemic milieu<sup>46</sup>. Interestingly, a key regulator of OCN's influence on memory is GPR158, an orphan GPCR expressed in neurons of the cornu ammonis 3 (CA3) region of the hippocampus<sup>18,19</sup>. Using primary hippocampal neurons, we first confirmed that the enhancement of AP-like structure formation by OCN is dependent on GPR158 (Extended Data Fig. 1d), by measuring lipidation levels of the LC3B protein (LC3B-II) (a key readout in AP-like structure biogenesis) in the presence or absence of a lysosomal blocker (bafilomycin). This observation was further confirmed in vivo showing that local stereotactic OCN injections enhance hippocampal LC3B lipidation in wild-type (WT) but not in *Gpr158*<sup>-/-</sup> mice (Extended Data Fig. 1E).

Remarkably, we identified five ciliary-targeting VxPx motifs at the C-terminal domain of GPR158, suggesting its possible localization in the PC (Extended Data Fig. 1f). Co-staining of endogenous GPR158 with neuronal ciliary protein adenylate cyclase 3 (ACIII) indicates its presence, at least partially, at the PC in hippocampal neurons, in cultures (in approximatively 75% of primary hippocampal neurons) and in mouse hippocampi (Fig. 1c and Extended Data Fig. 1g-j). Furthermore, we found that OCN treatment increases the number of GPR158-positive puncta associated with ACIII-positive ciliary plasma membrane (Fig. 1c). Endogenous GPR158 was further co-immunoprecipitated and co-localized with the ciliary protein Tubby-like protein 3 (TULP3), which is involved in the localization and trafficking of GPCRs to PC<sup>52</sup> (Fig. 1d). By contrast, mutagenesis of the five ciliary-targeting VxPx motifs at the C-terminal domain of GPR158 reduced the presence of GPR158-positive puncta in neuronal PC structure (Fig. 1e). Altogether, these data indicate that the induction of autophagy by OCN in hippocampal neurons is dependent on GPR158 and that OCN can enhance the localization of this receptor to PC.

### Core PC proteins are required for OCN to induce autophagy

Next, we found that treatment of primary hippocampal neurons with OCN, or local stereotactic injections of OCN in mouse hippocampus, enhance the level of core PC proteins (TULP3, KIF3A, IFT20 and IFT88), together with lipidated LC3B (LC3B-II levels) (Fig. 2a and Extended Data Fig. 2a). By contrast, IFT20 and KIF3A protein levels are lower in *Ocn*<sup>-/-</sup> and *Gpr158*<sup>-/-</sup> mice than in WT littermates, which is correlated with a reduction of LC3B-II levels (Extended Data Fig. 2b). At the mRNA levels, we did not observe any significant changes of these core PC proteins in *Ocn*<sup>-/-</sup> or *Gpr158*<sup>-/-</sup> hippocampi, but we did observe a slight increase of *Ift20* and *Tulp3* transcript levels after OCN treatment (Extended Data Fig. 3a-c). Then, using stereotactic injections of adeno-associated virus (AAV) expressing small hairpin RNA (shRNA), we generated mouse models in which the intraflagellar *Ift20* (AAV9-U6-shRNA-*Ift20*) or the kinesin *Kif3a* (AAV9-U6-shRNA-*Kif3a*) proteins are selectively down-regulated in the hippocampus. All in vivo experiments were performed on male mice in this study. These mice exhibit a significant reduction of hippocampal GPR158 levels in comparison to controls (AAV9-U6-shRNA-*scramble* injected mice) (Extended Data Fig. 3d,e). Similar results were obtained after *Ift20* downregulation in mature hippocampal neurons, generated by local stereotactic injections of AAVs expressing shRNA*mir for *Ift20* (AAV9-eSYN-*Ift20*-shRNA*mir* under the control of the neuronal eSynapsin 1 (e-SYN) promoter (Fig. 2b). Moreover, we found that *Ift20* downregulation in mature hippocampal
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<figure><img src="image_14.png" /><figcaption>**Fig. 2 |** OCN-GPR158 coupling system requires core PC proteins to induce autophagy machinery in hippocampal neurons. **a,** Western blot and quantification analysis of TULP3, KIF3A, IFT20, IFT88 and LCB-I/II protein levels. **b,** Top panels are representative fluorescent images of brain cross-sections 3 weeks after injections with AAV-eSYN-shRNAmir-scramble expressing eGFP. Left panel is a panoramic view; right panels are focus views on the DG, CA3 and cornu ammonis 1 (CA1) regions, respectively. Scale bar, 100 µm. Top right panel is the relative expression of *Ift20* mRNA level in the dorsal hippocampus of mice 3 weeks after injection with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNAmir-*Ift20*. Bottom panels represent western blot 3 weeks after hippocampal injections with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNAmir-*Ift20* and respective quantification of IFT20 (eSYN-Scrb *n* = 8 and eSYN-shRNA-*Ift20* *n* = 9) and GPR158 (eSYN-Scrb *n* = 8 and eSYN-shRNA-*Ift20* *n* = 7).</figcaption></figure>

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**c,** Western blot and quantification of IFT20 (eSYN-Scrb+veh *n* = 4, eSYN-Scrb+OCN *n* = 5, eSYN-shRNA-*Ift20*+OCN *n* = 5), LC3B-I/II (eSYN-Scrb+veh *n* = 4, eSYN-Scrb+OCN *n* = 5, eSYN-shRNA-*Ift20*+OCN, *n* = 5) and Beclin-1 (eSYN-Scrb+veh *n* = 4, eSYN-Scrb+OCN *n* = 4, eSYN-shRNA-*Ift20*+OCN, *n* = 5) proteins in hippocampi, 3 weeks after injections with AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNA-*Ift20*-mir and upon stereotactic injection of vehicle or OCN. Quantification is relative to injection with AAV-eSYN1-shRNA-scramble-mir treated with vehicle. β-actin was used as a loading control for each sample in all western blot analyses. All data are expressed as mean ± s.e.m., and *P* values were determined by one-tailed (**b**, top) or two-tailed (**a** and **b**, bottom) Student's *t*-tests compared to vehicle or eSYN-Scbr. **In c,** the *P* values were determined by one-way ANOVA followed by Tukey's multiple comparison test.

neurons is sufficient to abolish the induction of the autophagy machinery by OCN (Fig. 2c).

Altogether, these data demonstrate that core PC proteins are required to mediate the induction of autophagy machinery in hippocampal neurons, by the coupling of OCN and GPR158.

## Core PC proteins modulate autophagy in hippocampal neurons

We further investigated the link between core PC proteins and autophagy in hippocampal neurons. Downregulation of either *Ift20* or *Kif3a* decreases autophagy in the CA3 neurons, as shown by the

reduction of LC3B puncta, together with the increase of SQSTM1/p62-positive puncta in comparison to controls (Fig. 3a). Of note, this decrease was not observed in dentate gyrus (DG) (Extended Data Fig. 3f). Similar results were obtained after *Ift20* downregulation in mature hippocampal neurons (AAV9-eSYN-shRNA-*Ift20*-mir) (Fig. 3b,c). This reduction in autophagy observed after neuronal *Ift20* postnatal downregulation is associated with longer PC axonemes (Fig. 3d).

## Core PC proteins influence learning and memory

We previously found that neuronal autophagy in the hippocampus is essential to foster neuronal homeostasis and, thereby, memory$^{20,35}$.
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**Fig. 3 |** *Downregulation of PC core proteins impairs autophagy machinery in hippocampal neurons.*** a, LC3B and SQSTM1/p62 immunofluorescence and puncta quantification performed on brain cross-sections at the level of the hippocampal CA3 region from 3-month-old mice, 3 weeks after local stereotactic injections with AAV-U6-shRNA-*Ift20*, AAV-U6-shRNA-*Kif3a* or AAV-U6-shRNA-scramble. Scale bar, 20 µm. b, Representative western blot image and quantification of LC3B-II accumulation in 3-month-old mouse hippocampi, 3 weeks after local stereotactic injections with either eSYN-shRNA-*Ift20mir* or eSYN-shRNA-scramble-mir (two independent cohorts). β-actin was used as a loading control for each sample. c, SQSTM1/p62 immunofluorescence (blue) and punctae quantification in the CA3 region of the hippocampi of 3-month-old mice,

3 weeks after local stereotactic injections of AAVs expressing either eSYN-shRNA-*Ift20-mir* or eSYN-shRNA-scramble-mir. NeuN staining (purple) was used to label neuronal nucleus. Scale bar, 25 µm. Data were obtained from two independent cohorts. **d**, PC immunofluorescent staining (green, stained by ACIII) and PC length measurement in the CA3 region of mouse after stereotactic injections with either eSYN-shRNA-*Ift20-mir* or shRNA-scramble-mir. The length of the PC was measured and quantified in 3D using Imaris software. The length is expressed in µm, and 2–4 brain sections (n = 50–150 neurons per section) were analyzed per mouse. Scale bar, 10 µm. All data are expressed as mean ± s.e.m., and *P* values were determined by one-tailed (a) or two-tailed (b–d) Student's *t*-tests compared to Scrb.
Therefore, we investigated the functional impact of decreasing the levels of hippocampal core PC proteins for the regulation of cognitive functions. To that end, we subjected our mouse models of hippocampal *Ift20* and *Kif3a* downregulation to three independent behavioral tests assessing hippocampal-dependent learning and memory<sup>53,54</sup>: the novel object recognition (NOR) test, the three foot shocks contextual fear conditioning (CFC) paradigm and the Morris water maze (MWM) test (Fig. 4a–c). NOR and CFC tests assess associative memory and rely on two phases: a training phase, in which mice are exposed to novel memory stimulations (NOR: exposure to two similar objects; CFC: three foot shocks associated with a neutral environment) and a testing phase performed 24 h after the training phase, in which memory performances are evaluated (NOR: recognition of a novel exposed object; CFC: measurement of context-elicited freezing). In NOR, we found that *Ift20* or *Kif3a* downregulation impaired memory capacities, as the time spent to explore a novel object decreases compared to controls (Fig. 4a and Extended Data Fig. 4a). We confirmed these results in CFC, because mice downregulated for core PC proteins exhibited a decrease in freezing during contextual memory testing in comparison to controls (Fig. 4b). Spatial learning and memory were assessed using MWM, in which latency to locate the platform was recorded for each trial. Hippocampal downregulation of *Ift20* or *Kif3a* led to a decrease in memory performances, as shown by the increase in the latency to locate the escape platform in comparison to controls (Fig. 4c). By contrast, downregulation of *Ift20* or *Kif3a* did not affect performance in the open-field test (OFT) and the light/dark test (LDT), indicating that exploratory-like and anxiety-like behaviors are unaffected (Extended Data Fig. 4b,c). Notably, the downregulation of key core PC proteins in mature hippocampal neurons after stereotactic injections of AAV9-eSYN-*Ift20*-shRNAmir led to similar memory deficits, without affecting exploratory-like and anxiety-like behaviors (Fig. 4d,e and Extended Data Fig. 4f,g). Similar results were obtained with AAV9-eSYN-*Ift88*-shRNAmir (Extended Data Fig. 4d,e). By contrast, downregulation of *Ift20* in mature neurons did not induce any change in MWM (Fig. 4f), suggesting a potential implication of core PC proteins in neuronal progenitors, maturing neurons and/or other hippocampal resident cells (such as astrocytes and microglial cells). Taken together, these results demonstrate that reducing the levels of key core PC proteins in hippocampal neurons leads to memory impairments without affecting exploratory-like and anxiety-like behaviors.

## Autophagy induction counteracts cognitive deficits

To examine the functional role of the core PC-protein-dependent autophagy in the regulation of cognition, we tested whether inducing hippocampal autophagy could counteract the memory deficits induced upon neuronal downregulation of *Ift20*. To that end, we performed hippocampal daily infusion of either TAT-Beclin-1 peptide (pharmacological inducer of AP formation<sup>55</sup>) or TAT-scramble (control), after implantation of bilateral cannulas in *Ift20*-downregulated

animals. We validated that TAT-Beclin-1 infusions improve the defects in autophagy observed after neuronal *Ift20* downregulation (Fig. 4g), as LC3B-II significantly accumulates upon TAT-Beclin-1 treatment. Then, we found that the improved level of hippocampal autophagy is sufficient to restore the recognition and contextual memory deficits induced by neuronal *Ift20* downregulation (Fig. 4h).

## Restoring core PC protein levels improves memory deficits

Normal brain aging is characterized by a decline in autophagy machinery in hippocampal neurons, leading to memory deficits<sup>3,20</sup>. Notably, we observed that this alteration is correlated to a marked decrease of core PC constituents (IFT20, IFT88 and KIF3A), at both the mRNA and protein levels (Fig. 5a,b) in aged (16-month-old) mouse hippocampi. This decrease is associated with PC morphological abnormalities, characterized by a longer PC axoneme in hippocampal neurons of both CA3 (Fig. 5c) and DG regions (Extended Data Fig. 5a). These morphological abnormalities are similar to those observed in PC after *Ift20* downregulation in hippocampal neurons of 3-month-old mice (Fig. 3d). Then, we tested whether restoring core PC protein levels in old hippocampi could improve age-related neuronal autophagy decrease and memory deficits. To do so, we performed stereotactic injections of an AAV9 expressing *Ift20* cDNA in aged hippocampi (Fig. 5d). Three weeks after the injections, we confirmed that the IFT20 level was restored in old hippocampi (Fig. 5d). This restoration was accompanied by an improvement of the levels of other core PC proteins, such as IFT25, IFT88 and KIF3A (Fig. 5e). Notably, restoring *Ift20* levels was sufficient to increase the LC3B levels and reduce SQSTM1/p62 accumulation in old hippocampi, suggesting an improvement of autophagy activity in the CA3 of aged mice (Fig. 5f). Exposing these mice to behavioral tests, we observed that the restoration of neuronal IFT levels in old hippocampi, despite no change in PC size, is sufficient to reverse age-related cognitive deficits (Fig. 5g and Extended Data Fig. 5b,c). Together, these results indicate that the reduction of core PC protein levels in the hippocampus during aging is associated with cognitive decline. Their restoration is sufficient to improve memory as well as neuronal autophagy decline in old hippocampi.

## PC-dependent autophagy mediates the OCN effects on cognition

These findings prompted us to test whether core PC proteins are required to transduce the OCN effects on age-related cognitive decline. We showed that a local stereotactic injection of recombinant OCN in 16-month-old mice is sufficient to improve the reduction of core PC proteins, namely IFT20 and TULP3, in the hippocampus (Extended Data Fig. 6a). Conversely, the improvement of autophagy enfeeblement and memory alterations in aged mice after systemic OCN administration is abolished after *Ift20* downregulation in hippocampal neurons (Fig. 6a,b). However, although OCN treatment in 16-month-old mice is able to improve core PC protein levels (Extended Data Fig. 6a), it is

**Fig. 4 |** *Selective downregulation of PC core proteins in hippocampal neurons leads to severe learning and memory impairments.*** a–c, Behavioral analyses performed in 3-month-old mice after hippocampal stereotactic injections with AAV-U6-shRNA-*Ift20*, AAV-U6-shRNA-*Kif3a* or their respective AAV-U6-shRNA-scramble. NOR test (a), CFC (b) and MWM test (c) were assessed 3 weeks after local AAV injections. For the NOR, discrimination and preference indexes were measured 24 h after the training phase to assess memory performances. For the CFC, the percentage of freezing was measured for the training and testing phases. For the MWM, the graph shows the time to localize a submerged platform in the swimming area. All behavioral tests were performed in two independent cohorts of animals for each group and their respective control. d–f, The same behavioral analyses (NOR (d); CFC (e); MWM (f)) were performed in 3-month-old mice, 3 weeks after hippocampal stereotactic injections with either eSYN-shRNA-*Ift20mir* (eSYN-shRNA-*Ift20* or eSYN-shRNA-scramble-mir (eSYN-shRNA-Scrb.). This analysis was performed in two independent cohorts

of animals for each group, for each behavioral test. g, Representative western blot and quantification of LC3B-II accumulation (LC3B-II/β-actin ratio) in mouse hippocampi stereotactically injected with either eSYN-shRNA-*Ift20-mir* or eSYN-shRNA-scramble-mir and after five consecutive days of daily infusion with either TAT-scramble or TAT-Beclin-1. β-actin was used as a loading control for each sample. The quantification is relative to eSYN-shRNA-scramble-mir mice injected with TAT-scramble. h, NOR performed in the same experimental conditions as Fig. 4a, in 3-month-old mice after hippocampal stereotactic injections with eSYN-shRNA-scramble-mir + TAT-scramble, eSYN-shRNA-*Ift20-mir* + TAT-scramble or eSYN-shRNA-*Ift20mir* + TAT-Beclin-1. This analysis was performed in one cohort of animals for each group. Data are expressed as mean ± s.e.m. *P* values were determined by one-tailed (g) or two-tailed (a, b, d, e) Student's *t*-tests compared to shRNA-Scrb. The *P* values were determined by one-way (h) or two-way (c, f) ANOVA.
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Ift20

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Ift88

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DAPI + anti-ACIII

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**Fig. 5** | PC core protein levels are reduced during brain aging, and their restoration is sufficient to improve age-related memory decline. **a.** Relative expression of *Ift20*, *Ift25*, *Ift88* and *Kif3a* in WT dorsal mouse hippocampi at 3, 6, 12, 16 and 23 months of age. Quantification of mRNA expression is relative to the 3-month-old WT mouse group (n = 6 per group except for 23-month-old where n = 5 for *Ift20*, n = 5 for *Ift88* and n = 4 for *Kif3A*). **b.** Representative western blot of IFT20, IFT88 and KIF3A protein levels in WT dorsal mouse hippocampi at 3, 6, 12 and 16 months of age. **c.** PC immunofluorescent staining (green, stained by ACIII) in the CA3 region of the hippocampus of young (3-month-old) and aged (16-month-old) mice. The length of the PC was measured and quantified in 3D using Imaris software. Scale bar, 5 µm. **d.** Western blot image and quantification of IFT20 protein in 3-month-old or 16-month-old mice, 3 weeks after stereotactic injections with either AAV-GFP (control) or AAV-m*Ift20* (expressing mouse *Ift20* cDNA). **e.** Western blot images and quantification of IFT25, IFT88 and KIF3A levels

in the same experimental groups as in **d**. These measurements were performed in two independent experiments. **f**, LC3B and SQSTM1/p62 immunofluorescence (LC3B in red and p62 in green) and puncta quantification performed on brain cross-sections, at the level of the hippocampal CA3 region of 3-month-old or 16-month-old mice, 3 weeks after stereotactic injections with AAV-GFP (control) or AAV-m*Ift20* cDNA. Scale bars, 20 µm. **g**, NOR performed in 3-month-old or 16-month-old mice, after hippocampal stereotactic injections with AAV-GFP (control) or AAV-m*Ift20* cDNA. Discrimination and preference indexes were measured 24 h after the training phase to assess memory performances. The NOR was performed in two independent experiments for each group of mice. β-actin was used as a loading control for each sample in all western blot analyses. Data are expressed as mean ± s.e.m., and *P* values were determined by one-tailed (f) or two-tailed (c-e) Student's *t*-tests or by one-way ANOVA (a, g). Mo, month-old.

not sufficient to reverse the morphological abnormality in PC length (Extended Data Fig. 6b). Altogether, these data suggest a necessary contribution of core PC-protein-dependent autophagy to mediate the OCN's effects in fostering cognitive fitness.

### cAMP signaling pathway modulates OCN-induced autophagy
We further explored the signaling pathway linking PC and induced autophagy upon OCN treatment. To do so, we used our proteomic data (Fig. 1a) that we further analyzed with public differential gene expression datasets coupled with Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, using a graph theory approach (Fig. 6c-g). We elaborated three complementary parameters that we combined together to prioritize signaling pathways: (1) a distance-like measure that quantifies the pathway's absolute divergence from autophagy by measuring the difference in how gene expression within the pathway contributes to the network's overall structure; (2) strength of the connection between the pathway and autophagy; and (3) a key regulator score that describes the potential that the certain pathway could have the role of a regulator within the network. The pathways with combined coefficient greater than 0.35 were identified and ranked from the closest to the furthest pathway when compared to autophagy (Fig. 6d).

Among the signaling pathways selected, we revealed an interaction chain between core PC proteins (KIF3A linked to IFT20) - cAMP signaling (protein kinase cAMP-activated catalytic subunit alpha (Prkaca)) - autophagy (Fig. 6d,e). We further analyzed the patterns of differentially expressed proteins within the above-ranked pathways and revealed two distinct zones of interest (zones A and B). Zone A exhibited a more pronounced and stronger coordinated expression profile compared to zone B, as illustrated in the histogram profile of differentially expressed proteins (Fig. 6f). The enriched pathways the most closely associated with autophagy are the ones lying in front of those listed in zone A: Rap1, Ras and PI3K/Akt signaling pathways (Fig. 6d,f). Within the zone A, the most representative expressed proteins were those involved in the cAMP signaling pathway (Fig. 6d-g).

**Fig. 6** | The PC core protein IFT20 is required to mediate the rejuvenating effects of OCN on age-related memory deficits. **a, b.** Stereotactic injections with AAV-*eSYN*-shRNA-scramble-mir or AAV-*eSYN*-shRNA-*Ift20*-mir were performed on 3-month-old or 16-month-old mice before 28-d peripheral infusion with vehicle or OCN. In **a**, western blot analysis and quantification of LC3B-I/II and SQSTM1/ p62 accumulation from hippocampi. In **b**, discrimination and preference indexes of the NOR test. The NOR test was performed in two independent experiments for each group of mice. Data are expressed as mean ± s.e.m., and *P* values were determined by one-tailed Student's *t*-test compared to control groups (a) and by one-way ANOVA with Tukey's multiple comparison test (b). **c.** Graph theory and network analysis to determine elements within the network. Nodes represent pathways or protein expression. By analyzing connections between nodes, we can identify central and peripheral nodes within a cluster (or the entire network). Central nodes are associated with the core functionality of the cluster, whereas nodes positioned at the periphery of the cluster are associated with the nodes

### OCN regulates autophagy via CREB signaling in hippocampus
It is well described that activation of the transcription factor cAMP response element-binding protein (CREB) is decreased in aged mouse hippocampi⁵⁴⁻⁵⁶. These alterations in CREB signaling contribute to cognitive deficits in normal aging and various neuro-degenerative diseases⁵³⁻⁵⁵. Multiple reports showed that the rejuvenating effects of the young plasma administration on cognition are mediated, in part⁵⁷,⁵⁸, through the activation of CREB phosphorylation⁹,¹³. Moreover, we found that the cAMP signaling pathway is among the closest autophagy-related pathways regulated upon OCN treatment (Fig. 6d). Western blot and immunofluorescence analyses showed that OCN treatment is sufficient to enhance CREB phosphorylation and LC3B levels in the CA3, when no significant differences were observed in the CA1 and DG regions (Fig. 7a and Extended Data Fig. 7a,b).

Accordingly, we showed that the increase in LC3B-II accumulation in primary hippocampal neurons upon OCN is abolished after CREB inhibitor (666-15) treatment (Fig. 7b), suggesting that the CREB signaling pathway is required for the regulation of OCN-induced autophagy in hippocampal neurons. We next confirmed that CREB phosphorylation is lower in 16-month-old hippocampi and that this decrease could be reversed after OCN stereotactic injections (Fig. 7c). Hippocampal downregulation of either *Ift20* or *Kif3a* decreased neuronal pCREB/ CREB ratio (Fig. 7d). Conversely, the restoration of IFT20 levels (after local AAV9-*Ift20* cDNA injections) is sufficient to improve CREB phosphorylation deficits in the CA3 and DG of 16-month-old mice (Fig. 7e and Extended Data Fig. 7c). Moreover, we found that neuronal *Ift20* downregulation in the hippocampus is sufficient to counteract the induction of *c-FOS* expression, a major transcriptional target of pCREB, by OCN (Extended Data Fig. 7d). Downregulation of neuronal *Ift20* blocked the improvement of CREB phosphorylation by OCN in old mice (Fig. 7f). Altogether, these data support the necessary contribution of PC-dependent CREB signaling pathway to mediate the induction of autophagy machinery by OCN and, thereby, to the rejuvenating effects of OCN on age-related cognitive decline.

contributing to communication coordination. **d.** Pathway interaction graph based on the analysis of differentially expressed proteins from our proteomic data with KEGG databases, using a graph theory approach. Pathways were ordered based on their importance within the overall network using three parameters as detailed in the main text. Pathways with a combined coefficient greater than 0.35 are listed. *: pathways closely associated with those of zone A; **: pathways belonging to zone A. **e.** Graph illustrating the link between PC and autophagy-related pathways. A direct link could come through a KIF3A-PRKACA interaction. **f.** Identification of coordinated expression patterns within pathways in **d**. Only proteins with a significant impact within the network connectivity were taken into account. Rap1, Ras and PI3K/Akt pathways were the closest ones to localize near the identified zone A. Within the zone A, the most representative expressed proteins were involved in the cAMP signaling pathway. **g.** Histogram showing the strength of coordinated expression pattern in differentially expressed proteins from the ranked signaling pathway list in **d**. Mo, month-old.
a

AAV-shRNAs

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Hippocampus

3 Mo 16 Mo

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b

NOR

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Training

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Testing

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c

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d

<table><tr><td>0.01</td><td>8.0</td><td>0.5</td><td>Fc epsilon RI signaling pathway</td><td>0.4</td></tr><tr><td>0.02</td><td>7.0</td><td>0.7</td><td>Platelet activation</td><td>0.5</td></tr><tr><td>0.02</td><td>9.0</td><td>0.4</td><td>GnRH secretion</td><td>0.4</td></tr><tr><td>0.07</td><td>8.0</td><td>0.7</td><td>Sphingolipid signaling pathway</td><td>0.5</td></tr><tr><td>0.08</td><td>9.0</td><td>0.8</td><td>Relaxin signaling pathway</td><td>0.6</td></tr><tr><td>0.08</td><td>8.0</td><td>0.6</td><td>VEGF signaling pathway</td><td>0.5</td></tr><tr><td>0.10</td><td>10.0</td><td>0.7</td><td>Estrogen signaling pathway</td><td>0.6</td></tr><tr><td>0.10</td><td>9.0</td><td>0.7</td><td>Endocrine resistance</td><td>0.6</td></tr><tr><td>0.11</td><td>10.0</td><td>0.5</td><td>Cholinergic synapse</td><td>0.5</td></tr><tr><td>0.11</td><td>8.0</td><td>0.6</td><td>Progesterone-mediated oocyte maturation</td><td>0.4</td></tr><tr><td>0.12</td><td>9.0</td><td>0.8</td><td>Chemokine signaling pathway</td><td>0.6</td></tr><tr><td>0.12</td><td>9.0</td><td>0.6</td><td>Apoptosis</td><td>0.5</td></tr><tr><td>0.13</td><td>9.0</td><td>0.7</td><td>Insulin signaling pathway</td><td>0.5</td></tr><tr><td>0.13</td><td>7.0</td><td>0.9</td><td>cAMP signaling pathway **</td><td>0.5</td></tr><tr><td>0.16</td><td>8.0</td><td>0.6</td><td>ErbB signaling pathway</td><td>0.4</td></tr><tr><td>0.16</td><td>8.0</td><td>0.6</td><td>CREB1 phosphorylation **</td><td>0.4</td></tr><tr><td>0.17</td><td>7.0</td><td>0.7</td><td>Osteoclast differentiation</td><td>0.4</td></tr><tr><td>0.20</td><td>10.0</td><td>0.8</td><td>Growth hormone synthesis, secretion and action</td><td>0.6</td></tr><tr><td>0.20</td><td>9.0</td><td>0.9</td><td>Thyroid hormone signaling pathway</td><td>0.6</td></tr><tr><td>0.20</td><td>8.0</td><td>0.7</td><td>C-type lectin receptor signaling pathway</td><td>0.4</td></tr><tr><td>0.23</td><td>8.0</td><td>0.6</td><td>Phospholipase D signaling pathway</td><td>0.4</td></tr><tr><td>0.23</td><td>6.0</td><td>1.0</td><td>Neutrophil extracellular trap formation</td><td>0.4</td></tr><tr><td>0.25</td><td>9.0</td><td>0.8</td><td>Cellular senescence</td><td>0.5</td></tr><tr><td>0.33</td><td>8.0</td><td>0.9</td><td>Rap1 signaling pathway *</td><td>0.4</td></tr><tr><td>0.33</td><td>9.0</td><td>0.9</td><td>Ras signaling pathway *</td><td>0.5</td></tr><tr><td>0.41</td><td>8.0</td><td>1.0</td><td>PI3K-Akt signaling pathway *</td><td>0.4</td></tr></table>

Combined score

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e

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f

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a

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c

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d

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e

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f

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**Fig. 7** | CREB signaling pathway is required to mediate the autophagy machinery regulation by OCN. **a**, Representative western blot and quantification of phospho-CREB and CREB protein levels in the hippocampus of 3-month-old mice, after stereotactic injections of vehicle or OCN.
**b**, Representative western blot and quantification of LC3B-II/β-actin ratio) in mature primary hippocampal neurons treated for 4 h with vehicle, OCN, CREB inhibitor (666-15) or OCN + CREB inhibitor. These measurements were obtained from two independent experiments. **c**, Western blot analysis and quantification of phospho-CREB and CREB in the hippocampus of 3-month-old and 16-month-old mice after stereotactic injections of vehicle or OCN. **d**, Western blot and quantification of phospho-CREB and CREB in 3-month-old mice previously injected in the hippocampus with AAV-eSYN1-shRNA-*Ift20*, AAV-U6-shRNA-*Kif3a* or AAV-U6/eSYN1-shRNA-scramble. **e**, Representative images of phospho-CREB immunofluorescence staining (red) performed at the level of the hippocampal CA3 region. Scale bar, 20 µm. Brain cross-sections

of 3-month-old and 16-month-old mice collected 3 weeks after hippocampal stereotactic injections with either AAV-GFP (control) or AAV-*mlft20* cDNA were used. Relative intensity quantification was measured relative to the 3-month-old mice injected with AAV-CMV-GFP. **f**, Western blot analysis and quantification of phospho-CREB and CREB in the hippocampus of 3-month-old and 16-month-old mice, after hippocampal stereotactic injections of either AAV-eSYN-shRNA-scramble-mir or AAV-eSYN-shRNA-*Ift20*-mir and peripheral chronic infusion with vehicle or OCN. The quantification is relative to 3-month-old mice injected with AAV-eSYN-shRNA-scramble-mir and treated with vehicle. These measurements were obtained from two independent cohorts of mice for each group. β-actin was used as a loading control for each sample in all western blots. Data are expressed as mean ± s.e.m., and *P* values were determined by two-tailed Student's *t*-tests (**b**, **d**, **f**) or by one-way ANOVA followed by Tukey's multiple comparison test (e). Mo, month-old.

Discussion

In our study, we identified that a core PC protein-autophagy machinery axis could act as a gateway in hippocampal neurons to transmit the

effects of blood factors (OCN) on neuronal homeostasis and, thereby, allow for the maintenance of cognitive fitness. We found that GPR158 is present at the PC and that OCN can enhance the core PC protein
levels in hippocampal neurons. By contrast, downregulation of core PC proteins is sufficient to block the induction of hippocampal autophagy machinery by OCN. Altogether, these data led us to test the functional relevance of the link between core PC proteins and autophagy in hippocampal neurons and as mediators of the effects of systemic factors (OCN) in cognitive fitness. Accordingly, we demonstrated that downregulation of core PC proteins in hippocampal neurons leads to reduced autophagy and memory performance. Restoring autophagy is sufficient to counteract memory deficits of these mice. During aging, core PC protein levels are reduced, which might be correlated to a PC dysfunction in this region of the brain. In fact, we showed that restoring their levels improves age-impaired autophagy and memory in old mice. By contrast, core PC protein downregulation abolished the pro-cognitive effects of OCN in aged mice. Last, we found that the core PC proteins transduce the activation of autophagy by OCN via CREB phosphorylation in hippocampal neurons, a key transcriptional factor for the control of neuronal plasticity and memory. These results indicate that PC-dependent neuronal sensing is impaired during aging, resulting in the inability to sense and transduce signals from systemic factors, such as OCN, to maintain neuronal homeostasis, autophagy and, therefore, learning and memory.

As previously demonstrated, mice exhibited a significant impairment of memory and autophagy machinery from 16 months of age, compared to 3-month-old mice, indicating the progressive feature of aging¹⁸,²⁰. Chronic peripheral treatment with OCN in aged mice rescued age-related memory impairments, from 16 months of age¹⁸,²⁰. However, the molecular mechanisms of this OCN-dependent rescue were not well understood, despite its therapeutic potential. We previously reported that OCN controlled age-induced memory impairment through autophagy²⁰. Additionally, recent works have shown that autophagy regulation, among different factors, is downstream of the PC³⁶,³⁷,³⁹. Although it is important to note that all our in vivo experiments were performed using male mice, our results suggest that the pro-cognitive effects of OCN are reduced when core PC proteins are downregulated in neuronal PC.

Exploring the signaling pathway linking PC and induced autophagy upon OCN, we used a bioinformatic approach, based on a graph theory analysis and differential gene expression data coupled with the KEGG database. We revealed an interaction chain between core PC proteins (KIF3A and IFT20) - cAMP signaling - autophagy. These data reinforce the link between PC and autophagy and solidify further the importance of the cAMP signaling pathway in the OCN-dependent induction of autophagy in hippocampal neurons.

A correlation was shown between autophagy induction and activation of CREB phosphorylation to mediate neuronal survival and protection⁶⁰,⁶¹. Here, we showed that the effects of OCN on autophagic pathway are dependent on phospho-CREB, whose activation is reduced after core PC protein silencing. Notably, we demonstrated that restoring core PC proteins in aged mice is necessary and sufficient to correct the age-related phospho-CREB decline. Consistently, OCN treatment in aged mice requires core PC proteins to restore CREB activity. These data indicate that PC-dependent autophagic response is part of an integrated survival signaling network that activates the CREB pathway.

PC function as cellular antenna, enriched in specific GPCRs sensing and responding to neuromodulators in the extracellular milieu, being key in the transduction of the extracellular signals to regulate memory maintenance. Here, we showed that GPR158 localizes at the PC, where it interacts with PC proteins and transduces the OCN-mediated signal to regulate autophagy. Although the detailed molecular mechanism by which OCN boosts autophagy has not been detailed yet, we demonstrated the role of the core PC proteins in maintaining cognitive resilience.

In this study, we focused on the role of a PC-autophagy axis in neurons, but we cannot exclude that this axis could also play a role in other resident cell types in the brain. Indeed, OCN could also play a critical

role for the myelination of oligodendrocytes through its binding to GPR37 (ref. 62). Therefore, one can ask whether this axis could also be activated and mediate the OCN effects in this specific cell type. Lastly, beyond OCN, it is known that numerous receptors for systemic factors are present in the PC⁶³ and could modulate the autophagy process. Our findings may suggest that this PC-autophagy axis could constitute a key mechanism on how neurons communicate with the systemic milieu and enhance their adaptive response to environmental changes.

During aging, we found a reduction of key core PC protein levels in mouse hippocampi. This reduction is associated with a marked alteration of the transcriptional levels of genes encoding these core PC proteins. However, at the present time, it is difficult to determine whether the decline in core PC proteins is only due to transcriptional reduction or could be also associated with an age-related increase in core PC protein degradation. Further analyses are now needed to address this question and assess the degradative dynamics of these proteins in hippocampal neurons.

We showed that the structure of hippocampal neuronal PC is affected by aging, characterized by increased length. This result is consistent with the increased cilia length, but constant number of PC, observed in aged hippocampal neurons of rats⁶⁴. The importance of an optimal cilia length is underscored by the finding that its perturbation, generating shorter or longer cilia, can be observed in cilia-related diseases. In such cases, the location or trafficking of PC-localized receptors or additional scaffold proteins can be impaired, resulting in disruption of PC-dependent signaling cascades, such as those induced by OCN. We demonstrated that the increase in PC length, together with a decrease in core PC proteins, is associated with poor memory performance. These data suggest the importance of PC turnover in the maintenance of cognitive functions. This result is also consistent with previous findings showing the involvement of PC in cognition, as the disruption of cortical and hippocampal PC promotes alterations in behavior, learning and memory⁴,⁴³. Consistently, a recent study observed a decrease in ciliary genes in a mouse model of memory consolidation and demonstrated that failure to form lasting memories is accompanied by downregulation of PC-associated genes⁶⁵,⁶⁶. However, although we found that OCN injections or restoring IFT levels could correct age-related autophagy and memory decline, it is not sufficient to modify neuronal PC length in old mice. This observation may suggest that the age-related morphological PC alterations are irreversible and, consequently, that the PC morphology does not necessarily determine its functionality. Further experiments are needed to test whether prolonged OCN treatment could reverse this morphological defect or at least block the increase of PC length during the course of aging.

Neuronal sensing can be affected by age-related changes in systemic factor levels but also by dysfunctional detection of these hormones/signals, which finally impair signal transduction. Indeed, decrease in core PC proteins results in an incapacity to perceive systemic factor changes and induce the required signaling pathways, including autophagy, to modulate learning and memory behavior. Thus, a functional PC is required to integrate the pro-cognitive effects of OCN in aged mice. In young adult mice, OCN fails to promote neuronal hippocampal autophagy in a context of cilia impairment. Consistently, the OCN's pro-cognitive effects, dependent on autophagy²⁰, were deteriorated when core PC proteins were downregulated in neuronal PC. Despite that, we observed a partial amelioration of memory performances in OCN-treated mice with impaired IFT20, which can be attributed to the binding of OCN to non-ciliary-located GPR158 and IFT-independent functions.

Our study unravels a physiological role of the autophagy-PC axis in brain, where alteration of ciliary core PC proteins during aging leads to autophagy impairments and, thereby, memory deficits. This study identifies a paradigm in the mechanism controlling the communication between blood factors and neurons and its importance during brain aging for the maintenance of neuronal homeostasis in the
hippocampus. PC could constitute a gateway in hippocampal neurons through which systemic factors modulate neuronal autophagy and, thereby, cognitive resilience. By shedding light on this regulatory mechanism responsible for the decline of autophagy activity during aging, our findings provide the foundation for the development of more potent therapeutic targets to treat or even prevent age-related cognitive disorders as well as neurological disorders in ciliopathies.

# Methods

## Animals

All experiments were performed on C57BL/6J WT male mice (Janvier Laboratory). All mice were 3 months or 16 months of age at the start of experiments, and littermates were used as controls. Upon arrival, mice were housed at least 2 weeks before any behavioral or molecular testing. Male mice were housed five animals per cage in polycarbonate cages (35.5 × 18 × 12.5 cm), under a 12-h light/dark cycle with ad libitum access to food and water before experimentation. All behavioral experiments were performed in accordance with the European Communities for Experimental Animal Use (2010/63/EU) and ethical committee review procedures and protocols (APAFIS-25139).

## Proteomic analysis

Hippocampi lysates were prepared using BeatBox Tissue Kit 24x (Pro-Omics): samples were homogenized in 5% SDS 50 mM TEAB lysis buffer for 10 min, applying the high BeatBox setting. After 5-min heating at 95 °C and 10-min centrifugation at 14,000g, protein dosage was performed using a DC Protein Assay Kit (Bio-Rad). S-TrapTM micro spin column (ProtiFi) digestion was performed on 30 µg of cell lysates according to the manufacturer's instructions. After elution, peptides were vacuum dried and resuspended in 75 µl of 2% acetonitrile (ACN) and 0.1% formic acid in high-performance liquid chromatography (HPLC)-grade water before mass spectrometry (MS) analysis. Then, 400 ng of each sample was injected on a nanoElute2 HPLC system coupled to a timsTOF Pro (Bruker Daltonics) mass spectrometer. HPLC separation (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in ACN) was carried out at 250 nl min⁻¹ using a packed emitter column (C18, 25 cm × 75 µm, 1.6 µm) (Ion Optics) using a 70-min gradient elution (2% to 13% solvent B during 42 min; 13% to 20% during 23 min; 20% to 30% during 5 min; 30% to 85% for 5 min; and finally 85% for 5 min to wash the column).

Mass spectrometric data were acquired using the parallel accumulation serial fragmentation (PASEF) acquisition method in data-independent acquisition (DIA) mode. The measurements were carried out over the *m/z* range of 100 Th to 1,700 Th. The range of ion mobility values was from 0.6 Vs cm⁻² to 1.6 Vs cm⁻² (1/K₀). diaPASEF settings were as follows: mass range from 400 *m/z* to 1,200 *m/z*, mobility range from 0.60 Vs cm⁻² to 1.43 Vs cm⁻² (1/K₀), cycle time estimate of 1.79 s and 16 PASEF scans × two steps per PASEF scan (32 windows).

Data analysis was performed using DIA-NN software (version 1.8.1). A search against the UniProtKB/Swiss-Prot *Mus musculus* database (release July 2023, 17,167 entries) was performed using library-free workflow. For this purpose, 'FASTA digest for library free search/library generation' and 'Deep learning spectra, RTs and IMs prediction' options were checked for precursor ion generation. One trypsin missed cleavage was allowed, and the maximum variable modification was set to 2. Carbamidomethylation (Cys) was set as the fixed modification, and protein N-terminal methionine excision, methionine oxidation and N-terminal acetylation were set as variable modifications. The peptide length range was set to 7–30 amino acids, precursor charge range 2–4, precursor *m/z* range 300–1,300 and fragment ion *m/z* range 300–1,300. To search the parent mass and fragment ions, accuracy was set to 10 ppm manually. The false discovery rates (FDRs) at the protein and peptide level were set to 1%. Match between runs was allowed. For the quantification strategy, Robust LC (high precision) was used, whereas default settings were kept for the other algorithm parameters.

## Bioinformatic analyses

**Identification of the most relevant autophagy-related enriched pathways associated with OCN treatment.** Using custom-made scripts in Python and R languages, we constructed a gene co-expression network using a graph theory approach to analyze the network topology of genes in the hippocampus after OCN treatment. Leveraging the KEGG database, we built an initial network where nodes represented genes and/or pathways. We then enriched this network by retaining only statistically significant differentially expressed genes (adjusted *P* < 0.05). This resulted in a focused gene interaction map encompassing pathways relevant to our investigation.

We assessed how genes interact by looking at their connectivity within the network. We systematically removed the genes from the network components and measured how much the network's connectedness dropped. Genes causing a larger decrease were considered more central and crucial. Then, we ordered the genes based on their importance within the overall network structure (starting with the most important ones). From this list, only central ones (genes with negative scores) were chosen and fed to g:Profiler where the ordered query was run. Genes with a positive score had an increase of centrality upon their removal, meaning that the graph becomes more connected, thus indicating that these genes were positioned at the periphery of the obtained network. Finally, we constructed a network that specifically highlights how these important genes interact within the identified pathways.

To identify pathways functionally linked to autophagy, we employed a combined approach. Pathway impact on network structure and distance from autophagy (based on connectivity change) were assessed. We then evaluated pathway centrality (considering both connections and connected neighbors) to create a 'key regulator index'. Finally, a coefficient integrating connection strength with autophagy, network impact similarity and the key regulator index was used to prioritize the most relevant autophagy-connected pathways.

**Investigating the interactions of PC and autophagy.** To investigate the interaction between PC and autophagy, the initial gene pathway map, enriched with data from biological experiments, was used. Next, the minimum node distance between the genes known to be part of the PC (obtained from the KEGG pathway database - PC and associated proteins) and autophagy signaling pathway was identified.

**Integrating ordered gene list information with gene expression data.** The patterns of activity were determined using the initial ordered list of genes. We introduced a score that integrated both the information about the differential expression and the topographical importance of the node within the network. We iterated the initial ordered list of genes and either incremented the position-specific score by +1 if the current gene of interest was upregulated or decremented it by -1 if downregulated. Values around a constant value indicated a random distribution of pattern expression data within a certain region of the list, whereas sustained positive/negative slope of the score highlighted the regions of topographically related genes with coordinated expression levels. These hubs offered a more comprehensive picture of a network's functioning and likely represented a tightly controlled process, which was likely essential for the captured cellular response.

## Western blot analysis

Mouse dorsal hippocampi were dissected, snap frozen and lysed in RIPA lysis buffer with protease and phosphatase inhibitors. The lysates were sonicated, and the protein concentration was analyzed by BCA assay. The lysates were loaded on a 12.5% or 10% SDS polyacrylamide gradient gel and transferred onto a PVDF membrane. The blots were blocked in Tris-buffered saline with 0.1% Tween (TBST) and 5% BSA and incubated with the following: mouse anti-β-actin (1:5,000; Sigma-Aldrich, A-2228), rabbit anti-IFT20 (1:1,000; Proteintech, 13615), rabbit anti-IFT88
(1:1,000; Proteintech, 13967), rabbit anti-KIF3A (1:1,000; Proteintech, 13930), rabbit anti-IFT25 (1:1,000; Proteintech, 15732), rabbit anti-TULP3 (1:1,000; Proteintech, 13637), rabbit anti-GPR158 (1:1,000; Antibodies Online, ABIN6258978), mouse anti-Beclin-1 (1:1,000; BD Transduction Laboratories, 612113; only the top band corresponding to non-cleaved Beclin-1 was quantified in this study), rabbit anti-LC3B (1:10,000; Sigma-Aldrich, L7543), guinea pig anti-p62 (1:2,000; PROGEN, GP62-C), rabbit anti-phospho-CREB (1:1,000; Merck Millipore, 06-519) and rabbit anti-CREB (1:1,000; Cell Signaling Technology, 9197). Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-mouse IgG (Cell Signaling Technology, 7076), anti-rabbit IgG (Cell Signaling Technology, 7074) and anti-guinea pig IgG (Sigma-Aldrich, A5545)) were used for protein detection and revealed by Clarity Western ECL Substrate (Bio-Rad) or Immobilon Chemiluminescent HRP Substrate (Millipore). Selected films were scanned and quantified using Bio-Rad ChemiDoc Touch Imaging System version 1.0 and Image Lab software version 5.2, respectively. β-actin bands were used for normalization.

## Cell isolation and culture

Murine neuroblastoma N2a cells (American Type Culture Collection, Neuro-2a-CCL-131) were cultured in DMEM supplemented with 10% FBS and 100 U ml⁻¹ penicillin-streptomycin. N2a cells were treated for 4 d with 10 µM retinoic acid to induce differentiation before treatment.

Hippocampal neurons were isolated from mouse embryos (embryonic day 16.5). After dissection, hippocampi were digested for 15 min at 37 °C into 0.05% trypsin and 0.02% EDTA. After three washes with DMEM (Thermo Fisher Scientific, 6196505) supplemented with 10% FBS, 100 U ml⁻¹ penicillin-streptomycin and 1× GlutaMAX (Thermo Fisher Scientific), cells were dissociated by pipetting up and down and plated onto poly-L-lysine-coated plates or glass coverslips for microscopic examination. Twenty-four hours after plating, the media were replaced with Neurobasal medium (Thermo Fisher Scientific) containing B27 supplement (Thermo Fisher Scientific), GlutaMAX and MycoZap (Lonza) and refreshed twice a week. To decrease the proportion of glial cells, 1 µM Ara-C inhibitor (Sigma-Aldrich, C1768) was added in the pre-warmed complete Neurobasal medium, starting at day in vitro 5 (DIV5). Experiments were performed on cells after DIV15.

All cells were grown at 37 °C in a humidified incubator with 5% CO₂.

## Neuronal stimulation treatment of primary hippocampal neurons

Before treatments, neurons were starved for 4 h (Neurobasal medium without B27). Neurons were treated for 5 min or 4 h with Neurobasal medium containing 10 ng ml⁻¹ OCN. For the 4-h treatment, neurons were treated with Neurobasal medium containing 10 ng ml⁻¹ OCN, 100 mM Baf or both. For experiments using CREB inhibitor, neurons were treated for 4 h with Neurobasal medium containing 10 ng ml⁻¹ OCN, 73 nM CREB inhibitor 666-15 (Sigma-Aldrich, 5383410001) or both. After treatments, neurons were rinsed in PBS and proteins extracted in 1× Laemmli buffer containing phosphatase and protease inhibitors or fixed for 20 min in 4% paraformaldehyde (PFA)/4% glucose at room temperature.

## Recombinant OCN

Mouse uncarboxylated OCN was purified from BL21 bacteria transformed with pGEX2TK-mOCN as previously described¹⁷,¹⁸. In brief, GST-OCN fusion protein was bacterially produced in BL21 pLyS transformed with pGEX-2TK-mOCN after induction with IPTG. Bacteria were collected in lysis buffer (PBS, 10 mM Tris, pH 7.2, 2 mM EDTA, 1% Triton and 1× protease/phosphatase inhibitor cocktail; Thermo Fisher Scientific, 78443). After four freeze-thaw cycles and sonication, lysates were cleared by centrifugation. The supernatant was incubated for 4 h with Glutathione Sepharose 4B (GE Healthcare, 17075601) at 4 °C. After six washes with washing buffer (PBS 1× and 1% Triton) and

PBS, OCN was cleaved out from the GST moiety by using thrombin (GE Healthcare, 27-0846-01). Four fractions were collected, and each of them was incubated for 30 min with Benzamidine Sepharose (GE Healthcare, 17-5123-10) at room temperature to remove thrombin. Then, 10 ng of OCN (diluted in PBS) was injected in mouse hippocampi. Primary hippocampal neurons were treated with OCN with 10 ng ml⁻¹ in culture medium.

## Co-immunoprecipitation

We followed the manufacturer's protocol (Life Technologies, Dynabeads Protein G-10007D) for TULP3 co-immunoprecipitation. N2a cell lysates were prepared with RIPA and protease/phosphatase inhibitors as described above and sonicated, centrifuged and analyzed for protein concentration by BCA assay. We linked the rabbit anti-TULP3 antibody (1 µg; Proteintech, 13637) or control rabbit IgG (1 µg; Proteintech, 30000-0A) to magnetic beads by incubation on a rotatory wheel for 30 min at room temperature, washed and incubated with 400 µg of protein extract overnight at 4 °C on a rotatory wheel. After three washes, we eluted the proteins, added loading buffer and DTT, denatured at 95 °C and resolved by western blot.

## Immunostaining on cross-sections and quantification

Mice were deeply anesthetized with a mixture of ketamine and xylazine and transcardially perfused with cold PBS, followed by cold 4% PFA. Brains were post-fixed overnight in 4% PFA at 4 °C. Then, 30-µm serial coronal floating sections were obtained using a vibratome. Immunofluorescence experiments were performed on sections blocked for 1 h in 0.3% Triton X-100 and 10% donkey serum in TBS at room temperature and then incubated overnight at 4 °C with guinea pig anti-p62 (1:200; PROGEN, GP62C), mouse anti-NeuN (1:400; Sigma-Aldrich, MAB377), rabbit anti-LC3B (1:200; Sigma-Aldrich, L7543), rabbit anti-pCREB (1:500; Merck Millipore, 06-519) and rabbit anti-ACIII (1:400; Thermo Fisher Scientific, PA5-35382) diluted in 5% normal donkey serum in TBS-0.1% Tween.

For in vitro immunostaining, cell coverslips were permeabilized for 15 min with 0.2% Triton X-100 and then blocked for 30 min with 3% BSA at room temperature before being incubated with rabbit anti-TULP3 (1:100; Proteintech, 13637-1-AP), rabbit anti-ACIII (1:400; Thermo Fisher Scientific, PA5-35382) and mouse anti-GPR158 (1:100; Bio-Techne, MAB10286) overnight at 4 °C.

Sections and coverslips were washed with TBS-0.1% Tween before and after being incubated for 2 h with secondary antibodies (donkey anti-guinea pig IgG (1:200; Bioss, Alexa Fluor 555), donkey anti-rabbit IgG (1:200; Bioss, Alexa Fluor 647), donkey anti-rabbit IgG (1:200; Bioss, Alexa Fluor 546), donkey anti-mouse IgG (1:200; Bioss, Alexa Fluor 488) or donkey anti-rabbit IgG (1:200; Jackson ImmunoResearch, Cy3)) at room temperature in TBS-0.15% Triton. After PBS washes, Hoechst 33342 was used to stain nuclei (1:5,000; Thermo Fisher Scientific, 62249) for 5 min at room temperature. All sections and coverslips were mounted onto gelatin-subbed slides using Fluoromount G (Sigma-Aldrich). Images were obtained using a Zeiss Apotome 2 fluorescence microscope and analyzed using Zeiss ZEN lite LSM software under the same setting. The number of cells with SQSTM1/p62 puncta was quantified on digital images with Icy software (http://icy.bioimageanalysis.org). For phospho-CREB intensity quantification, images were acquired from five hippocampal sections of the different groups and transformed to a grayscale, and the signal was analyzed by calculating the integrated density of a region of interest after defining a threshold with ImageJ software (National Institutes of Health; available at http://rsb.info.nih.gov/ij/).

## PC counting and analysis

Cilia length analysis was performed on the microscopy analysis Imaris software program version 9.2.0 (Bitplane). Apotome images of the CA3 and DG hippocampal regions were imported and first converted to the
Bitplane format. PC were counted using Imaris Filament Tracer. Bitplane images were analyzed by manually tracing over PC using the Pencil tool in AutoPath mode. The start of the cilium was determined as the start of the red signal to the end of the same elongated signal. Cilia were traced only where Hoechst dye was apparent. Imaris functionality provided automatic centering of traced cilia once a cilium was delineated manually. Cilia were traced in 3D. Imaris statistics of cilia length, number of cilia per image and cilia straightness were then exported to Excel files for later data analysis. The number of cilia per image was normalized by dividing the number of cilia by the volume of the nuclei (µm³). The area was delimited by using the built-in Imaris surface tool. The detection of voxels threshold was manually adjusted to either increase or decrease coverage of nuclei that needed to be included in the volume of calculation. Between 49 and 116 cilia were examined for each image in the totality of the CA3 region, and between 85 and 368 cilia were examined for the upper layer of the DG. Three to four images were analyzed per mouse per region per age in the different experiments.

## GPR158 quantification and analysis

GPR158 analysis within the PC was performed on the microscopy analysis software program Imaris (version 9.2.0; Bitplane). Apotome images of the primary hippocampal neurons were converted to the Bitplane format. PC were first outlined with Imaris Filament Tracer in 3D, and Bitplane images were analyzed by manually tracing over PC using the Pencil tool in AutoPath mode following the same method as described above. The localization of GPR158 was then measured in 3D using the Spot Detection tool. We performed an automatic segmentation of objects, based on GPR158 signal intensity, with an estimated diameter of the spots of 0.280 µm. The Model PSF-elongation along the z axis and background subtraction options were selected. The Quality filter was manually adjusted to 57.6 on all images, so that the spots fit with GPR158 signal intensity. In the Spots menu, the filter 'Shortest distance to Cilia #' was added, and this process was repeated for all the cilia of the image. Then, the different distances to the cilia were set up (from 0 µm to 10 µm from the PC), and the number of GPR158 spots was read for all the distance groups.

## Semi-quantitative RT-PCR

Brain tissues were flash frozen after dissection, and total RNA was isolated with TRIzol reagent using a homogenizer. Single-stranded cDNA was synthesized from total RNA (2 µg) by using SuperScript II Reverse Transcriptase. qRT-PCR was performed using iTAQSYBR Green (Bio-Rad). The following primers were used: *Ift20* primers (5'-AGGCAGGGCT GCATTTTGAT-3' and 5'-CAAGCTCAATTAGACCACCAACA-3'), *Ift88* primers (5'-ACAGTGGCCAGAACAAATAGTG-3' and 5'-TCATCCTCGT CAATTCTTTTC-3'), *Kif3a* primers (5'-ATGCCGATCAATAAGTCGGAGA-3' and 5'-GTTCCCCTCAT TTCATCCACG-3'), *Ift25* primers (5'-TGATTCTG GCCACATCGAGTGATGA-3' and 5'-TCCTGGGGAAACATGCCTGTGG-3'), cFos primers (5'-CAGAGCGCAGAGCATCGCCA-3' and 5'-CGATTCCGC CACTTGGCTGC-3') and *Gapdh* primers (5'-AGGTCGGTGTG AACG GAT-3' and 5'-GGGTCGTTGATGGCAACA-3'). Data were analyzed with CFX Maestro software version 1.1.

## Stereotaxic injections and hippocampal cannula implantations

Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (100 mg kg⁻¹) (1000 Virbac) and xylazine (10 mg kg⁻¹) (Rompun 2%; Bayer) and placed in a stereotaxic frame (Kopf, 900SL). Ophthalmic eye ointment was applied to the cornea to prevent desiccation. The area around the incision was trimmed, and Vetoquinol was applied.

For the stereotactic injections, all drugs were administrated bilaterally into the dorsal hippocampi (coordinates from bregma; Paxinos and Franklin (2008): x = ±1.4 mm, y = 2.0 mm and z = -1.34 mm). Then, 1-µl volume of either AAV or drugs was injected over 4 min (injection rate, 0.25 µl min⁻¹). To limit reflux along the injection track, the needle

was maintained in situ for 4 min between each 1-µl injection. At the end of the surgical procedure, the skin was stitched over the brain, and an analgesic solution was administered subcutaneously (ketoprofen, 5 mg kg⁻¹).

For the cannula implantation, a hole was made at the following coordinates: anterior-posterior (AP) = 2.0, medial lateral (ML) = 1.4 (in mm, from bregma). A guide cannula (Plastics One, C315GMN/1.5, 26 gauge) was secured to the skull with dental cement (Sun Medical, Super-Bond C&B). Lastly, the scalp incision was sutured around the cannula, and an analgesic solution was administered subcutaneously (ketoprofen, 5 mg kg⁻¹).

## Drug preparation and hippocampal cannula infusion

TAT-Beclin-1 or TAT-scramble was dissolved to a concentration of 1 µg µl⁻¹ in PBS. For 7 d, the experimenter gently restrained the mouse and placed an injector cannula (C315IMN/1.5/0.5; 33 gauge, extending 0.5 mm beyond the 1.5-mm guide cannula). The drug was infused (1.0 µl per side at a rate of 1 µl min⁻¹) using a syringe pump (Harvard Apparatus) and a 10-µl Hamilton syringe connected via a PE-50 polyethylene catheter to the injector cannula.

## Osmotic pumps

Alzet micro-osmotic pumps (model 1002) were loaded 4 h before surgery with either vehicle saline solution (NaCl) or uncarboxylated OCN (30 ng h⁻¹). Mice were anesthetized with isoflurane, and osmotic pumps were surgically installed subcutaneously in the backs of the mice. The incision in the back of the mice was sutured, and the mouse was observed in a recovery chamber until it was fully mobile. Behavioral analyses started 1 week after osmotic pump installation.

## Pharmacological modulation of autophagy

We performed intra-hippocampal injections of either 1 µg of TAT-scramble (dissolved in PBS) or 1 µg of TAT-Beclin-1 (dissolved in PBS). The TAT-Beclin-1 (YGRKKRRQRRRGGGTNVFNATFEIWHDGEFGT) consisted of 11 amino acids of the TAT protein transduction domain (PTD) at the N-terminus, a GG linker to increase flexibility and 18 amino acids derived from Beclin-1-amino acids 267-284 containing three substitutions: H275E, S279D and Q281E. Control peptide, TAT-scramble (YGRKKRRQRRRGGVGNDFFINHETTGFATEW), consisted of the TAT PTD, a GG linker and a scrambled version of the C-terminal 18 amino acids from Tat-Beclin-1. All drugs were infused in a volume of 1 µl (bilaterally) in the dorsal hippocampus chronically using hippocampal cannulas for five consecutive days before behavioral tasks and maintained during the behavioral days (total, 10 d). For brain collection, hippocampal injections of TAT-scramble or TAT-Beclin-1 through cannulas were performed 12 h before euthanization.

## GPR158 mutagenesis and lentivirus constructs

Five putative ciliary localization sequences (VxPx) were identified on the C-terminal region of GPR158, at positions 212, 277, 300, 331 and 377, and mutated as follows: VCPW (212-215), (377-380) and (277-280) into ACAW; VLPG (300-303) into ALQG; and VAPK (331-334) into AAAK. The mutated GPR158 and the WT GPR158 fragments (purchased from Eurofins Genomics) were fused with the turbo EGFP and cloned into the pSicoR plasmid (by substitution of the original GFP). Lentiviral particles were produced for each construct (viral titer of 6.30 × 10⁸ TU ml⁻¹ or 7.60 × 10⁸ TU ml⁻¹ for the WT and mutated forms, respectively). Primary hippocampal neurons derived from Gpr158 knockout (KO) mice were infected (multiplicity of infection 10) at DIV1 with lentiviruses expressing either WT or mutated GPR158 and analyzed at DIV18 upon OCN treatment.

## AAVs expressing shRNA

All AAV9s expressing shRNA were purchased from Vector Biosystems. shRNAs specific to *Ift20* (AAV9-GFP-U6-m-*Ift20*-shRNA) (shRNA sequence (VBLKO-248329)):
5'-CCGGGGAGGAGTGCAAGGACTTTGTCTCGAGACAAAGTCCTT GCACTCCTCCTTTT-3', *Kif3a* (AAV9-GFP-U6-m-*Kif3a*-shRNA) (shRNA sequence (TRCN0000339511)): 5'-CCGGGGCCTGATGTGGGAGTA TATACTCGAGTATATACTCCCACATCAGGCCTTT TT-3' or scrambled non-targeting negative control (AAV9-GFP-U6-scrmb-shRNA) were bilaterally injected in a volume of 1 µl, 3 weeks before behavioral tests or brain tissue collection. The AAV titers were between 1.0 × 10¹³ GC ml⁻¹ and 4.3 × 10¹³ GC ml⁻¹. AAVs expressing shRNAmir target *IFT20* (AAV9-eSYN-GFP-*IFT20*-shRNAmir) with the following targeting sequence: GGAGGAGTGCAAGGACTTTGT or IFT88 (AAV9-eSYN-GFP-*IFT88*-shRNAmir) with the targeting sequence: GCAGGAAGACTGAAAGTGAAAT. These shRNAmir sequences are in the 3' untranslated region (UTR) of eGFP under the eSYN promoter, specifically active in neurons. The eSYN promoter is a hybrid promoter consisting of a 0.4-kb cytomegalovirus enhancer and the 0.45-kb human Synapsin 1 promoter fragment (hSYN1). AAV9-eSYN-GFP-shRNA155mir was used as control. The AAV titers were between 1.3 × 10¹³ GC ml⁻¹ and 6.2 × 10¹³ GC ml⁻¹. AAV overexpressing *IFT20* (AAV9-CMV-*mIFT20*-IRES-eGFP) and AAV9-CMV-eGFP, used as control, had titers between 5.0 × 10¹² GC ml⁻¹ and 6.0 × 10¹² GC ml⁻¹.

## Behavioral tests

For all behavioral assessments, mice were handled for at least 20 min over three consecutive days. Before the behavioral procedure, animals were transported a short distance from the holding mouse facility to the testing room in their home cages and left undisturbed for at least 1 h before testing.

**LDT.** Each animal was placed in a 43 × 43-cm open field also containing a dark compartment and tested for 10 min. Mice were placed individually into the dark chamber of the open-field arena and allowed to explore freely. Mice were monitored throughout each test session by infrared light beam activity monitor using actiMot2 software (TSE Systems, PhenoMaster Software). Anxiety was quantified by measuring the number of entries in the light compartment, the latency to reach the light compartment and the percentage of distance spent in the light versus dark chamber of the open-field box.

NOR paradigm, three foot shock CFC, MWM and OFTs were performed and analyzed exactly as described by Glatigny et al.²⁰.

## Statistics and reproducibility

All values are expressed as mean ± s.e.m. Alpha was set to 0.05 for all analyses. Statistical parameters, including the exact sample size (n), post hoc tests and statistical significance, are reported in every figure and figure legend. All micrographs or blots are representative of at least three replicates.

The number of mice was estimated to be sufficient on the basis of pilot experiments. Data distribution was assumed to be normal, but this was not formally tested. Data were estimated to be statistically significant when *P* ≤ 0.05 by Student's t-tests and one-way or two-way ANOVAs with repeated measures when appropriate. Significant ANOVA s were followed by a two-way repeated-measures ANOVA with a post hoc Tukey's honestly significant difference (HSD) multiple comparison test for pairwise differences between each mouse group. Data were analyzed using GraphPad Prism version 9 software. For each analyzed animal, the injection site was verified by immunohistochemistry or by qRT-PCR to check the downregulation of the targeted gene. In the case that these verifications were not correct (not injected in the hippocampus or downregulation <10%), animals were excluded.

Group sizes were determined after performing a power calculation to lead to an 80% chance of detecting a significant difference (*P* ≤ 0.05). To avoid cage effect, mice were allocated to different cages using simple randomization.

Behavioral experiments were carried out in a blinded fashion. Investigators were blinded during experimental procedures, data

collection and data analysis by assigning codes (prepared by other investigators irrelevant to this study) to mice, mouse cages, cell samples and images before processing, to ensure unbiased analysis.

## Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

## Data availability

Raw data of the proteomic analysis have been deposited in PRIDE under accession code PXD056975. All other data are available from the corresponding author upon reasonable request.

## Code availability

The code used for the analysis of the proteomic data is available on Dryad at https://doi.org/10.5061/dryad.dfn2z35bh.

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## Acknowledgements

We thank N. Dupont for critical suggestions during the course of the study. We thank the Viral Production, Imaging, Proteomics, Histology, Behavioral and Metabolism and Animal House Facility platforms of SFR Necker. This work was supported by FRM Labellisation (F.O. and M.R.), HFSP (F.O.), ANR (ciliophaging) (F.O. and E. Morel), the NRJ Foundation for Neuroscience (F.O.), the ATIP-AVENIR Program (F.O.), Emergence Ville de Paris (F.O.), AGEMED-INSERM (F.O.), INSERM-Transfer (F.O.), FSER (F.O.), MSCA (D.R.-G.) and the InterAging program (F.O. and V. Blanchet).

## Author contributions

V. Blanchet and F.M. contributed equally. M.R., D.R.-G., V. Blanchet, E. Morel, P.C., A.-S.A. and F.O. designed the experiments. M.R., D.R.-G., F.M., V. Blanchet and V. Boitez performed the experiments, with the help of N.G. and E.S. for the imagery, N.B. and M.R.B. for the preparation of primary hippocampal neurons, V. Boitez and A.-S.A. for the molecular analyses, S.M. and F.M. for the stereotactic surgeries and behavioral analyses, C.G. for the proteomic analyses and S.S. and D.M. for the bioinformatic analyses. M.R., D.R.-G., E. Morel, A.-S.A., M.L., C.G., S.S. and F.O. analyzed and interpreted the data. E. Morselli, P.C., E. Morel, A.-S.A. and F.O. conceived the study and wrote the manuscript, with input from all authors.

## Competing interests

The authors declare no competing interests.

## Additional information

**Extended data** is available for this paper at
https://doi.org/10.1038/s43587-024-00791-0.

**Supplementary information** The online version contains supplementary material available at
https://doi.org/10.1038/s43587-024-00791-0.

**Correspondence and requests for materials** should be addressed to Franck Oury.

**Peer review information** *Nature Aging* thanks Ji Eun Lee, Evandro Fang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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© The Author(s) 2025

<footnote n="1">¹Université Paris Cité, INSERM UMR-U1151, CNRS UMR-8253, Institut Necker Enfants Malades, Team 8, Paris, France. ²MedInsights SAS, Paris, France.</footnote>

<footnote n="3">³Platform for Image Analyses, Structure Fédérative de Recherche Necker, INSERM US24/CNRS UAR 3633, Paris, France. ⁴Platform for Neurobehavioral and Metaboblism, Institut Imagine, Structure Fédérative de Recherche Necker, 26 INSERM US24/CNRS UAR, Paris, France. ⁵Platform for Proteomic Analyses, Structure Fédérative de Recherche Necker, INSERM US24/CNRS UAR 3633, Paris, France. ⁶Université Paris Cité, INSERM UMR-U1151, CNRS UMR-8253, Institut Necker Enfants Malades, Team 6, Paris, France. ⁷Department of Basic Sciences, Faculty of Medicine and Sciences, Universidad San Sebastián, Santiago, Chile. ⁸Present address: Institute for Experimental and Clinical Pharmacology and Toxicology, Center of Brain, Behavior and Metabolism, University of Lübeck, Lübeck, Germany. ⁹These authors contributed equally: Manon Rivagorda, David Romeo-Guitart. ¹⁰These authors jointly supervised this work: Anne-Sophie Armand, Franck Oury. ✉e-mail: franck.oury@inserm.fr</footnote>
A

<figure><img src="image_66.png" /></figure>

B

<figure><img src="image_67.png" /></figure>

C

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D

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E

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F

<figure><img src="image_71.png" /></figure>

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G

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H

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I

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J

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**Extended Data Fig. 1** | GPR158 is present at the PC of hippocampal neurons.

A) PCA biplot showing that the proteomic datasets collected after stereotactic injections with either vehicle (NaCl) or OCN are orthogonal to each other. B) Bar graph representation of the top upregulated signaling pathways in differential analysis of hippocampi 4 h after stereotactic injections with either vehicle or OCN. C) Protein ontology network associated to PC-core proteins and PC functions enriched in the hippocampus after OCN treatment. D) Western blot analysis and quantification of LC3B-II accumulation in primary neurons infected with lentivirus expressing either shRNA-Gpr158 or shRNA-scramble. Independent neuronal cultures (n = 3) were treated with either vehicle, OCN, bafilomycin A1 (Baf), or OCN + Baf. E) Western blot analysis of LC3B-I, LC3B-II and IFT20 in WT and Gpr158/- mouse hippocampi 4 h after hippocampal stereotactic injections with either vehicle or OCN. F) Schematic representation

of the 5 discrete ciliary targeting (VXPX motif) sequences in the C-terminal tail of GPR158. We have generated a mutated Gpr158 version. G) Proportion of neuronal primary cilium presenting GPR158+ and ACIII+ puncta. H) Representative images of GPR158 staining in hippocampal cultures derived from either WT or Gpr158/- mice. Scale bar=10µm. I-J) Representative images of co-immunofluorescence for GPR158 (green) and ACIII (orange) in hippocampal neurons from 3 independent experiments (I) and in WT hippocampal cross-section of the CA3 region of mice treated with OCN from 2 independent experiments (J). 1, 2, and 3 squares in I are 3D rendering images of GPR158 localization in PC from mature hippocampal neurons treated with vehicle or OCN. Scale bar=5µm. In all western blots, β-actin was used as a loading control for each sample. Data are expressed as mean ± SEM. The p values were determined by a one-tailed Student's t-test.
<figure><img src="image_77.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_78.png" /></figure>

**Extended Data Fig. 2** | OCN enhances the level of core PC-proteins.

A) Representative Western blot and quantification of IFT20, IFT88, IFT25, KIF3A and TULP3 in the hippocampus 4 h after local stereotactic injections of vehicle or OCN. B) Representative Western blot and quantification of KIF3A, IFT20 and

LC3B-II accumulation in 3-month-old WT, *Ocn/-* or *Gpr158/-* mouse hippocampi. In all western blots, β-actin was used as a loading control for each sample. The p values were determined by a one-tailed (B) or two-tailed (A) Student's t-test.
<figure><img src="image_79.png" /><figcaption>A</figcaption></figure>

Ocn-/-

<figure><img src="image_80.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_81.png" /></figure>

Gpr158-/-

<figure><img src="image_82.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_83.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_84.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_85.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_86.png" /></figure>

Extended Data Fig. 3 | See next page for caption.
**Extended Data Fig. 3** | Reciprocal regulation of the GPR158-OCN coupling system and core PC protein levels. **A–C)** Relative gene expression of Ift20, Ift88, Ift25, Kif3a and Tulp3 in the hippocampus of either Ocn-/- (A), Gpr158-/- (B) or WT after stereotactic injections of OCN (C). The quantification is relative to WT (A-B) or WT after vehicle stereotactic injections (C). **D–E)** Relative gene expression (RT-qPCR) of either Ift20 or Kif3a and representative Western blots of IFT20, KIF3A and GPR158 in 3-month-old mice hippocampi, 3 weeks after hippocampal stereotactic injections with either AAV-U6-shRNA-Ift20 (D), AAV-U6-shRNA-Kif3a

(E) or their respective controls. β-actin was used as a loading control for WB analyses. **F)** LC3B and SQSTM1/p62 immunofluorescence and punctae quantification performed on brain cross sections at the level of the hippocampal Dentate Gyrus (DG) performed in 3-month-old mice, 3 weeks after local stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-U6-shRNA-Kif3a or AAV-U6-shRNA-Scramble. Scale bar: 7 µm. Data are expressed as mean ± SEM. The p-values were determined by a two-tailed Student's t-test compared to control (vehicle or U6-shRNA-Scrb depending on the experiment).
A

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Training

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Testing

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<figure><img src="image_90.png" /></figure>

B

<figure><img src="image_91.png" /></figure>

□ U6-shRNA-Scrb. (n=10)

■ U6-shRNA-*Ift20* (n=10)

<figure><img src="image_92.png" /></figure>

□ U6-shRNA-Scrb. (n=12)

■ U6-shRNA-*Kif3a* (n=9)

<figure><img src="image_93.png" /></figure>

C

<figure><img src="image_94.png" /></figure>

□ U6-shRNA-Scrb. (n=10)

■ U6-shRNA-*Ift20* (n=9)

<figure><img src="image_95.png" /></figure>

□ U6-shRNA-Scrb. (n=10)

■ U6-shRNA-*Kif3a* (n=10)

<figure><img src="image_96.png" /></figure>

D

eSYN-shRNA-*Ift88*

<figure><img src="image_97.png" /></figure>

<figure><img src="image_98.png" /></figure>

□ eSYN-shRNA-Scrb. (n=8)

■ eSYN-shRNA-*Ift88* (n=10)

<figure><img src="image_99.png" /></figure>

E

CFC

<figure><img src="image_100.png" /></figure>

Training

<figure><img src="image_101.png" /></figure>

Testing

<figure><img src="image_102.png" /></figure>

F

<figure><img src="image_103.png" /></figure>

□ eSYN-shRNA-Scrb. (n=6)

■ eSYN-shRNA-*Ift20-mir* (n=6)

G

<figure><img src="image_104.png" /></figure>

□ eSYN-shRNA-Scrb. (n=10)

■ eSYN-shRNA-*Ift20-mir* (n=10)

<figure><img src="image_105.png" /></figure>
**Extended Data Fig. 4** | Hippocampal downregulation of core-PC proteins does not affect exploratory- and anxiety-like behaviors. **A)** As control, preference index for the (left versus right) object location during the training phase of the NOR was measured in 3-month-old mice, 3 weeks after hippocampal stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-U6-shRNA-Kif3a, or their respective AAV-U6-shRNA-Scramble. The NOR was performed in two independent experiments for each group and its respective control. **B–C)** Open field test (OFT) (**B**) and Light-dark test (LDT) (**C**) performed in 3-month-old mice, 3 weeks after hippocampal stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-U6-shRNA-Kif3a, or their respective AAV-U6-shRNA-Scramble. For the OFT, time spent in the center of the arena (%), total ambulation (meter), and speed (cm/s) were measured. For the LDT, number of entries in the light compartment, latency to reach the light box (seconds) and distance spent in the light box (%) were measured. These analyses were performed on 1 cohort of mice for each group. **D)** Representative western blot and quantification of IFT88 in

mouse hippocampi, 3 weeks after stereotactic injections with either eSYN-shRNA-Ift88-mir or eSYN-shRNA-Scramble-mir. β-actin was used as a loading control for each sample. This analysis was performed on 2 independent cohorts of mice for each group. **E)** Contextual Fear Conditioning test (CFC) performed in 3-month-old mice, 3 weeks after stereotactic injections with either eSYN-shRNA-Ift88-mir or eSYN-shRNA-Scramble-mir. Percent freezing was measured for the training and testing phases. The CFC was performed in 2 independent cohorts of mice. **F–G)** Preference index for the (left versus right) object location during the training phase of the NOR (**F**), and number of entries in the light compartment and latency to reach the light box (seconds) during the Light-dark test (**LDT**) (**G**) performed in 3 month-old mice, 3 weeks after hippocampal stereotactic injections with either eSYN-shRNA-Ift20-mir or eSYN-shRNA-Scramble-mir. Data are expressed as mean ± SEM. The p values were determined by a two-tailed Student's t-test compared to U6-shRNA-Scrb in B and C or to eSYN-shRNA-Scrb in D, E and G.
<figure><img src="image_106.png" /><figcaption>A</figcaption></figure>

3 Mo (n=6)

16 Mo (n=6)

<figure><img src="image_107.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_108.png" /></figure>

<figure><img src="image_109.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_110.png" /><figcaption>**Extended Data Fig. 5** | Restoration of neuronal IFT20 levels in old hippocampi reverses age-related cognitive deficits, without changing PC size.</figcaption></figure>

A) Primary cilia immunofluorescent staining (ACIII antibody) in the DG region of the hippocampus of from young (3Mo) and aged (16Mo) mice. The length of the primary cilia was measured and quantified in 3D using the Imaris software. Scale bar: 5 µm.
B) Primary cilia length quantification obtained after immunofluorescent staining (ACIII) in the CA3 (left panel) and DG (right panel) regions of the hippocampus of 16 month-old mice, 3 weeks after local

stereotactic injections with either AAV-Ift20 cDNA (mIft20) or AAV-GFP (Control). The length of the primary cilia was measured and quantified in 3D using the Imaris software. Scale bar: 10 µm.
C) Morris water maze test (MWM) performed in 3- and 16-month-old mice, 3 weeks after local stereotactic injections with either AAV-Ift20 cDNA or AAV-GFP (Control). The MWM was performed in 1 cohort of mice. Data are expressed as mean ± SEM. The p values were determined by a two-tailed Student's t-test compared to 3 Mo in A and by a Two-way ANOVA with multiple comparison in C.
<figure><img src="image_111.png" /></figure>

<figure><img src="image_112.png" /></figure>

**Extended Data Fig. 6** | OCN treatment in old mice improves core PC-protein levels in hippocampus, without reversing PC length. A) Representative western blot image and quantification of IFT20, KIF3A, and TULP3 in 16-month-old mice 4 h after hippocampal stereotactic injection of either vehicle or OCN. β-actin was used as a loading control for each sample. The quantification is relative to Veh. injected group. B) Primary cilia length quantification obtained after immunofluorescent staining (ACIII antibody) in the CA3 (left panel) region of

the hippocampus of 3 and 16-month-old mice after peripheral chronic infusion (peripheral osmotic minipumps) with either vehicle or OCN. The length of the primary cilia was measured and quantified in 3D using the Imaris software. Scale bar: 10 µm. Data are expressed as mean ± SEM. The p values were determined by a two-tailed Student's t-test compared to veh in A and by a one-way ANOVA with multiple comparison followed by a Tukey test in B.
A

<figure><img src="image_113.png" /></figure>

B

<figure><img src="image_114.png" /></figure>

C

<figure><img src="image_115.png" /></figure>

D

<figure><img src="image_116.png" /></figure>

Extended Data Fig. 7 | OCN enhances CREB phosphorylation and LC3B levels in the CA3 region of hippocampus. A-B) Phospho-CREB (A) and LC3B (B) immunofluorescence and quantification (relative intensity) performed on brain cross-sections (hippocampal CA3, Dentate Gyrus (DG) and CA1 regions) of 3-month-old mice 4 h after hippocampal stereotactic injection with either vehicle or OCN. Puncta analysis was performed using Icy Software. Scale bar: 50 µm. C) Phospho-CREB immunofluorescence and quantification (relative intensity) performed on brain cross-sections (hippocampal DG region) of

3- and 16-month-old mice, 3 weeks after local stereotactic injections with either AAV-GFP (Control) or AAV-mlft20 cDNA. The analysis was performed in 1 cohort of mice. Scale bar: 20 µm. D) Relative gene expression of c-Fos in the hippocampi of 3-month-old mice previously injected in the hippocampus with either AAV-eSYN-shRNA-Ift20mir or eSYN-shRNA-Scramble-mir. The mice were subjected to stereotactic injection of either vehicle or OCN. Data are expressed as mean ± SEM. The p values were determined by a one-tailed (A, B, D) or a two-tailed (C) Student's t-test.
<figure><img src="image_117.png" /></figure>

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<table><tr><td>Study description</td><td>Briefly describe the study. For quantitative data include treatment factors and interactions, design structure (e.g. factorial, nested, hierarchical), nature and number of experimental units and replicates.</td></tr><tr><td>Research sample</td><td>Describe the research sample (e.g. a group of tagged Passer domesticus, all Stenocereus thurberi within Organ Pipe Cactus National Monument), and provide a rationale for the sample choice. When relevant, describe the organism taxa, source, sex, age range and any manipulations. State what population the sample is meant to represent when applicable. For studies involving existing datasets, describe the data and its source.</td></tr><tr><td>Sampling strategy</td><td>Note the sampling procedure. Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.</td></tr><tr><td>Data collection</td><td>Describe the data collection procedure, including who recorded the data and how.</td></tr><tr><td>Timing and spatial scale</td><td>Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale for these choices. If there is a gap between collection periods, state the dates for each sample cohort. Specify the spatial scale from which the data are taken</td></tr><tr><td>Data exclusions</td><td>If no data were excluded from the analyses, state so OR if data were excluded, describe the exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.</td></tr><tr><td>Reproducibility</td><td>Describe the measures taken to verify the reproducibility of experimental findings. For each experiment, note whether any attempts to repeat the experiment failed OR state that all attempts to repeat the experiment were successful.</td></tr><tr><td>Randomization</td><td>Describe how samples/organisms/participants were allocated into groups. If allocation was not random, describe how covariates were controlled. If this is not relevant to your study, explain why.</td></tr><tr><td>Blinding</td><td>Describe the extent of blinding used during data acquisition and analysis. If blinding was not possible, describe why OR explain why blinding was not relevant to your study.</td></tr></table>

Did the study involve field work?

Yes

No

## Field work, collection and transport

<table><tr><td>Field conditions</td><td>Describe the study conditions for field work, providing relevant parameters (e.g. temperature, rainfall).</td></tr><tr><td>Location</td><td>State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).</td></tr><tr><td>Access & import/export</td><td>Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).</td></tr></table>
# Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

## Materials & experimental systems

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☐</td><td>Antibodies</td></tr><tr><td>☐</td><td>Eukaryotic cell lines</td></tr><tr><td>☒</td><td>Palaeontology and archaeology</td></tr><tr><td>☒</td><td>Animals and other organisms</td></tr><tr><td>☒</td><td>Clinical data</td></tr><tr><td>☒</td><td>Dual use research of concern</td></tr><tr><td>☒</td><td>Plants</td></tr></tbody></table>

## Methods

<table><thead><tr><th>n/a</th><th>Involved in the study</th></tr></thead><tbody><tr><td>☒</td><td>ChIP-seq</td></tr><tr><td>☒</td><td>Flow cytometry</td></tr><tr><td>☒</td><td>MRI-based neuroimaging</td></tr></tbody></table>

## Antibodies

<table><tr><td>Antibodies used</td><td>Primary and secondary antibodies for Western Blot:<br/>Mouse monoclonal anti-β-actin (Sigma, Cat# A-2228, cloneAC-74, 1:5000 dilution); Rabbit polyclonal anti-IFT20 (Proteintech: Cat# 13615, 1:1000 dilution); Rabbit polyclonal anti-IFT88 (Proteintech: Cat# 13967-1-AP, 1:1000 dilution); Rabbit polyclonal anti-KIF3A (Proteintech, Cat# 13930, 1:1000 dilution); Rabbit polyclonal anti-IFT25 (Proteintech, Cat# 15732, 1:1000 dilution); Rabbit polyclonal anti-TULP3 (Proteintech, Cat# 13637 1:1000 dilution); Rabbit polyclonal anti-Gpr158 (Antibodies-Online, Cat# 6258978, 1:1000 dilution); Mouse monoclonal anti-Beclin1 (BD Transduction Laboratories, Cat# 612113, clone 20, 1:1000 dilution); Rabbit polyclonal anti-LC3b, (Sigma-Aldrich, Cat# L7543, 1:10000 dilution); Guinea Pig polyclonal anti-p62 (Progen, Cat# GP62-C, 1:2000 dilution); Rabbit monoclonal anti-CREB (Cell Signaling: Cat #9197, Clone: 48H2, 1:1000 dilution); Rabbit polyclonal anti-phospho-CREB (Ser133), Merck Millipore: Cat #06-519, 1:1000 dilution)(Horseradish peroxidase-conjugated secondary antibodies): Mouse IgG (HRP-linked antibody, Cell Signaling: Cat# 7076, 1:1000 dilution); Rabbit IgG (HRP-linked antibody, Cell Signaling: Cat# 7074, 1:1000 dilution); Guinea pig IgG (HRP-linked antibody, Sigma-Aldrich: Cat# A5545, 1:1000 dilution)<br/>Primary antibodies and secondary antibodies for Immunofluorescence:<br/>Rabbit polyclonal anti-TULP3 (Proteintech, Cat# 13637 1:100 dilution); Rabbit polyclonal anti-LC3b (Sigma-Aldrich, Cat# L7543 1:200 dilution); Guinea Pig polyclonal anti-p62 (Progen, Cat# GP62-C, 1:200 dilution); Mouse monoclonal anti-Gpr158 (Bio Techne, Cat# MAB10286-100, Clone # 1027604, 1:100 dilution); Rabbit polyclonal anti-phospho-CREB (Ser133) (Merck Millipore, Cat #06-519, 1:500 dilution); Rabbit polyclonal anti-ACIII (ThermoFisher, Cat# PAS-35382, 1:400 dilution); Mouse monoclonal anti-NeuN (Sigma-Aldrich, Cat# MAB377, clone A60, 1:400 dilution); Chicken polyclonal anti-MAP2 (ThermoFisher, Cat# PA1-10005, 1:500 dilution) Alexa Fluor 647 Donkey anti-rabbit IgG (Thermo Fisher: Cat# A-31573, 1:200); Alexa Fluor 647 Donkey anti-chicken IgY (Thermo Fisher Cat# A-78952, 1/200); Cy3 AffiniPure Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch, Cat# 711165152, 1:200); Alexa fluor 488 Donkey Anti-rabbit (Thermo Fisher: Cat# R37118, 1:200); Alexa Fluor 546 Donkey anti-Rabbit IgG (Thermo Fisher: Cat# A10040, 1:200); Alexa Fluor 555 Goat anti-Guinea Pig IgG (H+L) (Thermo Fisher: Cat# A-21435 1:200); Alexa fluor 488 Donkey Anti-mouse (Thermo Fisher: Cat# A-21202, 1:200)<br/>Co-Immunoprecipitation:<br/>Rabbit IgG control Polyclonal antibody ((Proteintech: Cat# 30000-0-AP, 1 µg)</td></tr><tr><td>Validation</td><td>All the antibodies used in this study are commercially available and validation were performed by the manufacturers and supported by the publications indicated in the manufacturer's websites</td></tr></table>

## Eukaryotic cell lines

Policy information about cell lines and Sex and Gender in Research

<table><tr><td>Cell line source(s)</td><td>Murine Neuroblastoma N2a cells were purchased from ATCC. This cell line was validated by the company. Primary neurons were obtained from digested mouse embryonic hippocampi as described in Methods. 1uM of Ara-C inhibitor was used to decrease the presence of glial cells in the culture.</td></tr><tr><td>Authentication</td><td>N2a cells were authenticated based on morphology and growth characteristics after treatment with retinoic acid. Primary Mature hippocampal neurons were authenticated by the presence of MAP2 by immunofluorescence</td></tr><tr><td>Mycoplasma contamination</td><td>N2a cell line was negative for mycoplasma</td></tr><tr><td>Commonly misidentified lines (See ICLAC register)</td><td>No commonly misidentified cell line was used in this study.</td></tr></table>
# Palaeontology and Archaeology

**Specimen provenance**

Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information). Permits should encompass collection and, where applicable, export.

**Specimen deposition**

Indicate where the specimens have been deposited to permit free access by other researchers.

**Dating methods**

If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.

☐ Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

**Ethics oversight**

Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance was required and explain why not.

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Animals and other research organisms

Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

**Laboratory animals**

All experiments were performed on C57BL/6J WT male mice (obtained from Janvier Laboratory, Le Genest St Isle, France). All mice were 3 or 16 months of age at the start of experiments. For all experiments, we used littermates as controls. Male mice were housed 5 animals polycarbonate cage (35.5 x 18 x 12.5 cm), under a 12-h light/dark cycle with ad libitum access to food and water prior to experimentation. Room temperature: 20-26°C; daily temperature difference: ≤4°C; relative humidity: 40%-70%

**Wild animals**

No wild animals were used for this study.

**Reporting on sex**

In vivo experiments (3 Mo and 16 Mo) were performed only in male mice.

**Field-collected samples**

No field-collected samples were used for this study.

**Ethics oversight**

All behavioral experiments were performed in accordance with the European Communities for Experimental animal use (2010/63/EU) and local ethical committee review procedures and protocols (APAFIS-25139).

Note that full information on the approval of the study protocol must also be provided in the manuscript.

# Clinical data

Policy information about clinical studies

All manuscripts should comply with the ICMJE guidelines for publication of clinical research and a completed CONSORT checklist must be included with all submissions.

**Clinical trial registration**

Provide the trial registration number from ClinicalTrials.gov or an equivalent agency.

**Study protocol**

Note where the full trial protocol can be accessed OR if not available, explain why.

**Data collection**

Describe the settings and locales of data collection, noting the time periods of recruitment and data collection.

**Outcomes**

Describe how you pre-defined primary and secondary outcome measures and how you assessed these measures.

# Dual use research of concern

Policy information about dual use research of concern

## Hazards

Could the accidental, deliberate or reckless misuse of agents or technologies generated in the work, or the application of information presented in the manuscript, pose a threat to:
No

Yes

<figure><img src="image_122.png" /></figure>

<figure><img src="image_123.png" /></figure>

Public health

<figure><img src="image_124.png" /></figure>

<figure><img src="image_125.png" /></figure>

National security

<figure><img src="image_126.png" /></figure>

<figure><img src="image_127.png" /></figure>

Crops and/or livestock

<figure><img src="image_128.png" /></figure>

<figure><img src="image_129.png" /></figure>

Ecosystems

<figure><img src="image_130.png" /></figure>

<figure><img src="image_131.png" /></figure>

Any other significant area

## Experiments of concern

Does the work involve any of these experiments of concern:

No

Yes

<figure><img src="image_132.png" /></figure>

<figure><img src="image_133.png" /></figure>

Demonstrate how to render a vaccine ineffective

<figure><img src="image_134.png" /></figure>

<figure><img src="image_135.png" /></figure>

Confer resistance to therapeutically useful antibiotics or antiviral agents

<figure><img src="image_136.png" /></figure>

<figure><img src="image_137.png" /></figure>

Enhance the virulence of a pathogen or render a nonpathogen virulent

<figure><img src="image_138.png" /></figure>

<figure><img src="image_139.png" /></figure>

Increase transmissibility of a pathogen

<figure><img src="image_140.png" /></figure>

<figure><img src="image_141.png" /></figure>

Alter the host range of a pathogen

<figure><img src="image_142.png" /></figure>

<figure><img src="image_143.png" /></figure>

Enable evasion of diagnostic/detection modalities

<figure><img src="image_144.png" /></figure>

<figure><img src="image_145.png" /></figure>

Enable the weaponization of a biological agent or toxin

<figure><img src="image_146.png" /></figure>

<figure><img src="image_147.png" /></figure>

Any other potentially harmful combination of experiments and agents

## Plants

Seed stocks

Report on the source of all seed stocks or other plant material used. If applicable, state the seed stock centre and catalogue number. If plant specimens were collected from the field, describe the collection location, date and sampling procedures.

Novel plant genotypes

Describe the methods by which all novel plant genotypes were produced. This includes those generated by transgenic approaches, gene editing, chemical/radiation-based mutagenesis and hybridization. For transgenic lines, describe the transformation method, the number of independent lines analyzed and the generation upon which experiments were performed. For gene-edited lines, describe the editor used, the endogenous sequence targeted for editing, the targeting guide RNA sequence (if applicable) and how the editor was applied.

Authentication

Describe any authentication procedures for each seed stock used or novel genotype generated. Describe any experiments used to assess the effect of a mutation and, where applicable, how potential secondary effects (e.g. second site T-DNA insertions, mosiacism, off-target gene editing) were examined.

## ChIP-seq

### Data deposition

<figure><img src="image_148.png" /></figure>

Confirm that both raw and final processed data have been deposited in a public database such as **GEO**.

<figure><img src="image_149.png" /></figure>

Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links

May remain private before publication.

For "Initial submission" or "Revised version" documents, provide reviewer access links. For your "Final submission" document, provide a link to the deposited data.

Files in database submission

Provide a list of all files available in the database submission.

Genome browser session

(e.g. UCSC)

Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, to enable peer review. Write "no longer applicable" for "Final submission" documents.

### Methodology

Replicates

Describe the experimental replicates, specifying number, type and replicate agreement.

Sequencing depth

Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads and whether they were paired- or single-end.

Antibodies

Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.

Peak calling parameters

Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.
Data quality

Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.

Software

Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.

# Flow Cytometry

## Plots

Confirm that:

<figure><img src="image_150.png" /></figure>

The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).

<figure><img src="image_151.png" /></figure>

The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).

<figure><img src="image_152.png" /></figure>

All plots are contour plots with outliers or pseudocolor plots.

<figure><img src="image_153.png" /></figure>

A numerical value for number of cells or percentage (with statistics) is provided.

## Methodology

Sample preparation

Describe the sample preparation, detailing the biological source of the cells and any tissue processing steps used.

Instrument

Identify the instrument used for data collection, specifying make and model number.

Software

Describe the software used to collect and analyze the flow cytometry data. For custom code that has been deposited into a community repository, provide accession details.

Cell population abundance

Describe the abundance of the relevant cell populations within post-sort fractions, providing details on the purity of the samples and how it was determined.

Gating strategy

Describe the gating strategy used for all relevant experiments, specifying the preliminary FSC/SSC gates of the starting cell population, indicating where boundaries between "positive" and "negative" staining cell populations are defined.

<figure><img src="image_154.png" /></figure>

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

# Magnetic resonance imaging

## Experimental design

Design type

Indicate task or resting state; event-related or block design.

Design specifications

Specify the number of blocks, trials or experimental units per session and/or subject, and specify the length of each trial or block (if trials are blocked) and interval between trials.

Behavioral performance measures

State number and/or type of variables recorded (e.g. correct button press, response time) and what statistics were used to establish that the subjects were performing the task as expected (e.g. mean, range, and/or standard deviation across subjects).

## Acquisition

Imaging type(s)

Specify: functional, structural, diffusion, perfusion.

Field strength

Specify in Tesla

Sequence & imaging parameters

Specify the pulse sequence type (gradient echo, spin echo, etc.), imaging type (EPI, spiral, etc.), field of view, matrix size, slice thickness, orientation and TE/TR/flip angle.

Area of acquisition

State whether a whole brain scan was used OR define the area of acquisition, describing how the region was determined.

Diffusion MRI

<figure><img src="image_155.png" /></figure>

Used

<figure><img src="image_156.png" /></figure>

Not used

## Preprocessing

Preprocessing software

Provide detail on software version and revision number and on specific parameters (model/functions, brain extraction, segmentation, smoothing kernel size, etc.).

Normalization

If data were normalized/standardized, describe the approach(es): specify linear or non-linear and define image types used for transformation OR indicate that data were not normalized and explain rationale for lack of normalization.
**Normalization template**

Describe the template used for normalization/transformation, specifying subject space or group standardized space (e.g. original Talairach, MNI305, ICBM152) OR indicate that the data were not normalized.

**Noise and artifact removal**

Describe your procedure(s) for artifact and structured noise removal, specifying motion parameters, tissue signals and physiological signals (heart rate, respiration).

**Volume censoring**

Define your software and/or method and criteria for volume censoring, and state the extent of such censoring.

## Statistical modeling & inference

**Model type and settings**

Specify type (mass univariate, multivariate, RSA, predictive, etc.) and describe essential details of the model at the first and second levels (e.g. fixed, random or mixed effects; drift or auto-correlation).

**Effect(s) tested**

Define precise effect in terms of the task or stimulus conditions instead of psychological concepts and indicate whether ANOVA or factorial designs were used.

**Specify type of analysis:**

<figure><img src="image_157.png" /></figure>

Whole brain

<figure><img src="image_158.png" /></figure>

ROI-based

<figure><img src="image_159.png" /></figure>

Both

**Statistic type for inference**

Specify voxel-wise or cluster-wise and report all relevant parameters for cluster-wise methods.

(See Eklund et al. 2016)

**Correction**

Describe the type of correction and how it is obtained for multiple comparisons (e.g. FWE, FDR, permutation or Monte Carlo).

## Models & analysis

n/a

Involved in the study

<figure><img src="image_160.png" /></figure>

<figure><img src="image_161.png" /></figure>

Functional and/or effective connectivity

<figure><img src="image_162.png" /></figure>

<figure><img src="image_163.png" /></figure>

Graph analysis

<figure><img src="image_164.png" /></figure>

<figure><img src="image_165.png" /></figure>

Multivariate modeling or predictive analysis

**Functional and/or effective connectivity**

Report the measures of dependence used and the model details (e.g. Pearson correlation, partial correlation, mutual information).

**Graph analysis**

Report the dependent variable and connectivity measure, specifying weighted graph or binarized graph, subject- or group-level, and the global and/or node summaries used (e.g. clustering coefficient, efficiency, etc.).

**Multivariate modeling and predictive analysis**

Specify independent variables, features extraction and dimension reduction, model, training and evaluation metrics.