Daniele Mattei

Parkinson’s disease (PD) and related α-synucleinopathies are defined by the spread of Lewy body (LB) pathology across vulnerable brain regions, yet how substantia nigra (SN) myeloid cells respond remains unresolved. Using SOX10-based oligodendrocyte depletion and CD11b-based myeloid enrichment, we profiled 169,799 brain myeloid cells and nuclei from 96 donors spanning the LB continuum, including 86,427 from the SN, and integrated them with AMP PD for a total of 298,592 profiles. Across eight conserved states, α-synucleinopathy is associated with a bidirectional signature: upregulated glycolytic and proteostatic-stress programs alongside downregulated lysosomal and mitochondrial pathways. Microglia shift from P2RY12-high sensome cells toward an HSP90AA1-high proteostatic stress state, a transition that intensifies at late Braak LB stages. Immunohistochemistry confirmed that HSP60 ⁺ proteostatic microglia state proportions increase with SN neuron loss. A rare NUCKS1-high state (∼1% of myeloid cells), more abundant in late-stage disease, carried the strongest PD polygenic-risk loading. The PD-protective rs3732765-A allele, a microglial cis-eQTL that lowers P2RY12 expression, was nominally associated with a higher proportion of P2RY12-high microglia and higher lysosomal program activity. These findings frame microglial involvement as structured and genetically tractable, nominating the P2RY12 genetic axis and NUCKS1-high microglia for mechanistic studies.

The role of peripheral immunity in Parkinson’s disease (PD) remains incompletely understood. Here, we performed paired single-cell RNA sequencing (scRNA-seq) and T-cell receptor (TCR) profiling of 630k peripheral blood mononuclear cells (PBMCs) from 168 donors spanning idiopathic and genetic PD, prodromal PD, and healthy controls. We identify broad changes in immune-cell composition across the disease spectrum, including decreased naive B cells and increased naive CD8+ T cells in idiopathic PD. Transcriptional profiling reveals disease stage-dependent remodeling of mitochondrial and oxidative phosphorylation programs, with increased activity in prodromal disease followed by reduced activity in manifest PD, consistent with a transition in peripheral immune state during disease progression. High-resolution T cell analysis further identifies sex-dependent changes in cytotoxic CD8+ T-cell states and TCR clonal diversity. TCR motif analysis reveals disease-associated receptor groups with predicted specificities spanning PD-related, viral and autoimmune antigens. Together, these findings provide a comprehensive single-cell map of peripheral immune dysregulation in PD, defining dynamic and sex-dependent remodeling of peripheral immunity across the PD spectrum.

Monocytes are increasingly implicated in Parkinson’s disease (PD) pathogenesis, with idiopathic cases showing mitochondrial and lysosomal dysfunction. However, the impact of PD-associated mutations on monocytes remains unclear. To address this, we investigated transcriptomic and functional disturbances in peripheral monocytes from patients with GBA1 - and LRRK2 -associated PD and idiopathic PD. Transcriptomic data revealed shared and mutation-specific signatures, including those related to immune dysregulation, and consistent defects in lysosomal and mitochondrial pathways. Network and pathway analyses further uncovered downregulation in protein translation and enrichment of integrated stress response (ISR) signatures, alongside aberrant expression of genes linked to ER stress, proteostasis, mitophagy and type-I interferon signaling. These data suggest that monocyte immune dysfunction in PD may be, at least in part, a consequence of impaired proteostasis, organelle stress and maladaptive ISR activation. We further interrogated these signatures in functional assays in patient-derived macrophages, which revealed impaired mitochondrial potential, proteolysosomal dysfunction, and defective phagocytosis. Our findings define convergent molecular and functional abnormalities in genetic PD monocytes, implicating proteostasis failure and maladaptive ISR activation as upstream drivers of immune dysfunction, highlighting novel targetable pathways for therapeutic intervention.

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Prenatal infections and activation of the maternal immune system have been proposed to contribute to causing neurodevelopmental disorders (NDDs), chronic conditions often linked to brain abnormalities. Microglia are the resident immune cells of the brain and play a key role in neurodevelopment. Disruption of microglial functions can lead to brain abnormalities and increase the risk of developing NDDs. How the maternal as well as the fetal immune system affect human neurodevelopment and contribute to NDDs remains unclear. An important reason for this knowledge gap is the fact that the impact of exposure to prenatal risk factors has been challenging to study in the human context. Here, we characterized a model of cerebral organoids (CO) with integrated microglia (COiMg). These organoids express typical microglial markers and respond to inflammatory stimuli. The presence of microglia influences cerebral organoid development, including cell density and neural differentiation, and regulates the expression of several ciliated mesenchymal cell markers. Moreover, COiMg and organoids without microglia show similar but also distinct responses to inflammatory stimuli. Additionally, IFN-γ induced significant transcriptional and structural changes in the cerebral organoids, that appear to be regulated by the presence of microglia. Specifically, interferon-gamma (IFN-γ) was found to alter the expression of genes linked to autism. This model provides a valuable tool to study how inflammatory perturbations and microglial presence affect neurodevelopmental processes.

Microglia, the innate immune cells of the central nervous system, have been genetically implicated in multiple neurodegenerative diseases. We previously mapped the genetic regulation of gene expression and mRNA splicing in human microglia, identifying several loci where common genetic variants in microglia-specific regulatory elements explain disease risk loci identified by GWAS. However, identifying genetic effects on splicing has been challenging due to the use of short sequencing reads to identify causal isoforms. Here we present the isoform-centric microglia genomic atlas (isoMiGA) which leverages the power of long-read RNA-seq to identify 35,879 novel microglia isoforms. We show that the novel microglia isoforms are involved in stimulation response and brain region specificity. We then quantified the expression of both known and novel isoforms in a multi-ethnic meta-analysis of 555 human microglia short-read RNA-seq samples from 391 donors, the largest to date, and found associations with genetic risk loci in Alzheimer’s disease and Parkinson’s disease. We nominate several loci that may act through complex changes in isoform and splice site usage.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI)-anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c.1022C > A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv^341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv^341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv^341E-hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor–related pathways in brain development. Significance Inherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated a viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using this model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37°C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4°C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

Pathogenic germline mutations in PIGV lead to glycosylphosphatidylinositol biosynthesis deficiency (GPIBD). Individuals with pathogenic biallelic mutations in genes of the glycosylphosphatidylinositol (GPI) anchor pathway exhibit cognitive impairments, motor delay, and often epilepsy. Thus far, the pathophysiology underlying the disease remains unclear, and suitable rodent models that mirror all symptoms observed in human patients have not been available. Therefore, we used CRISPR-Cas9 to introduce the most prevalent hypomorphic missense mutation in European patients, Pigv:c .1022C>A (p.A341E), at a site that is conserved in mice. Mirroring the human pathology, mutant Pigv ^341E mice exhibited deficits in motor coordination, cognitive impairments, and alterations in sociability and sleep patterns, as well as increased seizure susceptibility. Furthermore, immunohistochemistry revealed reduced synaptophysin immunoreactivity in Pigv ^341E mice, and electrophysiology recordings showed decreased hippocampal synaptic transmission that could underlie impaired memory formation. In single-cell RNA sequencing, Pigv ^341E -hippocampal cells exhibited changes in gene expression, most prominently in a subtype of microglia and subicular neurons. A significant reduction in Abl1 transcript levels in several cell clusters suggested a link to the signaling pathway of GPI-anchored ephrins. We also observed elevated levels of Hdc transcripts, which might affect histamine metabolism with consequences for circadian rhythm. This new mouse model will not only open the doors to further investigation into the pathophysiology of GPIBD, but will also deepen our understanding of the role of GPI-anchor–related pathways in brain development. Significance statement: Inherited GPI-anchor biosynthesis deficiencies (IGDs) explain many cases of syndromic intellectual disability. Although diagnostic methods are improving, the pathophysiology underlying the disease remains unclear. Furthermore, we lack rodent models suitable for characterizing cognitive and social disabilities. To address this issue, we generated the first viable mouse model for an IGD that mirrors the condition in human patients with a behavioral phenotype and susceptibility to epilepsy. Using the new model, we obtained neurological insights such as deficits in synaptic transmission that will facilitate understanding of the pathophysiology of IGDs.