With the world’s population ageing, the incidence of Parkinson’s disease (PD) is on the rise. In recent years, inflammatory processes have emerged as prominent contributors to the pathology of PD. There is great evidence that microglia have a significant neuroprotective role, and that impaired and over activated microglial phenotypes are present in brains of PD patients. Thereby, PD progression is potentially driven by a vicious cycle between dying neurons and microglia through the instigation of oxidative stress, mitophagy and autophagy dysfunctions, a-synuclein accumulation, and pro-inflammatory cytokine release. Hence, investigating the involvement of microglia is of great importance for future research and treatment of PD. The purpose of this review is to highlight recent findings concerning the microglia-neuronal interplay in PD with a focus on human postmortem immunohistochemistry and single-cell studies, their relation to animal and iPSC-derived models, newly emerging technologies, and the resulting potential of new anti-inflammatory therapies for PD.

In Alzheimer’s disease (AD), microglia form distinct cellular aggregates that play critical roles in disease progression, including Aβ plaque-associated microglia (PaM) and the newly identified coffin-like microglia (CoM). PaM are closely associated with amyloid-β (Aβ) plaques, while CoM are enriched in the pyramidal layer of the CA2/CA1 hippocampal subfields, where they frequently engulf neurons and associate with tau-positive tangles and phosphorylated α-synuclein. To elucidate the role of these microglial subtypes, we employed high-content neuropathology, integrating Deep Spatial Profiling (DSP), multiplex chromogenic immunohistochemistry and confocal microscopy, to comprehensively map and characterise their morphological and molecular signatures, as well as their neuropathological and astrocytic microenvironments, in AD and control post-mortem samples. PaM and PaM-associated astrocytes exhibited signatures related to complement system pathways, ErbB signalling, and metabolic and neurodegenerative processes. In contrast, CoM displayed markers associated with protein degradation and immune signalling pathways, including STING, TGF-β, and NF-κB. While no direct association between CD8 + T cells and either microglial type was observed, CD163 + perivascular macrophages were frequently incorporated into PaM. These findings provide novel insights into the heterogeneity of microglial responses, in particular their distinct interactions with astrocytes and infiltrating immune cells, and shed light on specific neurodegenerative hotspots and their implications for hippocampal deterioration in AD.

Astrocytes with their specialised morphology are essential for brain homeostasis as metabolic mediators between blood vessels and neurons. In neurodegenerative diseases such as Alzheimer’s disease (AD), astrocytes adopt reactive profiles with molecular and morphological changes that could lead to the impairment of their metabolic support and impact disease progression. However, the underlying mechanisms of how the metabolic function of human astrocytes is impaired by their morphological changes in AD are still elusive. To address this challenge, we developed and applied a metabolic multiscale modelling approach integrating the dynamics of metabolic energy pathways and physiological astrocyte morphologies acquired in human AD and age-matched control brain samples. The results demonstrate that the complex cell shape and intracellular organisation of energetic pathways determine the metabolic profile and support capacity of astrocytes in health and AD conditions. Thus, our mechanistic approach indicates the importance of spatial orchestration in metabolism and allows for the identification of protective mechanisms against disease-associated metabolic impairments.

Hippocampal alteration is at the centre of memory decline in the most common age-related neurodegenerative diseases: Alzheimer’s disease (AD) and Dementia with Lewy Bodies (DLB). However, the subregional deterioration of the hippocampus differs between both diseases with more severe atrophy in the CA1 subfield of the AD patients. How AD and DLB-typical pathologies compose the various local microenvironment of the hippocampus across AD and DLB needs to be further explored to understand this process. Additionally, microglia responses could further impact the atrophy rate. Some studies suggest that microglia react differently according to the underlying neurodegenerative disorder. How microglia are transformed across hippocampal subfields in AD and DLB, and how their changes are associated with disease-typical pathologies remains to be determined. To these purposes, we performed a volumetric analysis of phospho-Tau (P-Tau), Amyloid-ß (Aß), and phospho-α-Synuclein (P-Syn) loads, quantified and classified microglia according to distinct morphological phenotypes using high-resolution confocal 3D microscopy of hippocampal CA1, CA3 and DG/CA4 subfields of late-onset AD (n=10) and DLB (n=8) as well as age-matched control samples (n=11). We found that each of the Tau, Aß and Synuclein pathologies followed a specific subregional distribution, relatively preserved across AD and DLB. P-Tau, Aß and P-Syn burdens were significantly exacerbated in AD, with Tau pathology being particularly severe in the AD CA1. P-Tau and P-Syn burdens were highly correlated across subfields and conditions (R2Spear = 0.79; P < 0.001) and result from a local co-distribution of P-Tau and P-Syn inclusions in neighbouring neurons, with only a low proportion of double-positive cells. In parallel, we assessed the changes of the microglia responses by measuring 16 morphological features of more than 35,000 individual microglial cells and classifying them into seven-distinct morphological clusters. We found microglia features- and clusters-variations subfield- and condition-dependent. Two of the seven morphological clusters, with more amoeboid and less branched forms, were identified as disease-enriched and found to be further increased in AD. Interestingly, some microglial features or clusters were associated with one but more often with a combination of two pathologies in a subfield-dependent manner. In conclusion, our study shows a multimodal association of the hippocampal microglia responses with the co-occurrence, distribution and severity of AD and DLB pathologies. In DLB hippocampi, pathological imprint and microglia responses follow AD trends but with lesser severity. Our study suggests that the increased pathological burdens of P-Tau and P-Syn and associated microglia alterations are involved in a more severe deterioration of the CA1 in AD as compared to DLB.

Confocal fluorescent microscopy is a major tool to investigate the molecular orchestration of biomedical samples. The quality of the image acquisition depends critically on the tissue quality and thickness, the type, and concentration of antibodies used, as well as on microscope parameters. Due to these factors, intra-sample and inter-sample variability inevitably arises. Segmentation and quantification of targeted proteins can thus become a challenging process. Image processing techniques need therefore to address the acquisitions variability to minimize the risk of biases originating from changes in signal intensity, background noise, and parameterization. Here, we introduce PaFSe, a parameter-free segmentation algorithm for 3D fluorescent images. The algorithm is based on our established PRAQA approach, which evaluates the dispersion of several pixel intensity neighborhoods allowing for a statistical assessment whether individual subfields of an image can be considered as positive signal or background. PaFSe extends PRAQA by a fully automatic estimate for the segmentation parameters, and thereby provides a completely parameter-free and robust segmentation algorithm. By comparing PaFSe with Ilastik on synthetic examples, we show that our method achieves similar performances as a supervised approach in low-to-moderate noise environments without the need of tedious training. Furthermore, we validate the efficiency of PaFSe by segmenting and quantifying the abundance of hyperphosphorylated Tau protein in post-mortem human brain samples from Alzheimer’s disease patients and age-matched controls, where we obtain quantification values highly correlated with manual neuropathological segmentation. PaFSe is a parameter-free, fast, and adaptive approach for robust segmentation and quantification of protein abundance from complex 3D fluorescent images and is freely available at https://doi.org/10.17881/j20h-pa27.

Astrocytes with their specialized morphology are essential for brain homeostasis as metabolic mediators between blood vessels and neurons. In neurodegenerative diseases such as Alzheimer’s disease (AD), astrocytes adopt reactive profiles with molecular and morphological changes that could lead to the impairment of their metabolic support and impact disease progres-sion. However, the underlying mechanisms how metabolic function of human astrocytes is impaired by their morphological changes in AD is still elusive. To address this challenge, we developed and applied a metabolic multiscale modelling approach integrating the dynamics of metabolic energy pathways and physiological astrocyte morphologies acquired in human AD and age-matched control brain samples. The results demonstrate that the complex cell shape and intracellular organization of energetic pathways determine the metabolic profile and support capacity of astrocytes in health and AD conditions. Thus, our mechanistic approach indicates the importance of spatial orchestration in metabolism and allows for the identification of protective mechanisms against disease-associated metabolic impairments.