Microglia have been identified as key players in Alzheimer's disease pathogenesis, and other neurodegenerative diseases. Iba1, and more specifically TMEM119 and P2RY12 are gaining ground as presumedly more specific microglia markers, but comprehensive characterization of the expression of these three markers individually as well as combined is currently missing. Here we used a multispectral immunofluorescence dataset, in which over seventy thousand microglia from both aged controls and Alzheimer patients have been analysed for expression of Iba1, TMEM119 and P2RY12 on a single-cell level. For all markers, we studied the overlap and differences in expression patterns and the effect of proximity to β-amyloid plaques. We found no difference in absolute microglia numbers between control and Alzheimer subjects, but the prevalence of specific combinations of markers (phenotypes) differed greatly. In controls, the majority of microglia expressed all three markers. In Alzheimer patients, a significant loss of TMEM119⁺-phenotypes was observed, independent of the presence of β-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around β-amyloid plaques. No morphological features were conclusively associated with loss or gain of any of the markers or any of the identified phenotypes. All in all, none of the three markers were expressed by all microglia, nor can be wholly regarded as a pan- or homeostatic marker, and preferential phenotypes were observed depending on the surrounding pathological or homeostatic environment. This work could help select and interpret microglia markers in previous and future studies.

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Brain iron accumulation has been found to accelerate disease progression in amyloid-β(Aβ) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL⁺Iba1⁺-microglia, which were found to be the predominant Aβ-plaque infiltrating microglia. Finally, an increase of FTL⁺Iba1⁺-microglia was seen in patients with high Aβ load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with Aβ.

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Aims: Aceruloplasminemia is an ultra-rare neurodegenerative disorder associated with massive brain iron deposits, of which the molecular composition is unknown. We aimed to quantitatively determine the molecular iron forms in the aceruloplasminemia brain, and to illustrate their influence on iron-sensitive MRI metrics. Methods: The inhomogeneous transverse relaxation rate (R₂*) and magnetic susceptibility obtained from 7 T MRI were combined with Electron Paramagnetic Resonance (EPR) and Superconducting Quantum Interference Device (SQUID) magnetometry. The basal ganglia, thalamus, red nucleus, dentate nucleus, superior- and middle temporal gyrus and white matter of a post-mortem aceruloplasminemia brain were studied. MRI, EPR and SQUID results that had been previously obtained from the temporal cortex of healthy controls were included for comparison. Results: The brain iron pool in aceruloplasminemia detected in this study consisted of EPR-detectable Fe³⁺ ions, magnetic Fe³⁺ embedded in the core of ferritin and hemosiderin (ferrihydrite-iron), and magnetic Fe³⁺ embedded in oxidized magnetite/maghemite minerals (maghemite-iron). Ferrihydrite-iron represented above 90% of all iron and was the main driver of iron-sensitive MRI contrast. Although deep gray matter structures were three times richer in ferrihydrite-iron than the temporal cortex, ferrihydrite-iron was already six times more abundant in the temporal cortex of the patient with aceruloplasminemia compared to the healthy situation (162 µg/g vs. 27 µg/g), on average. The concentrations of Fe³⁺ ions and maghemite-iron in the temporal cortex in aceruloplasminemia were within the range of those in the control subjects. Conclusions: Iron-related neurodegeneration in aceruloplasminemia is primarily associated with an increase in ferrihydrite-iron, with ferrihydrite-iron being the major determinant of iron-sensitive MRI contrast.

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Iron accumulation in the brain is a phenomenon common to many neurodegenerative diseases, perhaps most notably Alzheimer’s disease (AD). We present here magnetic analyses of post-mortem brain tissue of patients who had severe Alzheimer’s disease, and compare the results with those from healthy controls. Isothermal remanent magnetization experiments were performed to assess the extent to which different magnetic carriers are affected by AD pathology and formalin fixation. While Alzheimer’s brain material did not show higher levels of magnetite/maghemite nanoparticles than corresponding controls, the ferrihydrite mineral, known to be found within the core of ferritin proteins and hemosiderin aggregates, almost doubled in concentration in patients with Alzheimer’s pathology, strengthening the conclusions of our previous studies. As part of this study, we also investigated the effects of sample preparation, by performing experiments on frozen tissue as well as tissue which had been fixed in formalin for a period of 5 months. Our results showed that the two different preparations did not critically affect the concentration of magnetic carriers in brain tissue, as observable by SQUID magnetometry.

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Animal studies suggest the involvement of natriuretic peptides (NP) in several brain functions that are known to be disturbed during Alzheimer's disease (AD). However, it remains unclear whether such findings extend to humans. In this study, we aimed to: (1) map the gene expression and localization of NP and their receptors (NPR) in human post-mortem brain tissue; (2) compare the relative amounts of NP and NPR between the brain tissue of AD patients and non-demented controls, and (3) compare the relative amounts of NP between the cerebrospinal fluid (CSF) of AD patients and non-demented controls. Using the publicly available Allen Human Brain Atlas dataset, we mapped the gene expression of NP and NPR in healthy humans. Using immunohistochemistry, we visualized the localization of NP and NPR in the frontal cortex of AD patients (n = 10, mean age 85.8 ± 6.2 years) and non-demented controls (mean age = 80.2 ± 9.1 years). Using Western blotting and ELISA, we quantified the relative amounts of NP and NPR in the brain tissue and CSF of these AD patients and non-demented controls. Our results showed that NP and NPR genes were ubiquitously expressed throughout the brain in healthy humans. NP and NPR were present in various cellular structures including in neurons, astrocyte-like structures, and cerebral vessels in both AD patients and non-demented controls. Furthermore, we found higher amounts of NPR type-A in the brain of AD patients (p = 0.045) and lower amounts of NP type-B in the CSF of AD patients (p = 0.029). In conclusion, this study shows the abundance of NP and NPR in the brain of humans suggesting involvement of NP in various brain functions. In addition, our findings suggest alterations of NP levels in the brain of AD patients. The role of NP in the development and progression of AD remains to be elucidated.@en

The ability to administer systemically high doses of manganese as contrast agent while circumventing its toxicity is of particular interest for exploratory MRI studies of the brain. Administering low doses either repeatedly or continuously over time has been shown to enable the acquisition of satisfactory MRI images of the mouse brain without apparent side effects. Here we have systematically compared the obtained MRI contrast and recorded potential systemic side effects such as stress response and muscle strength impairment in relation to the achieved contrast. We show in mice that administering MnCl2 via osmotic infusion pumps allows for a sideeffect free delivery of a high cumulative dose of manganese chloride (480 mg/kg bodyweight in 8 days). High contrast in MRI was achieved while we did not observe the weight loss or distress seen in other studies where mice received manganese via fractionated intraperitoneal injections of lower doses of manganese. As the normal daily conduct of the mice was not affected, this new manganese delivery method might be of particular use to study brain activity over several days. This may facilitate the phenotyping of new transgenic mouse models, the study of chronic disease models and the monitoring of changes in brain activity in long-term behavioral studies.@en