With the advent of spatial multi-omics, mosaic integration of diverse datasets with partially overlapping modalities enables the construction of comprehensive multimodal spatial atlases from heterogeneous sources. Here we present SpaMosaic, a tool that uses contrastive learning and graph neural networks to build a modality-agnostic, batch-corrected latent space for spatial domain identification and missing-modality imputation. We systematically benchmarked SpaMosaic against existing integration methods using simulated data and experimentally acquired datasets spanning RNA and protein abundance, chromatin accessibility and histone modifications from brain, embryo, tonsil and lymph node tissues. SpaMosaic consistently outperformed other methods in identifying coherent spatial domains by reducing noise and mitigating batch effects. We further challenged SpaMosaic with heterogeneous real-world datasets spanning different technologies, developmental stages, resolutions and modality compositions, where it consistently resolved fine anatomical structures and enabled comprehensive mouse embryo atlasing. Beyond integration, SpaMosaic enables accurate imputation of missing modalities. In a mosaic mouse brain dataset, the imputed histone modifications not only recapitulated expected transcriptome-epigenome correlations but also uncovered more region-specific regulatory links compared to the measured chromatin accessibility data, demonstrating the ability to infer relationships across modalities without coprofiling. Computationally, SpaMosaic is highly scalable, capable of integrating over 100 sections and processing a single section with more than 800,000 spots. In summary, SpaMosaic provides a versatile framework for unifying the rapidly accumulating heterogeneous spatial omics data into comprehensive biological atlases.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder marked by progressive motor neuron (MN) degeneration in the brain and spinal cord. Although neuroinflammation is increasingly recognized as a hallmark of ALS, the precise molecular programs linking immune responses to MN pathology remain poorly defined. Using an integrated approach that combines single-cell and bulk RNA sequencing with spatial proteogenomics, we characterized both shared and distinct immune dynamics in peripheral blood and spinal cord tissues from patients with sporadic ALS and those carrying C9orf72 repeat expansions. Our analysis revealed broad immune remodeling in C9orf72 ALS, ALS subtype-specific and progression-associated differences in monocyte activation and antigen-experienced CD8 effector memory T cells with clonal features consistent with antigen-driven responses. Spatial mapping revealed complement activation and lipid-programmed myeloid states converging at sites of MN loss and TDP-43 pathology. Together, these findings connect peripheral and central immune alterations to ALS heterogeneity and highlight stratified immunomodulation as a potential therapeutic strategy.

Positive CSF biomarkers have been shown to be a reliable predictor of post-mortem Alzheimer's disease neuropathologic change (ADNC) and are often incorporated into clinical diagnosis of neurodegenerative disease. However, less is known about the predictive value of borderline or inconclusive results, which are not uncommon in clinical practice. The current project determined correspondence between CSF AD biomarkers and post-mortem ADNC in a clinical research sample enriched for atypical dementia syndromes, focusing on borderline and inconclusive results. All participants in the Northwestern University Alzheimer's Disease Research Center (NUADRC) with available CSF AD biomarker results who donated their brain at the time of death were included ( N  = 74, Mean age at CSF = 64.99 [SD = 7.34], Mean years from CSF to death = 5.6 [range 1-14 years], 43% female, 95% non-Hispanic white; see Table 1 for breakdown of clinical syndromes and pathological diagnoses). CSF results were categorized as AD, Borderline, Inconclusive, or Not AD based on ADMark thresholds for amyloid-b 42 /total tau index (ATI) and phosphorylated tau (pTau; Figure 1). Across clinical phenotypes, results consistent with AD on CSF were highly specific for High ADNC on autopsy (97%) but only moderately sensitive (68%); positive predictive value was also high (97%). Within Borderline and Inconclusive cases, abnormal ATI reliably predicted High ADNC on autopsy (sensitivity = 100%, specificity = 80%), regardless of pTau results. In this sample, there were no cases with pTau in the AD range but normal ATI that had High ADNC on autopsy. Abnormal CSF ATI and/or pTau were seen in some cases of FTLD-tau, FTLD-TDP, and other primary neuropathologic diagnoses. Consistent with prior findings, CSF results categorized as consistent with AD were highly specific and predictive of primary ADNC pathology on post-mortem analysis, regardless of clinical presentation. In the setting of Borderline or inconclusive results, ATI in the AD range reliably predicted high ADNC, regardless of pTau range. These findings help clarify the prognostic utility of ATI and pTau values in Borderline and Inconclusive CSF results, which can help inform differential diagnosis between AD and other neurodegenerative etiologies.

Brain perivascular macrophages maintain brain physiology, yet their transcriptional regulators and functions in health and disease remain unclear. Using single-cell multi-omics and functional experiments, we identify cellular musculoaponeurotic fibrosarcoma oncogene (cMAF) as a key transcription factor for brain perivascular macrophages, and conditional deletion of cMAF disrupts their phenotype in vivo. Functionally, cMAF drives insulin-like growth factor-1 (IGF1) expression in perivascular macrophages, enabling communication with endothelial cells. Consistently, cMAF deletion in perivascular macrophages causes transcriptional alterations in cerebral arteries, affecting vascular functions. Notably, cMAF emerges as the main transcription factor for human perivascular macrophages, suggesting conservation of this transcriptional module. During Alzheimer’s disease (AD), human perivascular macrophages upregulate cMAF and IGF1 to enhance communication with vascular cells, and this response is abrogated in APOE4 carriers. Lastly, we explore an uncharacterized polymorphism in cMAF, providing evidence that the cMAF program is protective against AD. Targeting cMAF in perivascular macrophages may offer new therapeutic strategies for neurodegenerative and cerebrovascular diseases.

Genetically altered astrocytes reduce a cardinal pathological feature of Alzheimer's disease.

Alzheimer's disease (AD) therapies utilizing amyloid-β (Aβ) immunization have shown potential in clinical trials. Yet, the mechanisms driving Aβ clearance in the immunized AD brain remain unclear. Here, we use spatial transcriptomics to explore the effects of both active and passive Aβ immunization in the AD brain. We compare actively immunized patients with AD with nonimmunized patients with AD and neurologically healthy controls, identifying distinct microglial states associated with Aβ clearance. Using high-resolution spatial transcriptomics alongside single-cell RNA sequencing, we delve deeper into the transcriptional pathways involved in Aβ removal after lecanemab treatment. We uncover spatially distinct microglial responses that vary by brain region. Our analysis reveals upregulation of the triggering receptor expressed on myeloid cells 2 (TREM2) and apolipoprotein E (APOE) in microglia across immunization approaches, which correlate positively with antibody responses and Aβ removal. Furthermore, we show that complement signaling in brain myeloid cells contributes to Aβ clearance after immunization. These findings provide new insights into the transcriptional mechanisms orchestrating Aβ removal and shed light on the role of microglia in immune-mediated Aβ clearance. Importantly, our work uncovers potential molecular targets that could enhance Aβ-targeted immunotherapies, offering new avenues for developing more effective therapeutic strategies to combat AD.

Recently approved Alzheimer's disease (AD) therapies leverage immunization strategies targeting amyloid-β (Aβ). While most studies focus on microglial responses to these treatments, evidence suggests that sustained immune activity and Aβ clearance drive broader cellular changes. Astrocytes, which accumulate at microglia-targeted Aβ plaques, may play a complementary role in clearance. This study examines how astrocyte phenotypes are altered by Aβ immunization and their contribution to Aβ clearance. We performed single-cell RNA sequencing and spatial transcriptomics on post-mortem brain tissue from 16 AD patients—10 immunized (9 with AN1792 from the first active Aβ immunotherapy trial, 1 with lecanemab) and 6 non-immunized controls. We identified astrocyte states enriched in immunized brains and analyzed their transcriptomic profiles. Additionally, we mapped astrocyte states localized at microglia-targeted Aβ plaques. A distinct astrocyte population emerged following immunization with both AN1792 and lecanemab, characterized by upregulated CHI3L1 and other reactive astrocyte markers. Spatial analysis revealed these astrocytes preferentially localize at microglia-targeted Aβ plaques post-lecanemab treatment. Ongoing analyses aim to further define astrocyte phenotypes in terms of morphology, protein expression, Aβ uptake, and spatial relationships with Aβ pathology and microglia. We identify a unique astrocyte phenotype induced by Aβ immunization in AD, marked by high CHI3L1 expression. This astrocyte state may be driven by microglia at Aβ plaques and contribute to plaque clearance. Our findings highlight distinct astrocyte responses to Aβ immunization, shedding light on their role in therapeutic outcomes.

In this issue of Neuron, Reid et al. introduce MultiVINE-seq, a single-nucleus multi-omic platform for profiling human cerebrovascular and perivascular cells for paired transcriptomic and epigenomic profiling.1 By integrating genome-wide association data, they map neurodegenerative and cerebrovascular disease risk variants to specific vascular and immune cell types, uncovering distinct and convergent pathogenic pathways.

Angiotensin-converting enzyme ( ACE ) is a validated risk locus for developing late-onset Alzheimer’s disease (LOAD). ACE1 controls blood pressure through the renin-angiotensin system (RAS), but it is also present and acts locally in the brain. Hypertension is associated with an increased risk for developing AD, and people taking select RAS-targeting therapeutics have reduced incidence of AD. The ACE variant rs4980 (R1284Q murine mutation) was discovered in LOAD families through WGS. Our group previously showed that ACE1 R1284Q caused age-associated hippocampal neurodegeneration and gliosis in mutant knock-in (KI) mice, which was more aggressive in females. Importantly, these phenotypes were rescued by treatment with anti-hypertensive drugs. Our previous study showed that ACE1 R1284Q caused neuron death in mice, however the mechanism is still unknown. This work aims to identify vulnerable hippocampal cell populations and pathways which might clarify the mechanism of ACE1 R1284Q-mediated neurodegeneration. Single nuclei were extracted from 30mg of flash-frozen hippocampi from 6-, and 12-month-old R1284Q ACE +/+ (WT) and ACE KI/KI (KI) and processed using 10X Genomics 3’ Dual-Index chemistry. Libraries were sequenced using the NovaSeq 6000. Preprocessing, quality control, and integration was performed on reads before subjecting them to cluster, differential expression, and pathway analyses. Together, the samples were represented by 143,000 nuclei in 23 clusters encompassing all neuronal and glial populations in the hippocampus. We identified the expression of every RAS component, excluding renin, in the hippocampus. Notably, inhibitory and excitatory neurons had the most differentially expressed genes (DEGs) in 12-month KI samples compared to controls. Additionally, 12-month KI females had upregulated microglial C1q genes compared to males. We found expression of RAS genes in the hippocampus, which to our knowledge has not yet been characterized by single-nucleus RNA sequencing. Furthermore, inhibitory neuron transcriptomes are the most affected in 12-month KI mice, suggesting altered neuron communication that may lead to neuron loss. Additionally, we identified sex-specific differences in 12-month KI female microglia, which may explain the more aggressive gliosis seen in female KIs compared to males. Future directions include performing ingenuity pathway analysis to identify upstream regulators of DEGs and experimental perturbations to test our hypotheses.

Recent advances in Alzheimer’s disease (AD) therapeutics involve immunization against amyloid-β (Aβ). Post-mortem brain analysis from the first active Aβ immunotherapy trial indicated clearance of Aβ in some AD patients. Yet, the mechanisms regulating Aβ clearance following immunization remain unknown. Here, we utilized a novel spatial proteogenomics approach to study brain tissues from 13 AD patients immunized with Aβ. We compared these actively immunized patient brains to tissues from non-immunized AD patients and non-neurologic disease controls. Additionally, we used spatial proteogenomics and single-cell RNA sequencing technologies to investigate the effects of lecanemab, a passive anti-Aβ drug. We reveal the transcriptomic neuroimmune response in the Aβ plaque microenvironment following anti-Aβ immunization. This response is characterized by an increase in genes associated with the TREM2-APOE axis in microglia of the immunized AD cortex. Altogether, our data uncover immediate and lasting neuroimmune responses in the AD brain induced by active and passive Aβ vaccination.

Single-cell long-read sequencing was performed on immune cells from cerebrospinal fluid (CSF) and blood to assess isoform diversity in healthy aging individuals and those diagnosed with mild cognitive impairment (MCI) or Alzheimer's disease (AD). cDNA from single-cell experiments was subjected to long-read sequencing using Oxford Nanopore Technologies. The dataset included immune cells from CSF (45 controls, 13 MCI/AD) and blood (22 controls, 28 AD). Computational analysis incorporated scNanoGPS for extracting cell barcodes and mapping reads to the genome, IsoQuant for isoform modeling, and SQANTI3 for filtering artifacts. Single-cell long-read sequencing identified 97,920 unique transcripts, with half of all genes exhibiting multiple isoforms. Notably, 20% of the detected isoforms were previously unannotated in the human genome. Novel isoforms were observed in AD-associated genes, including BIN1 and PTK2B , with BIN1 displaying lymphoid cell-specific isoforms. Human immune cells demonstrate extensive isoform diversity, including previously unannotated isoforms, particularly in genes implicated in AD.

Spinal cord injury (SCI) increasingly affects aged individuals, where functional impairment and mortality are highest. However, the aging-dependent mechanisms underpinning tissue damage remain elusive. Here, we find that natural killer-like T (NKLT) cells seed the intact aged human and murine spinal cord and multiply further after injury. NKLT cells accumulate in the spinal cord via C-X-C motif chemokine receptor 6 and ligand 16 signaling to clonally expand by engaging with major histocompatibility complex (MHC)-I-expressing myeloid cells. NKLT cells expressing natural killer cell granule protein 7 (Nkg7) disrupt myeloid-cell-dependent wound healing in the aged injured cord. Nkg7 deletion in mice curbs NKLT cell degranulation to normalize the myeloid cell phenotype, thus promoting tissue repair and axonal integrity. Monoclonal antibodies neutralizing CD8+ T cells after SCI enhance neurological recovery by promoting wound healing. Our results unveil a reversible role for NKG7+CD8+ NKLT cells in exacerbating tissue damage, suggesting a clinically relevant treatment for SCI.

Accumulating evidence implicates the gut microbiome (GMB) in the pathogenesis and progression of Alzheimer's disease (AD). We recently showed that the GMB regulates reactive astrocytosis and Aβ plaque accumulation in a male APPPS1-21 AD mouse model. Yet, the mechanism(s) by which GMB perturbation alters reactive astrocytosis in a manner that reduces Aβ deposition remain unknown. Here, we performed metabolomics on plasma from mice treated with antibiotics (ABX) and identified a significant increase in plasma propionate, a gut-derived short-chain fatty acid, only in male mice. Administration of sodium propionate reduced reactive astrocytosis and Aβ plaques in APPPS1-21 mice, phenocopying the ABX-induced phenotype. Astrocyte-specific RNA-Seq on ABX- and propionate-treated mice showed reduced expression of proinflammatory and increased expression of neurotrophic genes. Next, we performed flow cytometry experiments, in which we found that ABX and propionate decreased peripheral RAR-related orphan receptor-γ+ (Rorγt+) CD4+ (Th17) cells and IL-17 secretion, which positively correlated with reactive astrocytosis. Last, using an IL-17 mAb to deplete IL-17, we found that propionate reduced reactive astrocytosis and Aβ plaques in an IL-17-dependent manner. Together, these results suggest that gut-derived propionate regulates reactive astrocytosis and Aβ amyloidosis by decreasing peripheral Th17 cells and IL-17 release. Thus, propionate treatment or strategies boosting propionate production may represent novel therapeutic strategies for the treatment of AD.

Microglia and border-associated macrophages (BAMs) are critical for brain health, and their dysfunction is associated to disease. Replacing brain macrophages holds substantial therapeutic promise but remains challenging. Here, we demonstrate that monocytes can efficiently replace all brain macrophages. Monocytes readily replaced embryonal BAMs upon their depletion and engrafted as monocyte-derived microglia (Mo-Microglia) upon more sustained niche availability. Mo-Microglia expanded comparably to their embryonic counterparts and showed similar longevity. However, monocytes were unable to replicate the distinct identity of embryonically derived BAMs and microglia. Using xenotransplantation, we found that human monocytes exhibited similar behavior, enabling identification of putative Mo-Microglia in Alzheimer's disease individuals. In mice and humans, monocyte ontogeny shaped their identity as brain macrophages. Importantly, mouse fetal liver monocytes exhibited a distinct epigenetic landscape and could develop a bona fide microglial identity. Our results illuminate brain macrophage development and highlight monocytes as an abundant progenitor source for brain macrophage replacement therapies.

The peripheral immune system in Alzheimer’s disease (AD) has not been thoroughly studied with modern sequencing methods. To investigate epigenetic and transcriptional alterations to the AD peripheral immune system, we used single-cell sequencing strategies, including assay for transposase-accessible chromatin and RNA sequencing. We reveal a striking amount of open chromatin in peripheral immune cells in AD. In CD8 T cells, we uncover a cis-regulatory DNA element co-accessible with the CXC motif chemokine receptor 3 gene promoter. In monocytes, we identify a novel AD-specific RELA transcription factor binding site adjacent to an open chromatin region in the nuclear factor kappa B subunit 2 gene. We also demonstrate apolipoprotein E genotype-dependent epigenetic changes in monocytes. Surprisingly, we also identify differentially accessible chromatin regions in genes associated with sporadic AD risk. Our findings provide novel insights into the complex relationship between epigenetics and genetic risk factors in AD peripheral immunity.

Single-cell and single-nucleus genomic approaches can provide unbiased and multimodal insights. Here, we discuss what constitutes a molecular cell atlas and how to leverage single-cell omics data to generate hypotheses and gain insights into cell transitions in development and disease of the nervous system. We share points of reflection on what to consider during study design and implementation as well as limitations and pitfalls.

Angiotensin I converting enzyme (ACE1) maintains blood pressure homeostasis by converting angiotensin I into angiotensin II in the renin-angiotensin system (RAS). ACE1 is expressed in the brain, where an intrinsic RAS regulates complex cognitive functions including learning and memory. ACE1 has been implicated in neurodegenerative disorders including Alzheimer's disease and Parkinson's disease, but the mechanisms remain incompletely understood. Here, we performed single-nucleus RNA sequencing to characterize the expression of RAS genes in the hippocampus and discovered that Ace is mostly expressed in CA1 region excitatory neurons. To gain a deeper understanding of the function of neuronal ACE1, we generated ACE1 conditional knockout (cKO) mice lacking ACE1 expression specifically in hippocampal and cortical excitatory neurons. ACE1 cKO mice exhibited hippocampus-dependent memory impairment in the Morris water maze, y-maze, and fear conditioning tests. Total ACE1 level was significantly reduced in the cortex and hippocampus of ACE1 cKO mice showing that excitatory neurons are the predominant cell type expressing ACE1 in the forebrain. Despite similar reductions in total ACE1 level in both the hippocampus and cortex, the RAS pathway was dysregulated in the hippocampus only. Importantly, ACE1 cKO mice exhibited age-related capillary loss selectively in the hippocampus. Here, we show selective vulnerability of the hippocampal microvasculature and RAS pathway to neuronal ACE1 knockout. Our results provide important insights into the function of ACE1 in the brain and demonstrate a connection between neuronal ACE1 and cerebrovascular function in the hippocampus.

AbstractPatients aged 65 years and older account for an increasing proportion of patients with traumatic brain injury (TBI). Older TBI patients experience increased morbidity and mortality compared to their younger counterparts. Our prior data demonstrated that by blocking α4 integrin, anti-CD49d antibody (aCD49d Ab) abrogates CD8+ T-cell infiltration into the injured brain, improves survival, and attenuates neurocognitive deficits. Here, we aimed to uncover how aCD49d Ab treatment alters local cellular responses in the aged mouse brain. Consequently, mice incur age-associated toxic cytokine and chemokine responses long-term post-TBI. aCD49d Ab attenuates this response along with a T helper (Th)1/Th17 immunological shift and remediation of overall CD8+ T cell cytotoxicity. Furthermore, aCD49d Ab reduces CD8+ T cells exhibiting higher effector status, leading to reduced clonal expansion in aged, but not young, mouse brains with chronic TBI. Together, aCD49d Ab is a promising therapeutic strategy for treating TBI in the older people.

INTRODUCTION:The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimer's disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research. METHODS:In 2023, the Alzheimer's Association convened multidisciplinary researchers at the "AAIC Advancements: APOE" conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function. RESULTS:This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field. DISCUSSION:Understanding apoE's multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine. HIGHLIGHTS:APOE is a central player in the pathogenesis of Alzheimer's disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE.

Angiotensin I converting enzyme (ACE1) maintains blood pressure homeostasis by converting angiotensin I (angI) into angiotensin II (angII) in the renin-angiotensin system (RAS). ACE1 is expressed in the brain, where an intrinsic RAS regulates complex cognitive functions including learning and memory. ACE1 has been implicated in neurodegenerative disorders including Alzheimer’s disease (AD) and Parkinson’s disease (PD), but the mechanisms remain incompletely understood. Here, we performed single-nucleus RNA sequencing to characterize the expression RAS genes in the hippocampus and discovered that Ace is mostly expressed in CA region excitatory neurons. To gain a deeper understanding of the function of neuronal ACE1, we generated ACE1 conditional knockout (cKO) mice lacking ACE1 expression specifically in hippocampal and cortical excitatory neurons. Interestingly, ACE1 cKO mice exhibited hippocampus-dependent memory impairment in the Morris water maze, y-maze, and fear conditioning tests, but exhibited normal motor skills in rotarod. Total ACE1 level was significantly reduced in the cortex and hippocampus of ACE1 cKO mice showing that excitatory neurons are the predominant cell type expressing ACE1 in the forebrain. Despite similar reductions in total ACE1 level in both the hippocampus and cortex, the RAS pathway was dysregulated in the hippocampus only. Importantly, ACE cKO mice exhibited exacerbated age-related capillary loss selectively in the hippocampus. Here, we show selective vulnerability of the hippocampal microvasculature and RAS pathway to neuronal ACE1 knockout. Our results provide important insights into the function of ACE1 in the brain and demonstrate a connection between neuronal ACE and cerebrovascular function in the hippocampus.### Competing Interest StatementThe authors have declared no competing interest.* ACE1 : angiotensin I converting enzyme angI : angiotensin I angII : angiotensin II RAS : renin-angiotensin system AD : Alzheimer’s disease cKO : conditional knockout AT1R : angII type 1 receptor ARBs : AT1R blockers ACEis : ACE1 inhibitors CNS : central nervous system snRNA-seq : single nucleus RNA-sequencing RER : respiratory exchange ratio PV : parvalbumin NPY : neuropeptide Y NVC : neurovascular coupling CBF : cerebral blood flow BBB : blood brain barrier SMCs : smooth muscle cells