Inactive variants of death receptor p75 NTR reduce Alzheimer’s neuropathology by interfering with APP internalization
2020; Springer Nature; Volume: 40; Issue: 2 Linguagem: Inglês
10.15252/embj.2020104450
ISSN1460-2075
AutoresChenju Yi, Ket Yin Goh, Lik‐Wei Wong, Ajeena Ramanujan, Kazuhiro Tanaka, Sreedharan Sajikumar, Carlos F. Ibáñez,
Tópico(s)Computational Drug Discovery Methods
ResumoArticle1 December 2020free access Source DataTransparent process Inactive variants of death receptor p75NTR reduce Alzheimer's neuropathology by interfering with APP internalization Chenju Yi Chenju Yi Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ket Yin Goh Ket Yin Goh Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Lik-Wei Wong Lik-Wei Wong Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ajeena Ramanujan Ajeena Ramanujan Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Kazuhiro Tanaka Kazuhiro Tanaka Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Sreedharan Sajikumar Sreedharan Sajikumar Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Carlos F. Ibáñez Corresponding Author Carlos F. Ibáñez [email protected] orcid.org/0000-0003-4090-0794 Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Department of Neuroscience, Karolinska Institute, Stockholm, Sweden Stellenbosch Institute for Advanced Study, Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa Search for more papers by this author Chenju Yi Chenju Yi Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ket Yin Goh Ket Yin Goh Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Lik-Wei Wong Lik-Wei Wong Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Ajeena Ramanujan Ajeena Ramanujan Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Kazuhiro Tanaka Kazuhiro Tanaka Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Sreedharan Sajikumar Sreedharan Sajikumar Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Search for more papers by this author Carlos F. Ibáñez Corresponding Author Carlos F. Ibáñez [email protected] orcid.org/0000-0003-4090-0794 Department of Physiology, National University of Singapore, Singapore City, Singapore Life Sciences Institute, National University of Singapore, Singapore City, Singapore Department of Neuroscience, Karolinska Institute, Stockholm, Sweden Stellenbosch Institute for Advanced Study, Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa Search for more papers by this author Author Information Chenju Yi1,2,†, Ket Yin Goh1,2, Lik-Wei Wong1,2, Ajeena Ramanujan1,2, Kazuhiro Tanaka1,2, Sreedharan Sajikumar1,2 and Carlos F. Ibáñez *,1,2,3,4 1Department of Physiology, National University of Singapore, Singapore City, Singapore 2Life Sciences Institute, National University of Singapore, Singapore City, Singapore 3Department of Neuroscience, Karolinska Institute, Stockholm, Sweden 4Stellenbosch Institute for Advanced Study, Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa †Present address: The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, China *Corresponding author. Tel: +65 6516 5889; E-mail: [email protected] The EMBO Journal (2021)40:e104450https://doi.org/10.15252/embj.2020104450 Correction(s) for this article Inactive variants of death receptor p75NTR reduce Alzheimer's neuropathology by interfering with APP internalization01 September 2021 Correction added on September 1st 2021, after first online publication: Figure 6A has been corrected; see the associated Corrigendum at https://10.15252/embj.2021109067. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract A prevalent model of Alzheimer's disease (AD) pathogenesis postulates the generation of neurotoxic fragments derived from the amyloid precursor protein (APP) after its internalization to endocytic compartments. The molecular pathways that regulate APP internalization and intracellular trafficking in neurons are incompletely understood. Here, we report that 5xFAD mice, an animal model of AD, expressing signaling-deficient variants of the p75 neurotrophin receptor (p75NTR) show greater neuroprotection from AD neuropathology than animals lacking this receptor. p75NTR knock-in mice lacking the death domain or transmembrane Cys259 showed lower levels of Aβ species, amyloid plaque burden, gliosis, mitochondrial stress, and neurite dystrophy than global knock-outs. Strikingly, long-term synaptic plasticity and memory, which are completely disrupted in 5xFAD mice, were fully recovered in the knock-in mice. Mechanistically, we found that p75NTR interacts with APP at the plasma membrane and regulates its internalization and intracellular trafficking in hippocampal neurons. Inactive p75NTR variants internalized considerably slower than wild-type p75NTR and showed increased association with the recycling pathway, thereby reducing APP internalization and co-localization with BACE1, the critical protease for generation of neurotoxic APP fragments, favoring non-amyloidogenic APP cleavage. These results reveal a novel pathway that directly and specifically regulates APP internalization, amyloidogenic processing, and disease progression, and suggest that inhibitors targeting the p75NTR transmembrane domain may be an effective therapeutic strategy in AD. Synopsis 5xFAD mice, a severe animal model of Alzheimer's disease (AD), expressing signaling-deficient variants of the p75 neurotrophin receptor (p75NTR) show greater neuroprotection from AD neuropathology than animals completely lacking this receptor. The results reveal a novel pathway that directly and specifically regulates APP internalization, amyloidogenic processing and disease progression, and suggest that inhibitors targeting the p75NTR transmembrane domain may be an effective therapeutic strategy in AD. p75NTR knock-in mice lacking the death domain or conserved transmembrane Cys259 were crossed to 5xFAD mice and compared to 5xFAD animals in p75NTR knock-out and wild type backgrounds. Knock-in mice showed lower levels of Aβ species, amyloid plaque burden, gliosis, mitochondrial stress and neurite dystrophy than global knock-outs. Long-term synaptic plasticity and memory, which are completely disrupted in 5xFAD mice, were fully recovered in the knock-in mice. p75NTR interacted with APP at the plasma membrane and regulated its internalization and intracellular trafficking in hippocampal neurons. Inactive p75NTR variants internalized slower than wild type p75NTR and showed increased association with the recycling pathway, thereby reducing APP internalization and colocalization with BACE1, favoring non-amyloidogenic APP cleavage. Introduction A central tenet of the amyloid hypothesis of AD pathogenesis is the generation of neurotoxic fragments of APP through a series of proteolytic cleavages (Karran et al, 2011; Selkoe & Hardy, 2016). APP cleavage by BACE (beta-site APP cleaving enzyme) at an extracellular site close to the plasma membrane leaves a transmembrane C-terminal stub (beta-carboxyterminal fragment or CTFβ) that serves as a substrate for further intramembrane cleavage by gamma-secretase, a multisubunit complex that includes the aspartyl protease presenilin-1 (PS-1) as its catalytic subunit. Cleavage by the gamma-secretase complex liberates a soluble CTFβ (referred to as either sCTFβ or APP intracellular domain, AICD) and a small N-terminal fragment of 40 or 42 amino acids in length known as the amyloid beta peptide or Aβ, the main component of the amyloid plaques that accumulate in the AD brain (Karran et al, 2011; Selkoe & Hardy, 2016). The majority of familial AD cases are caused by mutations in the genes encoding APP or PS-1 (Karran et al, 2011; Selkoe & Hardy, 2016), supporting the amyloid hypothesis of AD pathogenesis. APP can also be cleaved by cell surface alpha-secretases, most notably ADAM10, in an extracellular site very close to the plasma membrane, but C-terminal to the site of BACE cleavage. Thus, alpha-secretase cleavage precludes the generation of all BACE-derived products, including Aβ, and constitutes the non-amyloidogenic pathway in APP processing. Cleavage by alpha-secretase generates a soluble N-terminal fragment (sAPPα) and a C-terminal stub (sCTFα) that can be further processed by gamma-secretase. Recent studies have indicated that, while cleavage by alpha-secretases occurs at the plasma membrane, proteolytic processing by BACE requires APP internalization from the cell surface and thus mainly takes places in intracellular, endocytic compartments (Schneider et al, 2008; Sannerud et al, 2011; Haass et al, 2012). Several studies have linked APP internalization to Aβ production (Koo & Squazzo, 1994; Selkoe et al, 1996), and complementary lines of evidence support this notion, including the requirement of low pH for BACE optimal catalytic activity (Vassar et al, 2009), the fact that genetic or pharmacological inhibition of APP internalization reduces Aβ generation (Perez et al, 1999; Carey et al, 2005), and evidence that neuronal activity enhances Aβ production by inducing APP internalization and trafficking to BACE-containing endosomes (Das et al, 2013, 2016). Thus, the localization and intracellular trafficking of APP appear to be critical for the balance between competing amyloidogenic and non-amyloidogenic pathways of APP processing. Aside from main components of the endocytic machinery, such as dynamin, our knowledge of the molecular pathways that can regulate APP internalization and intracellular trafficking in neurons is incomplete. p75NTR is a member of the death receptor superfamily, characterized by the presence of a death domain (DD) in their intracellular region (Liepinsh et al, 1997), which also includes the tumor necrosis factor receptor 1 (TNFR1), CD40, Fas, and others (Ibáñez & Simi, 2012). Many of these receptors can induce cell death pathways as a mechanism for clearing damage produced after a lesion or insult. However, after severe injury or disease, they can also amplify tissue damage as a result of overactivation and/or overexpression. Upon neural injury or cellular stress, p75NTR signaling can contribute to neuronal death, axonal degeneration, and synaptic dysfunction (Ibáñez & Simi, 2012). p75NTR can function as a receptor of neurotrophins, a family of neurotrophic growth factors, that includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and others, as well as other ligands unrelated to the neurotrophins, including Aβ (for review, see (Underwood & Coulson, 2008)). Notably, expression of p75NTR is increased in the brain of AD patients (Ernfors et al, 1990; Mufson & Kordower, 1992; Hu et al, 2002; Chakravarthy et al, 2012) as well as animal models of AD (Chakravarthy et al, 2010; Wang et al, 2011). Aβ can induce rapid cell death in cultured neurons through direct interaction with p75NTR and downstream activation of cell death pathways (Rabizadeh et al, 1994; Yaar et al, 1997; Perini et al, 2002; Sotthibundhu et al, 2008; Knowles et al, 2009), but the relevance of in vitro overnight effects is unclear, as neuronal degeneration occurs during long periods of time in AD patients, and it is in fact seldom observed in animal models of AD. In line with this, elimination of p75NTR affords a rather limited improvement in those models (Knowles et al, 2009). In this study, we used the 5xFAD model of AD (Oakley et al, 2006) to investigate neuropathological effects in different strains of knock-in and knock-out p75NTR mutant mice. Unexpectedly, we found that knock-in mutations that inactivate p75NTR signaling, but leave normal levels of receptor expression, conferred much higher protection from AD-associated neuropathology than a global knock-out. In our efforts to understand how an inactive receptor can afford greater neuroprotection than the absence of the receptor, we discovered a novel mechanism by which p75NTR regulates APP internalization, intracellular trafficking, and its localization to endocytic compartments containing BACE. Results Reduced Aβ content and histopathology in the hippocampus of 5xFAD mice carrying inactive p75NTR variants The transmembrane domain of p75NTR contains a highly conserved cysteine residue (Cys259 in mouse) which is critical for p75NTR activation and signaling in response to neurotrophin ligands (Vilar et al, 2009). Neurons from knock-in mice carrying a Cys to Ala mutation at this position (C259A), or lacking the receptor death domain (ΔDD), are resistant to cell death induced by pro-neurotrophins and neurodegeneration induced by epileptic seizures, to a similar extent as knock-out (KO) neurons lacking p75NTR entirely (Tanaka et al, 2016). Cortical neurons derived from these 3 lines also show comparable resistance to Aβ-mediated toxicity in vitro (Fig 1A–C). In order to study the contribution of p75NTR activity to AD neuropathology in vivo, we crossed each of the 3 lines of p75NTR mutant mice to the 5xFAD mouse model of AD. These mice express a human APP transgene carrying three mutations found in AD patients and a human PS-1 transgene with two AD mutations, both under regulatory sequences of a Thy1 transgene, thus primarily directing expression to neurons (Oakley et al, 2006). 5xFAD mice develop cerebral amyloid plaques starting at 2 months of age, achieve substantial Aβ burden with gliosis and neurite degeneration by 4 months, and show significant memory impairment by 6 months. The severity and accelerated progression of AD pathology displayed by this model served as a stringent test for assessing potential protective effects of the different p75NTR mutations. At 5 months of age, 5xFAD mice showed increased levels of p75NTR in the hippocampus compared to wild-type mice, with prominent expression in dendrites of pyramidal neurons (Fig 2A). At 6 months, Western blots of hippocampal extracts indicated approximately 3-fold increase in p75NTR protein levels in 5xFAD mice compared to wild-type controls (Fig 2B and C). Figure 1. Neurons lacking p75NTR transmembrane Cys259, the death domain, or the whole receptor are equally resistant to Aβ neurotoxicity in vitro Representative photomicrographs of MAP2 (green) and cleaved caspase-3 (red) immunocytochemistry of embryonic cortical neurons from wild-type (upper panels) or C259A mice treated with vehicle or 10 µM Aβ oligomers for 14 h. Scale bar 15 µm. Quantification of active (cleaved) caspase-3 in cultured cortical neurons of the indicated genotypes treated for 14 h with vehicle or Aβ oligomers (2.5, 5, or 10 µM). Data are expressed as percentage cleaved caspase-3 and MAP2 double positive cells over the total number of MAP2 positive cells. Error bars indicate SD. N = 3 independent experiments each performed in duplicate. *P < 0.05; **P < 0.01 versus vehicle (Student's t-test). Quantification of neurite length in cultured cortical neurons of the indicated genotypes treated for 24 h with vehicle or Aβ oligomers (2.5, or 10 µM). Data are expressed as mean neurite length in individual neurons, relative to wild-type vehicle (set to 100 units). Error bars indicate SD. N = 3 independent experiments, n ≥ 35 neurons per experiment. *P < 0.05 versus vehicle (Student's t-test). Download figure Download PowerPoint Figure 2. Reduced Aβ content in the hippocampus of 5xFAD mice carrying inactive p75NTR variants A. Immunohistochemistry for p75NTR in hippocampal CA1 of 5-month-old wild type, 5xFAD, and p75NTR knock-out (KO) mice. Scale bar, 20 μm. B. Western blot analysis of p75NTR expression in total lysates of hippocampus from 6-month-old wild-type (WT) and 5xFAD mice assessed using antibody GT15057 against p75NTR extracellular domain (Table S1). The lower panel shows reprobing for GAPDH to control for equal gel loading. C. Quantification of p75NTR expression in mouse hippocampus relative to GAPDH. Mean ± SEM; **P < 0.01; N = 5 mice per group (one-way ANOVA followed by post hoc test). D. Immunostaining for Aβ plaques with 6E10 antibody in coronal sections through the hippocampus of 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A mice of the indicated ages. Scale bar, 400 μm. E. Quantification of Aβ plaque burden in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Aβ plaques (mean ± SEM, N = 5 mice per group). *P < 0.05 and **P < 0.01 versus 5XFAD. #P < 0.05 versus 5XFAD/KO (one-way ANOVA followed by post hoc test). F. Quantification of the number of Aβ plaques larger than 30 µm in diameter per µm2 in coronal sections through the hippocampus of 9-month-old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A mice. Color codes are as in panel (E). Histogram shows mean ± SEM, N = 4 mice per group; *P < 0.05 KO versus knock-in genotypes. G–I. ELISA determinations of Aβ1-42 content in hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Aβ monomers (G) refer to the soluble fraction after Tris-buffered saline extraction, Aβ oligomers (H) to the soluble fraction after RIPA buffer extraction of the Tris-buffered saline pellet, and Aβ fibrils (I) to the soluble fraction after formic acid treatment of the RIPA pellet. See Materials and Methods for details. Shown is mean ± SEM; squares denote individual animals. *P < 0.05 and **P < 0.01 versus 5XFAD. #P < 0.05 versus 5XFAD/KO (one-way ANOVA followed by post hoc test). Source data are available online for this figure. Download figure Download PowerPoint 5xFAD mice developed progressively increased Aβ plaque burden as detected histologically in sections through the hippocampus; at 12 months of age, 5% of the area of the hippocampus was occupied by Aβ plaques (Fig 2D and E). No Aβ immunoreactivity could be detected at any age in wild-type mice (not shown). In the 5xFAD background, all three p75NTR mutants (5xFAD/KO, 5xFAD/C259A, and 5xFAD/ΔDD, respectively) showed reduced levels of Aβ plaque burden at all the ages examined (6, 9, and 12 months) compared to 5xFAD mice expressing wild-type p75NTR (Fig 2E). However, the two knock-in mutants afforded greater protection than the knock-out, with differences against 5xFAD/KO mice reaching statistical significance, specially at 9 and 12 months of age (Fig 2E). In addition, all p75NTR mutants showed significantly reduced number of large Aβ plaques (> 30 μm in diameter) compared to 5xFAD mice that were wild type for p75NTR (Fig 2F). Next, we quantified the levels of Aβ1–42 monomers, oligomers, and fibrils by ELISA, after differential detergent and acid extraction from hippocampus of 9-month-old mice. All three p75NTR mutations significantly lowered the levels of Aβ1–42 species in 5xFAD mice, with, again, the knock-in variants showing a stronger effect than the knock-out (Fig 2G–I). Astrogliosis and microgliosis were significantly increased between 6 and 12 months of age in the hippocampus of 5xFAD mice, reaching 15–20% and 6–10% of the hippocampus area, respectively, compared to 5–10% and 2–4% in wild-type controls (Fig 3A–D). Astrocytes and microglial cells with morphology indicative of an activated state were found concentrated around Aβ plaques in the hippocampus of 5xFAD animals (Fig 3A and C). All three p75NTR mutations significantly reduced both forms of gliosis in the 5xFAD hippocampus. However, the knock-in strains showed the lowest levels of gliosis, reaching statistically significant differences compared to 5xFAD/KO at 9 and 12 months (Fig 3B and D). Next, we assessed the extent of neurite dystrophy in dendritic arbors of pyramidal hippocampal neurons, as assessed by accumulation of reticulon 3 (RTN3). Previous studies have shown RTN3 aggregates to be markedly accumulated in dystrophic neurites in the brains of AD patients and APP transgenic mice (Hu et al, 2007). At 9 months, RTN3-positive neurites appeared as bright spots concentrated in and around Aβ plaques in the hippocampus of 5xFAD mice (Fig 3E). No RTN3 immunoreactivity could be detected in wild-type mice (not shown). RTN3 immunoreactivity in 5xFAD hippocampus was significantly reduced by all three p75NTR mutations, with the strongest effects observed in the knock-in strains (Fig 3F). We note that, while soluble RTN3 has been reported to negatively regulate BACE1 activity (He et al, 2004), its aggregation appears to correlate with enhanced amyloidogenic cleavage of APP and production of β-amyloid (Shi et al, 2009). Finally, we looked at mitochondrial dysfunction, a well-known feature of AD neuropathology (Swerdlow, 2018), as assessed by MitoSOX staining, a mitochondrial superoxide indicator widely used to assess mitochondrial stress (Dikalov & Harrison, 2014). MitoSOX staining was significantly increased at 2 and 6 months in neurons of the pyramidal layer of the hippocampus of 5xFAD mice compared to wild-type controls (Fig 3G and H). At 2 months of age, all three p75NTR mutations reduced MitoSOX levels significantly in the 5xFAD background, almost to the low levels found in wild-type mice (Fig 3H). However, at 6 months, there was a significant advantage of the p75NTR knock-in strains over 5xFAD/KO mice (Fig 3H). Figure 3. Reduced histopathology in the hippocampus of 5xFAD mice carrying inactive p75NTR variants Immunostaining for glial fibrillary acidic protein (GFAP), a marker of astrocytes, and Aβ plaques in coronal sections through the hippocampus of 6-month-old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A mice. Scale bar, 300 μm. Right-hand panels show high magnification of the indicated areas. Scale bar, 50 μm. Quantification of GFAP area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by GFAP immunostaining (mean ± SEM, N = 5 mice per group). *P < 0.05 and **P < 0.01 versus 5XFAD. #P < 0.05 and ##P < 0.01 versus 5XFAD/KO (one-way ANOVA followed by post hoc test). Immunostaining for Ionized calcium binding adaptor molecule 1 (Iba1), a marker of microglial cells, and Aβ plaques in coronal sections through the hippocampus of 6-month-old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A. Scale bar, 300 μm. Right-hand panels show high magnification of the indicated areas. Scale bar, 10 μm. Quantification of Iba1 area in the hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of hippocampal area occupied by Iba1 immunostaining (mean ± SEM, N = 5 mice per group). *P < 0.05 and **P < 0.01 versus 5XFAD. #P < 0.05 versus 5XFAD/KO. Other P values are indicated (one-way ANOVA followed by post hoc test). Immunostaining of reticulon 3 (RTN3), a marker of dystrophic neurites, and Aβ plaques in coronal sections through the hippocampus of 6-month-old 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A mice. Scale bar, 40 μm. Quantification of RTN3-positive dystrophic neurite area in the hippocampus of 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows the percentage of Aβ plaque area that overlapped with RTN3 immunostaining (mean ± SEM, N = 5 mice per group). *P < 0.05 and **P < 0.01 versus 5XFAD. Other P values are indicated (one-way ANOVA followed by post hoc test). MitoSOX staining, a mitochondrial superoxide indicator, and DAPI in coronal sections through the hippocampus of 6-month-old wild type, 5xFAD, 5xFAD/KO, 5xFAD/ΔDD, and 5xFAD/C259A mice. Scale bar, 60 μm. Quantification of MitoSOX signal in the pyramidal cell layer of hippocampus of wild type and 5xFAD mouse strains carrying different p75NTR variants as indicated. Histogram shows MitoSOX mean fluorescence intensity in arbitrary units (mean ± SEM, N = 5 mice per group). **P < 0.01 and ***P < 0.001 versus 5XFAD. #P < 0.05 versus 5XFAD/KO. (one-way ANOVA followed by post hoc test). Download figure Download PowerPoint Together, these studies indicated a significantly higher level of neuroprotection in mouse strains carrying signaling-deficient p75NTR variants compared to knock-out mice lacking the receptor. This was in contrast to the results of the in vitro assay of Aβ neurotoxicity (Fig 1), in which neurons from all three strains were equally resistant, suggesting that additional mechanisms must operate to account for the differences observed in vivo. Inactive p75NTR variants, but not the knock-out, fully rescue synaptic deficits and memory impairment in 5xFAD mice We asked whether the beneficial effects on AD histopathology afforded by the different p75NTR mutants had an impact on synaptic function and cognitive behavior of 5xFAD mice. Previous studies have shown that AD is associated with deficits in various forms of synaptic plasticity, including hippocampal long-term potentiation (LTP), in human patients and animal models (Selkoe, 2002; Palop & Mucke, 2010; Koch et al, 2012; Mango et al, 2019). In our studies, we used a form of late LTP induced by theta-burst stimulation (TBS-LTP) in hippocampal area CA1. Hippocampal slices from 6-month-old wild-type mice showed pronounced potentiation induced by TBS that was sustained for at least 4 h (Fig 4A). In contrast, synaptic potentiation was not maintained and declined rapidly in slices derived from 5xFAD mice, indicating a profound deficit in LTP induction (Fig 4A). Slices from 5xFAD/KO mice showed intermediate levels of potentiation that declined slowly over the course of the 4h recording, indicating a partial recovery (Fig 4A). Remarkably, TBS-LTP in slices from 5xFAD/C259A and 5xFAD/ΔDD mice was strong in terms of induction, persistent, and maintained throughout the whole recording period of 4h, and was essentially indistinguishable from that recorded in wild-type slices (Fig 4A). Quantification of the change in field excitatory post-synaptic potential (fEPSP) revealed a stronger rescue of synaptic function in the p75NTR knock-in strains, reaching statistically significant differences from 5xFAD mice throughout the recording period, unlike the knock-out (Fig 4B). We note that TBS-LTP in the absence of the 5xFAD transgene was normal in all three mutant strains and identical to that of wild-type animals (Fig EV1A and B). Figure 4. Inactive p75NTR variants, but not the knock-out, fully rescue synaptic deficits and memory impairment of 5xFAD mice Percentage of change in field excitatory post-synaptic potential (fEPSP) recorded after theta-burst stimulation (TBS) in Schaffer collaterals of hippocampal slices from 6-month-old wild type, 5xFAD, and 5xFAD/p75NTR mutant mice, as indicated. Results are presented as mean % change normalized to t = 0 ± SEM. N = 6 (wild type), 5 (5xFAD), 9 (5xFAD/KO), 7 (5xFAD/ΔDD), and 8 (5xFAD/C259A) slices from 3 mice per genotype, respectively. Quantification of fEPSP (mean % change ± SEM) in the indicated genotypes at 3 time points. *P < 0.05; **P < 0.01 versus 5xFAD (two-way ANOVA followed by post hoc test). N numbers as in (A). Behavior in the novel object recognition (NOR) test of 6-month-old wild type, 5XFAD, and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean recognition index ± SEM during training, and 30 min, 24 h, and 14 days after training, corresponding to measures of short-term, long-term, and remote memory, respectively. Bar color codes are as in panel (A). *P < 0.05 versus 5xFAD (two-way ANOVA followed by post hoc test). N = 12 (wild type, 5xFAD/ΔDD, and 5xFAD/C259A), 10 (5xFAD), and 8 (5xFAD/KO) mice per genotype, respectively. Training latency in the Barnes maze test of 6-month-old wild type, 5XFAD, and 5XFAD/p75NTR mutant mice, as indicated. Histograms show mean latency in seconds to find the platform hole ± SEM in 4 consecutive training sessions. Bar color codes are as in panel (A). *P < 0.05 versus 5xFAD; #P < 0.05 versus wild type, 5xFAD/ΔDD or 5xFAD/C259A (two-way ANOVA followed by post hoc test). N = 14 (wild type and 5xFAD/ΔDD), 10 (5xFAD, 5xFAD/KO and 5xFAD/C259A) mice per genotype, respectively. Percentage of time spent (mean ± SEM) in the target quadrant of t
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