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• W3195606798 abstract "Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-β precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid β (Aβ), a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles and produce human Aβ. Here, we studied young Itm2bD rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2bD rats not only present subtle changes in human Aβ levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor–mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aβ and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia. Familial British dementia and familial Danish dementia are neurodegenerative disorders caused by mutations in the gene integral membrane protein 2B (ITM2b) encoding BRI2, which tunes excitatory synaptic transmission at both presynaptic and postsynaptic termini. In addition, BRI2 interacts with and modulates proteolytic processing of amyloid-β precursor protein (APP), whose mutations cause familial forms of Alzheimer's disease (AD) (familial AD). To study the pathogenic mechanisms triggered by the Danish mutation, we generated rats carrying the Danish mutation in the rat Itm2b gene (Itm2bD rats). Given the BRI2/APP interaction and the widely accepted relevance of human amyloid β (Aβ), a proteolytic product of APP, to AD, Itm2bD rats were engineered to express two humanized App alleles and produce human Aβ. Here, we studied young Itm2bD rats to investigate early pathogenic changes in these diseases. We found that periadolescent Itm2bD rats not only present subtle changes in human Aβ levels along with decreased spontaneous glutamate release and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor–mediated responses but also had increased short-term synaptic facilitation in the hippocampal Schaeffer-collateral pathway. These alterations in excitatory interneuronal communication can impair learning and memory processes and were akin to those observed in adult mice producing rodent Aβ and carrying either the Danish or British mutations in the mouse Itm2b gene. Collectively, the data show that the pathogenic Danish mutation alters the physiological function of BRI2 at glutamatergic synapses across species and early in life. Future studies will determine whether this phenomenon represents an early pathogenic event in human dementia. Model organisms that reproduce the pathogenesis of human diseases are useful to dissect disease mechanisms, identify therapeutic targets, and test therapeutic strategies. Because genetic manipulation has been easier in mice, mice have overtaken rats as the major rodent-based model organism in neurodegeneration research. Thus, to study familial Danish dementia (FDD) and familial British dementia (FBD), 15 years ago, we generated mice carrying the pathogenic Danish and British dementia mutations (integral membrane protein 2B [ITM2b]; Itm2bD and Itm2bB mice) into the Itm2b mouse gene (1Tamayev R. Matsuda S. Fa M. Arancio O. D'Adamio L. Danish dementia mice suggest that loss of function and not the amyloid cascade causes synaptic plasticity and memory deficits.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20822-20827Crossref PubMed Scopus (48) Google Scholar, 2Tamayev R. Giliberto L. Li W. D'Abramo C. Arancio O. Vidal R. D'Adamio L. Memory deficits due to familial British dementia BRI2 mutation are caused by loss of BRI2 function rather than amyloidosis.J. Neurosci. 2010; 30: 14915-14924Crossref PubMed Scopus (42) Google Scholar, 3Giliberto L. Matsuda S. Vidal R. D'Adamio L. Generation and initial characterization of FDD knock in mice.PLoS One. 2009; 4e7900Crossref PubMed Scopus (28) Google Scholar). We choose a knock-in (KI) approach rather than the more common transgenic overexpression approach for several reasons. KIs mimic the genetic of FDD and FBD and make no assumption about pathogenic mechanisms (except the unbiased genetic one), whereas the transgenic approach aims to reproduce pathology (plaques, neurofibrillary tangles [NFTs], etc.), under the assumption that this “pathology” is pathogenic. In KI models, expression of mutant genes is controlled by endogenous regulatory elements, avoiding issues related to overexpression of disease proteins in a nonphysiological quantitative–spatial–temporal manner. Finally, potential confounding “insertion” effects of transgenes are avoided. Because rats are better suited to study neurodegenerative diseases, we took advantage of recent developments in gene-editing technologies and introduced the familial Danish mutation into the genomic Itm2b rat locus (Itm2bD rats). The rat was the organism of choice for most behavioral, memory, and cognitive research, which is critical when studying neurodegenerative diseases—because physiological processes are similar in rats and humans and the rat is an intelligent and quick learner (4Deacon R.M. Housing, husbandry and handling of rodents for behavioral experiments.Nat. Protoc. 2006; 1: 936-946Crossref PubMed Scopus (156) Google Scholar, 5Whishaw I.Q. Metz G.A. Kolb B. Pellis S.M. Accelerated nervous system development contributes to behavioral efficiency in the laboratory mouse: A behavioral review and theoretical proposal.Dev. 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Finally, gene-expression differences suggest that rats may be advantageous model of neurodegenerative diseases over mice. For example, alternative spicing of tau (12Andreadis A. Tau gene alternative splicing: Expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases.Biochim. Biophys. Acta. 2005; 1739: 91-103Crossref PubMed Scopus (204) Google Scholar, 13Janke C. Beck M. Stahl T. Holzer M. Brauer K. Bigl V. Arendt T. Phylogenetic diversity of the expression of the microtubule-associated protein tau: Implications for neurodegenerative disorders.Brain Res. Mol. Brain Res. 1999; 68: 119-128Crossref PubMed Scopus (76) Google Scholar, 14Hong M. Zhukareva V. Vogelsberg-Ragaglia V. Wszolek Z. Reed L. Miller B.I. Geschwind D.H. Bird T.D. McKeel D. Goate A. Morris J.C. Wilhelmsen K.C. Schellenberg G.D. Trojanowski J.Q. Lee V.M. 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Goedert M. Schellenberg D.G. et al.A novel mutation at position +12 in the intron following exon 10 of the tau gene in familial frontotemporal dementia (FTD-Kumamoto).Ann. Neurol. 2000; 47: 422-429Crossref PubMed Scopus (93) Google Scholar, 22Kowalska A. Hasegawa M. Miyamoto K. Akiguchi I. Ikemoto A. Takahashi K. Araki W. Tabira T. A novel mutation at position +11 in the intron following exon 10 of the tau gene in FTDP-17.J. Appl. Genet. 2002; 43: 535-543PubMed Google Scholar, 23Grover A. England E. Baker M. Sahara N. Adamson J. Granger B. Houlden H. Passant U. Yen S.H. DeTure M. Hutton M. A novel tau mutation in exon 9 (1260V) causes a four-repeat tauopathy.Exp. Neurol. 2003; 184: 131-140Crossref PubMed Scopus (52) Google Scholar), leads to expression of tau isoforms with three or four microtubule-binding domains (3R and 4R, respectively). Adult human and rat brains express both 3R and 4R tau isoforms (24Hanes J. Zilka N. Bartkova M. Caletkova M. Dobrota D. Novak M. Rat tau proteome consists of six tau isoforms: Implication for animal models of human tauopathies.J. Neurochem. 2009; 108: 1167-1176Crossref PubMed Scopus (62) Google Scholar): in contrast, adult mouse brains express only 4R tau (25McMillan P. Korvatska E. Poorkaj P. Evstafjeva Z. Robinson L. Greenup L. Leverenz J. Schellenberg G.D. D'Souza I. Tau isoform regulation is region- and cell-specific in mouse brain.J. Comp. Neurol. 2008; 511: 788-803Crossref PubMed Scopus (111) Google Scholar), suggesting that the rat may be a better model organism for dementias with tauopathy, such as FDD and FBD. BRI2 physically interacts with and modulates processing of amyloid-β precursor protein (APP), which bears relevance to Alzheimer's disease (AD) pathogenesis (26Matsuda S. Giliberto L. Matsuda Y. Davies P. McGowan E. Pickford F. Ghiso J. Frangione B. D'Adamio L. The familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and inhibits amyloid-beta production.J. Biol. Chem. 2005; 280: 28912-28916Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 27Fotinopoulou A. Tsachaki M. Vlavaki M. Poulopoulos A. Rostagno A. Frangione B. Ghiso J. Efthimiopoulos S. BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production.J. Biol. Chem. 2005; 280: 30768-30772Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Matsuda S. Giliberto L. Matsuda Y. McGowan E.M. D'Adamio L. BRI2 inhibits amyloid beta-peptide precursor protein processing by interfering with the docking of secretases to the substrate.J. Neurosci. 2008; 28: 8668-8676Crossref PubMed Scopus (74) Google Scholar, 29Matsuda S. Matsuda Y. Snapp E.L. D'Adamio L. Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles.Neurobiol. Aging. 2011; 32: 1400-1408Crossref PubMed Scopus (51) Google Scholar, 30Matsuda S. Tamayev R. D'Adamio L. Increased AβPP processing in familial Danish dementia patients.J. Alzheimers Dis. 2011; 27: 385-391Crossref PubMed Scopus (28) Google Scholar). In addition, APP processing mediates long-term potentiation and memory deficits of Danish and British KI mice (31Tamayev R. Matsuda S. Giliberto L. Arancio O. D'Adamio L. APP heterozygosity averts memory deficit in knock in mice expressing the Danish dementia BRI2 mutant.EMBO J. 2011; 30: 2501-2509Crossref PubMed Scopus (40) Google Scholar, 32Tamayev R. D'Adamio L. Inhibition of γ-secretase worsens memory deficits in a genetically congruous mouse model of Danish dementia.Mol. Neurodegener. 2012; 7: 19Crossref PubMed Scopus (66) Google Scholar, 33Tamayev R. D'Adamio L. Memory deficits of British dementia knock-in mice are prevented by Aβ-precursor protein haploinsufficiency.J. Neurosci. 2012; 32: 5481-5485Crossref PubMed Scopus (18) Google Scholar, 34Tamayev R. Matsuda S. Arancio O. D'Adamio L. β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia.EMBO Mol. Med. 2012; 4: 171-179Crossref PubMed Scopus (101) Google Scholar, 35Lombino F. Biundo F. Tamayev R. Arancio O. D'Adamio L. An intracellular threonine of amyloid-β precursor protein mediates synaptic plasticity deficits and memory loss.PLoS One. 2013; 8e57120Crossref PubMed Scopus (16) Google Scholar, 36Biundo F. Ishiwari K. Del Prete D. D'Adamio L. Deletion of the γ-secretase subunits Aph1B/C impairs memory and worsens the deficits of knock-in mice modeling the Alzheimer-like familial Danish dementia.Oncotarget. 2016; 7: 11923-11944Crossref PubMed Scopus (10) Google Scholar). Aggregated forms of Aβ, a product of APP processing, are by and large considered the main pathogenic molecule in AD. Rat and human APP differ by three amino acids in the Aβ region: given that human Aβs are believed to have higher propensity to form toxic Aβ species as compared with rodent Aβs, we produced rats carrying the humanized Aβ sequence (Apph rats) (37Tambini M.D. Yao W. D'Adamio L. Facilitation of glutamate, but not GABA, release in familial Alzheimer's APP mutant knock-in rats with increased β-cleavage of APP.Aging Cell. 2019; 18e13033Crossref PubMed Scopus (16) Google Scholar, 38Tambini M.D. Norris K.A. D'Adamio L. Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation.Elife. 2020; 9e52612Crossref PubMed Scopus (19) Google Scholar). Thus, to study possible interactions between the Danish mutation and human Aβ, Itm2bD rats were backcrossed to Apph rats. Hence, all rats used in this study produce human and not rodent Aβ species. Here, we studied periadolescent Itm2bD rats, with the purpose of investigating early dysfunctions that may underlie initial pathogenic mechanisms leading to dementia later in life. The KI founder F0-Itm2bD rat, which is carrying FDD mutation on Itm2b rat gene, was generated by CRISPR/Cas–mediated genome engineering as described in the Experimental procedures section and Supporting information. The F0-Itm2bD rat, which is a chimera for the Itm2b gene, was crossed to WT (Itm2bw/w) Long–Evans rats to generate F1-Itm2bD/w rats. F1-Itm2bD/w rats were crossed to WT Long–Evans to generate F2-Itm2bD/w rats. These crossings were repeated three more times to obtain F5-Itm2bD/w rats. The probability that F5 rats carry unidentified off-target mutations (except those, if present, on chromosome 15) is ∼1.5625%. Male and female F5-Itm2bD/w rats were crossed to obtain Itm2bD/w, Itm2bD/D, and Itm2bw/w rats. The FDD mutation consists of a ten nucleotides duplication one codon before the normal stop codon (39Vidal R. Revesz T. Rostagno A. Kim E. Holton J.L. Bek T. Bojsen-Møller M. Braendgaard H. Plant G. Ghiso J. Frangione B. A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4920-4925Crossref PubMed Scopus (259) Google Scholar). This produces a frameshift in the BRI2 sequence generating a precursor protein 11 amino acids larger than normal (Fig. 1A). To verify that the Danish mutation was correctly inserted into Itm2b exon 6, we amplified by PCR the Itm2b gene exon 6 Itm2bD/w, Itm2bD/D, and Itm2bw/w rats. Sequencing of the PCR products shows that the Danish mutation was correctly inserted in the Itm2b gene exon 6 (Fig. 1B) and encoded for the COOH terminus of the Danish BRI2 mutant. When we generated FDD KI mice, we humanized the mouse COOH-terminal region of BRI2 by introducing an alanine (A) substituted for threonine (T) at codon 250 (3Giliberto L. Matsuda S. Vidal R. D'Adamio L. Generation and initial characterization of FDD knock in mice.PLoS One. 2009; 4e7900Crossref PubMed Scopus (28) Google Scholar). Since that humanization did not result into deposition of ADan peptides in amyloid plaques in KI mice (3Giliberto L. Matsuda S. Vidal R. D'Adamio L. Generation and initial characterization of FDD knock in mice.PLoS One. 2009; 4e7900Crossref PubMed Scopus (28) Google Scholar), that modification was not repeated in rats. To generate Itm2bD/w, Itm2bD/D, and Itm2bw/w rats on a background in which rat App has a humanized Aβ region, Itm2bD/w and Apph/h rats were crossed to generate Itm2bD/w;Apph/w rats. The Appw allele was removed in subsequent crosses. Henceforth, Itm2bD/D, Itm2bD/w, and Itm2bw/w rats used in this study have an Apph/h background and produce human and not rodent Aβ species. To determine whether Itm2b expression is disrupted by the introduced mutations, we examined Itm2b mRNA levels in p21 Itm2bD/D and Itm2bw/w rats by standard RNA-Seq analysis on total brain RNA. The mRNA expression of Itm2b shows no significant difference between Itm2bD/D and Itm2bw/w rats (Fig. 1C). BRI2 is type II membrane protein that is synthesized as an immature precursor (imBRI2). imBRI2 is cleaved at the COOH terminus by proprotein convertase to produce the NH2-terminal mature BRI2 (mBRI2) and the 23 amino acid–long COOH-terminal peptide called Bri23 (40Choi S.I. Vidal R. Frangione B. Levy E. Axonal transport of British and Danish amyloid peptides via secretory vesicles.FASEB J. 2004; 18: 373-375Crossref PubMed Scopus (42) Google Scholar). As noted previously, in the Danish patients, a frameshift caused by a ten nucleotides duplication 5′ to the stop codon leads to the synthesis of a BRI2 precursor protein 11 amino acids larger than normal (39Vidal R. Revesz T. Rostagno A. Kim E. Holton J.L. Bek T. Bojsen-Møller M. Braendgaard H. Plant G. Ghiso J. Frangione B. A decamer duplication in the 3' region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4920-4925Crossref PubMed Scopus (259) Google Scholar). Convertase-mediated cleavage of Danish imBRI2 generates a WT-like mBRI2 and a 34 amino acid–long peptide called ADan, which codeposits with Aβ species in amyloid fibrils in patients. For clarity, we will refer to the WT imBri2 as Bri2–Bri23 and to the Danish mutant imBri2 as Bri2-ADan. To determine whether the Itm2bD allele codes for Bri2-ADan, we examined Bri2 expression in total neuronal lysates isolated from male and female 2-month-old Itm2bD/w, Itm2bD/D, and Itm2bw/w rats. However, the Bri2 antibody tested identified many nonspecific bands (Fig. S1), making a rigorous assessment of Bri2 expression in rat brains challenging. Analysis of mouse Itm2bw/w and Itm2bD/D primary neurons showed that the mBri2/Bri2–Bri23 ratio in Itm2bw/w primary neurons was significantly higher than the mBri2/Bri2-ADan ratio in Itm2bD/D primary neurons (41Yin T. Yao W. Lemenze A.D. D'Adamio L. Danish and British dementia ITM2b/BRI2 mutations reduce BRI2 protein stability and impair glutamatergic synaptic transmission.J. Biol. Chem. 2021; 296: 100054Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). In addition, lysosomal inhibition caused accumulation of mBri2 but not Bri2–Bri23 in Itm2bw/w primary neurons; in contrast, both mBri2 and Bri2-ADan accumulated in Itm2bD/D primary neurons (41Yin T. Yao W. Lemenze A.D. D'Adamio L. Danish and British dementia ITM2b/BRI2 mutations reduce BRI2 protein stability and impair glutamatergic synaptic transmission.J. Biol. Chem. 2021; 296: 100054Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). These observations indicated that the Danish mutation reduced maturation of the mutant precursor Bri2 in mouse neurons. Based on these observations, we probed whether primary neurons could be used to assess mBri2, Bri2–Bri23, and Bri2-ADan expression in KI rats. Primary neurons are a simpler system compared with total brain; this, per se, may reduce the number of nonspecific bands identified by anti-Bri2 antibodies. Moreover, inhibition of lysosome-mediated degradation of Bri2 species in primary neurons may help identify specific Bri2 molecules. Thus, primary neurons derived from Itm2bw/w and Itm2bD/D rats were treated with the lysosomal inhibitor chloroquine and analyzed by Western blot. The anti-Bri2 antibody identified a band of ∼34 kDa in all samples, which was increased by chloroquine (Fig. 2, A and C). These observations are consistent with the ∼34 kDa corresponding to mBri2. A second band of ∼36 kDa was detected in Itm2bw/w primary neurons (Fig. 2A). In contrast, a slightly larger second band (∼37 kDa) that was increased by chloroquine treatment was detected in Itm2bD/D primary neurons (Fig. 2, A and C). These observations are consistent with the ∼36 and ∼37 kDa bands corresponding to Bri2–Bri23 and Bri2-ADan, respectively.Table 1Results of the statistical analysis of data shown in Figure 2Ordinary two-way ANOVA analysis of Figure 2imBri2Source of variationF (DFn, DFd)pInteractionF (1, 20) = 272.2<0.0001TreatmentF (1, 20) = 354.3<0.0001GenotypeF (1, 20) = 1966<0.0001Sidak's multiple comparisons testSummaryAdjusted pItm2bw/w (Veh) versus Itm2bD/D (Veh)∗∗∗∗<0.0001Itm2bw/w (Veh) versus Itm2bw/w (Chlo)NS0.5229Itm2bD/D (Veh) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001Itm2bw/w (Chlo) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001mBri2Source of variationF (DFn, DFd)pInteractionF (1, 20) = 207.4<0.0001TreatmentF (1, 20) = 186.9<0.0001GenotypeF (1, 20) = 228.5<0.0001Sidak's multiple comparisons testSummaryAdjusted pItm2bw/w (Veh) versus Itm2bD/D (Veh)NS0.9969Itm2bw/w (Veh) versus Itm2bw/w (Chlo)∗∗∗∗<0.0001Itm2bD/D (Veh) versus Itm2bD/D (Chlo)NS0.9966Itm2bw/w (Chlo) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001imBri2/mBri2Source of variationF (DFn, DFd)pInteractionF (1, 20) = 108.3<0.0001TreatmentF (1, 20) = 104.2<0.0001GenotypeF (1, 20) = 627.1<0.0001Sidak's multiple comparisons testSummaryAdjusted pItm2bw/w (Veh) versus Itm2bD/D (Veh)∗∗∗∗<0.0001Itm2bw/w (Veh) versus Itm2bw/w (Chlo)NS>0.9999Itm2bD/D (Veh) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001Itm2bw/w (Chlo) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001LC3AI/IISource of variationF (DFn, DFd)pInteractionF (1, 20) = 37.36<0.0001TreatmentF (1, 20) = 353.5<0.0001GenotypeF (1, 20) = 36.16<0.0001Sidak's multiple comparisons testSummaryAdjusted pItm2bw/w (Veh) versus Itm2bw/w (Chlo)∗∗∗∗<0.0001Itm2bD/D (Veh) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001LC3B I/IISource of variationF (DFn, DFd)pInteractionF (1, 20) = 8.1110.0099TreatmentF (1, 20) = 26.75<0.0001GenotypeF (1, 20) = 8.2830.0093Sidak's multiple comparisons testSummaryAdjusted pItm2bw/w (Veh) versus Itm2bw/w (Chlo)NS0.2185Itm2bD/D (Veh) versus Itm2bD/D (Chlo)∗∗∗∗<0.0001Abbreviation: NS, not significant.∗∗∗∗ indicates P < 0.0001. Open table in a new tab Table 2Results of the statistical analysis of data shown in Figure 3COrdinary one-way ANOVA analysis of Figure 3CProteinF (DFn, DFd)psAPPαF (2, 27) = 0.10840.8977sAPPβF (2, 27) = 0.76660.4744Aβ38F (2, 27) = 0.11210.8943Aβ40F (2, 27) = 2.0300.1509Aβ42F (2, 27) = 4.7640.0169∗Aβ43F (2, 26) = 2.6540.0893Aβ42/Aβ40F (2, 27) = 4.0740.0284∗Aβ43/Aβ40F (2, 26) = 4.0310.0299∗Aβ43/Aβ42F (2, 23) = 3.2810.0558Tukey's multiple comparisons testSummaryAdjusted pAβ42Itm2bw/w versus Itm2bD/wNS0.6966Itm2bw/w versus Itm2bD/D∗0.0159Itm2bD/w versus Itm2bD/DNS0.0948Aβ42/Aβ40Itm2bw/w versus Itm2bD/wNS0.8326Itm2bw/w versus Itm2bD/D∗0.0301Abbreviation: NS, not significant.∗ indicates P < 0.05. Open table in a new tab Abbreviation: NS, not significant. ∗∗∗∗ indicates P < 0.0001. Abbreviation: NS, not significant. ∗ indicates P < 0.05. Without treatment, the levels of Bri2-ADan in Itm2bD/D primary neurons were significantly higher than the levels of Bri2–Bri23 in Itm2bw/w primary neurons (Fig. 2, A and C), and the mBri2/Bri2–Bri23 ratio in Itm2bw/w primary neurons was significantly higher than the mBri2/Bri2-ADan ratio in Itm2bD/D primary neurons (Fig. 2, A and C). LC3A and LC3B are autophagosome membrane proteins: the LC3A I/LC3A II and LC3B I/LC3B II ratios for each provides an independent measure of the impact of chloroquine on lysosomal degradation. Chloroquine significantly reduced the LC3A I/LC3A II and LC3B I/LC3B II ratios (Fig. 2, B and C), confirming inhibition of lysosome-mediated degradation. Sequential processing of APP by α-/γ-secretase and β-/γ-secretase generates the following APP-derived peptides and polypeptides: sAPPβ, sAPPα, β-CTF, α-CTF, AID/AICD, P3, and Aβ. Since BRI2 interacts with APP and modulates APP processing by α-, β-, and γ-secretase (26Matsuda S. Giliberto L. Matsuda Y. Davies P. McGowan E. Pickford F. Ghiso J. Frangione B. D'Adamio L. The familial dementia BRI2 gene binds the Alzheimer gene amyloid-beta precursor protein and inhibits amyloid-beta production.J. Biol. Chem. 2005; 280: 28912-28916Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 27Fotinopoulou A. Tsachaki M. Vlavaki M. Poulopoulos A. Rostagno A. Frangione B. Ghiso J. Efthimiopoulos S. BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production.J. Biol. Chem. 2005; 280: 30768-30772Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 28Matsuda S. Giliberto L. Matsuda Y. McGowan E.M. D'Adamio L. BRI2 inhibits amyloid beta-peptide precursor protein processing by interfering with the docking of secretases to the substrate.J. Neurosci. 2008; 28: 8668-8676Crossref PubMed Scopus (74) Google Scholar, 29Matsuda S. Matsuda Y. Snapp E.L. D'Adamio L. Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles.Neurobiol. Aging. 2011; 32: 1400-1408Crossref PubMed Scopus (51) Google Scholar, 30Matsuda S. Tamayev R. D'Adamio L. Increased AβPP processing in familial Danish dementia patients.J. Alzheimers Dis. 2011; 27: 385-391Crossref PubMed Scopus (28) Google Scholar), we determined the steady-state levels of several of these APP metabolites in the central nervous system of young male and female Itm2bD KI rats. Full-length APP, α-CTF, and β-CTF were measured by Western blot: soluble APPs (sAPPα/sAPPβ) were detected by ELISA, and human Aβ species (Aβ38, Aβ40, Aβ42, and Aβ43) were detected by human Aβ-specific ELISA. These measurements have previously been used for other KI rats generated in our laboratory (38Tambini M.D. Norris K.A. D'Adamio L. Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation.Elife. 2020; 9e52612Crossref PubMed Scopus (19) Google Scholar, 42Tambini M.D. D'Adamio L. Trem2 splicing and expression are preserved in a human Abeta-producing, rat knock-in model of Trem2-R47H Alzheimer's risk variant.Sci. Rep. 2020; 10: 4122Crossref PubMed Scopus (6) Google Scholar, 43Ren S. Breuillaud L. Yao W. Yin T. Norris K.A. Zehntner S.P. D'Adamio L. TNF-α-mediated reduction in inhibitory neurotransmission precedes sporadic Alzheimer's disease pathology in young Trem2 R47H rats.J. Biol. Chem. 2020; 296: 100089Abstract F" @default.
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• W3195606798 title "A familial Danish dementia rat shows impaired presynaptic and postsynaptic glutamatergic transmission" @default.
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