Detection of SARS-CoV-2-Specific Humoral and Cellular Immunity in COVID-19 Convalescent Individuals.

The World Health Organization has declared SARS-CoV-2 virus outbreak a worldwide pandemic. However, there is very limited understanding on the immune responses, especially adaptive immune responses to SARS-CoV-2 infection. Here, we collected blood from COVID-19 patients who have recently become virus-free, and therefore were discharged, and detected SARS-CoV-2-specific humoral and cellular immunity in eight newly discharged patients. Follow-up analysis on another cohort of six patients 2 weeks post discharge also revealed high titers of immunoglobulin G (IgG) antibodies. In all 14 patients tested, 13 displayed serum-neutralizing activities in a pseudotype entry assay. Notably, there was a strong correlation between neutralization antibody titers and the numbers of virus-specific T cells. Our work provides a basis for further analysis of protective immunity to SARS-CoV-2, and understanding the pathogenesis of COVID-19, especially in the severe cases. It also has implications in developing an effective vaccine to SARS-CoV-2 infection.

Structural basis for intrinsic transcription termination.

Efficient and accurate termination is required for gene transcription in all living organisms1,2. Cellular RNA polymerases in both bacteria and eukaryotes can terminate their transcription through a factor-independent termination pathway3,4-called intrinsic termination transcription in bacteria-in which RNA polymerase recognizes terminator sequences, stops nucleotide addition and releases nascent RNA spontaneously. Here we report a set of single-particle cryo-electron microscopy structures of Escherichia coli transcription intrinsic termination complexes representing key intermediate states of the event. The structures show how RNA polymerase pauses at terminator sequences, how the terminator RNA hairpin folds inside RNA polymerase, and how RNA polymerase rewinds the transcription bubble to release RNA and then DNA. These macromolecular snapshots define a structural mechanism for bacterial intrinsic termination and a pathway for RNA release and DNA collapse that is relevant for factor-independent termination by all RNA polymerases.

Vaccination with Glycan-Modified HIV NFL Envelope Trimer-Liposomes Elicits Broadly Neutralizing Antibodies to Multiple Sites of Vulnerability.

The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination.

Targeting and monitoring ovarian cancer invasion with an RNAi and peptide delivery system.

RNA interference (RNAi) therapeutics are an emerging class of medicines that selectively target mRNA transcripts to silence protein production and combat disease. Despite the recent progress, a generalizable approach for monitoring the efficacy of RNAi therapeutics without invasive biopsy remains a challenge. Here, we describe the development of a self-reporting, theranostic nanoparticle that delivers siRNA to silence a protein that drives cancer progression while also monitoring the functional activity of its downstream targets. Our therapeutic target is the transcription factor SMARCE1, which was previously identified as a key driver of invasion in early-stage breast cancer. Using a doxycycline-inducible shRNA knockdown in OVCAR8 ovarian cancer cells both in vitro and in vivo, we demonstrate that SMARCE1 is a master regulator of genes encoding proinvasive proteases in a model of human ovarian cancer. We additionally map the peptide cleavage profiles of SMARCE1-regulated proteases so as to design a readout for downstream enzymatic activity. To demonstrate the therapeutic and diagnostic potential of our approach, we engineered self-assembled layer-by-layer nanoparticles that can encapsulate nucleic acid cargo and be decorated with peptide substrates that release a urinary reporter upon exposure to SMARCE1-related proteases. In an orthotopic ovarian cancer xenograft model, theranostic nanoparticles were able to knockdown SMARCE1 which was in turn reported through a reduction in protease-activated urinary reporters. These LBL nanoparticles both silence gene products by delivering siRNA and noninvasively report on downstream target activity by delivering synthetic biomarkers to sites of disease, enabling dose-finding studies as well as longitudinal assessments of efficacy.

Salt stress-induced chloroplastic hydrogen peroxide stimulates pdTPI sulfenylation and methylglyoxal accumulation.

High salinity, an adverse environmental factor affecting about 20% of irrigated arable land worldwide, inhibits plant growth and development by causing oxidative stress, damaging cellular components, and disturbing global metabolism. However, whether and how reactive oxygen species disturb the metabolism of salt-stressed plants remain elusive. Here, we report that salt-induced hydrogen peroxide (H2O2) inhibits the activity of plastid triose phosphate isomerase (pdTPI) to promote methylglyoxal (MG) accumulation and stimulates the sulfenylation of pdTPI at cysteine 74. We also show that MG is a key factor limiting the plant growth, as a decrease in MG levels completely rescued the stunted growth and repressed salt stress tolerance of the pdtpi mutant. Furthermore, targeting CATALASE 2 into chloroplasts to prevent salt-induced overaccumulation of H2O2 conferred salt stress tolerance, revealing a role for chloroplastic H2O2 in salt-caused plant damage. In addition, we demonstrate that the H2O2-mediated accumulation of MG in turn induces H2O2 production, thus forming a regulatory loop that further inhibits the pdTPI activity in salt-stressed plants. Our findings, therefore, illustrate how salt stress induces MG production to inhibit the plant growth.

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach.

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

Structural basis for regulation of SOS response in bacteria.

In response to DNA damage, bacterial RecA protein forms filaments with the assistance of DinI protein. The RecA filaments stimulate the autocleavage of LexA, the repressor of more than 50 SOS genes, and activate the SOS response. During the late phase of SOS response, the RecA filaments stimulate the autocleavage of UmuD and λ repressor CI, leading to mutagenic repair and lytic cycle, respectively. Here, we determined the cryo-electron microscopy structures of Escherichia coli RecA filaments in complex with DinI, LexA, UmuD, and λCI by helical reconstruction. The structures reveal that LexA and UmuD dimers bind in the filament groove and cleave in an intramolecular and an intermolecular manner, respectively, while λCI binds deeply in the filament groove as a monomer. Despite their distinct folds and oligomeric states, all RecA filament binders recognize the same conserved protein features in the filament groove. The SOS response in bacteria can lead to mutagenesis and antimicrobial resistance, and our study paves the way for rational drug design targeting the bacterial SOS response.

An SI3-σ arch stabilizes cyanobacteria transcription initiation complex.

Multisubunit RNA polymerases (RNAPs) associate with initiation factors (σ in bacteria) to start transcription. The σ factors are responsible for recognizing and unwinding promoter DNA in all bacterial RNAPs. Here, we report two cryo-EM structures of cyanobacterial transcription initiation complexes at near-atomic resolutions. The structures show that cyanobacterial RNAP forms an "SI3-σ" arch interaction between domain 2 of σA (σ2) and sequence insertion 3 (SI3) in the mobile catalytic domain Trigger Loop (TL). The "SI3-σ" arch facilitates transcription initiation from promoters of different classes through sealing the main cleft and thereby stabilizing the RNAP-promoter DNA open complex. Disruption of the "SI3-σ" arch disturbs cyanobacteria growth and stress response. Our study reports the structure of cyanobacterial RNAP and a unique mechanism for its transcription initiation. Our data suggest functional plasticity of SI3 and provide the foundation for further research into cyanobacterial and chloroplast transcription.

Structural insights into the transcription activation mechanism of the global regulator GlnR from actinobacteria.

In actinobacteria, an OmpR/PhoB subfamily protein called GlnR acts as an orphan response regulator and globally coordinates the expression of genes responsible for nitrogen, carbon, and phosphate metabolism in actinobacteria. Although many researchers have attempted to elucidate the mechanisms of GlnR-dependent transcription activation, progress is impeded by lacking of an overall structure of GlnR-dependent transcription activation complex (GlnR-TAC). Here, we report a co-crystal structure of the C-terminal DNA-binding domain of GlnR (GlnR_DBD) in complex with its regulatory cis-element DNA and a cryo-EM structure of GlnR-TAC which comprises Mycobacterium tuberculosis RNA polymerase, GlnR, and a promoter containing four well-characterized conserved GlnR binding sites. These structures illustrate how four GlnR protomers coordinate to engage promoter DNA in a head-to-tail manner, with four N-terminal receiver domains of GlnR (GlnR-RECs) bridging GlnR_DBDs and the RNAP core enzyme. Structural analysis also unravels that GlnR-TAC is stabilized by complex protein-protein interactions between GlnR and the conserved ß flap, σAR4, αCTD, and αNTD domains of RNAP, which are further confirmed by our biochemical assays. Taken together, these results reveal a global transcription activation mechanism for the master regulator GlnR and other OmpR/PhoB subfamily proteins and present a unique mode of bacterial transcription regulation.

A spatiotemporal barrier formed by Follistatin is required for left-right patterning.

How left-right (LR) asymmetry emerges in a patterning field along the anterior-posterior axis remains an unresolved problem in developmental biology. Left-biased Nodal emanating from the LR organizer propagates from posterior to anterior (PA) and establishes the LR pattern of the whole embryo. However, little is known about the regulatory mechanism of the PA spread of Nodal and its asymmetric activation in the forebrain. Here, we identify bilaterally expressed Follistatin (Fst) as a regulator blocking the propagation of the zebrafish Nodal ortholog Southpaw (Spaw) in the right lateral plate mesoderm (LPM), and restricting Spaw transmission in the left LPM to facilitate the establishment of a robust LR asymmetric Nodal patterning. In addition, Fst inhibits the Activin-Nodal signaling pathway in the forebrain thus preventing Nodal activation prior to the arrival, at a later time, of Spaw emanating from the left LPM. This contributes to the orderly propagation of asymmetric Nodal activation along the PA axis. The LR regulation function of Fst is further confirmed in chick and frog embryos. Overall, our results suggest that a robust LR patterning emerges by counteracting a Fst barrier formed along the PA axis.

Genome sequences and population genomics provide insights into the demographic history, inbreeding, and mutation load of two 'living fossil' tree species of Dipteronia.

'Living fossils', that is, ancient lineages of low taxonomic diversity, represent an exceptional evolutionary heritage, yet we know little about how demographic history and deleterious mutation load have affected their long-term survival and extinction risk. We performed whole-genome sequencing and population genomic analyses on Dipteronia sinensis and D. dyeriana, two East Asian Tertiary relict trees. We found large-scale genome reorganizations and identified species-specific genes under positive selection that are likely involved in adaptation. Our demographic analyses suggest that the wider-ranged D. sinensis repeatedly recovered from population bottlenecks over late Tertiary/Quaternary periods of adverse climate conditions, while the population size of the narrow-ranged D. dyeriana steadily decreased since the late Miocene, especially after the Last Glacial Maximum (LGM). We conclude that the efficient purging of deleterious mutations in D. sinensis facilitated its survival and repeated demographic recovery. By contrast, in D. dyeriana, increased genetic drift and reduced selection efficacy, due to recent severe population bottlenecks and a likely preponderance of vegetative propagation, resulted in fixation of strongly deleterious mutations, reduced fitness, and continuous population decline, with likely detrimental consequences for the species' future viability and adaptive potential. Overall, our findings highlight the significant impact of demographic history on levels of accumulation and purging of putatively deleterious mutations that likely determine the long-term survival and extinction risk of Tertiary relict trees.

Attractor neural networks with double well synapses.

It is widely believed that memory storage depends on activity-dependent synaptic modifications. Classical studies of learning and memory in neural networks describe synaptic efficacy either as continuous or discrete. However, recent results suggest an intermediate scenario in which synaptic efficacy can be described by a continuous variable, but whose distribution is peaked around a small set of discrete values. Motivated by these results, we explored a model in which each synapse is described by a continuous variable that evolves in a potential with multiple minima. External inputs to the network can switch synapses from one potential well to another. Our analytical and numerical results show that this model can interpolate between models with discrete synapses which correspond to the deep potential limit, and models in which synapses evolve in a single quadratic potential. We find that the storage capacity of the network with double well synapses exhibits a power law dependence on the network size, rather than the logarithmic dependence observed in models with single well synapses. In addition, synapses with deeper potential wells lead to more robust information storage in the presence of noise. When memories are sparsely encoded, the scaling of the capacity with network size is similar to previously studied network models in the sparse coding limit.

Structural Basis of Mycobacterium tuberculosis Transcription and Transcription Inhibition.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, which kills 1.8 million annually. Mtb RNA polymerase (RNAP) is the target of the first-line antituberculosis drug rifampin (Rif). We report crystal structures of Mtb RNAP, alone and in complex with Rif, at 3.8-4.4 Å resolution. The results identify an Mtb-specific structural module of Mtb RNAP and establish that Rif functions by a steric-occlusion mechanism that prevents extension of RNA. We also report non-Rif-related compounds-Nα-aroyl-N-aryl-phenylalaninamides (AAPs)-that potently and selectively inhibit Mtb RNAP and Mtb growth, and we report crystal structures of Mtb RNAP in complex with AAPs. AAPs bind to a different site on Mtb RNAP than Rif, exhibit no cross-resistance with Rif, function additively when co-administered with Rif, and suppress resistance emergence when co-administered with Rif.

The structural mechanism for transcription activation by Caulobacter crescentus GcrA.

Canonical bacterial transcription activators bind to their cognate cis elements at the upstream of transcription start site (TSS) in a form of dimer. Caulobacter crescentus GcrA, a non-canonical transcription activator, can activate transcription from promoters harboring its cis element at the upstream or downstream of TSS in a form of monomer. We determined two cryo-EM structures of C. crescentus GcrA-bound transcription activation complexes, GcrA TACU and GcrA TACD, which comprise GcrA, RNAP, σ70 and promoter DNA with GcrA cis elements at either the upstream or downstream of TSS at 3.6 and 3.8 Å, respectively. In the GcrA-TACU structure, GcrA makes bipartite interactions with both σ70 domain 2 (σ702) and its cis element, while in the GcrA-TACD structure, GcrA retains interaction with σ702 but loses the interaction with its cis element. Our results suggest that GcrA likely forms a functionally specialized GcrA-RNAP-σA holoenzyme, in which GcrA first locates its cis element and then facilitates RNAP to load on core promoter at its proximal region. The sequence-specific interaction of GcrA and DNA is disrupted either at the stage of RPo formation or promoter escape depending on the location of GcrA cis elements relative to TSS.

The N-Myc-responsive lncRNA MILIP promotes DNA double-strand break repair through non-homologous end joining.

The protooncoprotein N-Myc, which is overexpressed in approximately 25% of neuroblastomas as the consequence of MYCN gene amplification, has long been postulated to regulate DNA double-strand break (DSB) repair in neuroblastoma cells, but experimental evidence of this function is presently scant. Here, we show that N-Myc transcriptionally activates the long noncoding RNA MILIP to promote nonhomologous end-joining (NHEJ) DNA repair through facilitating Ku70-Ku80 heterodimerization in neuroblastoma cells. High MILIP expression was associated with poor outcome and appeared as an independent prognostic factor in neuroblastoma patients. Knockdown of MILIP reduced neuroblastoma cell viability through the induction of apoptosis and inhibition of proliferation, retarded neuroblastoma xenograft growth, and sensitized neuroblastoma cells to DNA-damaging therapeutics. The effect of MILIP knockdown was associated with the accumulation of DNA DSBs in neuroblastoma cells largely due to decreased activity of the NHEJ DNA repair pathway. Mechanistical investigations revealed that binding of MILIP to Ku70 and Ku80 increased their heterodimerization, and this was required for MILIP-mediated promotion of NHEJ DNA repair. Disrupting the interaction between MILIP and Ku70 or Ku80 increased DNA DSBs and reduced cell viability with therapeutic potential revealed where targeting MILIP using Gapmers cooperated with the DNA-damaging drug cisplatin to inhibit neuroblastoma growth in vivo. Collectively, our findings identify MILIP as an N-Myc downstream effector critical for activation of the NHEJ DNA repair pathway in neuroblastoma cells, with practical implications of MILIP targeting, alone and in combination with DNA-damaging therapeutics, for neuroblastoma treatment.

Genome-wide analyses of individual differences in quantitatively assessed reading- and language-related skills in up to 34,000 people.

The use of spoken and written language is a fundamental human capacity. Individual differences in reading- and language-related skills are influenced by genetic variation, with twin-based heritability estimates of 30 to 80% depending on the trait. The genetic architecture is complex, heterogeneous, and multifactorial, but investigations of contributions of single-nucleotide polymorphisms (SNPs) were thus far underpowered. We present a multicohort genome-wide association study (GWAS) of five traits assessed individually using psychometric measures (word reading, nonword reading, spelling, phoneme awareness, and nonword repetition) in samples of 13,633 to 33,959 participants aged 5 to 26 y. We identified genome-wide significant association with word reading (rs11208009, P = 1.098 × 10-8) at a locus that has not been associated with intelligence or educational attainment. All five reading-/language-related traits showed robust SNP heritability, accounting for 13 to 26% of trait variability. Genomic structural equation modeling revealed a shared genetic factor explaining most of the variation in word/nonword reading, spelling, and phoneme awareness, which only partially overlapped with genetic variation contributing to nonword repetition, intelligence, and educational attainment. A multivariate GWAS of word/nonword reading, spelling, and phoneme awareness maximized power for follow-up investigation. Genetic correlation analysis with neuroimaging traits identified an association with the surface area of the banks of the left superior temporal sulcus, a brain region linked to the processing of spoken and written language. Heritability was enriched for genomic elements regulating gene expression in the fetal brain and in chromosomal regions that are depleted of Neanderthal variants. Together, these results provide avenues for deciphering the biological underpinnings of uniquely human traits.

Recent advances in super-resolution optical imaging based on aggregation-induced emission.

Super-resolution imaging has rapidly emerged as an optical microscopy technique, offering advantages of high optical resolution over the past two decades; achieving improved imaging resolution requires significant efforts in developing super-resolution imaging agents characterized by high brightness, high contrast and high sensitivity to fluorescence switching. Apart from technical requirements in optical systems and algorithms, super-resolution imaging relies on fluorescent dyes with special photophysical or photochemical properties. The concept of aggregation-induced emission (AIE) was proposed in 2001, coinciding with unprecedented advancements and innovations in super-resolution imaging technology. AIE probes offer many advantages, including high brightness in the aggregated state, low background signal, a larger Stokes shift, ultra-high photostability, and excellent biocompatibility, making them highly promising for applications in super-resolution imaging. In this review, we summarize the progress in implementation methods and provide insights into the mechanism of AIE-based super-resolution imaging, including fluorescence switching resulting from photochemically-converted aggregation-induced emission, electrostatically controlled aggregation-induced emission and specific binding-regulated aggregation-induced emission. Particularly, the aggregation-induced emission principle has been proposed to achieve spontaneous fluorescence switching, expanding the selection and application scenarios of super-resolution imaging probes. By combining the aggregation-induced emission principle and specific molecular design, we offer some comprehensive insights to facilitate the applications of AIEgens (AIE-active molecules) in super-resolution imaging.

Pharmacological activation of STAT1-GSDME pyroptotic circuitry reinforces epigenetic immunotherapy for hepatocellular carcinoma.

BACKGROUND: Genomic screening uncovered interferon-gamma (IFNγ) pathway defects in tumours refractory to immune checkpoint blockade (ICB). However, its non-mutational regulation and reversibility for therapeutic development remain less understood. OBJECTIVE: We aimed to identify ICB resistance-associated druggable histone deacetylases (HDACs) and develop a readily translatable combination approach for patients with hepatocellular carcinoma (HCC). DESIGN: We correlated the prognostic outcomes of HCC patients from a pembrolizumab trial (NCT03419481) with tumourous cell expressions of all HDAC isoforms by single-cell RNA sequencing. We investigated the therapeutic efficacy and mechanism of action of selective HDAC inhibition in 4 ICB-resistant orthotopic and spontaneous models using immune profiling, single-cell multiomics and chromatin immunoprecipitation-sequencing and verified by genetic modulations and co-culture systems. RESULTS: HCC patients showing higher HDAC1/2/3 expressions exhibited deficient IFNγ signalling and poorer survival on ICB therapy. Transient treatment of a selective class-I HDAC inhibitor CXD101 resensitised HDAC1/2/3high tumours to ICB therapies, resulting in CD8+T cell-dependent antitumour and memory T cell responses. Mechanistically, CXD101 synergised with ICB to stimulate STAT1-driven antitumour immunity through enhanced chromatin accessibility and H3K27 hyperacetylation of IFNγ-responsive genes. Intratumoural recruitment of IFNγ+GZMB+cytotoxic lymphocytes further promoted cleavage of CXD101-induced Gasdermin E (GSDME) to trigger pyroptosis in a STAT1-dependent manner. Notably, deletion of GSDME mimicked STAT1 knockout in abolishing the antitumour efficacy and survival benefit of CXD101-ICB combination therapy by thwarting both pyroptotic and IFNγ responses. CONCLUSION: Our immunoepigenetic strategy harnesses IFNγ-mediated network to augment the cancer-immunity cycle, revealing a self-reinforcing STAT1-GSDME pyroptotic circuitry as the mechanistic basis for an ongoing phase-II trial to tackle ICB resistance (NCT05873244).

Progressive Excitability Changes in the Medial Entorhinal Cortex in the 3xTg Mouse Model of Alzheimer's Disease Pathology.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder characterized by memory loss and progressive cognitive impairments. In mouse models of AD pathology, studies have found neuronal and synaptic deficits in hippocampus, but less is known about changes in medial entorhinal cortex (MEC), which is the primary spatial input to the hippocampus and an early site of AD pathology. Here, we measured neuronal intrinsic excitability and synaptic activity in MEC layer II (MECII) stellate cells, MECII pyramidal cells, and MEC layer III (MECIII) excitatory neurons at 3 and 10 months of age in the 3xTg mouse model of AD pathology, using male and female mice. At 3 months of age, before the onset of memory impairments, we found early hyperexcitability in intrinsic properties of MECII stellate and pyramidal cells, but this was balanced by a relative reduction in synaptic excitation (E) compared with inhibition (I; E/I ratio), suggesting intact homeostatic mechanisms regulating MECII activity. Conversely, MECIII neurons had reduced intrinsic excitability at this early time point with no change in synaptic E/I ratio. By 10 months of age, after the onset of memory deficits, neuronal excitability of MECII pyramidal cells and MECIII excitatory neurons was largely normalized in 3xTg mice. However, MECII stellate cells remained hyperexcitable, and this was further exacerbated by an increased synaptic E/I ratio. This observed combination of increased intrinsic and synaptic hyperexcitability suggests a breakdown in homeostatic mechanisms specifically in MECII stellate cells at this postsymptomatic time point, which may contribute to the emergence of memory deficits in AD.SIGNIFICANCE STATEMENT AD causes cognitive deficits, but the specific neural circuits that are damaged to drive changes in memory remain unknown. Using a mouse model of AD pathology that expresses both amyloid and tau transgenes, we found that neurons in the MEC have altered excitability. Before the onset of memory impairments, neurons in layer 2 of MEC had increased intrinsic excitability, but this was balanced by reduced inputs onto the cell. However, after the onset of memory impairments, stellate cells in MEC became further hyperexcitable, with increased excitability exacerbated by increased synaptic inputs. Thus, it appears that MEC stellate cells are uniquely disrupted during the progression of memory deficits and may contribute to cognitive deficits in AD.