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- W3103396319 abstract "Alzheimer’s disease (AD) is a neurodegenerative dementia associated with deposition of amyloid plaques and neurofibrillary tangles, formed by amyloid β (Aβ) peptides and phosphor-tau, respectively, in the central nervous system. Approximately 2% of AD cases are due to familial AD (FAD); ∼98% of cases are sporadic AD (SAD). Animal models with FAD are commonly used to study SAD pathogenesis. Because mechanisms leading to FAD and SAD may be distinct, to study SAD pathogenesis, we generated Trem2R47H knock-in rats, which carry the SAD risk factor p.R47H variant of the microglia gene triggering receptor expressed on myeloid cells 2 (TREM2). Trem2R47H rats produce human-Aβ from a humanized-App rat allele because human-Aβ is more toxic than rodent-Aβ and the pathogenic role of the p.R47H TREM2 variant has been linked to human-Aβ–clearing deficits. Using periadolescent Trem2R47H rats, we previously demonstrated that supraphysiological tumor necrosis factor-α (TNF-α) boosts glutamatergic transmission, which is excitatory, and suppresses long-term potentiation, a surrogate of learning and memory. Here, we tested the effect of the p.R47H variant on the inhibitory neurotransmitter γ-aminobutyric acid. We report that GABAergic transmission is decreased in Trem2R47H/R47H rats. This decrease is due to acute and reversible action of TNF-α and is not associated with increased human-Aβ levels and AD pathology. Thus, the p.R47H variant changes the excitatory/inhibitory balance, favoring excitation. This imbalance could potentiate glutamate excitotoxicity and contribute to neuronal dysfunction, enhanced neuronal death, and neurodegeneration. Future studies will determine whether this imbalance represents an early, Aβ-independent pathway leading to dementia and may reveal the AD-modifying therapeutic potential of TNF-α inhibition in the central nervous system. Alzheimer’s disease (AD) is a neurodegenerative dementia associated with deposition of amyloid plaques and neurofibrillary tangles, formed by amyloid β (Aβ) peptides and phosphor-tau, respectively, in the central nervous system. Approximately 2% of AD cases are due to familial AD (FAD); ∼98% of cases are sporadic AD (SAD). Animal models with FAD are commonly used to study SAD pathogenesis. Because mechanisms leading to FAD and SAD may be distinct, to study SAD pathogenesis, we generated Trem2R47H knock-in rats, which carry the SAD risk factor p.R47H variant of the microglia gene triggering receptor expressed on myeloid cells 2 (TREM2). Trem2R47H rats produce human-Aβ from a humanized-App rat allele because human-Aβ is more toxic than rodent-Aβ and the pathogenic role of the p.R47H TREM2 variant has been linked to human-Aβ–clearing deficits. Using periadolescent Trem2R47H rats, we previously demonstrated that supraphysiological tumor necrosis factor-α (TNF-α) boosts glutamatergic transmission, which is excitatory, and suppresses long-term potentiation, a surrogate of learning and memory. Here, we tested the effect of the p.R47H variant on the inhibitory neurotransmitter γ-aminobutyric acid. We report that GABAergic transmission is decreased in Trem2R47H/R47H rats. This decrease is due to acute and reversible action of TNF-α and is not associated with increased human-Aβ levels and AD pathology. Thus, the p.R47H variant changes the excitatory/inhibitory balance, favoring excitation. This imbalance could potentiate glutamate excitotoxicity and contribute to neuronal dysfunction, enhanced neuronal death, and neurodegeneration. Future studies will determine whether this imbalance represents an early, Aβ-independent pathway leading to dementia and may reveal the AD-modifying therapeutic potential of TNF-α inhibition in the central nervous system. Sporadic Alzheimer's disease (SAD) represents ∼95% of Alzheimer's disease (AD) cases. Yet, the most commonly used animal models are with familial AD (FAD) mutations, which only represent ∼5% of AD cases. This may be an issue because if FAD and SAD present significant pathogenic differences, therapeutic strategies effective in animals with FAD may have limited therapeutic efficacy in patients with SAD. Thus, model organisms that reproduce the pathogenesis of SAD would be helpful to identify therapeutic targets and test SAD-modifying therapeutics. The p.R47H variant of the microglia gene triggering receptor expressed on myeloid cells 2 (TREM2) triples the risk of SAD in heterozygous carriers (1Guerreiro R. Wojtas A. Bras J. Carrasquillo M. Rogaeva E. Majounie E. Cruchaga C. Sassi C. Kauwe J.S. Younkin S. Hazrati L. Collinge J. Pocock J. Lashley T. Williams J. et al.Alzheimer Genetic Analysis, G.TREM2 variants in Alzheimer's disease.N. Engl. J. Med. 2013; 368: 117-127Crossref PubMed Scopus (1561) Google Scholar). Like other SAD-associated TREM2 variants, the p.R47H variant impairs the amyloid β (Aβ)-clearing activities of microglia, presumably hampering elimination of toxic Aβ peptide forms (2Zhou Y. Ulland T.K. Colonna M. TREM2-Dependent effects on microglia in Alzheimer's disease.Front. Aging Neurosci. 2018; 10: 202Crossref PubMed Scopus (29) Google Scholar, 3Zhou S.L. Tan C.C. Hou X.H. Cao X.P. Tan L. Yu J.T. TREM2 variants and neurodegenerative diseases: a systematic review and meta-analysis.J. Alzheimers Dis. 2019; 68: 1171-1184Crossref PubMed Scopus (15) Google Scholar). To study mechanisms by which this variant promotes SAD, we generated Trem2R47H knock-in (KI) rats. These rats carry the R47H mutation in the rat Trem2 gene and exhibit normal Trem2 splicing and expression (4Tambini 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 (5) Google Scholar). Trem2 is processed by A Disintegrin and Metalloproteinase 10; this cleavage releases a soluble N-terminal ectodomain (5Wunderlich P. Glebov K. Kemmerling N. Tien N.T. Neumann H. Walter J. Sequential proteolytic processing of the triggering receptor expressed on myeloid cells-2 (TREM2) protein by ectodomain shedding and gamma-secretase-dependent intramembranous cleavage.J. Biol. Chem. 2013; 288: 33027-33036Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 6Kleinberger G. Yamanishi Y. Suarez-Calvet M. Czirr E. Lohmann E. Cuyvers E. Struyfs H. Pettkus N. Wenninger-Weinzierl A. Mazaheri F. Tahirovic S. Lleo A. Alcolea D. Fortea J. Willem M. et al.TREM2 mutations implicated in neurodegeneration impair cell surface transport and phagocytosis.Sci. Transl. Med. 2014; 6: 243ra286Crossref Scopus (392) Google Scholar). Levels of soluble N-terminal ectodomain were not changed in the brains of young Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats (4Tambini 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 (5) Google Scholar). Trem2R47H KI rats express two humanized App rat alleles that drive production of human Aβ (4Tambini 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 (5) Google Scholar). Hence, this KI model is useful to study both human Aβ–dependent and human Aβ–independent effects of the R47H mutation. In summary, Trem2R47H KI rats represent a genetically faithful model organism of SAD. Pathogenic mechanisms leading to SAD may start early in life. To reveal early dysfunctions that may lead, over time, to neurodegeneration, we have studied young Trem2R47H KI rats. Preadolescent (4 weeks old) and periadolescent (6- to 8-weeks old) Trem2R47H rats showed no alteration in steady-state levels of central nervous system (CNS) and cerebrospinal fluid (CSF) Aβ peptides (4Tambini 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 (5) Google Scholar, 7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar), suggesting that Aβ-clearance deficits caused by the p.R47H TREM2 variant may manifest with aging. Yet, young Trem2R47H rats present significant increased CNS and CSF concentrations of tumor necrosis factor-α (TNF-α), which cause augmented glutamatergic transmission and suppression of long-term potentiation (LTP) (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar), an electrophysiological surrogate of learning and memory. Physiological levels of TNF-α produced by microglia are necessary to maintain normal surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors at postsynaptic termini; increased TNF-α concentrations promote rapid exocytosis of AMPA receptors in hippocampal pyramidal neurons, increasing the strength of glutamatergic synaptic responses (8Ogoshi F. Yin H.Z. Kuppumbatti Y. Song B. Amindari S. Weiss J.H. Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons.Exp. Neurol. 2005; 193: 384-393Crossref PubMed Scopus (118) Google Scholar, 9Beattie E.C. Stellwagen D. Morishita W. Bresnahan J.C. Ha B.K. Von Zastrow M. Beattie M.S. Malenka R.C. Control of synaptic strength by glial TNFalpha.Science. 2002; 295: 2282-2285Crossref PubMed Scopus (985) Google Scholar, 10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar, 11Stellwagen D. Malenka R.C. Synaptic scaling mediated by glial TNF-alpha.Nature. 2006; 440: 1054-1059Crossref PubMed Scopus (1142) Google Scholar). The alterations in glutamatergic transmission found in Trem2R47H rats are consistent with these effects of TNF-α and establish a direct link between a pathogenic variant of the microglia-specific TREM2 gene and neuronal dysfunction of glutamatergic transmission and LTP. In addition to boosting excitatory transmission, TNF-α decreases inhibitory synaptic strength by promoting endocytosis of γ-aminobutyric acid (GABA) receptors, hence reducing surface GABA receptors (10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar). Thus, in this study, we tested whether the p.R47H TREM2 variant may reduce GABA transmission via increased brain TNF-α levels, with the purpose of determining whether the p.R47H TREM2 variant changes the excitatory/inhibitory balance between glutamate and GABA transmission, favoring excitation. This unbalance could potentiate glutamate excitotoxicity and, over time, contribute to neuronal dysfunction, enhanced neuronal cell death, and neurodegeneration. We examined the effects of the Trem2R47H variant on GABAergic synaptic transmission in the hippocampal Schaffer-collateral pathway. First, we examined paired-pulse facilitation (PPF) of GABAA receptor postsynaptic current, which is inversely correlated to the presynaptic GABA release probability. Our results show that PPF with 50- and 200-ms intervals is significantly increased in Trem2R47H/R47H rats with SAD (Fig. 1, A–C), indicating GABA release is undermined in Trem2R47H/R47H rats. Second, we analyzed miniature inhibitory postsynaptic currents (mIPSCs). The frequency of mIPSCs that largely reflects presynaptic GABA release probability is slightly decreased in both Trem2R47H/w and Trem2R47H/R47H rats; however, this decrease fails to achieve statistical significance (Fig. 1, D, E and H). Interestingly, the amplitude of mIPSCs that is dependent on levels of postsynaptic ionotropic GABAA receptors was significantly decreased in both Trem2R47H/w and Trem2R47H/R47H rats (Fig. 1D, F and I), suggesting a reduction of GABAA receptors on the postsynaptic surface. Moreover, we found that in Trem2R47H/R47H rats, the decay time of mIPSC is shorter than in WT rats (Fig. 1G). As the subunit composition of the GABAA receptor determines the decay time of mIPSCs (12Eyre M.D. Renzi M. Farrant M. Nusser Z. Setting the time course of inhibitory synaptic currents by mixing multiple GABA(A) receptor α subunit isoforms.J. Neurosci. 2012; 32: 5853-5867Crossref PubMed Scopus (66) Google Scholar), it is possible that the subunit composition of the GABAA receptor is altered in Trem2R47H/R47H rats. Altogether, these results suggest that the pathogenic variant p.R47H of the microglia gene TREM2 leads to the reduction of GABAergic transmission to CA1 pyramidal neurons, and this effect is gene-dosage dependent. TNF-α produced by glia is necessary for physiological postsynaptic surface expression of both AMPA and GABA receptors. These two opposite effects of physiological TNF-α cooperate in maintaining physiological excitatory/inhibitory balance and the excitatory synaptic strength. Increased TNF-α concentration causes a swift surface AMPA receptor expression at postsynaptic termini and endocytosis of GABA receptors in hippocampal pyramidal neurons, changing the excitatory/inhibitory balance and favoring excitation (8Ogoshi F. Yin H.Z. Kuppumbatti Y. Song B. Amindari S. Weiss J.H. Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons.Exp. Neurol. 2005; 193: 384-393Crossref PubMed Scopus (118) Google Scholar, 9Beattie E.C. Stellwagen D. Morishita W. Bresnahan J.C. Ha B.K. Von Zastrow M. Beattie M.S. Malenka R.C. Control of synaptic strength by glial TNFalpha.Science. 2002; 295: 2282-2285Crossref PubMed Scopus (985) Google Scholar, 10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar, 11Stellwagen D. Malenka R.C. Synaptic scaling mediated by glial TNF-alpha.Nature. 2006; 440: 1054-1059Crossref PubMed Scopus (1142) Google Scholar). Consistently, young Trem2R47H/R47H rats show increased levels of TNF-α in the brain and CSF, which leads to enhanced glutamatergic transmission (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar). To test whether TNF-α mediates the reduced inhibitory synaptic transmission at hippocampal SC–CA3>CA1 synapses of periadolescent rats carrying the Trem2R47H variant, we treated hippocampal slices with a neutralizing antibody to rat TNF-α (anti–TNF-α), which functions as a TNF-α antagonist. To control for off-target effects of the antibody, we used a goat immunoglobulin (Ig)G isotype control. The 50% neutralization dose of this anti–TNF-α antibody against the cytotoxic effect of recombinant rat TNF-α (0.25 ng/ml) is about 500 ng/ml. Because physiological levels of TNF-α are necessary for normal glutamatergic transmission and most of the activities of TNF-α can be rapidly reversed (8Ogoshi F. Yin H.Z. Kuppumbatti Y. Song B. Amindari S. Weiss J.H. Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons.Exp. Neurol. 2005; 193: 384-393Crossref PubMed Scopus (118) Google Scholar, 9Beattie E.C. Stellwagen D. Morishita W. Bresnahan J.C. Ha B.K. Von Zastrow M. Beattie M.S. Malenka R.C. Control of synaptic strength by glial TNFalpha.Science. 2002; 295: 2282-2285Crossref PubMed Scopus (985) Google Scholar, 10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar, 11Stellwagen D. Malenka R.C. Synaptic scaling mediated by glial TNF-alpha.Nature. 2006; 440: 1054-1059Crossref PubMed Scopus (1142) Google Scholar), we tested the acute effects of 10 ng/ml of anti–TNF-α, a concentration ∼50 times lower than the 50% neutralization dose. At this concentration, anti–TNF-α occluded the increased PPF (Fig. 2, A–C), the decreased mIPSC amplitude, and decay time (Fig. 2D, F, G and I) in Trem2R47H/R47H rats. The goat IgG isotype control did not restore inhibitory GABAergic transmission alterations observed in the mutant rats (Fig. 2D, F, G and I) indicating that the effects of anti–TNF-α are specific. In addition, these low doses of anti–TNF-α do not alter inhibitory transmission in Trem2w/w rats (Fig. 2, A–I), indicating that at least at this dosage, anti–TNF-α only targets GABAergic transmission alterations triggered by excess TNF-α set off by the Trem2R47H variant. Overall, these data indicate that the decrease of GABAergic transmission at SC–CA3>CA1 synapses of Trem2R47H/R47H rats is due to the acute action of supraphysiological TNF-α concentrations prompted by the Trem2R47H variant and are rapidly reversible. Analysis of brain homogenates from preadolescent rats (4Tambini 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 (5) Google Scholar) showed no significant alterations in levels of human Aβ40, Aβ42, and the Aβ42-to-Aβ40 ratio in Trem2R47H rats, although the Trem2R47H variant reduces binding and clearance of human Aβ in vitro (13Zhao Y. Wu X. Li X. Jiang L.L. Gui X. Liu Y. Sun Y. Zhu B. Pina-Crespo J.C. Zhang M. Zhang N. Chen X. Bu G. An Z. Huang T.Y. et al.TREM2 is a receptor for beta-amyloid that mediates microglial function.Neuron. 2018; 97: 1023-1031.e7Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Previously, we found no changes in Aβ levels in the CSF of periadolescent animals as compared with Trem2w/w rats (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar). However, CSF concentrations of Aβ may not reflect the brain Aβ levels because aggregation of Aβ peptides in brain parenchyma may influence Aβ levels in the CSF. Thus, we assessed further Aβ levels in the brains of Trem2R47H periadolescent animals, which were tested for glutamatergic (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar) and GABAergic transmission (Figs. 1 and 2). No differences were seen in Aβ38, Aβ40, and Aβ42 levels and the Aβ42-to-Aβ40 ratio between periadolescent Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats (Fig. 3A), further suggesting that the reduced Aβ clearance caused by the Trem2R47H variant in vitro does not result in significant alterations of Aβ steady-state levels in vivo, at least in preadolescent and periadolescent rats. It has been postulated that toxic forms of Aβ are oligomers (14Shankar G.M. Li S. Mehta T.H. Garcia-Munoz A. Shepardson N.E. Smith I. Brett F.M. Farrell M.A. Rowan M.J. Lemere C.A. Regan C.M. Walsh D.M. Sabatini B.L. Selkoe D.J. Amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.Nat. Med. 2008; 14: 837-842Crossref PubMed Scopus (2665) Google Scholar); we tested whether toxic oligomers are augmented in periadolescent Trem2R47H/w and Trem2R47H/R47H rats as compared with Trem2w/w animals. To this end, we used the prefibrillar oligomer-specific antibody A11 to perform dot blots (15Kayed R. Head E. Thompson J.L. McIntire T.M. Milton S.C. Cotman C.W. Glabe C.G. Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis.Science. 2003; 300: 486-489Crossref PubMed Scopus (3278) Google Scholar). We found no evidence supporting an increase in neurotoxic brain oligomer levels in periadolescent Trem2 mutant rats with SAD as compared with WT rats (Fig. 3B). The Aβ ELISA may not efficiently measure aggregated insoluble Aβ species. These species may trigger TNF-α release and impact neurodevelopment and/or cause overt neuropathology. To test these possibilities, we used histology and immunohistochemistry (IHC) to characterize brains from 3-month-old male and female Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats (see Table 1). We tested 3-month-old rats to increase the possibility of detecting pathology that may start in preadolescent rats, which may be detectable by histology and IHC only weeks later. Regions of analysis included the frontal cortex, cingulate cortex, whole hippocampus, and entorhinal cortex. No gross morphological changes were evident by H&E staining in any of the rats analyzed (Fig. 4). Qualitative inspection of NeuN staining showed no appreciable changes in the neuronal density in any of the regions analyzed. Qualitative analysis performed on the hippocampus (CA1) and the somatosensory cortex did not indicate overt neuronal loss in Trem2R47H/w and Trem2R47H/R47H rats as compared with Trem2w/w rats (Fig. 5). Despite the presence of elevated proinflammatory cytokines in the brain and CSF of Trem2R47H/R47H rats and to a lesser extent of Trem2R47H/w rats, no evidence of significant astrocytosis or microgliosis was observed (Figure 4, Figure 5, Figure 6), a evaluated by the staining intensity (Fig. 5) and cellular morphology of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 (IBA1)-stained tissues (Fig. 6, A–B). Although a trend toward higher IBA-1 staining intensity was observed in the Trem2R47H/R47H rats than in Trem2w/w and Trem2R47H/w rats, this was not statistically significant. The microglia presented numerous fine processes, characteristic of the resting state, and did not present obvious intermediate or amoeboid morphologies with enlarged processes or cell bodies (Fig. 6A) in all three genotypes. Similarly, astrocytes did not present hypertrophy of soma and processes in any of the groups (Fig. 6B). Amyloid plaques, as measured by simultaneous costaining with the anti-Aβ antibodies 6E10 and 4G8, were absent in all tissues analyzed (Fig. 4). Those results are consistent with the similar Aβ40 or Aβ42 levels in Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats as well as the absence of plaque in 3-month-old rats with humanized Aβ (16Tambini 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 beta-cleavage of APP.Aging Cell. 2019; 18e13033Crossref PubMed Scopus (12) Google Scholar). Moreover, Tau phosphorylation as measured by AT8 immunostaining was absent in all the groups (Fig. 4), and a modified Bielschowsky silver staining did not reveal plaques, dystrophic neurites, or axonal pathologies in any of the tissues analyzed (Fig. 4). Overall, histological analysis of these rats shows no obvious evidence of neurodevelopmental impairments, neurodegeneration, neuroinflammation, or AD-like pathology at 3 months of age.Table 1Primary and amplification antibodies used for IHCTargetAntigen antibody/clone (supplier)Antigen retrievalDilutionSecondary and amplificationNeuronsNeuN,Mouse monoclonal A60 (Millipore)Citrate, pH 6.0HIER1:3000RbαM and GtαRb-HRPAmyloid beta1–16 and 17–24 beta amyloid,Mouse monoclonal 6E10, and4G8 (Biolegend)80% Formic acid1:1000RbαM and GtαRb-HRPMicrogliaIBA-1,Rabbit polyclonal (Wako)Citrate, pH 6.0HIER1:2000DkαRb-bio and SA-HRPPhospho-TauPhospho-Tau (Ser202, Thr205), mouse monoclonal AT8 (ThermoFisher)Citrate, pH 6.0HIER + 10-min PK1:1000RbαM and GtαRb-HRPAstrocytesGFAP,Rabbit polyclonal (Thermo Scientific)Citrate, pH 6.0HIER1:200DkαRb-bio and SA-HRPGFAP, glial fibrillary acidic protein; IHC, immunohistochemistry; HIER, heat-induced antigen retrieval; PK, proteinase K; M, mouse; Rb, rabbit; Gt, goat; Dk, donkey; bio, biotin; SA, streptavidin; HRP, horseradish peroxidase. Open table in a new tab Figure 5Qualitative assessment of the NeuN, IBA-1, and GFAP staining in 3-month-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats. Immunohistochemistry staining was scored in Trem2w/w (4 male and 5 female rats), Trem2R47H/w (4 male and 4 female rats), and Trem2R47H/R47H (4 male and 4 female rats). Data are represented as mean ± SD of the qualitative score within the cortex and hippocampus-CA1 regions. Data were analyzed by ordinary one-way ANOVA within each brain region. No statistically significant differences were seen in NeuNCx [F (2, 21) = 1.736, p = 0.200], NeuNHC-CA1 [F (2, 21) = 0.533, p = 0.594], IBA-1Cx [F (2, 22) = 0.375, p = 0.692], IBA-1HC-CA1 [F (2, 22) = 0.507, p = 0.609], GFAPCx [F (2, 22) = 0.159, p = 0.854], or GFAPHC-CA1 [F (2, 22) = 0.0237, p = 0.977]. GFAP, glial fibrillary acidic protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Astrocyte and microglia immunohistochemistry staining in 3-month-old Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rats. Representative images illustrate microglia (A, IBA-1) and astrocyte (B, GFAP) staining with a red-brown chromagen in the dorsal hippocampus-CA1, hippocampus-CA3, and hippocampus-dentate gyrus (DG), of 3-month-old male Trem2w/w, Trem2R47H/w, and Trem2R47H/R47H rat brains (left to right). The scale bar is equivalent to 500 microns. GFAP, glial fibrillary acidic protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) GFAP, glial fibrillary acidic protein; IHC, immunohistochemistry; HIER, heat-induced antigen retrieval; PK, proteinase K; M, mouse; Rb, rabbit; Gt, goat; Dk, donkey; bio, biotin; SA, streptavidin; HRP, horseradish peroxidase. Proinflammatory cytokines, especially TNF-α, are significantly increased in the brain and CSF of young Trem2R47H rats (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar). Consistent with the evidence that (1) TNF-α produced by glia controls postsynaptic physiological expression of AMPA receptors and (2) increased concentrations of TNF-α cause rapid AMPA receptor exocytosis increasing excitatory synaptic strength (8Ogoshi F. Yin H.Z. Kuppumbatti Y. Song B. Amindari S. Weiss J.H. Tumor necrosis-factor-alpha (TNF-alpha) induces rapid insertion of Ca2+-permeable alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA)/kainate (Ca-A/K) channels in a subset of hippocampal pyramidal neurons.Exp. Neurol. 2005; 193: 384-393Crossref PubMed Scopus (118) Google Scholar, 9Beattie E.C. Stellwagen D. Morishita W. Bresnahan J.C. Ha B.K. Von Zastrow M. Beattie M.S. Malenka R.C. Control of synaptic strength by glial TNFalpha.Science. 2002; 295: 2282-2285Crossref PubMed Scopus (985) Google Scholar, 10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar, 11Stellwagen D. Malenka R.C. Synaptic scaling mediated by glial TNF-alpha.Nature. 2006; 440: 1054-1059Crossref PubMed Scopus (1142) Google Scholar), we found that supraphysiological TNF-α concentrations boost glutamatergic transmission and suppress LTP, a surrogate of learning and memory, in periadolescent Trem2R47H rats with SAD (7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar). TNF-α also physiologically regulates, in an opposite manner, inhibitory synaptic strength by promoting endocytosis GABA receptors, hence reducing surface GABA receptors at inhibitory synapses (10Stellwagen D. Beattie E.C. Seo J.Y. Malenka R.C. Differential regulation of AMPA receptor and GABA receptor trafficking by tumor necrosis factor-alpha.J. Neurosci. 2005; 25: 3219-3228Crossref PubMed Scopus (632) Google Scholar). In accord, we show here that young Trem2R47H rats have reduced GABA responses (Fig. 1). Low doses of a neutralizing anti–TNF-α antibody occlude these alterations (Fig. 2) indicating that supraphysiological TNF-α concentrations impair GABAergic transmission in Trem2R47H rats. This evidence also indicates that GABAergic deficits must be due to an acute and constant action of supraphysiological TNF-α. TNF-α–dependent synaptic transmission alterations occur in the absence of changes in steady-state levels of soluble Aβ (Fig. 3 and (4Tambini 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 (5) Google Scholar, 7Ren S. Yao W. Tambini M.D. Yin T. Norris K.A. D'Adamio L. Microglia.Elife. 2020; 9Google Scholar)) and obvious evidence of neurodevelopmental impairments, neurodegeneration, neuroinflammation, or AD-like pathology (Figure 4, Figure 5, Figure 6). Overall, these data suggest that the TNF-α–dependent synaptic transmission alterations caused by the p.R47H TREM2 variant are independent of, and perhaps precede, changes in Aβ steady-state levels and brain pathology. TNF-α binds 2 receptors: TNFR1 is ubiquitously expressed, whereas TNFR2 is mainly expressed by immune and endothelial cells (17Sedger L.M. McDermott M.F. TNF and TNF-receptors: from mediators of cell death and inflammation to therapeutic giants - past, present and future.Cytokine Growth Factor. Rev. 2014; 25: 453-472Crossref PubMed Scopus (420) Google Scholar). Future studies will be needed to address the contribution of TNFR1" @default.
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- W3103396319 title "TNF-α–mediated reduction in inhibitory neurotransmission precedes sporadic Alzheimer’s disease pathology in young Trem2 rats" @default.
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