Autophagy protein NRBF2 has reduced expression in Alzheimer’s brains and modulates memory and amyloid-beta homeostasis in mice
Dysfunctional autophagy is implicated in Alzheimer’s Disease (AD) pathogenesis. The alterations in the expression of many autophagy related genes (ATGs) have been reported in AD brains; however, the disparity of the changes confounds the role of autophagy in AD.
To further understand the autophagy alteration in AD brains, we analyzed transcriptomic (RNAseq) datasets of several brain regions (BA10, BA22, BA36 and BA44 in 223 patients compared to 59 healthy controls) and measured the expression of 130 ATGs. We used autophagy-deficient mouse models to assess the impact of the identified ATGs depletion on memory, autophagic activity and amyloid-β (Aβ) production.
We observed significant downregulation of multiple components of two autophagy kinase complexes BECN1-PIK3C3 and ULK1/2-FIP200 specifically in the parahippocampal gyrus (BA36). Most importantly, we demonstrated that deletion of NRBF2, a component of the BECN1-PIK3C3 complex, which also associates with ULK1/2-FIP200 complex, impairs memory in mice, alters long-term potentiation (LTP), reduces autophagy in mouse hippocampus, and promotes Aβ accumulation. Furthermore, AAV-mediated NRBF2 overexpression in the hippocampus not only rescues the impaired autophagy and memory deficits in NRBF2-depleted mice, but also reduces β-amyloid levels and improves memory in an AD mouse model.
Our data not only implicates NRBF2 deficiency as a risk factor for cognitive impairment associated with AD, but also support the idea of NRBF2 as a potential therapeutic target for AD.
AMP-activated protein kinase
Autophagy-related protein 13
Contextual fear conditioning
Enzyme-linked immunosorbent assay
FAK family kinase-interacting protein of 200 kDa
Microtubule-associated proteins 1A/1B light chain 3
Mammalian Target of Rapamycin
Nuclear Receptor Binding Factor 2
Object location task
Class III PI3-kinase
Spontaneous alternation task
Unc-51 Like Autophagy Activating Kinase 1
Alzheimer’s disease (AD) is the leading cause of dementia affecting our elders and the seventh cause of death worldwide. While genetic variants contribute to a subset of AD cases, aging persist to be the primary risk factor for AD. In addition, the pathological hallmarks of AD are the excessive β-amyloid deposits (Aβ) and intraneuronal neurofibrillary tangles containing hyperphosphorylated-tau (pTau) [1, 2, 3]. The aberrant accumulation of Aβ and pTau suggests a failure of protein handling system during the course of the disease. In fact, loss of the proteostasis network — including the autophagy pathway — is implicated in the pathogenesis of AD [4, 5, 6, 7, 8].
Over the past decades, many studies have documented the dysregulation of autophagy in AD postmortem brains and experimental models. Early ultrastructural analysis of AD brains showed accumulation of autophagic vacuoles (AVs) in dystrophic neurites  and examination of autophagy pathway showed upregulation of mTOR activity, a negative regulator of autophagy signaling , and reduced expression of Beclin 1, a core component of class III PI3-kinase (PIK3C3) that controls autophagy initiation , therefore suggesting that autophagy is impaired in AD. However, a genome-wide analysis indicated a transcriptional upregulation of autophagy in entorhinal cortex of AD patients , and others reported hyperactivation of AMPK, a positive autophagy signaling kinase, thus supporting an enhanced autophagic activity in AD [13, 14, 15]. A recent finding showed that hippocampal neurons isolated from AD subjects contained greater expression of genes or proteins related to autophagosomes and lysosomes biogenesis. However, the same study suggests an impediment of autophagy flux despite the enhanced autophagy biogenesis . Thus, the available evidence for autophagy alteration in AD appears conflicting, obscuring the role of autophagy in the disease’s onset and progression. It is conceivable that multiple factors may contribute to the discrepancies in these results, such as the small sample size, the disease stages, the distinct brain regions and the ATGs examined. Hence, studies with increased sample size and improved approaches are necessary to comprehend the precise function of autophagy in AD. Herein, we examined the expression of over 100 autophagy related (ATG) genes in multiple brain regions from more than 200 AD postmortem brains. Our analysis revealed significant downregulation of genes encoding autophagy kinase complexes in the parahippocampal gyrus and hippocampus. Our data suggest that loss of NRBF2 functions in the hippocampus impairs memory in mice and may contribute to the cognitive impairment associated with AD. Our study also supports NRBF2 as a potential therapeutic target.
A list of 130 core ATGs was manually curated based on literature reviews [17, 18] and public database (www.tanpaku.org/autophagy/) . Expression of these genes was examined at the mRNA level in multiple brain regions of healthy control and AD patient samples from the Mount Sinai Brain Bank https://www.synapse.org/#!Synapse:syn3157743) . Differential gene analysis was performed using Bioconductor R Limma Package  with Benjamini-Hochberg correction for multiple testing. Spearman correlation analysis was performed to examine the relationship between ATG expression and CDR score. Adjusted p value< 0.05 was considered statistically significant.
Subjects were housed in groups of two to five. Food and water were supplied ad libitum in an animal facility with a regular 12 h light/dark cycle (light on at 7:00 A.M.). Tails were cut and ears were notched when pups were 7–14 days for genotyping and identification purposes respectively. NRBF2-KO mouse genotyping was performed as mentioned in  and the standard protocol of Jackson Laboratory was used for the 5XFAD mouse genotyping (stock number 008730). Mice were weaned at 21 days. Unless mentioned, mice used in this study were from three different cohorts, aged around 3–4 months. For all test conditions, the male:female ratio was ~ 1:1 and compare to WT littermate controls.
NuPAGE® MOPS SDS Running Buffer (20X), (#NP0001–02), NuPAGE® MES SDS Running Buffer (20X), (#NP0002–02), NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mmX15well, (#NP0323BOX), NuPAGE™ 4–12% Bis-Tris Protein Gels, 1.0 mmX26well, (#WG1403BOX) and PierceTM BCA Protein Assay Kit (#23225) and ProLong Diamond antifade mountant with or without DAPI (#P36962 or #P36961) were from Thermo Scientific. Western Lightning Plus ECL (#NEL105001EA) was from PerkinElmer. Immobilon®-FL Transfer membrane 0.45 um, Polyvinylidene Difluoride (PVDF) membrane (#ISEQ00010) was from Merck Millipore. HyBlot CL films (#E3012) were from Denville Scientific, Inc. Non-fat dry milk (#M0841) was from Lab Scientific. Protease and Phosphatase inhibitors tablets (#88669) were from Thermo Scientific. Dynabeads Protein G was from Novex (Life Technologies, #10004D). Ponceau S Solution (#P7170) was from Sigma-Aldrich. OCT compound (#23–730-571) and microscope slides (# 12–550-15) were from Fisher Scientific. Liquid blocker pap pen (#71310) was from Electron Microscopy Sciences (EMS).
AMPKα (D5A2) Rabbit mAb (#5831, 1:1000), phospho- AMPKα (Thr172) (40H9) Rabbit mAb (#2535, 1:500), Raptor (24C12) Rabbit mAb (#2280, 1:500), phospho-Raptor (Ser792) Rabbit polyclonal antibody (pAb) (#2083, 1:500), mTOR (7C10) Rabbit mAb (#2983, 1:500), phospho-mTOR (Ser2481) Rabbit pAb (#2974, 1:500), 4E-BP1 (#9452, 1:500), phospho-4E-BP1 (Thr37/46) (236B4) Rabbit mAb (#2855, 1:1000), ULK1 (D8H5) Rabbit mAb (#8054, 1:500), FIP200 (D10D11) Rabbit mAb (#12436, 1:250), LC3B Rabbit pAb (#2775, 1:1000) and Rabbit (DA1E) mAb IgG XP® Isotype Control (#3900) were from Cell Signaling Technology. Goat anti-Rabbit IgG-HRP pAb (sc-2004, 1:1000) were from Santa Cruz Biotechnology, Inc. NRBF2 Rabbit pAb (A301-851A) was from Bethyl Laboratories, Inc. P62 Guinea pig pAb (#GP62-C, 1:4000) was from PROGEN. P62 Guinea pig pAb (PM066, 1:4000) was from Medical and Biological Laboratories Co., LTD. (MBL). Goat anti-Mouse IgG-HRP (#A28177, 1:1000), goat anti-Guinea Pig IgG-HRP (#A18775, 1:1000) antibodies were from Thermo Scientific. PSD95 (6G6-1C9) mAb (#MA1–045, 1:1000) was from Thermo Scientific. VAMP2 Rabbit pAb (#104202, 1:5000) was from Synaptic Systems.
Phophos- AMPKα (Thr172) Rabbit pAb (#AP0432, 1:200 IHC) was from ABclonal Technology. β-Amyloid (D54D2) Rabbit monoclonal antibody (mAb) (#8243, 1:500) and cleaved Caspase-3 (Asp175) Rabbit pAb (#9661, 1:100) were from Cell Signaling Technology. Goat anti-Rabbit IgG-Alx647 (#A21246, 1:500) antibody was from Thermo Scientific.
Perfusion and Cryo-sectioning
Mice were transcardially perfused with 25–30 ml (6–7 ml/min.) of ice-cold PBS 1X to remove excess blood, then perfused with 25–30 ml of ice-cold 4% paraformaldehyde. After perfusion, the brain was removed from the skull and fixed overnight at 4 °C in 15 ml falcon tube filled with 4% PFA. The next morning, the brain were washed 3x with 15 ml of ice-cold PBS 1X and incubate with 15 ml of 30% sucrose solution for at least 24 h or up until the brain has sunk at the bottom of the tube. Left and right sagittal hemisphere were divided, embedded in OCT compound, gradually froze in liquid nitrogen, and stored at − 80 °C. Cryo-sectioning was performed using Leica CM3050 S cryostat. 40 μm sagittal sections were conserved at − 20 °C in anti-freezing medium (25% glycerol, 30% ethylene glycol, 50 mM phosphate buffer pH 7.4).
Heat-induced epitope retrieval
The protocol used is based on the following reference . Briefly, sections were wash 3 × 10 min. With 500 μl PBS 1X at RT on microscope slides and allowed to dry for 20–30 min. in the dark. Next, we incubate the slides in pre-warmed citrate buffer (10 mM Sodium Citrate dihydrate pH 6.0, 0.05% Tween 20) in a water bath heated at 65 °C for 45 min. After incubation, slides were washed 3 × 10 min. at RT with PBS 1X while shaking. A final wash was executed for 10 min. at room temperature (RT) with PBS 1X containing 0.1% Triton-X-100.
Immunofluorescence and confocal imaging
Brain sections were encircled with liquid blocker pap pen and blocked with 150 μl/section PBS containing 5% goat serum and 0.1% Triton X-100 for 1 h at RT. Sections were incubated in a humid chamber with 100 μl of primary antibody diluted in blocking buffer overnight at 4 °C. After washing 3 × 10 min. With 150 μl PBS, sections were incubated with 100 μl Alexa-conjugated secondary antibody for 1 h at RT. After 4 washes with PBS, sections were mounted with ProLong Diamond antifade reagent. Sections were examined under Carl Zeiss upright confocal microscope (LSM780 system). Images were taken sequentially with 40X oil immersion objective lens at RT. Single or tile scan acquisitions were performed by Zen2012 software.
Confocal image analysis
Basic Intensity Quantification was performed with Image J. RGB pictures were converted into single 16-bit grayscale images. A duplicate of the grayscale picture was generated and further processed into a binary picture. The background was subtracted with the rolling ball (radius of 50.0 pixels) tool with light background and sliding paraboloid options selected. The threshold was finally adjusted to highlight all the structures having signal. Next, we calibrated the scale, set the measurements, and redirect it to the original grayscale image. The particles were analyzed and stated as mean gray intensity over the total area (μm2). We finally normalized these data to the WT mean values and reported the fold change.
Once the tissue harvested and flash frozen, 400 μl of homogenization buffer (0.32 M sucrose, 1 mM NaHCO3, 20 mM HEPES, 0.25 mM CaCl2, 1 mM MgCl2 and protease/phosphatase inhibitors) were added to 0.1 g of tissue and homogenized with blue pestle and cordless pestle motor in 1.5 or 2.0 ml Eppendorf tube. Using insulin syringe, 20 up and down were performed to disrupt the tissue. The homogenates were incubated for 30 min. at 4 °C using end over end mixing. Then the samples were centrifuged at 1500×g for 10 min. at 4 °C, the supernatant harvested, and the pellet discarded. Samples were diluted 1:10 and protein concentration was measured.
Immunoprecipitations (IPs) were performed using 150 μg of proteins extracted from hippocampal homogenates diluted in 300 μl of homogenization buffer. Anti-ULK1 antibody (1:150, v/v) or isotype control (same concentration than ULK1 Ab) were added to each tube and incubated overnight at 4 °C with end over end agitation. The next morning, 30 μL of Dynabeads Protein G was added, followed by an 1 h. incubation at 4 °C. Samples were then centrifuge 1 min at 4000 RPM in a microcentrifuge and washed three times with 1 mL of homogenization buffer, immunoprecipitated proteins were eluted by addition of 30 μL of 4X SDS sample buffer, followed by a 10 min. Incubation at 95 °C. Initial lysates and immunoprecipitated proteins were analyzed by SDS-PAGE and immunoblotting with specific antibodies.
All biological samples have been analyzed at least in duplicate in two independent experiments. Samples were diluted to 1 mg/ml with buffer and SB4X denaturation buffer (200 mM 4X Tris-HCl/SDS ph 6.8, 8% SDS, 400 mM DTT, 40% glycerol (v/v) 0.4% Bromophenol Blue) diluted to 1X. Samples were denatured at 95 °C for 10 min and spin at RT for 30s at 14000 rpm. 10μg of protein samples were separated on 4–12% Bis-Tris NuPAGE gels for 70-80 min. at 150 V at room temperature (RT) using 1X MOPS or 1X MES buffer (Invitrogen). The proteins were transferred to a PVDF membrane for 1 h at 100 V at 4 °C. The membranes were dried and stained for 10 min. With Ponceau S. Excess stain was removed for 2 min. With Milli-Q water. The membranes were scanned, cut and block in TBS 1X containing 0.1% Tween 20 (TBST) and 5% non-fat dry milk for 1 h at RT. Primary antibodies were applied, and membranes were incubated overnight at 4 °C. The membranes were washed 3 × 8 min. With TBST. Secondary antibodies were applied, and membranes were incubated for 1 h at RT. The membranes were washed 3 × 8 min. With TBST and twice with TBS 1X. The proteins were visualized using ECL detection kit.
Immunoblot membrane stripping
After phospho-antibodies detection, membranes were washed once during 10 min. in distilled water to remove ECL. Membranes were incubated 3 × 10 min. in NaOH (0.2 M) solution to strip off the antibodies. Membranes were finally incubated in TBST buffer for 10 min. and the blotting procedure was started over.
Western blot quantification was performed based on the recommendations of Gassmann et al. . All quantified immunoblots were revealed using the same type of films and carefully exposed to avoid saturation. Films were scanned using an Epson Perfection v500 or v800 Photo scanner. Acquisition was performed at 600 dpi in 16-bits grayscale with auto-exposure and colour-correction options turned off. Images were analyzed using the ImageJ software. Lanes were selected and plotted using the ‘Gel analyzer’ functions. Peaks on the plots were individually closed to the background level of each lane using the Straight line’ tool and the enclosed area was measured using the ‘Wand’ tool.
Stereotaxic delivery of recombinant adeno-associated virus, serotype 9 (AAV) for expression of a mCherry or NRBF2-mCherry fusion protein under control of the CMV promoter was done as follows: mice were anesthetized with 2% isoflurane and 1 μl of virus for each hemisphere (∼5.8 × 1010 viral genomic copies) was injected at a rate of 0.2 μl/min using a Hamilton syringe, a micro pump, and stereotaxic instrument (Stoelting). Syringe remained in place for an additional 2 min. After completion of the injection. Coordinates for injection were as follows: − 2.0 mm anterior/posterior, ±1.5 mm medial/lateral, and − 1.75 mm dorsal/ventral. Viruses were produced and purified by Vigene Biosciences Inc. (Rockville, MD, USA).
Mouse Aβ42 ELISA
Aβ42 level was quantify from hippocampal extracts. Fractions were analyzed in duplicate. Same protein amount was loaded into each well, and the plate was incubated overnight at 4 °C with gentle agitation. ELISA was performed according to the manufacturer’s instructions (#KMB3441, Thermo Scientific).
Object Location Task (OLT) 
On habituation day (day 1), mice were individually placed into an open-field box (40 × 40 cm) surrounded by 40 cm high walls made of transparent plastic and allowed to freely explore the arena for 5 min in an infrared-lit room. On training day (day 2), mice were placed into the previously explored box now containing two similar objects and allowed to explore for 10 min. On testing day (day 3), one object was moved forward, and the mice were placed back into the arena and allowed to explore for 10 min. Videos were recorded by the EthoVision video tracking system (Noldus, Wageningen, The Netherlands) and were manually scored. Object explorations were counted once the following criteria have been met: the snout of the mouse is oriented toward and close to the object and the animal’s body is beyond the object (no climbing). To assess object bias we evaluate the time spent sniffing each object on day 2 relative to the total time spent exploring (Time spent sniffing object 1/ Total time spent sniffing object 1 and 2 X 100%). Mice exhibiting an object bias score below 0.2 or above 0.8 were excluded. Discrimination ratio was calculated as follows: time spent sniffing the object divided by the total time spent sniffing both objects, Score equivalent to 0.5 indicates equal time spent exploring the displaced and familiar objects.
Contextual fear conditioning (CFC)
CFC experiments were conducted in sound attenuating chambers with automated stimulus delivery software (Med Associates, St. Albans, VT, USA). On training day, mice were exposed to a 218 s period of acclimation to the conditioning arena (context A) followed by three consecutive foot shocks (0.5 mA, 2 s, 100 s interval between shocks) and a final 30 s resting period. On testing day, mice were re-exposed to context A for 3 min. One hour after re-exposure to context A, mice were placed in a modified arena (context B) and allowed to explore for 3 min. Percentage time freezing was quantified by automated motion-sensitive software (Video Freeze; Med Associates).
Radial-arm maze (RAM) 
The maze consisted of eight arms (7.5 × 35 cm, 17.5 cm high walls) assembled radially around a circular starting platform. Mice were placed onto the starting platform and were free to enter the arms. Mice were tested until all eight arms were visited once. Each repeated entry in arm was counted as an error. Mice were trained on day 1 and tested on day 2.
Spontaneous Alternation Task (SAT) 
The Y-Maze (Maze Engineers) consisted of three gray acrylic closed arms measuring 35 cm L × 5 cm W × 20 cm H. Mice were placed in the center of the maze and were free to explore for 10 min. The number of alternation and the number of entries were recorded and scored by the EthoVision video tracking system (Noldus, Wageningen, The Netherlands). Percentage of alternation was calculated as the number of alternation/ (total number of entries - 2) × 100. Total number of entries was reported to control any potential hyperactivity.
Hippocampal slice preparation and field electrophysiology
Hippocampal slices (350–400 μm) were prepared from NRBF2-deficient mice and wild type littermates. Slices were perfused with Ringer’s solution containing (in mM): NaCl, 125.0; KCl, 2.5; MgSO4, 1.3; NaH2PO4, 1.0; NaHCO3, 26.2; CaCl2, 2.5; glucose, 11.0, and bubbled with 95% O2/5% CO2, at 32 °C during extracellular recordings. Slices were maintained for 1–2 h prior to establishment of a baseline of field excitatory postsynaptic potentials (fEPSPs) recorded from stratum radiatum in area CA1, evoked by stimulation of the Schaffer collateral-commissural afferents (100 μs pulses every 30 s) with bipolar tungsten electrodes placed into area CA3 . The EPSP initial slope (mV/ms) was determined from the average waveform of four consecutive responses. After determining the input/output relationship, long-term potentiation (LTP) was induced by a high-frequency stimulus (four trains of 100 Hz, 1 s stimulation separated by 5 min) with a success rate > 90%. Field EPSP initial slopes from averaged traces after LTP induction were normalized to baseline.
Statistical analyses were performed using GraphPad Prism version 8.1 for Windows (GraphPad Software) using the unpaired one- or two-tailed Student’s t test and Regular or Row-Matched Two-way ANOVA test followed by Bonferroni’s multiple comparisons test. Data were considered significant when P values were < 0.05 (*), < 0.01(**) or < 0.001 (***).
Analysis of AD brains reveals autophagy alterations and reduced NRBF2 expression in the parahippocampal gyrus and hippocampus
Description of the dataset used in this study
Brain Bank (Template)
BA10-Anterior prefrontal cortex
BA22-Superior Temporal Gyrus
BA44-Inferior Frontal Gyrus
Medial Temproral Gyrus
Superior Frontal Gyrus
NRBF2 depletion reduces autophagy, causes memory deficits, and impairs long-term potentiation
To further understand the basis of the memory deficits in NRBF2-KO mice, we examine long-term potentiation (LTP), a well-known cellular mechanism linked to learning and memory. Results of the field recordings from the Schaffer collateral pathway showed a reduced maintenance of LTP in NRBF2-KO animals when compared to WT (Fig. 2d), while no change in basal synaptic transmission (Fig. 2e) or expression of pre- and post-synaptic markers (Additional file 2: Figure S6A-C) were observed. Given that AMPK signaling modulates autophagy , memory and LTP processes [14, 39, 40], we decided to examine the phosphorylation of AMPK-Thr172 as well as the phosphorylation of Raptor-Ser792 — an AMPK substrate  — to understand further the molecular changes occurring in NRBF2-KO hippocampus that could relate to LTP deficit. Western blot analysis showed higher phosphorylation levels of AMPK-Thr172 and Raptor-Ser792 in NRBF2-KO animals, whereas no change of their total protein levels was detected (Additional file 2: Figure S7A-C). We also performed immunofluorescence imaging experiments and showed that CA1 and CA3 pyramidal neurons of NRBF2-KO mice had enhanced phospho-AMPK-T172 signal, thus validating the hyperactivation of AMPK in NRBF2-KO hippocampus (Additional file 2: Figure S7D-E). While increased phosphorylation of Raptor-Ser792 is known to inhibit mTOR kinase function, we examined mTOR catalytic activity through assessment of its autophosphorylation site, i.e. Ser2481,  and phosphorylation of 4E-BP1-Thr37/46 , a known mTOR substrate, while both are crucial for LTP and memory consolidation [44, 45, 46]. Immunoblot analysis demonstrated that phosphorylation of mTOR-Ser2481 and 4E-BP1-Thr37/46 were decreased in hippocampal extracts of NRBF2-KO mice when compared to the littermate controls, while no change in these proteins basal expression was observed (Additional file 2: Figure S7F-H). Together, these data suggest that hyperactivation of AMPK and reduced mTOR activity could be related to LTP and memory deficits in NRBF2-KO mice.
Loss of NRBF2 promotes accumulation of APP C-terminal fragments and Aβ in mouse hippocampus
AAV transduction of NRBF2 into the dorsal hippocampus rescues autophagy and memory impairments of NRBF2-KO and reduces β-amyloid load in 5XFAD mice
Several studies have attempted to characterize the changes of autophagy genes in AD brains. However, the results lack an agreement on the exact nature of the change in autophagy. By leveraging the transcriptomic dataset of a large AD cohort, our analysis reveals altered expression of specific functional groups of autophagy genes in the PHG of AD patients. Our data shows that downregulation of various autophagy kinase complex components, e.g. BECN1-PIK3C3, ULK1/2-ATG13-FIP200, and NRBF2, is prominent and coincides with the disease progression (CDR). Indeed, dysregulation of Beclin 1-PIK3C3 and ULK1 have already been reported in AD brains [10, 11, 13, 14, 48]. However, the significance of NRBF2 or its cellular functions in learning and memory has never been demonstrated.
Our study is the first to demonstrate that loss of NRBF2 and NRBF2-associated protein complex integrities promote memory impairments in young animals. Our findings of reduced expression of NRBF2 (along with other ATGs) in AD brains and characterization of NRBF2-KO mice support the role of impaired NRBF2-associated function in promoting memory dysfunctions and AD risk. Specifically, our study demonstrates that NRBF2-KO mice develop memory deficits through multiple cognition assays, i.e. working memory (RAM), reference memory (CFC) and recognition memory (OLT), while displaying minor change in anxiety-related behavior based on OF, LD and EPM studies. Thus, our data may suggest selective vulnerability of hippocampal regions responsible for memory function caused by deletion of NRBF2. Interestingly, these cognitive domains are known to be impaired in different AD mouse models and are recognized to model the preclinical behavioral changes observed in AD . Our work therefore provide insight into how autophagy related processes, mediated by NRBF2, could potentially modulate pathogenic pathways in AD.
Furthermore, we showed that depletion of NRBF2 alters ULK1-FIP200 complex, in addition to Beclin 1-PIK3C3 complex as we previously reported . Reestablishing NRBF2 expression by viral transduction into dorsal hippocampus of KO mice rescues memory impairment, autophagy flux, ULK1-FIP200 interaction, thus supporting that memory deficits are unlikely caused by developmental effect or neurodegeneration. Therefore, the lack of NRBF2-related functions in the hippocampus primarily accounts for the memory impairment in the mutant mice. Our work shows an impairment of LTP while observing no change in basal synaptic transmission in the hippocampus of NRBF2-KO mice. However, the precise mechanism that contributes to LTP disruption remains to be clarified. One could suspect that a modification in AMPA receptors (AMPAR) trafficking is causing the LTP impairment or cognitive deficits in the mutant mice. In fact, BECN1-PIK3C3 and ULK1/2 complexes have been shown to promote endocytosis and ER-to-Golgi trafficking respectively [50, 51]. Therefore, dysregulation of the functions of these kinases beyond autophagy in the hippocampus of NRBF2-KO mice could disrupt the AMPAR trafficking and explain the LTP inhibition. Nevertheless, deeper mechanistic analysis is required to precisely define the molecular components contributing to the LTP impairment observed in NRBF2-KO mice.
In summary, our findings identify progressive decline in the expression of NRBF2 and NRBF2-associated autophagy complex in specific brain regions of AD patients, which correlates with clinical dementia progression. Our investigation reveals the impact of dysfunctional NRBF2-related pathways in promoting Aβ accumulation and memory deficits in experimental animal models. Our study also provides evidence that restoration or modulation of NRBF2 and perhaps its associated kinase complexes activities may represent a new therapeutic strategy for improving memory impairment related to AD.
We thank members of the Yue, Lu, Sweet, Blitzer and Bozdagi-Gunal labs for technical assistance.
J-HL initiated the project. ES and OB-G performed and analyzed the electrophysiological experiments. QW executed the bio-informatics analysis. JLJ and YZ help to score the videos recorded during the OLT task. C-ZC and X-XZ performed and analyzed the experiments with aged mice. IC executed IHC experiments with 5XFAD mice injected with AAV viruses. VL designed, executed, and analyzed all the remaining experiments. VL and ZY wrote the manuscript. OB-G, J-HL, QW, VL and ZY reviewed and edited the manuscript. All authors read and approved the final manuscript.
This work was supported by NIH/NINDS R01-NS060123 (Z.Y.), NIH/NIMH R01-MH103455 (O.B-G.), 2U01AG046170–06 (B.Z.), YS2017YFGH000899, FDCT-024/2017/AMJ, FDCT-022/2015/A1 (J-H.L), and CIHR postdoctoral fellowship MFE-146812 (V.L.).
All animal studies were conducted in compliance with the IACUC committee of Icahn School of Medicine at Mount Sinai.
Consent for publication
All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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