V). Underlying alterations in calcium channel localization and function are KV2.1 channels whose interactions drive calcium channel clustering to enhance calcium entry and fuel neurotoxic elevations in mitochondrial calcium. Targeted disruption of KV2–CaV interactions rescues aberrant CaV1.2 clustering, elevated mitochondrial calcium, and neurotoxicity in vitro. Our findings provide evidence that NPC is a nanostructural ion channel clustering disease, characterized by altered distribution and activity of ion channels at membrane contacts, which contribute to neurodegeneration.Lysosomes communicate through cholesterol transfer at endoplasmic reticulum (ER) contact sites. At these sites, the Niemann Pick C1 cholesterol transporter (NPC1) facilitates the removal of cholesterol from lysosomes, which is then transferred to the ER for distribution to other cell membranes. Mutations in NPC1 result in cholesterol buildup within lysosomes, leading to Niemann-Pick Type C (NPC) disease, a progressive and fatal neurodegenerative disorder. The molecular mechanisms connecting NPC1 loss to NPC-associated neuropathology remain unknown. Here we show both in vitro and in an animal model of NPC disease that the loss of NPC1 function alters the distribution and activity of voltage-gated calcium channels (Ca The molecular mechanisms connecting Niemann Pick C1 cholesterol transporter (NPC1) loss to Niemann-Pick Type C neuropathology remain unknown. Here, the authors demonstrate that loss of NPC1 function alters the nanoscale distribution and function of ion channels to promote abnormal calcium entry, mitochondrial dysfunction, and neurotoxicity. Given the importance of maintaining cholesterol homeostasis for controlled neuronal function, it is not surprising that mutations that disrupt cholesterol production or transport result in severe abnormalities of the central nervous system (CNS) and neurodegeneration9.Cholesterol, an essential sterol, is particularly abundant in the brain10 with neurons drawing their homeostatic requirements from two sources (i) de novo synthesis in the Endoplasmic Reticulum (ER), and (ii) uptake of the externally derived cholesterol-conjugated lipoproteins. External cholesterol is imported into neurons in a receptor-dependent manner and trafficked to late endosomes /lysosomes11. At these vesicular structures, the Niemann Pick type C (NPC) proteins are key players in mobilizing cholesterol to the ER and other cellular membranes13. Specifically, following liberation by acidic lipases in the lysosome lumen, cholesterol binds NPC2, which subsequently interacts with the transmembrane NPC1 cholesterol transporter to facilitate the egress of cholesterol across the lysosome membrane to its cytoplasmic leaflet before subsequent transfer to the ER at lysosome–ER Membrane Contact Sites (MCSs). The importance of the NPC1 protein is clear; physiologically, it is a key gatekeeper for cholesterol homeostasis and in doing so tunes mTORC1 activity, lipid transfer at membrane contact sites, and Ca2+ signaling17. Pathophysiologically, missense mutations in NPC1 lead to the progressive neurodegenerative disorder NPC disease. This terminal condition has no cure and is characterized by the progressive neurodegeneration of several brain regions and a host of devastating symptoms including seizures, psychiatric problems, and dementia. Despite its ubiquitous expression, key role in cholesterol transport, and fundamental requirement for human health, the molecular mechanism(s) linking loss of NPC1 function to neurodegeneration are unknown.In the brain, all cholesterol is made locally22 that use lipids and Ca2+ as “currency” to communicate information. A key signaling lipid that is transferred at ER–lysosome MCSs is cholesterol. The NPC1 protein not only influences cholesterol transport at lysosome–ER MCSs but is also a master regulator of other MCSs within mammalian cells24. Loss of function or disease mutations in NPC1 cholesterol transporter results in the remodeling of ER–lysosome15, ER–Golgi16 and ER–Mitochondria17 MCSs to influence mTORC1 signaling, anterograde trafficking to the Plasma Membrane (PM), and mitochondrial Ca2+, respectively. That said, there is little information as to how lysosomal cholesterol transport alters the molecular choreography of neuronal ER–PM junctions. This is important because (i) 40–90% of cellular cholesterol is found in the PM25, therefore disruption of NPC1-mediated cholesterol homeostasis would be expected to result in modification of ER–PM MCSs, and (ii) ER–PM MCSs are critical platforms for regulating lipid and Ca2+ homeostasis in neurons31. Despite such importance, we do not know how the loss of cholesterol transport from the lysosome alters ER–PM MCSs, which proteins within these contacts have altered distribution/activity, and the cellular consequences of such changes.A key mechanism through which the lysosome communicates instructions to other organelles is through physical membrane contacts. These intracellular synapses are intimate sites (~10–30 nm) of membrane contact between two or more organellesV2.1. Gating of this voltage-dependent ion channel provides an inhibitory brake on excitability33. However, over the past few years it has been appreciated that a subset of non-conducting KV2 ion channels perform a physical function by generating ER–PM junctions through an association between their C-terminus and ER-localized VAP proteins in a phosphorylation-dependent manner35. Moreover, at ER–PM MCSs, KV2 channels physically interact with and cluster Ca2+ signaling proteins like voltage-gated L-type Ca2+ channels (CaV1)36. CaV1 channels are key neuronal Ca2+ signaling proteins which serve as a principal route of Ca2+ entry at depolarized membrane potentials. Such biophysical properties give CaV1 a prominent role in neuronal excitability and gene expression, which are crucial for neuronal signaling and synaptic plasticity37. Moreover, several neurodegenerative diseases are characterized by excessive Ca2+ influx through CaV1 channels39 with their targeted knockdown or inhibition being neuroprotective42. While KV2-mediated targeting and regulation of CaV1 channels at ER–PM MCSs serves a physiologically important role in neurons, there is no information on how this form of regulation may be altered in disease, including how lysosomal cholesterol may regulate KV2 clustering to tune excitability and Ca2+ signaling at ER–PM MCSs to impact neurodegeneration in NPC disease.In neurons, a key regulator of ER–PM MCSs is the voltage-gated potassium channel KV2.1 clustering at the PM. We show that enhanced Kv2.1-containing ER-PM MCSs are enriched in SERCA, CaV1.2, and RyR resulting in enhanced Ca2+ signaling at KV2.1 associated ER–PM nanodomains. In addition to remodeling of ER–PM MCSs, we also show that Ca2+ handling proteins at ER–Mitochondrial MCSs are modified following loss of NPC1 function. Collaboratively, this molecular transformation of MCSs leads to deviant elevations in mitochondrial Ca2+ and neurotoxicity. Importantly, we demonstrate that uncoupling upstream KV2–CaV1 interactions rescues mitochondrial Ca2+ and neurotoxicity and thus represents a potential therapeutic target in NPC1 disease. Collectively, these data demonstrate that NPC is a nanostructural ion channel clustering disease with altered ion channel distribution/activity at MCSs contributing to neurodegeneration.In this current study, we report that loss of NPC1 function results in phosphorylation-dependent increases in K2+ channels (CaV) permits movement of Ca2+ down its concentration gradient to increase intracellular Ca2+ levels and represents a major mechanism through which neurons translate electrical signals into biochemical ones. Elevations in Ca2+ are tightly regulated in mammalian neurons44 with dysregulation linked to several neuropathologies51. Recently, we have reported that NPC1 inhibition, NPC1 mutations (NPC1I1061T) or NPC1 knockout (NPC1−/−) neurons are hyperexcitable, firing significantly more Action Potentials (APs) compared to age and sex-matched Wild-Type (WT) littermates52. Given the importance of the membrane potential for controlling Ca2+ entry through CaV channels53, we wanted to test if NPC1 loss of function alters voltage-dependent Ca2+ activity. To test this hypothesis, we incubated cortical neurons overnight using a specific pharmacological inhibitor of NPC155 , which increases spontaneous and stimulated voltage responses as well as internal cholesterol accumulation  elevations in Fluo-4 intensity relative to controls  not only abrogated any NPC1-driven increase in basal and electrically evoked Fluo-4 intensities, but also reduced both spontaneous and stimulated cytosolic Ca2+ amplitude elevations compared to control neurons , such that adjacent channels can functionally interact with one another permitting cooperative gating61. As a result, the activity of physically interacting channels in a cluster is driven by the highest open probability channel, thus enhanced channel clustering facilitates the amplification of Ca2+ currents. To test if NPC1 loss of function alters CaV distribution we treated cortical with U18 before fixing and immunolabeling for CaV1.2 and performing super-resolution AiryScan or single molecule localization TIRF microscopy (super-resTIRF). Quantification of total intensity, cluster density, and cluster size from AiryScan confocal regions close to the PM demonstrated that U18-treatment enhanced CaV1.2 clustering in both the soma and dendrites  further corroborated our AiryScan analysis that U18-treatment increased the size and number of PM CaV1.2 clusters  and found that total CaV1.2 protein levels were similar between control and U18-treated neurons  mice for KV2.1. Similar to treatment with U18, mice harboring the NPC1I1061T mutation displayed increased KV2.1 clustering in cortical . The PLA technique allows the detection and quantification of those CaV1.2 and KV2.1 channels that are within 40 nm of one another63. Like super-resTIRF imaging, PLA experiments revealed an increase in the number of CaV1.2–KV2.1 channel clusters within 40 nm of one another, with each hetero-cluster being closer to each other in the neuronal soma following loss of NPC1 function  located in the proximal cytoplasmic C-terminus of KV2.12. To test if CCAD-dependent interactions between KV2.1–CaV1.2 drive increases in CaV1.2 channel clusters following loss of NPC1 function we took advantage of a cell permeant synthetic peptide “HA-TAT-C1aB” (CCAD)66 which selectively uncouples KV2.1–CaV1.2 channel complexes2. Using this synthetic tool, and a corresponding peptide with a scrambled CCAD sequence “TAT-HA-C1aB-Scr” (SCRBL), we compared CaV1.2 distribution in isolated mouse  promoting KV2.1 clustering67  and/or Torin-1 (to inhibit mTORC1) before fixing and labeling for CaV1.2 and KV2.1. Subsequent super-res imaging revealed that U18-dependent increases CaV1.2 and KV2.1 clusters were sensitive to mTORC1 inhibition with KV2.1–CaV1.2 nanocomplexes reduced back to control levels  domain generates a noncanonical FFAT  in an Acidic Tract) motif that facilitates interactions with ER VAP proteins leading to the formation of ER–PM MCSs35. Given the increase in KV2.1 clusters following loss of NPC1 function, we asked if ER–PM MCSs are also altered  MAPPER74, which enable visualization and quantification of ER–PM MCSs in living neurons. The SPLICSS/L-P2AER-PM tool is based on the split-YFP protein fused to either an ER or PM target so it only emits fluorescence upon self-assembly at the ER–PM interface. A short or long spacer placed at the split-YFP ER targeted protein allows for the study of narrow (8–10 nm) or wide (40–50 nm) ER–PM MCSs73. Transfection of this ER-PM sensor into cortical neurons revealed a punctate distribution within either 40 nm or 10 nm of the TIRF footprint that represents the cortical ER in close apposition to the PM  and Sacro/Endoplasmic Reticulum Ca2+-ATPase (SERCA)75. Considering the increases in KV2.1–CaV1.2 complexes  which resulted in neurons being refractory to U18 and having significantly decreased responses relative to control . High-speed imaging of ArchLight-Q239 revealed that U18-treated neurons had similar fluctuations in intensity when compared to U18 and CCAD  at ER–Mito MCSs ensures a steep local Ca2+ gradient for Ca2+ to move into mitochondria to maintain energy production and cell health78. However, deviant mitochondrial Ca2+ (Ca2+Mito) levels can lead to altered bioenergetics, ATP production, and Reactive Oxygen Species (ROS) production, triggering necrosis or apoptosis81. Therefore, we tested the hypothesis that NPC1-dependent elevations in cytoplasmic Ca2+ increases Ca2+Mito and leads to neurotoxicity  Caity Fig. .Fig. 9NP2+ domains were affected by NPC1 function. At ER–Mito MCSs the IP3R and VDAC1 are coupled through interactions with the chaperone protein GRP7582. To determine if the IP3R–GRP75–VDAC1 signaling axis, which has been implicated in the progression of other neurodegenerative diseases like Alzheimer’s, Parkinson’s, and Amyotrophic Lateral Sclerosis, undergoes reorganization in NPC disease we took a two-pronged approach leveraging NPC1−/− cells and neurons treated with U18. First, using control and NPC1-/- cells, or vehicle and U18-treated neurons, we immunolabeled for the GRP75 protein and acquired super-resolution AiryScan confocal images. Fig. −/− cells also showed higher GPR75 intensity levels and larger clusters , and blinded neuronal experiments  levels due to increased ER Ca2+ leak through IP3R123. To test if similar results occur in neurons, we transfected neurons with the ER-targeted GCaMP indicator, GCaMP6-15084, and treated with vehicle control or U18. Aligned with published results, loss of NPC1 function resulted in about a 50 % decrease in GCaMP intensity . To evaluate Ca2+Mito we transfected cortical neurons with a mitochondria-targeted RCaMP plasmid (pCAG-mito-RCaMP1h)85, and incubated neurons with vehicle or U18 for 24 h. Consistent with the model that leaky IP3R1 on ER membranes drive Ca2+ entry into mitochondria, normalized pCAG-mito-RCaMP1h intensities were significantly elevated following the loss of NPC1 function  and found that U18-dependent elevations in Ca2+Mito were abrogated . Consistent with the model that CaV1 channels play a central role in mediating increases in Ca2+Mito in NPC disease, treatment with nifedipine abolished increases in Ca2+Mito  and changes in mitochondrial membrane potential (MMP) therefore we tested if they were also altered in models of NPC. Like Ca2+Mito, both ROS and MMP were differentially changed following NPC1 inhibition with levels of ROS increased  supports that KV2–CaV1 dependent Ca2+ entry is either a stimulus for stabilizing ER–PM MCS or an instructive signal to induce the formation of new ER–PM MCSs in neurons. The latter model that Kv2–CaV1 Ca2+ entry nucleates the formation of new MCSs aligns with previous reports that a number of lipid binding ER–PM MCS forming proteins are sensitive to local changes in cytoplasmic Ca2+ concentrations94 and provides evidence that KV2 channels are a crucial foundational element that regulates multiple sub-populations of ER–PM MCSs.In addition to physically interacting to organize CaV1.2–RyR and KV2.1–SERCA, suggesting that KV2.1-associated ER–PM MCSs are enriched in RyR, SERCA and CaV1.2, consistent with previous proteomics analyses of Kv2.1-containing ER-PM MCSs purified from mouse brain31. Having more CaV1.2 juxtaposed to RyR would amplify Ca2+ entry into the cytoplasm through Ca2+-Induced Ca2+-Release (CICR). Such amplification of Ca2+ signals is important in normal neuronal function as it facilitates excitation-transcription coupling2, however, in NPC disease it is further increased and together with the hyperexcitability phenotype results in aberrant and ultimately neurotoxic (see below) increases in Ca2+. An intuitive prediction of enhanced KV2–CaV1–RyR Ca2+ entry in NPC disease would be an increase in ER Ca2+ levels. The statement is supported by the increase in clustering at ER–PM MCSs of two ER Ca2+ uptake mediator proteins, KV2.1 and SERCA . The molecular mechanism underlying reduced ER Ca2+ in NPC disease is enhanced spontaneous Ca2+ release from IP3R1 in ER membranes17. Evidence herein suggests that heightened RyR CICR activity may also contribute to the leaky ER Ca2+ phenotype in NPC disease.In the present study, we find that NPC1 deficiency leads to increased proximity between CaRCA Fig. 75, and i2+ overload leading to mitochondrial membrane rupture and release of pro-apoptotic factors98. The IP3R–GRP75–VDAC1 signaling axis is a hotspot for Ca2+ flux from the ER towards the mitochondria. Our data provide evidence that ER Ca2+ stores are low whereas mitochondrial Ca2+ is significantly increased in NPC neurons. Furthermore, we find that increases in mitochondrial Ca2+ correlate with alterations in MMP, increased ROS generation, and cell death supporting the model that mitochondrial Ca2+ overload is a mechanism for neurodegeneration in NPC disease. Mechanistically, increased interactions between IP3R–GRP75–VDAC1 at ER–Mito MCSs coupled with more spontaneously active IP3R117 creates the opportunity for destructive funneling of Ca2+ into mitochondria. We propose that in NPC disease, excess KV2–CaV1 Ca2+ entry at ER–PM MCSs is transported into the ER through increased SERCA activity23 and clustering but instead of accumulating to increase ER Ca2+ concentrations, is quickly released by IP3R1 receptors at ER–Mito MCSs to promote neurotoxic increases in mitochondrial Ca2+  and non-conducting (Ca2+ nanodomain organization) KV2.1 subpopulations by protein kinases and phosphatases to ensure the maintenance of neuronal health.A common feature in neurodegenerative disorders is mitochondrial Caa2+ Fig. . Support2+ phenotype across multiple organelles in NPC disease? Our working model is that alterations in Ca2+ gradients across MCSs represent cellular programs attempting to redistribute cholesterol rapidly and efficiently to cellular membranes to restore cholesterol homeostasis103. In this model, unless new homeostatic set points are found quickly, gross alterations in Ca2+ gradients, as well as lipids52, provide a substrate for neurodegeneration23. To design rational targets to slow or abrogate NPC-linked pathology, further quantitative characterization of the structural and functional alterations to ER–PM contacts and the downstream consequences linking mitochondrial Ca2+ elevations to neurodegeneration are required.Why is there such a prominent Ca2+ handling proteins at two membrane contact sites: ER–PM and ER–Mito. Our data demonstrate that NPC is a nanostructural ion channel clustering disease with altered ion channel distribution/activity at membrane contacts contributing to neurodegeneration.To conclude, this study details a molecular link between cholesterol egress from lysosomes and the reorganization of CaKey resources are documented in Supplementary Table I1061T mice were purchased from The Jackson Laboratory and kindly provided by Daniel Ory64, respectively. Mice were maintained under standard light-dark cycles, fed standard chow and water ad libitum, and housed in a vivarium with controlled conditions.All experiments were performed in strict compliance with the University of California Davis ethical regulations for studies involving animals as approved by the University of California Davis Animal Care and Use Committee (protocol #: 22644). C57BL/6 WT and NPC1−/− cells were a kind gift from Dr. Ory , HeLa WT and NPC1−/− cells were a kind gift from Dr. Judith Storch (Rutgers). tsA201 and HeLa cells were cultured in DMEM supplemented with 10% FBS , with 2 mM L-glutamine  and 0.2% Penicillin/Streptomycin . CHO cells were cultured in DMEM/F12 (1:1)  supplemented with 10% FBS  and 0.2% Penicillin/Streptomycin . All cell lines were passaged twice weekly and incubated in 5% CO2 at 37 °C. NPC1−/−tsA201 cells were purchased from Sigma (Cat #96121229), CHO WT and NPC1104 and meninges, cerebellum, hippocampus, and striatum were discarded. Cortical tissue was pelleted and incubated in papain for 20 min at 37 °C (agitating every 5 mins), followed by trituration. Consequently, cells were centrifuged 5′ at 1000g and resuspended in Earle’s Balanced Salt Solution (EBSS), ovomucoid (papain inhibitor) and DNase. Neurons were plated in Neurobasal  supplemented with B27 , Glutamax  and 0.2% penicillin/streptomycin at a density of 650,000 cells / mL. Neurons were incubated in 5% CO2 at 37 °C and half of the media was changed every 4 days. Experiments were performed at DIV 6-8, except for the GCaMP3-Kv2.1P404W Ca2+ imaging which was conducted at DIV 14–16. Rat cortical neurons were dissected and isolated at gestation day 18 as previously described in Vierra et al.31.Cortical neurons from mice of both sexes were dissociated at embryonic day 15–18 (E15–18) using the Papain Dissociation System purchased from Worthington (Cat #LK003150). All stock solutions were prepared as per manufacturer’s recommendations. Dissection was conducted in sterile PBS at 4 °C as previously described inXestospongin C (XestoC) was dissolved in DMSO and used at a final concentration of 10 µM and incubated overnight (O/N) or for 48 h. Torin-1 was dissolved in DMSO and used at a final concentration of 10 µM and incubated O/N. U18666A (U18) was dissolved in DMSO to a final concentration of 10 µM and incubated O/N or for 48 h. Tetrodotoxin (TTX) was dissolved in DMSO and incubated O/N at a final concentration of 200 nM. roscovitine (Rosc) was dissolved in DMSO and incubated O/N or for 48 h at a final concentration 10 µM. Ionomycin was dissolved in DMSO and used at a final concentration of 2.5 µM for ∼6 min. TAT-FFAT-HA (472-481) (FFAT), TAT-HA-C1aB (CCAD) and their respective control scramble peptides TAT-HA-C1aB-Scr and TAT-FFAT-HA (472-481)-Scr (SCBRL) were dissolved in molecular biology grade water and used at a final concentration of 1 µM for 48 h.Cells were washed with PBS and fixed in 4% paraformaldehyde  for 20 min at 21 °C. Cells were stained with fillipin (100 µg/ml in PBS) for 2 h at room temperature . During the fillipin staining process cells were protected from light exposure. Cells were excited using a 405-nm LED and light collected using a Plan-Apochromat 63×/1.40 oil-immersion lens and a Zeiss LSM 880 Airyscan microscope. Images were acquired in PBS solution at RT using Zen v2.3 software.V2.1 at 4 μg/mL , CaV1.2 at 1:250 , CDK5 at 1:100 , p35 at 1:100 , p39 at 1:100  or GAPDH at 1:1000 . Blot bands were detected using fluorescent secondary antibodies goat anti-rabbit 680RD at 1:1000  and goat anti-Mouse 800CW at 1:1000 . Signals were detected using Azure SapphireTM Biomolecular Imager (Azure Biosystems) and quantified using the ImageJ software. Abundance of proteins was normalized to GAPDH.Protein was harvested from neuronal cultures in RIPA buffer  containing Complete/Mini/EDTA-free protease inhibitor cocktail , sodium fluoride 1 mM , SDS 0.05%, sodium deoxycholate 0.4% (DOC) and Microcystin 4 µg/mL . Cells were scraped in 100 μL of lysis buffer and centrifuged at 13,200 rpm for 20 min at 4 °C to isolate the postnuclear supernatant. Protein concentration was quantified using a Pierce BCA protein assay kit . 15 μg of each protein sample was added to 4%–12% Bis-Tris gels and electrophoresed under reducing conditions for approximately 75 min at 155 V. Proteins were transferred onto nitrocellulose membranes  using the Mini-Blot system  for 2 h 30 min at 12 V (∼200 mA). After 30 min incubation at RT in Tris-Buffered Saline (TBS) buffer supplemented with 0.05% Tween‐20 (TBS-T) and 5% non‐fat dry milk, membranes were exposed O/N at 4 °C to the following primary antibodies: K76, 2 µg GFP-MAPPER74, 1 µg SPLICSL-P2AER-PM , 1 µg SPLICSS-P2AER-PM  and 1.1 µg GCaMP3-Kv2.1P4O4W31. Plasmid transfection mixtures for CHO cells were prepared using 7 µl of Lipofectamine LTX and 7 µl of Plus  and were imaged the day after transfection. The following plasmids were used: 1 µg per 960,000 cells rat CaV1.2α1c , 1 µg per 960,000 cells rat CaVα2δ , 1 µg per 960,000 cells CaVβ3  and 1 µg per 960,000 cells DsRed-KV2.135.Plasmid transfection mixtures were prepared using 2.5 µl/ µg DNA of Lipofectamine 2000 , added to neurons and incubated for 2 days. The following plasmids  were used in this study: 4 µg pCAG-mito-RCaMP1h , 4 µg ER-GCaMP6-150 , 2 ug ArcLight-Q239 2, 1 MgCl2, 10 HEPES, and 8 D‐Glucose) with the ZEN software v2.3, respectively. Images were taken at 0.5 μm depth intervals within the cell (see PLA assay and ER staining in live neurons) or as a single internal or plasma membrane plane for HA, GRP75, IP3R and VDAC1, or, CaV1.2, CaV1.3, KV2.1, CaV2.1 and RyR immunolabeling, respectively. 3D analysis for the ER morphology in neurons was conducted with the IMARIS software (Oxford Instruments); all other datasets were carried out following background subtraction and similar thresholding using ImageJ . Colocalization/overlap analyses were performed by multiplying binary masks of CaV1.2, KV2.1, RyR, IP3R, VDAC1, ER and mitochondria images. For neuronal analyses, the soma area and a 15 µm2 ROI from proximal neuronal dendrites were used. For protein clustering and ER–PM MAPPER puncta studies in the soma and cell body, sum intensity and numbers of clusters were normalized to their respective areas.Imaging was conducted at RT using a Zeiss LSM880 confocal laser scanning microscope equipped with a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a super‐resolution Airyscan detection unit. Images of GFP/Alexa‐488, Alexa-568/mCherry and Alexa‐647 were collected using 488-, 594- and 633-nm excitation lasers, respectively, with multicolor images sequentially acquired. Fixed and live cells were acquired in PBS or Ringer’s solution , respectively. For fixed cell imaging  50,000 cycles with an exposure time of 10 ms were acquired and imaging analysis was performed using IMARIS software (Oxford Instruments). For all analyses, cluster number was normalized to the respective soma area. Colocalization/overlapping analyses were performed by creating a 2D KV2.1 mask and measuring CaV1.2, CDK5, VAPA/B or SERCA areas within said mask. Live cell imaging of GCaMP3-KV2.1P4O4W analyses were performed using the ImageJ and ClampFit (Molecular Devices) softwares.Images were acquired using a Leica Infinity TIRF microscope equipped with a 163× 1.49 (fixed cells) and a 100× 1.47 (live cells) CORR TIRF oil immersion objective and a Hamamatsu orca flash 4.0 camera. Leica LAS X software was employed for both image acquisition and processing. GFP/YFP, Alexa‐647 and Alexa-568 images were collected with the 488 nm, 638 nm and 561 nm excitation lines, respectively. Fixed or live cells were imaged in a GLOX–MEA  or Ringer’s solution , CaV1.2 1:333 , KV2.1(pS603) 1:5 (L61/14.2), VDAC1 1:100 , SERCA 10 µg/mL , CaV1.3 10 µg/mL , pan-RyR 1:100 , IP3R 18 µg/mL , CaV2.1 1:200 , and KV2.1 pS603 1:5 (L61/14). After primary antibody incubation, neurons were washed 3 × 5 min and subsequently incubated for 1 h at RT with the following secondary antibodies: Goat anti-Mouse-647 and −568 nm 1:1000 , anti-Rabbit-647 and −555 nm 1:1000 , anti-Mouse IgG1-568 nm 1:1000  and anti-Mouse IgG1-CF568 1:250 .Antibody dilutions/amounts, validation, company names, catalogue numbers and clone numbers can be found either below, in the reporting summary, and/or supplementary table Fixation: Postnatal day 60 (P60) wild-type and NPC1I1016T mice were anesthetized and transcardially perfused with 4% phosphate buffered (PB) formaldehyde (pH 7.4) made from powdered paraformaldehyde using a peristaltic pump. Perfused brains were collected and cryoprotected with 30% sucrose/PB solution. Sectioning: Sagittal brains sections (30 µM) were generated using a freezing microtome and immunolabeling was performed on free floating sections. Briefly, tissue was blocked in 10% normal goat serum (NGS) with 0.3% TritonX-100 in 0.1 M PB at RT for 1 h followed by incubation in primary antibody O/N at 4 °C. Primary antibody incubation was performed in 5% NGS/0.3% TritinX-100/0.1 M PB. Antibodies: the following primary antibodies were used for immunolabeling: Calbindin 1:200 (Sigma-Aldrich #C9848),Calbindin, 2 µg/mL , KV2.1 10 µg/mL , CaV1.2 1:200 , CaV1.2 5 µg/mL . Species-specific Alexa Fluor 488, 555 and 647conjugated secondary antibodies  were incubated for 60 min at RT. Images were taken using a Zeiss ApoTome Fluoview FV3000 confocal microscope (Olympus) or a Zeiss Axioscope 2 widefield microscope with ApoTome. All images were assembled using FIJI and Illustrator (Adobe). CaV1.2–KV2.1 colocalization volumes were calculated using IMARIS software by quantifying the KV2.1-positive pixels inside a 3D CaV1.2 mask.V1.2-KV2.1 proximity in fixed cortical neurons treated with U18 or vehicle control (DMSO). Following fixation, as described in Immunofluorescence Immunocytochemistry above, cells were incubated for 15 min with 100 mM glycine at RT. Subsequently, neurons were washed in PBS (2 ×3 min) and permeabilized in 0.1% Triton-X100 for 20’ at RT. Neurons were blocked in a 20% SEA BLOCK solution  for 1 h and incubated at 4 °C O/N with the following primary antibodies: KV2.1 10 µg/mL  and CaV1.2 1:333 . Secondary oligonucleotide-conjugated antibodies (PLA probes: anti-mouse MINUS and anti-rabbit PLUS) were used at 1:5 dilution in Duolink Antibody Diluent. PLA assay was performed as per the manufacturer’s recommendations. Coverslips were mounted with DAPI Fluoromount-G . The fluorescence signal was detected using a Zeiss LSM880 confocal laser scanning microscope equipped with a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a super‐resolution Airyscan detection unit. 405- and 488-nm lasers were employed to visualize the DAPI-stained nucleus and PLA signal, respectively. For each neuron, optical z-axis sections were acquired at 0.5 μm intervals and combined to a single maximum intensity projection using ImageJ.The Duolink In Situ PLA kit  was used to quantify CaCells were incubated for 20 min using Mito Tracker  and ER Tracker . Cells were subsequently washed twice (5 min each time) before being imaged with a Zeiss LSM880 confocal laser scanning microscope equipped with a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a super‐resolution Airyscan detection unit; 633- and 546-nm excitation lasers were used to image mitochondria and ER, respectively. For tSA201, cells single planes near the PM were acquired and overlapping areas between both organelles were analyzed by multiplying their respective binary masks in ImageJ and normalized to the respective cell body area. For neurons, z-stacks were taken at 0.5 μm depth intervals within the cell and analysis was performed in the IMARIS software (Oxford instruments).2+ Ringer’s solution using a LSM880 confocal laser scanning microscope equipped with a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a super‐resolution Airyscan detection unit. A 546 nm laser was used to excite pCAG-mito-RCamPh1, while a 488 nm laser was used to excite ER-GCaMP6-150. After collection of a baseline image, cells were perfused with a 20 mM Ringer’s solution containing 2.5 µM Ionomycin for 6 min. The mean intensity ratio of pre-ionomycin/post-ionomycin signals was calculated for each cell. Higher ratios were taken to indicate higher resting mitochondrial or ER Ca2+ concentrations.48 h post-transfection with pCAG-mito-RCamPh1 or ER-GCaMP6-150, neurons were imaged in a 2 mM Ca2+ Ringer’s solution containing 2.5 µM Fluo-4 and 0.1% Pluronic acid for 30 min. Cells were then moved to a Fluo-4–free Ringer’s solution to deesterify the Fluo-4 for 30 min. Following deesterification, Fluo-4–loaded cells were excited with a 488-nm laser and the resulting fluorescence monitored using an inverted microscope with a Plan-Apochromat 63×/1.40 oil objective, connected to an Andor W1 spinning-disk confocal with a Photometrics Prime 95B camera. Images were acquired every 50 ms for a total of 50 s in a 2 mM Ca2+ Ringer’s solution at RT using Micromanager (1.4.21) software. Electrical stimulation at 40 V was performed at 1 Hz for 5 s with a Field Stimulator (IonOptix). Intracellular Ca2+ activity was measured as Ca2+ peak frequency and Ca2+ peak amplitude in a Region of Interest (ROI) localized in the soma or proximal neuronal dendrites using ImageJ or ClamPfit software.Cells were incubated in a 2 mM Ca2, 1 MgCl2, 10 HEPES, and 8 D‐Glucose) containing B-Kay 500 nM every 100 ms for a total time of 50–100 s by exciting with a 488-nm laser. Ca2+ activity was measured as GCaMP3-Kv2.1P4O4W peak amplitude in a ROI localized in the soma or proximal neuronal projections using ImageJ and ClamPfit softwares.48 h post-transfection, TIRF imaging was performed using a Leica Infinity TIRF super-resolution microscope equipped with a 100× 1.47 TIRF oil immersion objective and a Hamamatsu Orca Flash 4.0 camera. Images were acquired in Ringer’s solution  at 488 nm using a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a Zeiss 880 Airyscan microscope at RT. Images were analyzed using ImageJ and mean intensities were taken to plot ROS production.The ROS production assay was conducted as per the manufacturer’s recommendations . Live DIV 6-8 cortical neurons were incubated O/N with U18 and CCAD or SCRBL peptides. Single-plane images were collected in Ringer’s solution  using a Plan‐Apochromat 63× 1.4 Oil DIC M27 objective and a Zeiss 880 Airyscan microscope at RT. Images were analyzed using ImageJ software.Mitochondrial membrane potential was conducted as per the manufacturer’s recommendations  and plotted as a ratio of aggregated JC-1 form/monomer JC-1 form, with higher ratios indicating more polarized membranes. JC-1 aggregates were imaged at a 618 nm emission and a 488 nm excitation, and JC-1 monomers at a 536 nm emission and 488 nm excitation in Ringer’s solution  to dead (564 nm) was taken for each single neuron every captured image, with higher ratios indicating increased neuronal viability.The cell viability assay was conducted as per the manufacturer’s recommendations . Live DIV 6–8 cortical neurons were incubated 48 h with U18, XestoC, Rosc or CCAD or SCRBL peptides before being washed once with 2 mM Ca107. When comparing two or three independent groups, normality was determined with the Agostino-Pearson omnibus test and a parametric, unpaired t test, or nonparametric, Mann–Whitney t test, was employed. When comparing the means of 4 groups with two or more categorical independent variables, the two-way ANOVA for multiple comparisons with appropriate post hoc test was used. When quantifying fold changes respective to the control condition, one sample t test was employed. P < 0.05 was considered statistically significant ; # shows significance between sample groups and the negative control when ANOVA was employed. To determine whether values within a dataset have significant outliers, a two‐sided Grubbs’ test, with an alpha value of 0.05 was performed. All the data values are presented as means ± SEM and the number of technical and biological replicates is detailed in each figure legend, which is based on previously published observations and is consistent with the general number of replicates and conditions commonly accepted in the field. For each neuronal dataset experiments were performed from 1-4 independent neuronal cultures with each isolation containing 6–8 pups. Several key datasets were blinded and outcome accessed.The number of replicates is based on previously published observations to reached statistical differences between datasetsFurther information on research design is available in the Supplementary informationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary


Soluble amyloid-beta oligomers (AβOs), the main synaptotoxins engaged in the pathogenesis of Alzheimer’s disease, generate aberrant Ca2+ signals in primary hippocampal neurons, increase their oxidative tone and disrupt structural plasticity. Here, we explored the effects of sub-lethal AβOs concentrations on activity-generated nuclear Ca2+ signals and on the Ca2+-dependent expression of neuroprotective genes. To induce neuronal activity, neuron-enriched primary hippocampal cultures were treated with the GABAA receptor blocker gabazine (GBZ), and nuclear Ca2+ signals were measured in AβOs-treated or control neurons transfected with a genetically encoded nuclear Ca2+ sensor. Incubation (6 h) with AβOs significantly reduced the nuclear Ca2+ signals and the enhanced phosphorylation of cyclic AMP response element-binding protein (CREB) induced by GBZ. Likewise, incubation (6 h) with AβOs significantly reduced the GBZ-induced increases in the mRNA levels of neuronal Per-Arnt-Sim domain protein 4 (Npas4), brain-derived neurotrophic factor (BDNF), ryanodine receptor type-2 (RyR2), and the antioxidant enzyme NADPH-quinone oxidoreductase (Nqo1). Based on these findings we propose that AβOs, by inhibiting the generation of activity-induced nuclear Ca2+ signals, disrupt key neuroprotective gene expression pathways required for hippocampal-dependent learning and memory processes.Hippocampal neuronal activity generates dendritic and somatic Ca Alzheimer’s disease (AD), the most common form of age-associated dementia worldwide, is a progressive and fatal neurodegenerative illness manifested by severe and progressive deterioration of cognitive and memory processes . Extrace2+ signaling are central features of this disease [Uncontrolled neuronal activity, as occurs in AD, affects neural network homeostasis and initiates synaptotoxic processes . Current disease ,19. Seve disease ). Even s disease ,22,23. M disease  and was  disease ,24.2+ signals, defined as transient, controlled increments in intracellular Ca2+ concentration, play essential roles in hippocampal synaptic plasticity and memory processes [2+ signals lead to neuronal dysfunction and eventually cause neuronal death [2+ homeostasis and signaling, locally in dendritic spines, and in somatic and nuclear compartments [2+ influx through N-methyl-D-aspartate (NMDA) glutamate receptors, which induces Ca2+ release mediated by ryanodine receptor (RyR) Ca2+ channels [2+ signals, which are long-lasting but of low amplitude, engage Ca2+-dependent pathways that hinder BDNF-induced dendritic spine growth and induce excitotoxicity, leading to the loss of synaptic structure and function [2⁺ levels induce instantaneous uncoupling of a protein called Jacob from LaminB1 at the nuclear lamina and promote the association of Jacob with the transcription factor cAMP-responsive element-binding protein (CREB) [2+ signaling and the related excitable and synaptic dysfunctions are consolidated hallmarks of AD onset and possibly of other neurodegenerative diseases [Neuronal Carocesses ,26. Howeal death . Accordiartments ,28; thesartments ,29. In pchannels . The ensfunction ,29,30. On (CREB) . Interesn (CREB) . In summdiseases ,34.2+-dependent gene expression [2+ signals that, in addition to promoting the activation of cytoplasmic signaling cascades, readily reach and enter the nucleus via passive diffusion through nuclear pores [2+ entry requires Ca2+ signals to reach the vicinity of the nucleus, which does not occur by simple Ca2+ diffusion since this process is highly restricted in the cytoplasm [2+ signals to the nucleus [2+ signals contribute to activity-dependent transcription by promoting Ca2+-dependent phosphorylation of CREB-binding protein (CBP), which allows the formation of the transcriptionally active CREB-CBP complex [Essentially, all activity-induced, long-term modifications of brain functions require appropriate synapse-to-nucleus communication to precisely control nuclear Capression . Robust ar pores ,36. Howeytoplasm ,38. The  nucleus . Once in complex ,39,40.A receptor function with gabazine (GBZ) stimulates the activity of rat primary hippocampal neurons [2+ transients, which propagate from the cytoplasm to the neuronal nucleus, where they promote CREB phosphorylation [2+ signals, synaptic plasticity, and antioxidant defense mechanisms in hippocampal neurons remain undefined.Blocking GABA neurons ,41,42, w neurons . The GBZrylation —a key evrylation ,45. The rylation , all of rylation ,47,48. Irylation . Moreoverylation . However2+ signals induced by GBZ addition to rat primary hippocampal neurons; AβOs also decreased the GBZ-induced CREB phosphorylation increase, and the expression of genes engaged in memory and neuroprotective processes, such as BDNF, RyR2, and Npas4. Importantly, AβOs also reduced the expression of NADPH-quinone oxidoreductase-1 (Nqo1), an enzyme with a key role in the neuronal antioxidant response that is significantly impaired in AD [2+-signaling processes and suppress the expression of genes associated with neuroprotection in primary hippocampal cultures, we present novel results that advance our current knowledge of the noxious effects of AβOs on rodent hippocampal function. Hence, this work provides a framework for a deeper understanding of how AD-related mechanisms impact synaptic plasticity and neuronal health. Moreover, it offers promising avenues for therapeutic interventions in AD and related neurodegenerative conditions.In this work, we report that AβOs inhibited the generation of nuclear Caed in AD ,50. In tReagents: The Aβ1–42 peptide was from Bachem Inc. , gabazine SR95531 was from Tocris Bioscience ; cytoplasmic GCaMP5 and nuclear GCaMP3-NLS were kindly provided by Dr. H. Bading, University of Heidelberg, Germany; DMSO was from Sigma , and hexafluoro-2-propanol (HFIP) was from Merck . The serum-free neurobasal medium, B27 supplement, GlutaMAX™, DAPI, Lipofectamine 2000 and TRIzol reagent were from Gibco™/Thermofisher Scientific . Brilliant III Ultra-Fast SYBR® Green QPCR Master Mix and DAKO mounting medium were from Agilent Technologies . The DNAase Turbo DNA-freeTM kit was from Ambion™/Thermofisher Scientific . Improm II TM reverse transcriptase was from Promega . The protein content was determined with a BCA kit from Thermofisher Scientific .Antibodies: Specific antibodies against p-CREB were from Cell Signaling Technologies . Antibodies against MAP2 were from Abcam  and those against β-tubulin were from Sigma-Aldrich . Alexa Fluor 488 anti-rabbit and Alexa Fluor 635 anti-mouse were from Invitrogen .Primary rat hippocampal cultures: Primary hippocampal cultures were prepared from Sprague Dawley rats at a gestation age of 18 days, as described in [2. In all experiments, cultures were used between 12–15 days in vitro (DIV). For most of the experiments, hippocampal neurons were pre-incubated with 500 nM AβOs for 6 h before incubation with 5 μM gabazine for 30 min for Ca2+ measurements and CREB immunofluorescence; for determinations by qPCR, cultures were incubated for 6 h with 500 nM AβOs before incubation with 5 μM gabazine for 2 h. Compounds were maintained during the respective incubation period, as stated. All experimental protocols used in this work complied with the “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society and were approved by the Bioethics Committee on Animal Research, Faculty of Medicine, Universidad de Chile.ribed in . BrieflyPreparation of AβOs: The Aβ1–42 peptide was prepared as a dried hexafluoro-2-propanol (HIFP) film and stored at −20 °C, as described in [g for 10 min at 4 °C to remove protofibrils and fibrils (insoluble aggregates). The supernatants that contained Aβ oligomers were transferred to clean tubes and stored at 4 °C; an aliquot was used to determine AβOs content with a BCA kit. Fresh AβOs preparations (up to 48 h) were used in all experiments.ribed in ,13,15,242+ Signals:Cytoplasmic Ca To detect cytoplasmic Ca2+ signals, primary hippocampal cultures were transfected with the genetically encoded cytoplasmic Ca2+ sensor GCaMP5. Transfected neurons were recorded in Tyrode’s solution . As an additional strategy to detect neuronal Ca2+ signals, primary cultures were loaded for 15 min at 37 °C with the Ca2+ probe Fluo 4-AM, prepared in Tyrode’s extracellular medium. Cells were placed in the microscopy stage of a wide-field Zeiss Cell Observer epifluorescence microscope , using, as objectives, Plan-Neofluar 20×/0.4 or Plan Apochromat, 40×/1.3 water, with a light source 470 nm Colibri 2 LED-based module and a digital camera EMCCD Evolve 512 delta . All settings were adjusted to minimize bleaching and maximize acquisition frequency. After recording a stable baseline, 10 μM of gabazine was added to the culture; ionomycin (3 μM) was added at the end of the experiment to visualize the maximum fluorescence signals.2+ levels, cultures were incubated at 37 °C for 20 min in the dark with 2 µM Fura-2 AM. Subsequently, cells were washed three times with Tyrode’s solution . Cells were placed in the microscopy stage of a Spinning Disc IX81 microscope , using a xenon lamp as the excitation source, with excitation filters for 340/402 nm wavelengths, and a 40× objective. The first minutes of the record yielded the fluorescence levels corresponding to the resting Ca2+ concentration. Then, ionomycin was added to saturate the Ca2+ probe. For data analysis, the background fluorescence was subtracted and the ratio of the 340 nm and 380 nm fluorescence intensity  was calculated.To determine basal Ca2+ Signals:Nuclear Ca To detect nuclear Ca2+ signals, primary hippocampal cultures (14–16 DIV) were transfected with the genetically encoded Ca2+ sensor GCaMP3-NLS, which has a sequence of nuclear destination and thus accumulates in the neuronal nucleus. The GCaMP3-transfected neurons were recorded in Tyrode’s solution . To assess the direct effect of AβOs on nuclear Ca2+ signals, cultures were incubated at the microscope stage with 500 nM AβOs and acquired by image time-lapse for 15 min. To determine the effect of AβOs on activity-induced nuclear Ca2+ signals, neurons were incubated for 6 h with 500 nM AβOs before the addition of 5 μM GBZ at the microscope stage. A wide-field Zeiss Cell Observer epifluorescence microscope , using Plan-Neofluar 20 Å~/0.4 or Plan Apochromat as objectives, 40 Å~/1.3 water, with a light source, 470-nm Colibri 2 light-emitting diode (LED)-based module, and a digital camera, an electron-multiplying charge-coupled device (EMCCD) Evolve 512 delta  was used. All settings were adjusted to minimize bleaching and maximize acquisition frequency. After recording a stable baseline, 5 μM GBZ was added to the culture; 3 μM ionomycin was added at the end of the experiment to visualize the maximum fluorescence signals. The analysis of Ca2+ signals was performed as described in [max − F0/F0) or as F/F0, where Fmax and F represent, respectively, the maximal recorded fluorescence intensity and the recorded fluorescence intensity; F0 corresponds to the intensity at the initial time (mean intensity of 20 to 50 frames recorded before the stimulus).ribed in . BrieflyCREB phosphorylation and nuclear staining: To determine whether AβOs interrupt the GBZ-induced increase in synaptic activity and CREB phosphorylation, hippocampal cultures were pre-incubated for 6 h with 500 nM AβOs before the addition of 5 μM GBZ for 30 min. Cells were then fixed and CREB phosphorylation was detected by immunofluorescence using an antibody against the phosphorylated form of CREB (Ser-133 p-CREB). In addition, cultures were probed with an antibody against the microtubule-associated protein MAP2 to identify neuronal cells and with DAPI to detect all cell nuclei. All images were recorded with a 40× objective in the z-axis of all confocal planes acquired by spinning disk microscopy . Quantification of the relative levels of p-CREB fluorescence was detected in different conditions by background subtraction  and segmentation by Otsu threshold, using DAPI staining as the total nuclear staining reference. Mean fluorescence intensity was quantified only in neuronal cells (MAP2 positive) and expressed as a fold change relative to the control cells.RNA Extraction and qPCR: Total RNA was isolated using TRIzol reagent following the manufacturer’s recommendations; to remove any contamination with genomic DNA; a digestion step with DNAase  was included to remove residual contaminating DNA. The RNA purity was determined by the 260/280 absorbance ratio in nanodrop . Complementary DNA (cDNA) was synthesized with Improm II TM reverse transcriptase using 2 μg of total RNA. The mRNA levels of RyR2, BDNF exon IV, Npas4, and NqoI were amplified using specific primers  were added at the microscope stage to hippocampal cultures transfected with the nuclear Ca2+ sensor GCaMP3-NLS  with AβOs were treated with the GABAA receptor antagonist GBZ, which, by decreasing the inhibitory GABAergic tone, produces an excitatory/inhibitory imbalance and a rapid increase in neuronal activity [2+ signals .To test the effects of AβOs on the Caactivity ,52. As r signals C,D, measaMP3-NLS C, displaaMP3-NLS D. Data a2+ signals elicited by GBZ ; at least 50 neurons in total were analyzed per condition. Statistical analysis was performed with Student’s t-test. *, the exact p values were p = 0.040 in p = 0.037 in 2+ signals caused by the increase in neuronal activity induced by GBZ. Of note, treatment with 500 nM AβOs (6 h) did not modify the basal cytoplasmic Ca2+ levels  with 500 nM AβOs before GBZ addition displayed a conspicuous decrease in the emergence of the oscillatory and synchronic nuclear Cad by GBZ . Primarymplitude A and themplitude B induced+ levels . These rt 30 min , did notn = 3. **: p < 0.01 Statistically significant differences were evaluated by one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. **: p < 0.01. From left to right, the exact p values are p = 0.003; p = 0.030 and p = 0.005. These results show that incubation for 6 h with AβOs abolished the subsequent GBZ-induced CREB phosphorylation increase.The increase in hippocampal neuronal activity induced by GBZ triggers the early induction of gene expression changes commanded initially, among other factors, by the phosphorylation of the transcription factor CREB and of its binding protein CBP by different nuclear kinases ,28. To dn = 5. Statistical analysis was performed with ANOVA followed by Tukey’s post hoc test for multiple comparisons. *: p < 0.05; **: p < 0.01. From left to right, the exact p values are p = 0.0002 and p = 0.0311 for p = 0.009; p = 0.013 and p = 0.025 for p < 0.001; p = 0.039 for p = 0.013 and p = 0.039 for The increase in neuronal activity induced by GBZ, together with promoting CREB phosphorylation also increases the expression of the early-response gene Npas4 . In agre2+ signals induced by neuronal activity and reduced or abolished the GBZ-induced increases in CREB phosphorylation and of Npas4, RyR2, BDNF, and Nqo1 mRNA levels.Taken together, these results show that incubation with AβOs inhibited the nuclear Ca2+ signals and the phosphorylation of the transcription factor CREB, and reduced or abolished the enhanced expression of Npas4, BDNF, RyR2, and Nqo1 induced by GBZ.Alterations in synapse-to-nucleus communication, which occur in different central nervous system disorders, contribute to the development of neurodegenerative diseases . The res2+ signals have a wide range of functions in the activity-dependent synaptic plasticity processes that underlie hippocampal spatial memory [2+ signaling, the endoplasmic reticulum resident inositol 1,4,5-trisphosphate receptors and RyR channels have central roles. Both Ca2+ channel types amplify Ca2+ entry signals via Ca2+-induced Ca2+ release and thus contribute to regulating the function of neuronal Ca2+-dependent signaling pathways [2+ release plays an essential role in hippocampal synaptic plasticity, and the formation/consolidation of spatial memory [2+, whereas oxidizing agents have the opposite effect [2+ release and the functional defects associated with AD and age-related hippocampal dysfunction [Neuronal Cal memory ,55,56. Apathways ,58,59,60l memory ,46,61,62e effect ,58,59,60function ,64,65,66A receptors [A receptor antagonist than bicuculline, pentobarbital, and alfaxalone [In this work, to induce neuronal activity, primary hippocampal cultures were treated with 5 µM GBZ, which at this concentration effectively inhibits GABAeceptors ,52,67; mfaxalone . Additiofaxalone  that the2+ signaling produced by AβOs in primary hippocampal neurons provide a foundation for future research exploring synaptic plasticity and electrophysiological aspects in more detail.Previous studies have reported that AβOs are synaptotoxins that lead to cognitive impairment in AD  and impe2+ signaling that occur during aging and in response to Aβ peptide toxicity [2+ homeostasis failures and increased β-amyloid and p-Tau levels; negative allosteric modulators of RyR channels reverse these effects, reaffirming the importance of regulating RyR-mediated Ca2+ release in AD-affected neurons [2+ release for neutralizing the AD disease phenotype [2+ release enhances Tau phosphorylation and is associated with reduced neuronal activation and cognitive impairment [Our in vitro rat model of AβOs associated synaptotoxicity, which entails acute treatment of primary hippocampal cultures with non-lethal AβOs concentrations , adequattoxicity . In agre neurons . Additiohenotype . Moreovepairment . Additiopairment ,74.2+ signals induced by neuronal activity, a process that engages RyR2-mediated Ca2+ release [2+ signals that are RyR-mediated [2+ signals were generated by AβOs in the neuronal nucleus. Interestingly, basal cytoplasmic Ca2+ levels, as determined by the ratiometric fluorescence indicator Fura-2, were not significantly different between control neurons and neurons treated for 6 h with AβOs. These results indicate that primary hippocampal neurons can restore, with time, cytoplasmic Ca2+ levels within a normal range, despite the low-intensity and long-lasting Ca2+ increases that AβOs promote for at least the first 30 min in the cytoplasm. Nevertheless, by mechanisms that are not fully elucidated, incubation for 6 h with AβOs significantly reduced the nuclear Ca2+ signal generation induced by GBZ. Hence, neuronal stimulation seems to potentiate the inhibitory effects of AβOs on Ca2+ dynamics, particularly on nuclear Ca2+ signal generation, a result which may have implications for the deleterious effects of AβOs on hippocampal neuronal function during the performance of tasks that entail neuronal activation and engage RyR-mediated Ca2+ release. The development of pharmacological strategies that modulate the activity of RyR channels will continue to attract interest in the development of new drugs to regulate neurodegenerative processes [Adding to these studies, the present results show that acute AβOs treatment inhibited the generation of nuclear Ca release . Of notemediated . As reporocesses .2+ signals, is selectively induced by neuronal activity [The inhibitory effects of AβOs on the enhanced expression of Npas4 triggered by GBZ-induced neuronal activity reported here may contribute to understand the novel and central role of Npas4 in the context of AD. The immediate–early gene Npas4 is expressed only in neurons and is considered as a molecular link between neuronal activity and memory . Moreoveactivity ,78. By oactivity . Recent activity . The decactivity . In contactivity . In the activity .2+ signaling [Moreover, our results, which indicate that AβOs reduced the increase in Npas4 gene expression induced by gabazine in primary hippocampal neurons, open a new direction for extensive future research, including neuroinflammation. Of note, neuroinflammation, which is closely related to neurodegenerative processes, also seems to play an important role in the regulation of the excitatory/inhibitory tone; infiltration of CD8+ T lymphocytes into the hippocampus of a transgenic AD model (APP-PS1) alters the expression of Npas4 and Arc along with other genes related to synaptic plasticity and Caignaling . Moreoveignaling ,85.2+-mediated signaling might prevent uncontrolled activation of Npas4 and neurodegenerative processes [Altogether, these studies position Npas4 and BDNF as outstanding therapeutic targets against AβOs-induced synaptotoxicity and may constitute targets to prevent the development of AD and other neurodegenerative diseases ,87,88. Irocesses .2+ release [2+-dependent kinase pathways that contribute to inducing and maintaining hippocampal LTP [2+ signals and the ensuing expression of both BDNF and RyR2 mRNA levels, may add to our current understanding of AD pathology. In particular, the AβOs-induced decrease of BDNF mRNA levels induced by neuronal activity may compromise BDNF-induced intracellular signaling pathways relevant to hippocampal function [Many studies have established that BDNF, a neurotrophin synthesized and released from neurons in an activity-dependent manner , mediate release , includimpal LTP . In thismpal LTP ,96. In vmpal LTP ,97,98,99function ,100,101.2+ release and ROS [2+-dependent expression of Nqo1 induced by GBZ, indicating that these aggregates disrupt neuroprotective pathways induced by neuronal activity that regulate neuronal oxidative tone.Of note, coupled with an increase in neuronal activity, BDNF induces the nuclear translocation of the transcription factor Nrf2 in neurons , a maste and ROS . One of  and ROS . Here, w2+ signals and reduce the expression of genes engaged in synaptic plasticity and the antioxidant response, we propose that these disruptions are likely to contribute to the cognitive decline and the neurodegeneration that characterize Alzheimer’s disease. Moreover, this work adds to the current understanding of the toxic effects of AβOs on hippocampal neuronal function and reveals new possible therapeutic targets to treat AD. Hence, one potential therapeutic approach to slow or prevent AD progression would be to restore activity-induced nuclear Ca2+ signaling and early gene expression. More research is needed to develop and test these therapeutic approaches. Of note, a recent report analyzes how AβOs can become potential therapeutic targets for AD [Based on the results of this study, which provides the first integrative evidence that AβOs disrupt the generation of activity dependent nuclear Cas for AD . Recentls for AD ,109,110.


DNA double-strand breaks (DSBs) occur every cell cycle and must be efficiently repaired. Non-homologous end joining (NHEJ) is the dominant pathway for DSB repair in G1-phase. The first step of NHEJ is to bring the two DSB ends back into proximity (synapsis). Although synapsis is generally assumed to occur through passive diffusion, we show that passive diffusion is unlikely to produce the synapsis speed observed in cells. Instead, we hypothesize that DNA loop extrusion facilitates synapsis. By combining experimentally constrained simulations and theory, we show that a simple loop extrusion model constrained by previous live-cell imaging data only modestly accelerates synapsis. Instead, an expanded loop extrusion model with targeted loading of loop extruding factors (LEFs), a small portion of long-lived LEFs, and LEF stabilization by boundary elements and DSB ends achieves fast synapsis with near 100% efficiency. We propose that loop extrusion contributes to DSB repair by mediating fast synapsis. DNA double-strand breaks (DSBs) occur every cell cycle and must be repaired. Here the authors combine theory and simulations to establish a likely role for loop extrusion in bringing the DSB ends back into proximity for repair. For example, normal metabolism has been estimated to cause ~1–50 DSBs per human cell per day7. Consequently, fast and reliable DNA repair is necessary to prevent deleterious chromosomal rearrangements such as translocations, inversions, amplifications, and deletions8. The three major DSB repair pathways are non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR)11. The choice of DSB pathway depends on sequence, chromatin context, cell cycle phase, and the complexity of DSB ends12. Here we focus on NHEJ, which is operational throughout the cell cycle and the dominant DSB repair pathway in G1-phase13.DNA double-strand breaks (DSBs) can be caused by environmental agents such as radiation and drugs15, what all recruitment mechanisms have in common is that they are reactive: recruitment of DSB repair factors begins only after the DSB has occurred and been sensed. This introduces a time delay16 during which the DSB ends can diffuse apart17, which may delay the repair, prevent repair, or result in aberrant ligation between distinct chromosomes, causing translocations18. Indeed, prior experimental work has demonstrated that, in human cells, DSB ends can move several hundreds of nanometers apart within minutes after a DSB has occurred20. The DNA DSB repair process through NHEJ, therefore, requires two major steps: (1) bringing the DSB ends back into proximity  and (2) recruiting the necessary proteins to covalently ligate the synapsed DSB ends  is the cohesin complex, which is thought to extrude DNA bidirectionally at a rate of ~0.5–2 kb/s until cohesin is blocked by a Boundary Element (BE)30. The primary BE in mammalian interphase is the insulator protein, CTCF31. By extruding the genome until it encounters CTCF boundaries, cohesin-mediated loop extrusion folds the genome into loops and domains known as topologically associating domains (TADs)33  cells20. Consistently, the average synapsis time is estimated to be around 6–17 min in mammalian cells based on prior experimental data43. To see if 3D diffusion-mediated synapsis is consistent with these values, we performed 3D polymer simulations to determine the kinetics of synapsis mediated by passive diffusion alone, using parameters from live-cell imaging experiments without cohesin and loop extrusion41. We found that passive diffusion takes ~160 min to achieve 95% DSB synapsis, almost an order of magnitude longer than the experimentally observed value of 20 min  facilitates DSB synapsis and repair in two ways, in parallel to 3D diffusion.Given the unphysiologically slow rates of DSB end synapsis mediated by passive diffusion alone, we hypothesize that DNA loop extrusion by loop extruding factors (LEFs) , where we define a gap as the DNA segment between the constraining LEF and the broken DNA end  Can the process of DNA loop extrusion accelerate synapsis? (2) If so, can we identify physiologically plausible conditions for loop extrusion (e.g. LEF density on DNA or LEF processivity) that achieve synapsis kinetics and efficiency observed in vivo?41 that incorporate DSBs, LEFs, and boundary elements (BEs). We simulated one chromosome 70 Mb in length that could be bound by LEFs, where the chain was discretized into 1 kb DNA segments. BEs were placed on the chromosome to create TADs of various sizes within the experimentally expected range48 . The mean-synapsis time is modestly but significantly reduced compared with passive diffusion alone . Surprisingly, however, for unconstrained DSBs, active extrusion was slightly detrimental to synapsis  in Supplementary Note 54.We note that loop extrusion only facilitates synapsis when its constraining role is in effect Fig. c–f. As sPend-joining∣constrained modulates synapsis efficiency. We derived a general analytical expression for Pend-joining∣constrained that accounts for gap-bridging LEFs that load and finish extruding gaps on both sides of the DSB before the constraining LEF unloads. With simplifying assumptions (Eqs. (67–70), see Supplementary Note Pend-joining∣constrained: τloading/τconstrained and τextrusion/τconstrained . Since unconstrained DSBs have synapsis kinetics slower than passive diffusion alone , we examined the necessary conditions that have to be met to achieve synapsis. One key requirement for synapsis is the simultaneous bridging of the gaps on both sides of the DSB. In other words, a gap-bridging LEF that has bridged the gap on one side of the DSB needs to remain on DNA until the gap on the other side of the DSB is also bridged  is not the bottleneck of synapsis. Instead, this suggests that the establishment of the end products of gap-bridging are the limiting step; this led us to search for mechanisms that accelerate the rate of gap-bridging.59 possibly mediated by the MRN/MRX  complex59, though two other studies reported cohesin enrichment only for S/G2 cells but not G1 cells via laser induction61, suggesting that further experimental work is needed. However, there are also indications that other LEFs may function as gap-bridging LEFs, such as MRE1162, and evidence of targeted loaded LEFs helping γH2AX spreading near a DSB end64.Guided by the objective of finding mechanisms that accelerate gap-bridging product formation, we turned to biological mechanisms that increase the loading rate of LEFs to the gap. One such mechanism is the targeted loading of LEFs to the DSB ends   the fold stabilization of LEFs at BEs, (2) the fraction of long-lived LEFs, (3) the fold stabilization of LEFs at DSB ends, (4) the fold increase in loading rate at the DSB, (5) the ratio of LEF processivity to LEF separation. This yielded a total of 768 different parameter combinations -like contact maps from 1D simulations with the models, which achieve high synapsis efficiency. Consistent with the experimental Hi-C maps reported by ref. In summary, our simulations show that with experimentally plausible parameter values, loop extrusion can achieve fast and ≥95% efficient synapsis. We thus propose that loop extrusion plays a previously unrecognized role in mediating DSB synapsis as part of NHEJ in mammalian cells.65. However, our 3D polymer simulations show that passive diffusion would lead to unphysiologically slow synapsis in mammalian nuclei.Synapsis is the first step of DSB repair by NHEJ, which is the dominant repair pathway in the G1-phase. Synapsis has largely been assumed to occur by passive 3D diffusionFbn2 locus fell short of the synapsis speed observed in vivo, but did accelerate synapsis compared with passive diffusion alone. Guided by our analytical theory, we explored four plausible extensions to the loop extrusion model that constituted mechanistically distinct ways to improve synapsis outcome and tested the theory with 1D simulations of the extrusion-mediated DSB synapsis process, finding they were in good agreement. We found that while each mechanistic extension could moderately improve synapsis efficiency, it was by combining all four mechanisms that we found a regime with synapsis kinetics consistent with experimental observations. Our theory demonstrates that loop extrusion is a viable and efficient way to mediate the first step of the NHEJ process, DSB end synapsis, by co-opting the cell’s chromosome organization machinery. Altogether, the action of constraining LEFs and gap-bridging LEFs may constitute a new paradigm for thinking about the process of DSB end synapsis.Here we propose that protein-mediated DNA loop extrusion may promote fast and efficient synapsis in cells. We emphasize that loop extrusion can be a preemptive mechanism facilitating synapsis, in contrast to the reactive recruitment of DSB repair machinery after the DSB has occurred, where DSB ends may diffuse apart during the recruitment period. We built a probabilistic theoretical framework to understand LEF-mediated DSB end synapsis. The loop extrusion model with dynamic parameters confined by experimental data measured at the γH2AX spreading64 and by forming structural scaffolds with 53BP1 and RIF1 to protect DSB ends from aberrant processing66. In addition, loop extrusion by cohesins (one of the best-studied LEFs) has been proposed to facilitate V(D)J recombination69 and class switch recombination (CSR)70 by aligning the genomic loci to be recombined. It is worth noting that loop extrusion’s role in V(D)J recombination likely precedes DSB occurrence69, since the DSB ends are already in proximity when DSB occurs and are kept close by the post-cleavage complex along and other DDR repair factors71. Our model can be readily generalized to encompass CSR, since CSR can be considered a special case of NHEJ synapsis where two instead of one DSBs are induced70. In addition, the proposed role of LEFs in mediating γH2AX spreading64 is synergistic with our proposed gab-bridging mechanism. Our work, therefore, provides a framework to extend, integrate, and test several models.A broader role for loop extrusion in DNA repair is beginning to emerge. Recent experimental studies have proposed that DNA loop extrusion may facilitate DSB repair foci formation by mediating 72. Furthermore, it would be interesting to incorporate LEF bypass, as recent work has shown that condensins can bypass each other74, which would help improve genome coverage by DNA loops. We have also not considered the effect of DNA-damage response (DDR) foci80 on synapsis. Repair factors recruited to the DDR foci, such as 53BP1, could drive microphase separation to help constrain the DSB ends and promote synapsis83. The shift to a repressive chromatin state at the DSB through the accumulation of repressive complexes, including HP1, might also help constrain the two DSB ends to facilitate synapsis84. Other chromatin-binding proteins, such as multivalent transcription factors, could also constrain DSB end movement by bridging two sides of a DSB85. The long protein filaments formed around DSB ends by downstream NHEJ factors XRCC4, XLF, and DNA ligase IV89 could also facilitate dynamic synapsis90, potentially by reducing the dimensionality of the system and allowing bridging of DSB ends at a larger distance89. Such dynamic synapsis structures at DSB sites could efficiently transition to NHEJ ligation products92. Enhanced mobility of chromatin around DSBs mediated by nuclear actin and microtubules has also been hypothesized to promote synapsis94. We have assumed an extrusion speed of 1 kb/s based on in vitro studies95, though we note that the in vivo extrusion speed is associated with large uncertainty and was estimated to be ~0.2–0.25 kb/s for cohesin at the Fbn2 locus in mESCs41. Furthermore, the extrusion speed may vary with different genomic loci, cell types, and LEF types, and may change upon the recruitment of DSB repair factors. Finally, we have assumed a uniform distribution of LEFs throughout the genome, while previous work suggests that cohesin loading may not be uniform across the genome99. In summary, a limitation of our study is that it does not account for all possible mechanisms. Instead, our focus here is to specifically evaluate if and how loop extrusion can mediate synapsis and contribute above and beyond passive 3D diffusion. Nevertheless, as our detailed quantitative knowledge of these additional mechanisms improves, it will be important to extend our model to incorporate these additional mechanisms in the future.Importantly, we note some limitations of our study. First, we have not exhausted the space of possible mechanisms that could facilitate DSB end synapsis. For example, we do not consider alternative extensions of loop extrusion based on the diffusive sliding of LEFs57 or ESCO-mediated acetylation56. Second, we suggest that the LEF processivity/separation ratio may increase following a DSB, perhaps through ATM-mediated phosphorylation of cohesin subunits SMC1 and SMC3100, consistent with the global stabilization of cohesins observed experimentally44. Third, long-lived LEFs may correspond to acetylated STAG1-cohesins56, or LEFs of a different kind that exhibits higher processivity. Fourth, we suggest that targeted LEF loading at DSBs may be mediated by the MRN/MRX complex as the knockdown of MRX led to a significant decrease in cohesin loaded at DSBs59. Importantly, given MRN’s hypothesized loop extrusion activity101 and the enrichment of MRN subunit NBS1 at DSB sites102, MRN itself could also function as gap-bridging LEFs, effectively achieving targeting loading of LEFs while alleviating the required fold increase in loading of cohesin at DSBs. Fifth, we speculate that LEF stabilization by DSBs may be mediated by the ATM complex, which phosphorylates cohesins and accumulates cohesins in the DSB-containing TAD103. Finally, we note that our mechanistic understanding of loop extrusion and DSB repair is advancing rapidly, such that other factors and mechanisms are likely to be found to play a role beyond the ones mentioned above.Here, we speculate on candidate biological processes that could execute the required tasks for (1) stabilization of LEFs at BEs, (2) creating a mixture of long-lived and short-lived LEFs, (3) generating high LEF processivity/separation ratio, (4) facilitating targeted loading, and (5) DSB stabilization. First, we suggest that the stabilization of LEFs at BEs could be mediated by CTCF-mediated cohesin protection from its unloader WAPLτconstrained would most strongly decrease synapsis efficiency. Indeed, our 1D simulations predict the loss of stabilization of LEFs at BEs to have the strongest effect: loss of stabilization of LEFs at BEs would reduce LEF-mediated synapsis efficiency to ~60%. Experimentally, this may be tested by mutating Y226 and F228 in the N-terminus of CTCF since this is predicted to eliminate CTCF-mediated stabilization of cohesin without affecting CTCF binding to DNA57. Next, we predict that eliminating long-lived LEFs would have the second-strongest effect, reducing LEF-mediated synapsis efficiency to ~80%. For example, this could be achieved through acute auxin-inducible degron (AID) depletion of ESCO1 or STAG1. Knocking out targeted loading would have the third-strongest effect, reducing LEF-mediated synapsis efficiency to ~87% and drastically increasing the mean-synapsis time by ~181%. Experimentally, acute depletion of MRN would be one way of testing this. Lastly, although we predict lowering the processivity/separation ratio from 2 to 0.5 and knock-out of DSB stabilization to be relatively mild, reducing LEF-mediated synapsis efficiency to ~89 and ~94%, respectively, their knock-out would slow down synapsis substantially increasing the mean-synapsis time by ~67 and ~39%, respectively. Finally, given the high redundancy between the mechanisms considered in our study, we also predicted the quantitative effect of double knock-outs and alteration of the extrusion processivity/separation ; for 1D simulations, we defined a single long chromosome as a lattice of G = 2,164,800 sites (corresponding to 2164.8 Mbp of DNA). Loop extruding factors (LEFs) were comprised of two motor subunits that move bidirectionally away from each other one lattice site at a time. Like most cohesin simulations37, we assumed LEFs cannot bypass each other upon encounter. LEFs also could not extrude past the first and the last lattice sites, so LEFs could not “walk off" the chromosome.We used a fixed-time-step Monte Carlo algorithm for 1D simulations as described in previous workb. Unless specified otherwise, a boundary strength of b = 0.5 was used, in line with experimental estimates of CTCF binding site occupancy40. Once a motor subunit was stalled by a BE , no further movement of the subunit was allowed until the LEF dissociated from the locus and reloaded back onto the chromosome somewhere else. Only one motor subunit could occupy a BE lattice site at a time. We placed pairs of BEs on the chromosome (each pair consisting of two divergently oriented BEs) with the spacing between each pair ranging from 200 to 1200 kb Fig. 106.Each boundary element (BE) occupied one lattice site. BEs were directional (indicated by the red arrow in schematics) and only if the LEF motor subunit’s extrusion direction was convergent with the direction of a BE, would the motor subunit be stalled by the BE with a probability equivalent to boundary strength Each DSB site occupied two lattice sites on the chromosome, each of which corresponded to a DSB end. We introduced DSBs approximately every 3 Mb for 3D polymer simulations and 10 Mb for 1D simulations. We first randomly picked the DSB site in the very first TAD on the chromosome, and then we found the TAD 3/10 Mb to the right of the first DSB site, and randomly induced the second DSB in the TAD (so that the distance between DSBs and BEs were randomized), and so forth. This results in altogether 22–28 DSB sites for 3D polymer simulations and 216–218 DSB sites for 1D simulations on the chromosomes. We first ran 100 thousand time steps of the 1D simulations, and then introduced all the DSBs simultaneously. After DSB occurrence, LEFs were not allowed to extrude past DSB ends. We allowed multiple motor subunits to occupy the DSB end lattice site at the same time, to enable simulations with targeted loading of LEFs to DSB sites.G and LEF separation d . The dissociation rate was linked to the LEF processivity λ  , r is the 3D distance between adjacent monomers, and bo is one monomer size (the mean 3D distance between adjacent monomer). Monomers connected by LEFs were bonded via the same potential. The excluded volume interactions between monomers were implemented with a weak polynomial repulsive potentialr < σ = 1.05 monomer, where ϵm = 46,656/823,543 and ϵexc = 50kbT.The chromosome used in 3D polymer simulations consisted of 70,000 consecutive monomers bonded with pairwise potentialFor each simulation run, the polymer was initialized as a compact conformation on a cubic lattice with normally distributed velocities. The error tolerance for the variable time step Langevin integrator was set to 0.01, and the collision rate was set to 1. Simulations were performed with spherical confinement to achieve a DNA volume fraction of 20% per simulation volume. The initial cubic lattice conformation was allowed to relax into a steady-state conformation for 15 million simulation time steps. Subsequently, harmonic bonds between LEFs were added (where LEF starting positions were taken from a steady-state loop extrusion 1D simulation), and we performed a further local energy minimization to relax the structure with the LEFs constraints. We allowed for at least 173,690 additional simulation steps for the new conformations to relax with the LEF constraints before introducing DSBs (i.e. removing the harmonic bond between the two monomers corresponding to the DSB ends).41. Using inference methods described previously in ref. 41, we inferred that each simulation time step corresponds to ~0.014 s and each monomer corresponds to ~30 nm in diameter. We used a total LEF extrusion speed of 1 kb/s on chromatin (0.5 kb/s for each motor subunit)95. The chromatin diffusion coefficient was 0.7 × 10−3 μm2/s41. We note there is a wide range of estimates for the chromatin diffusion coefficient111, and the value used in this study is consistent with estimates on the order of 1 × 10−3 μm2/s based on recent live-cell imaging experiments in mammalian cells112.The simulation time and distances were calibrated in real-time and distances using MSDs determined from microscopy data113. We then calculated the 3D distance between the two DSB ends at every simulation time step following the initial 1-min lag and recorded a synapsis event when the two DSB ends were within our contact radius. Given the average DSB end displacement per time step was around 1 to 2 monomers, we used a contact radius of four monomers unless otherwise specified so that we did not miss any synapsis events. Similar trends were observed with capture radii of three or five monomers  to allow the DSB ends to diffuse freely, and then started to record synapsis events. We motivated the 1-min time scale as the mean expected time for the recruitment of DSB repair factors such as Ku70/80, which are necessary to facilitate synapsisFor the 1D simulations that were not coupled to 3D polymer simulations, at every time step during the simulation (and for each DSB site), we first checked whether there was at least one LEF whose two motor subunits were on opposite sides of the DSB . For the constrained DSB sites, we counted the number of (unextruded) lattice sites between the innermost constraining LEF and the DSB ends ; if there were only two lattice sites (corresponding to 2 kb DNA), we considered synapsis was achieved and we stopped monitoring this site. We scored all DSB sites that were not initially constrained by LEFs as having failed to achieve synapsis, and did not continue monitoring these sites in later time steps . When there was a subpopulation of long-lived LEFs, the separation d referred to the separation of long-lived LEFs and normal LEFs combined. With targeted loading of LEFs at DSB, the loading probability at the lattice sites representing DSB ends was F-fold higher than anywhere else on the chromosome.With the stabilization of LEFs at BEs, the LEFs with at least one motor subunit at the lattice sites representing BE had 115 to generate the ChIP-seq heatmaps shown in Supplementary Fig. 44.We first divided the genome into 5-kb bins, and then we counted the number of LEF motor subunits in each bin using the stored LEF positions 10-min post-DSB, and wrote this to a BED file containing each bin’s score . Along with another BED file containing the DSB coordinates, we used the plotHeatmap command from deepTools53. An iterative correction was then applied to the calculated contact maps to generate the final contact maps116. Note in our 1D simulations, unlike experiments, targeted loading of LEFs to DSB ends continued even if they are already synapsed (this was so that the LEF abundance at each DSB site did not depend on when the DSB sites are synapsed). Moreover, in experiments, the stripe pattern measured by Hi-C44 might be weaker because certain DSBs sites could have already been synapsed/repaired in a fraction of the cells, whereas we did not simulate the DNA repair process downstream of synapsis.We first normalized the LEF positions on the lattice sites by subtracting the positions of the closest DSB sites, so that the LEF positions were all relative to the closest DSB sites. We only included LEFs that were within ±2.5 Mb of the DSB sites. We then calculated the contact probability maps directly from the LEF positions, by utilizing a Gaussian approximation developed previously to simulate bacterial Hi-C mapsFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Reporting Summary