Ashley S. Harms

Microglial activation in response to aggregated α-synuclein (α-syn) accumulation is a pathological hallmark of Parkinson’s disease (PD). A distinct activated microglial state, disease-associated microglia (DAM), has been identified across multiple neurodegenerative disorders, including PD. However, the transcriptional and epigenetic programs governing DAM differentiation remain unclear. Here, we show that neuronal α-syn overexpression drives progressive microglial state transitions toward DAM, accompanied by increased phagocytic activity and enhanced cytokine and chemokine production. Integrative transcriptomic and chromatin accessibility analyses reveal a hierarchical NF-κB–AP-1–IRF regulatory program underlying these state transitions. Microglial NF-κB p65 (RelA) or IRF4 deficiency impairs microglial progression toward DAM, with RelA and IRF4 governing early microglial activation and subsequent DAM differentiation, respectively. Enrichment of NF-κB, AP-1, and IRF motifs in DAM in human PD supports conservation of this regulatory program across species. Together, our findings establish RelA and IRF4 as sequential, stage-specific regulators that coordinate α-syn-induced DAM differentiation in PD.

Microglial activation in response to aggregated α -synuclein ( α -syn) accumulation is a pathological hallmark of Parkinson’s disease (PD). A distinct activated microglial state, disease-associated microglia (DAM), has been identified across multiple neurodegenerative disorders, including PD. However, the transcriptional and epigenetic programs governing DAM differentiation remain unclear. Here, we show that neuronal α -syn overexpression drives progressive microglial state transitions toward DAM, accompanied by increased phagocytic activity and enhanced cytokine and chemokine production. Integrative transcriptomic and chromatin accessibility analyses reveal a hierarchical NF-κB–AP-1–IRF regulatory program underlying these state transitions. Microglial NF-κB p65 (RelA) or IRF4 deficiency impairs microglial progression toward DAM, with RelA and IRF4 governing early microglial activation and subsequent DAM differentiation, respectively. Enrichment of NF-κB, AP-1, and IRF motifs in DAM in human PD supports conservation of this regulatory program across species. Together, our findings establish RelA and IRF4 as sequential, stage-specific regulators that coordinate α -syn-induced DAM differentiation in PD.

Co-expression of tau and amyloid-beta with α-syn pathology is shown in over 50% of Parkinson’s Disease postmortem brains, and these patients present with enhanced cognitive decline and disease phenotype. This review integrates the known immune response in PD with co-pathology incidence, highlighting synergistic interactions between α-syn, tau and Aβ and how this promotes a distinct neuroinflammatory response leading to severe neuronal loss and cognitive symptoms. Based on this, we support the need for combinatorial therapeutic strategies to address this debilitating, yet common factor of disease.

This adapted stereology protocol allows users to count objects in an unbiased fashion in layers V/VI of the secondary motor cortex (M2 cortex) of mouse brain tissue. This particular protocol uses SatB2+ neurons as the marker of interest but could be used to count a variety of markers in the M2 cortex. Changes were made to the origin protocol to account for differences in sampling region and marker of interest, other minor changes were made for clarity.

A collection of protocols included in the study assessing the role of co-pathologies on neuroinflammation and neuropathology in vivo. This collection includes optimized methods for immunohistochemistry (IHC), immunofluorescence (IF), flow cytometry, behavior techniques and quantification methods (stereology for example) developed by members of the Harms Lab and collaborators.

Parkinson's Disease (PD) is an incurable neurodegenerative disease that causes movement disorders. Neurons in PD aggregate α-synuclein and are depleted from the substantia nigra (SN), which is a movement control hub. The presence of α-synuclein-reactive T cells in PD patient blood suggests a role for adaptive immunity in the pathogenesis of PD. However, the characteristics of this response within the brain are not well understood. Here, we employed single-nucleus RNAseq, spatial transcriptomics, and T cell receptor (TCR) sequencing to analyze T cell and glial cell states in post-mortem PD brain tissue. CD8 + T cells were enriched in the PD SN and characterized by clonal expansion and TCR sequences with homology to those reactive to α-synuclein. Furthermore, PD T cells were spatially correlated with CD44+ astrocytes, which increased in the PD SN. Silencing CD44 in cultured astrocytes attenuated neuroinflammatory signatures, suggesting a potential therapeutic target. These findings provide insight into the neurodegenerative niche underlying T cell-mediated neuroinflammation in PD.

Growing evidence indicates that persons with Parkinson disease (PD), have a unique composition of indigenous gut microbes. Given the long prodromal or pre-diagnosed period, longitudinal studies of the human and rodent gut microbiome before symptomatic onset and for the duration of the disease are currently lacking. PD is partially characterized by the accumulation of the protein α-synuclein (α-syn) into insoluble aggregates, in both the central and enteric nervous systems. As such, several experimental rodent and non-human primate models of α-syn overexpression recapitulate some of the hallmark pathophysiologies of PD. These animal models provide an opportunity to assess how the gut microbiome changes with age under disease-relevant conditions. Here, we used a transgenic mouse strain, which overexpress wild-type human α-syn to test how the gut microbiome composition responds in this model of PD pathology during aging. Using shotgun metagenomics, we find significant, age and genotype-dependent bacterial taxa whose abundance becomes altered with age. We reveal that α-syn overexpression can drive alterations to the gut microbiome composition and suggest that it limits diversity through age. Taxa that were most affected by genotype-age interaction were Lactobacillus and Bifidobacteria. In a mouse model, we showed direct link between alpha synuclein geneotype (hallmark of PD), a dysbiotic and low-diversity gut microbiome, and dysbiotic levels of Bifidobacteria and Lactobacillus (most robust features of PD microbiome). Given emerging data on the potential contributions of the gut microbiome to PD pathologies, our data provide an experimental foundation to understand how the PD-associated microbiome may arise as a trigger or co-pathology to disease.

Disease-associated microglia (DAM) are a subset of microglia that appear at various stages of central nervous system neurodegenerative diseases. DAM were identified using single-cell RNA sequencing within Alzheimer’s Disease (AD) where they were characterized by their unique localization near amyloid-β plaques and their phagocytic and lipid-metabolizing features. Unfortunately, activation and etiology of DAM are only understood within the context of AD where Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), a receptor for amyloid-β, appears to be the key regulator in microglial transition to a DAM state. Despite this reliance on TREM2 in AD, DAM appear across other neurodegenerative diseases in which TREM2 may not be a critical player. This begs the question of if DAM are truly the same across all neurodegenerative diseases or if there exists a heterogeneity to DAM across neurodegenerative pathologies. Investigation into this critical gap in the field regarding DAM etiology and activation, as well as DAM function, could be delineated utilizing models of Parkinson’s disease (PD) to complement studies in models of AD. Though highly underexplored regarding DAM, PD with its pattern of protein aggregation-associated pathology like AD could serve as the spatiotemporal comparison against AD findings to ascertain the nature of DAM. The experimental vehicle that could guide the future of such investigation is the multi-omics model. With a compound approach focusing on exploring triggers for DAM at the chromatin or mRNA level and related protein output, it becomes possible to strongly characterize and firmly answer the question of what is a DAM.

Alzheimer’s (AD) and Parkinson disease (PD) pathology often co-occur. Amyloid-β and phosphorylated tau are found in 30-50% of idiopathic PD cases, while α-synuclein inclusions are present in 50% of AD cases. These co-pathologies are linked to increased mortality and earlier onset of cognitive decline. Immune activation is a hallmark of these neurodegenerative diseases, but current models primarily examine each pathology in isolation. How these co-pathologies drive inflammation and neuronal loss remains poorly understood. We therefore developed a mouse model combining tau, amyloid-β, and α-synuclein. We found that co-pathologies synergistically trigger an amplified neuroimmune response, with expanded populations of CD4 ⁺ and CD8 ⁺ tissue-resident memory T cells and CD68 ⁺ microglia, compared to single pathologies. These changes were abundant in the hippocampus and cortex, regions with elevated protein pathology load and enhanced neuronal loss. Our findings demonstrate that co-pathologies enhance proteinopathy and synergistically enhance immune activation and neurodegeneration, suggesting that combinatorial therapeutic strategies that target both co-pathologies and inflammation, may be disease modifying. Summary: Webster et al. demonstrate that co-occurring Alzheimer’s and Parkinson disease protein pathologies, common in cognitively impaired patient populations, amplify proteinopathy and synergistically enhance CNS neuroinflammatory responses and neurodegeneration. This work supports the need for combinatorial therapeutic strategies and positions neuroinflammation as an important link for co-pathology enhanced neurodegeneration.

Multiple system atrophy (MSA) is rare, fast progressing, and fatal synucleinopathy with alpha-synuclein (α-syn) inclusions located within oligodendroglia called glial cytoplasmic inclusions (GCI). Along with GCI pathology there is severe demyelination, neurodegeneration, and neuroinflammation. In post-mortem tissue, there is significant infiltration of CD8+ T cells into the brain parenchyma, however their role in disease progression is unknown. To determine the role of CD8+ T cells, a modified AAV, Olig001-SYN, was used to selectively overexpress α-syn in oligodendrocytes modeling MSA in mice. Four weeks post transduction, we observed significant CD8+ T cell infiltration into the striatum of Olig001-SYN transduced mice recapitulating the CD8+ T cell infiltration observed in post-mortem tissue. To understand the role of CD8+ T cells, a CD8 knockout mice were transduced with Olig001-SYN. Six months post transduction into a mouse lacking CD8+ T cells, demyelination and neurodegeneration were unchanged. Four weeks post transduction, neuroinflammation and demyelination were enhanced in CD8 knockout mice compared to wild type controls. Applying unbiased spectral flow cytometry, CD103+, CD69+, CD44+, CXCR6+, CD8+ T cells were identified when α-syn was present in oligodendrocytes, suggesting the presence of tissue resident memory CD8+ T (Trm) cells during MSA disease progression. This study indicates that CD8+ T cells are not critical in driving MSA pathology but are needed to modulate the neuroinflammation and demyelination response.