Inflammation-induced TRPV4 channels exacerbate blood-brain barrier dysfunction in multiple sclerosis.

BACKGROUND: Blood-brain barrier (BBB) dysfunction and immune cell migration into the central nervous system (CNS) are pathogenic drivers of multiple sclerosis (MS). Ways to reinstate BBB function and subsequently limit neuroinflammation present promising strategies to restrict disease progression. However, to date, the molecular players directing BBB impairment in MS remain poorly understood. One suggested candidate to impact BBB function is the transient receptor potential vanilloid-type 4 ion channel (TRPV4), but its specific role in MS pathogenesis remains unclear. Here, we investigated the role of TRPV4 in BBB dysfunction in MS. MAIN TEXT: In human post-mortem MS brain tissue, we observed a region-specific increase in endothelial TRPV4 expression around mixed active/inactive lesions, which coincided with perivascular microglia enrichment in the same area. Using in vitro models, we identified that microglia-derived tumor necrosis factor-α (TNFα) induced brain endothelial TRPV4 expression. Also, we found that TRPV4 levels influenced brain endothelial barrier formation via expression of the brain endothelial tight junction molecule claudin-5. In contrast, during an inflammatory insult, TRPV4 promoted a pathological endothelial molecular signature, as evidenced by enhanced expression of inflammatory mediators and cell adhesion molecules. Moreover, TRPV4 activity mediated T cell extravasation across the brain endothelium. CONCLUSION: Collectively, our findings suggest a novel role for endothelial TRPV4 in MS, in which enhanced expression contributes to MS pathogenesis by driving BBB dysfunction and immune cell migration.

Cortical iron accumulation in MAPT- and C9orf 72-associated frontotemporal lobar degeneration.

Neuroinflammation has been implicated in frontotemporal lobar degeneration (FTLD) pathophysiology, including in genetic forms with microtubule-associated protein tau (MAPT) mutations (FTLD-MAPT) or chromosome 9 open reading frame 72 (C9orf72) repeat expansions (FTLD-C9orf72). Iron accumulation as a marker of neuroinflammation has, however, been understudied in genetic FTLD to date. To investigate the occurrence of cortical iron accumulation in FTLD-MAPT and FTLD-C9orf72, iron histopathology was performed on the frontal and temporal cortex of 22 cases (11 FTLD-MAPT and 11 FTLD-C9orf72). We studied patterns of cortical iron accumulation and its colocalization with the corresponding underlying pathologies (tau and TDP-43), brain cells (microglia and astrocytes), and myelination. Further, with ultrahigh field ex vivo MRI on a subset (four FTLD-MAPT and two FTLD-C9orf72), we examined the sensitivity of T2*-weighted MRI for iron in FTLD. Histopathology showed that cortical iron accumulation occurs in both FTLD-MAPT and FTLD-C9orf72 in frontal and temporal cortices, characterized by a diffuse mid-cortical iron-rich band, and by a superficial cortical iron band in some cases. Cortical iron accumulation was associated with the severity of proteinopathy (tau or TDP-43) and neuronal degeneration, in part with clinical severity, and with the presence of activated microglia, reactive astrocytes and myelin loss. Ultra-high field T2*-weighted MRI showed a good correspondence between hypointense changes on MRI and cortical iron observed on histology. We conclude that iron accumulation is a feature of both FTLD-MAPT and FTLD-C9orf72 and is associated with pathological severity. Therefore, in vivo iron imaging using T2*-weighted MRI or quantitative susceptibility mapping may potentially be used as a noninvasive imaging marker to localize pathology in FTLD.

How iron can drive neurodegeneration.

Iron overload in neurodegenerative diseases is well established but of uncertain significance. In a recent article, Ryan et al. reveal that microglia are especially vulnerable to iron overload-induced ferroptosis. Their evidence for microglial ferroptosis in clinical specimens indicates that ferroptosis inhibitors may hold therapeutic promise for these diseases.

Iron accumulation induces oxidative stress, while depressing inflammatory polarization in human iPSC-derived microglia.

Iron accumulation in microglia has been observed in Alzheimer's disease and other neurodegenerative disorders and is thought to contribute to disease progression through various mechanisms, including neuroinflammation. To study this interaction, we treated human induced pluripotent stem cell-derived microglia (iPSC-MG) with iron, in combination with inflammatory stimuli such as interferon gamma (IFN-γ) and amyloid ß. Both IFN-γ and iron treatment increased labile iron levels, but only iron treatment led to a consistent increase of ferritin levels, reflecting long-term iron storage. Therefore, in iPSC-MG, ferritin appeared to be regulated by iron revels rather than inflammation. Further investigation showed that while IFN-γ induced pro-inflammatory activation, iron treatment dampened both classic pro- and anti-inflammatory activation on a transcriptomic level. Notably, iron-loaded microglia showed strong upregulation of cellular stress response pathways, the NRF2 pathway, and other oxidative stress pathways. Functionally, iPSC-MG exhibited altered phagocytosis and impaired mitochondrial metabolism following iron treatment. Collectively, these data suggest that in MG, in contrast to current hypotheses, iron treatment does not result in pro-inflammatory activation, but rather dampens it and induces oxidative stress.

Co-expression patterns of microglia markers Iba1, TMEM119 and P2RY12 in Alzheimer's disease.

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 ß-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 ß-amyloid plaques in its proximity. Contrary, phenotypes showing loss of P2RY12, but consistent Iba1 expression were increasingly prevalent around ß-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.

Quantitative susceptibility mapping in the thalamus and basal ganglia of systemic lupus erythematosus patients with neuropsychiatric complaints.

Systemic lupus erythematosus (SLE) is an auto-immune disease characterized by multi-organ involvement. Although uncommon, central nervous system involvement in SLE, termed neuropsychiatric SLE (NPSLE), is not an exception. Current knowledge on underlying pathogenic mechanisms is incomplete, however, neuroinflammation is thought to play a critical role. Evidence from neurodegenerative diseases and multiple sclerosis suggests that neuroinflammation is correlated with brain iron accumulation, making quantitative susceptibility mapping (QSM) a potential hallmark for neuroinflammation in vivo. This study assessed susceptibility values of the thalamus and basal ganglia in (NP)SLE patients and further investigated the in vivo findings with histological analyses of postmortem brain tissue derived from SLE patients. We used a 3T MRI scanner to acquire single-echo T2*-weighted images of 44 SLE patients and 20 age-matched healthy controls. Of the 44 patients with SLE, all had neuropsychiatric complaints, of which 29 were classified as non-NPSLE and 15 as NPSLE (seven as inflammatory NPSLE and eight as ischemic NPSLE). Mean susceptibility values of the thalamus, caudate nucleus, putamen, and globus pallidus were calculated. Formalin-fixed paraffin-embedded post-mortem brain tissue including the putamen and globus pallidus of three additional SLE patients was obtained and stained for iron, microglia and astrocytes. Susceptibility values of SLE patients and age-matched controls showed that iron levels in the thalamus and basal ganglia were not changed due to the disease. No subgroup of SLE showed higher susceptibility values. No correlation was found with disease activity or damage due to SLE. Histological examination of the post-mortem brain showed no increased iron accumulation. Our results suggest that neuroinflammation in NPSLE does not necessarily go hand in hand with iron accumulation, and that the inflammatory pathomechanism in SLE may differ from the one observed in neurodegenerative diseases and in multiple sclerosis.

Iron loading is a prominent feature of activated microglia in Alzheimer's disease patients.

Brain iron accumulation has been found to accelerate disease progression in amyloid-ß(Aß) 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 Aß-plaque infiltrating microglia. Finally, an increase of FTL+Iba1+-microglia was seen in patients with high Aß 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 Aß.

Visual cohort comparison for spatial single-cell omics-data.

Spatially-resolved omics-data enable researchers to precisely distinguish cell types in tissue and explore their spatial interactions, enabling deep understanding of tissue functionality. To understand what causes or deteriorates a disease and identify related biomarkers, clinical researchers regularly perform large-scale cohort studies, requiring the comparison of such data at cellular level. In such studies, with little a-priori knowledge of what to expect in the data, explorative data analysis is a necessity. Here, we present an interactive visual analysis workflow for the comparison of cohorts of spatially-resolved omics-data. Our workflow allows the comparative analysis of two cohorts based on multiple levels-of-detail, from simple abundance of contained cell types over complex co-localization patterns to individual comparison of complete tissue images. As a result, the workflow enables the identification of cohort-differentiating features, as well as outlier samples at any stage of the workflow. During the development of the workflow, we continuously consulted with domain experts. To show the effectiveness of the workflow, we conducted multiple case studies with domain experts from different application areas and with different data modalities.

Generation of pure monocultures of human microglia-like cells from induced pluripotent stem cells.

Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y12+ 95% TMEM119+ ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease.

Pathological characterization of T2*-weighted MRI contrast in the striatum of Huntington's disease patients.

Previous MRI studies consistently reported iron accumulation within the striatum of patients with Huntington's disease (HD). However, the pattern and origin of iron accumulation is poorly understood. This study aimed to characterize the histopathological correlates of iron-sensitive ex vivo MRI contrast change in HD brains. To this end, T2*-weighted 7T MRI was performed on postmortem tissue of the striatum of three control subjects and 10 HD patients followed by histological examination. In addition, formalin-fixed paraffin-embedded material of three control subjects and 14 HD patients was selected for only histology to identify the cellular localization of iron using stainings for iron, myelin, microglia and astrocytes. As expected HD striata showed prominent atrophy. Compared to controls, the striatum of HD patients was in general more hypointense on T2*-weighted high-field MRI and showed a more intense histopathological staining for iron. In addition, T2*-weighted MRI identified large focal hypointensities within the striatum of HD patients. Upon histological examination, these large focal hypointensities frequently colocalized with enlarged perivascular spaces and iron was found within the vessel wall and reactive astrocytes. In conclusion, we show that the striatum of HD patients has a distinctive phenotype on T2*-weighted MRI compared to control subjects. On ex vivo MRI, these contrast changes are heavily biased by enlarged perivascular spaces from which it is currently unknown whether this is a fixation artefact or a disease specific observation. Clinically, the observation of iron within reactive astrocytes is of importance for the interpretation and understanding of the potential underlying mechanisms of T2*-weighted MRI results in HD patients.

Post-Mortem MRI and Histopathology in Neurologic Disease: A Translational Approach.

In this review, combined post-mortem brain magnetic resonance imaging (MRI) and histology studies are highlighted, illustrating the relevance of translational approaches to define novel MRI signatures of neuropathological lesions in neuroinflammatory and neurodegenerative disorders. Initial studies combining post-mortem MRI and histology have validated various MRI sequences, assessing their sensitivity and specificity as diagnostic biomarkers in neurologic disease. More recent studies have focused on defining new radiological (bio)markers and implementing them in the clinical (research) setting. By combining neurological and neuroanatomical expertise with radiological development and pathological validation, a cycle emerges that allows for the discovery of novel MRI biomarkers to be implemented in vivo. Examples of this cycle are presented for multiple sclerosis, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. Some applications have been shown to be successful, while others require further validation. In conclusion, there is much to explore with post-mortem MRI and histology studies, which can eventually be of high relevance for clinical practice.

7T MRI allows detection of disturbed cortical lamination of the medial temporal lobe in patients with Alzheimer's disease.

Using 7T T2⁎-weighted imaging, we scanned post-mortem hemispheres of Alzheimer patients and age-matched controls to describe the patterns of appearance of cortical lamination on T2*-weighted MRI in the medial temporal lobe and to assess the changes in Alzheimer patients versus controls. While controls showed a hypointense line of Baillarger in the majority of the cases, appearance of cortical lamination varied to a greater extent in the Alzheimer patients. Severely distorted cortical lamination was also observed in advanced stage Alzheimer patients and presented itself as a broad hypointense inhomogeneous band, covering a large part of the cortical width. Histology indicated that the changes in the appearance of visible cortical lamination were not only associated with myelin changes, but also with diffuse cortical iron alterations and depositions. Therefore, imaging cortical lamination alterations in Alzheimer patients using T2*-weighted MRI might provide new information on involved neuroanatomical structures in an advanced neurodegenerative stage.

Postmortem T2*- Weighted MRI Imaging of Cortical Iron Reflects Severity of Alzheimer's Disease.

The value of iron-based MRI changes for the diagnosis and staging of Alzheimer's disease (AD) depends on an association between cortical iron accumulation and AD pathology. Therefore, this study determined the cortical distribution pattern of MRI contrast changes in cortical regions selected based on the known distribution pattern of tau pathology and investigated whether MRI contrast changes reflect the underlying AD pathology in the different lobes. T2*-weighted MRI was performed on postmortem cortical tissue of controls, late-onset AD (LOAD), and early-onset AD (EOAD) followed by histology and correlation analyses. Combining ex vivo high-resolution MRI and histopathology revealed that: 1) LOAD and EOAD have a different distribution pattern of AD pathological hallmarks and MRI contrast changes over the cortex, with EOAD showing more severe MRI changes; 2) per lobe, severity of AD pathological hallmarks correlates with iron accumulation, and hence with MRI. Therefore, iron-sensitive MRI sequences allow detection of the cortical distribution pattern of AD pathology ex vivo.