Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Continued dysfunction of capillary pericytes promotes no-reflow after experimental stroke in vivo.

Incomplete reperfusion of the microvasculature ('no-reflow') after ischaemic stroke damages salvageable brain tissue. Previous ex vivo studies suggest pericytes are vulnerable to ischaemia and may exacerbate no-reflow, but the viability of pericytes and their association with no-reflow remains under-explored in vivo. Using longitudinal in vivo two-photon single-cell imaging over 7 days, we showed that 87% of pericytes constrict during cerebral ischaemia and remain constricted post reperfusion, and 50% of the pericyte population are acutely damaged. Moreover, we revealed ischaemic pericytes to be fundamentally implicated in capillary no-reflow by limiting and arresting blood flow within the first 24 h post stroke. Despite sustaining acute membrane damage, we observed that over half of all cortical pericytes survived ischaemia and responded to vasoactive stimuli, upregulated unique transcriptomic profiles and replicated. Finally, we demonstrated the delayed recovery of capillary diameter by ischaemic pericytes after reperfusion predicted vessel reconstriction in the subacute phase of stroke. Cumulatively, these findings demonstrate that surviving cortical pericytes remain both viable and promising therapeutic targets to counteract no-reflow after ischaemic stroke.

The pseudoprotease iRhom1 controls ectodomain shedding of membrane proteins in the nervous system.

Proteolytic ectodomain shedding of membrane proteins is a fundamental mechanism to control the communication between cells and their environment. A key protease for membrane protein shedding is ADAM17, which requires a non-proteolytic subunit, either inactive Rhomboid 1 (iRhom1) or iRhom2 for its activity. While iRhom1 and iRhom2 are co-expressed in most tissues and appear to have largely redundant functions, the brain is an organ with predominant expression of iRhom1. Yet, little is known about the spatio-temporal expression of iRhom1 in mammalian brain and about its function in controlling membrane protein shedding in the nervous system. Here, we demonstrate that iRhom1 is expressed in mouse brain from the prenatal stage to adulthood with a peak in early postnatal development. In the adult mouse brain iRhom1 was widely expressed, including in cortex, hippocampus, olfactory bulb, and cerebellum. Proteomic analysis of the secretome of primary neurons using the hiSPECS method and of cerebrospinal fluid, obtained from iRhom1-deficient and control mice, identified several membrane proteins that require iRhom1 for their shedding in vitro or in vivo. One of these proteins was 'multiple-EGF-like-domains protein 10' (MEGF10), a phagocytic receptor in the brain that is linked to the removal of amyloid ß and apoptotic neurons. MEGF10 was further validated as an ADAM17 substrate using ADAM17-deficient mouse embryonic fibroblasts. Taken together, this study discovers a role for iRhom1 in controlling membrane protein shedding in the mouse brain, establishes MEGF10 as an iRhom1-dependent ADAM17 substrate and demonstrates that iRhom1 is widely expressed in murine brain.

Probing intracellular potassium dynamics in neurons with the genetically encoded sensor lc-LysM GEPII 1.0 in vitro and in vivo.

Neuronal activity is accompanied by a net outflow of potassium ions (K+) from the intra- to the extracellular space. While extracellular [K+] changes during neuronal activity are well characterized, intracellular dynamics have been less well investigated due to lack of respective probes. In the current study we characterized the FRET-based K+ biosensor lc-LysM GEPII 1.0 for its capacity to measure intracellular [K+] changes in primary cultured neurons and in mouse cortical neurons in vivo. We found that lc-LysM GEPII 1.0 can resolve neuronal [K+] decreases in vitro during seizure-like and intense optogenetically evoked activity. [K+] changes during single action potentials could not be recorded. We confirmed these findings in vivo by expressing lc-LysM GEPII 1.0 in mouse cortical neurons and performing 2-photon fluorescence lifetime imaging. We observed an increase in the fluorescence lifetime of lc-LysM GEPII 1.0 during periinfarct depolarizations, which indicates a decrease in intracellular neuronal [K+]. Our findings suggest that lc-LysM GEPII 1.0 can be used to measure large changes in [K+] in neurons in vitro and in vivo but requires optimization to resolve smaller changes as observed during single action potentials.

Inadequate food and water intake determine mortality following stroke in mice.

Experimental stroke models producing clinically relevant functional deficits are often associated with high mortality. Because the mechanisms that underlie post-stroke mortality are largely unknown, results obtained using these models are often difficult to interpret, thereby limiting their translational potential. Given that specific forms of post-stroke care reduce mortality in patients, we hypothesized that inadequate food and water intake may underlie mortality following experimental stroke. C57BL/6 mice were subjected to 1 h of intraluminal filament middle cerebral artery occlusion. Nutritional support beginning on the second day after filament middle cerebral artery occlusion reduced the 14-day mortality rate from 59% to 15%. The surviving mice in the post-stroke support group had the same infarct size as non-surviving control mice, suggesting that post-stroke care was not neuroprotective and that inadequate food and/or water intake are the main reasons for filament middle cerebral artery occlusion-induced mortality. This notion was supported by the presence of significant hypoglycemia, ketonemia, and dehydration in control mice. Taken together, these data suggest that post-filament middle cerebral artery occlusion mortality in mice is not primarily caused by ischemic brain damage, but secondarily by inadequate food and/or water intake. Thus, providing nutritional support following filament middle cerebral artery occlusion greatly minimizes mortality bias and allows the study of long-term morphological and functional sequelae of stroke in mice.