Apolipoprotein E aggregation in microglia initiates Alzheimer's disease pathology by seeding ß-amyloidosis.

The seeded growth of pathogenic protein aggregates underlies the pathogenesis of Alzheimer's disease (AD), but how this pathological cascade is initiated is not fully understood. Sporadic AD is linked genetically to apolipoprotein E (APOE) and other genes expressed in microglia related to immune, lipid, and endocytic functions. We generated a transgenic knockin mouse expressing HaloTag-tagged APOE and optimized experimental protocols for the biochemical purification of APOE, which enabled us to identify fibrillary aggregates of APOE in mice with amyloid-ß (Aß) amyloidosis and in human AD brain autopsies. These APOE aggregates that stained positive for ß sheet-binding dyes triggered Aß amyloidosis within the endo-lysosomal system of microglia, in a process influenced by microglial lipid metabolism and the JAK/STAT signaling pathway. Taking these observations together, we propose a model for the onset of Aß amyloidosis in AD, suggesting that the endocytic uptake and aggregation of APOE by microglia can initiate Aß plaque formation.

Myelin dysfunction drives amyloid-ß deposition in models of Alzheimer's disease.

The incidence of Alzheimer's disease (AD), the leading cause of dementia, increases rapidly with age, but why age constitutes the main risk factor is still poorly understood. Brain ageing affects oligodendrocytes and the structural integrity of myelin sheaths1, the latter of which is associated with secondary neuroinflammation2,3. As oligodendrocytes support axonal energy metabolism and neuronal health4-7, we hypothesized that loss of myelin integrity could be an upstream risk factor for neuronal amyloid-ß (Aß) deposition, the central neuropathological hallmark of AD. Here we identify genetic pathways of myelin dysfunction and demyelinating injuries as potent drivers of amyloid deposition in mouse models of AD. Mechanistically, myelin dysfunction causes the accumulation of the Aß-producing machinery within axonal swellings and increases the cleavage of cortical amyloid precursor protein. Suprisingly, AD mice with dysfunctional myelin lack plaque-corralling microglia despite an overall increase in their numbers. Bulk and single-cell transcriptomics of AD mouse models with myelin defects show that there is a concomitant induction of highly similar but distinct disease-associated microglia signatures specific to myelin damage and amyloid plaques, respectively. Despite successful induction, amyloid disease-associated microglia (DAM) that usually clear amyloid plaques are apparently distracted to nearby myelin damage. Our data suggest a working model whereby age-dependent structural defects of myelin promote Aß plaque formation directly and indirectly and are therefore an upstream AD risk factor. Improving oligodendrocyte health and myelin integrity could be a promising target to delay development and slow progression of AD.

Myelinated peripheral axons are more vulnerable to mechanical trauma in a model of enlarged axonal diameters.

The velocity of axonal impulse propagation is facilitated by myelination and axonal diameters. Both parameters are frequently impaired in peripheral nerve disorders, but it is not known if the diameters of myelinated axons affect the liability to injury or the efficiency of functional recovery. Mice lacking the adaxonal myelin protein chemokine-like factor-like MARVEL-transmembrane domain-containing family member-6 (CMTM6) specifically from Schwann cells (SCs) display appropriate myelination but increased diameters of peripheral axons. Here we subjected Cmtm6-cKo mice as a model of enlarged axonal diameters to a mild sciatic nerve compression injury that causes temporarily reduced axonal diameters but otherwise comparatively moderate pathology of the axon/myelin-unit. Notably, both of these pathological features were worsened in Cmtm6-cKo compared to genotype-control mice early post-injury. The increase of axonal diameters caused by CMTM6-deficiency thus does not override their injury-dependent decrease. Accordingly, we did not detect signs of improved regeneration or functional recovery after nerve compression in Cmtm6-cKo mice; depleting CMTM6 in SCs is thus not a promising strategy toward enhanced recovery after nerve injury. Conversely, the exacerbated axonal damage in Cmtm6-cKo nerves early post-injury coincided with both enhanced immune response including foamy macrophages and SCs and transiently reduced grip strength. Our observations support the concept that larger peripheral axons are particularly susceptible toward mechanical trauma.

NMDAR1 autoantibodies amplify behavioral phenotypes of genetic white matter inflammation: a mild encephalitis model with neuropsychiatric relevance.

Encephalitis has an estimated prevalence of ≤0.01%. Even with extensive diagnostic work-up, an infectious etiology is identified or suspected in <50% of cases, suggesting a role for etiologically unclear, noninfectious processes. Mild encephalitis runs frequently unnoticed, despite slight neuroinflammation detectable postmortem in many neuropsychiatric illnesses. A widely unexplored field in humans, though clearly documented in rodents, is genetic brain inflammation, particularly that associated with myelin abnormalities, inducing primary white matter encephalitis. We hypothesized that "autoimmune encephalitides" may result from any brain inflammation concurring with the presence of brain antigen-directed autoantibodies, e.g., against N-methyl-D-aspartate-receptor NR1 (NMDAR1-AB), which are not causal of, but may considerably shape the encephalitis phenotype. We therefore immunized young female Cnp-/- mice lacking the structural myelin protein 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) with a "cocktail" of NMDAR1 peptides. Cnp-/- mice exhibit early low-grade inflammation of white matter tracts and blood-brain barrier disruption. Our novel mental-time-travel test disclosed that Cnp-/- mice are compromised in what-where-when orientation, but this episodic memory readout was not further deteriorated by NMDAR1-AB. In contrast, comparing wild-type and Cnp-/- mice without/with NMDAR1-AB regarding hippocampal learning/memory and motor balance/coordination revealed distinct stair patterns of behavioral pathology. To elucidate a potential contribution of oligodendroglial NMDAR downregulation to NMDAR1-AB effects, we generated conditional NR1 knockout mice. These mice displayed normal Morris water maze and mental-time-travel, but beam balance performance was similar to immunized Cnp-/-. Immunohistochemistry confirmed neuroinflammation/neurodegeneration in Cnp-/- mice, yet without add-on effect of NMDAR1-AB. To conclude, genetic brain inflammation may explain an encephalitic component underlying autoimmune conditions.

Autoantibodies against NMDA receptor 1 modify rather than cause encephalitis.

The etiology and pathogenesis of "anti-N-methyl-D-aspartate-receptor (NMDAR) encephalitis" and the role of autoantibodies (AB) in this condition are still obscure. While NMDAR1-AB exert NMDAR-antagonistic properties by receptor internalization, no firm evidence exists to date that NMDAR1-AB by themselves induce brain inflammation/encephalitis. NMDAR1-AB of all immunoglobulin classes are highly frequent across mammals with multiple possible inducers and boosters. We hypothesized that "NMDAR encephalitis" results from any primary brain inflammation coinciding with the presence of NMDAR1-AB, which may shape the encephalitis phenotype. Thus, we tested whether following immunization with a "cocktail" of 4 NMDAR1 peptides, induction of a spatially and temporally defined sterile encephalitis by diphtheria toxin-mediated ablation of pyramidal neurons ("DTA" mice) would modify/aggravate the ensuing phenotype. In addition, we tried to replicate a recent report claiming that immunizing just against the NMDAR1-N368/G369 region induced brain inflammation. Mice after DTA induction revealed a syndrome comprising hyperactivity, hippocampal learning/memory deficits, prefrontal cortical network dysfunction, lasting blood brain-barrier impairment, brain inflammation, mainly in hippocampal and cortical regions with pyramidal neuronal death, microgliosis, astrogliosis, modest immune cell infiltration, regional atrophy, and relative increases in parvalbumin-positive interneurons. The presence of NMDAR1-AB enhanced the hyperactivity (psychosis-like) phenotype, whereas all other readouts were identical to control-immunized DTA mice. Non-DTA mice with or without NMDAR1-AB were free of any encephalitic signs. Replication of the reported NMDAR1-N368/G369-immunizing protocol in two large independent cohorts of wild-type mice completely failed. To conclude, while NMDAR1-AB can contribute to the behavioral phenotype of an underlying encephalitis, induction of an encephalitis by NMDAR1-AB themselves remains to be proven.

Ketogenic diet ameliorates axonal defects and promotes myelination in Pelizaeus-Merzbacher disease.

Pelizaeus-Merzbacher disease (PMD) is an untreatable and fatal leukodystrophy. In a model of PMD with perturbed blood-brain barrier integrity, cholesterol supplementation promotes myelin membrane growth. Here, we show that in contrast to the mouse model, dietary cholesterol in two PMD patients did not lead to a major advancement of hypomyelination, potentially because the intact blood-brain barrier precludes its entry into the CNS. We therefore turned to a PMD mouse model with preserved blood-brain barrier integrity and show that a high-fat/low-carbohydrate ketogenic diet restored oligodendrocyte integrity and increased CNS myelination. This dietary intervention also ameliorated axonal degeneration and normalized motor functions. Moreover, in a paradigm of adult remyelination, ketogenic diet facilitated repair and attenuated axon damage. We suggest that a therapy with lipids such as ketone bodies, that readily enter the brain, can circumvent the requirement of a disrupted blood-brain barrier in the treatment of myelin disease.

Correction to: Ketogenic diet ameliorates axonal defects and promotes myelination in Pelizaeus-Merzbacher disease.

The original article was published.

Oligodendrocytes produce amyloid-ß and contribute to plaque formation alongside neurons in Alzheimer's disease model mice.

Amyloid-ß (Aß) is thought to be neuronally derived in Alzheimer's disease (AD). However, transcripts of amyloid precursor protein (APP) and amyloidogenic enzymes are equally abundant in oligodendrocytes (OLs). By cell-type-specific deletion of Bace1 in a humanized knock-in AD model, APPNLGF, we demonstrate that OLs and neurons contribute to Aß plaque burden. For rapid plaque seeding, excitatory projection neurons must provide a threshold level of Aß. Ultimately, our findings are relevant for AD prevention and therapeutic strategies.

Spatial Transcriptomics-correlated Electron Microscopy maps transcriptional and ultrastructural responses to brain injury.

Understanding the complexity of cellular function within a tissue necessitates the combination of multiple phenotypic readouts. Here, we developed a method that links spatially-resolved gene expression of single cells with their ultrastructural morphology by integrating multiplexed error-robust fluorescence in situ hybridization (MERFISH) and large area volume electron microscopy (EM) on adjacent tissue sections. Using this method, we characterized in situ ultrastructural and transcriptional responses of glial cells and infiltrating T-cells after demyelinating brain injury in male mice. We identified a population of lipid-loaded "foamy" microglia located in the center of remyelinating lesion, as well as rare interferon-responsive microglia, oligodendrocytes, and astrocytes that co-localized with T-cells. We validated our findings using immunocytochemistry and lipid staining-coupled single-cell RNA sequencing. Finally, by integrating these datasets, we detected correlations between full-transcriptome gene expression and ultrastructural features of microglia. Our results offer an integrative view of the spatial, ultrastructural, and transcriptional reorganization of single cells after demyelinating brain injury.

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease.

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack.

Local cholesterol metabolism orchestrates remyelination.

Cholesterol is an essential component of all cell membranes and particularly enriched in myelin membranes. Myelin membranes are a major target of immune attacks in the chronic neurological disorder multiple sclerosis (MS). During demyelinating insults, cholesterol is released from damaged myelin, increasing local levels of this unique lipid and impeding tissue regeneration. Here, we summarize the current knowledge of cholesterol-dependent processes during demyelination and remyelination, emphasizing cell type-specific responses. We discuss cellular lipid/cholesterol metabolism during early and late disease phases and highlight the concept of lipid-based pharmacological interventions. We propose that knowledge of the interplay between cell type-specific cholesterol handling, inflammation, and blood-brain barrier (BBB) integrity will unravel disease processes and facilitate development of strategies for therapies to promote remyelination.

Propofol versus midazolam sedation in patients with cardiogenic shock - an observational propensity-matched study.

PURPOSE: Benzodiazepines are recommended as first line sedative agent in ventilated cardiogenic shock patients, although data regarding the optimal sedation strategy are sparse. The aim of this study was to investigate the hemodynamic effects of propofol versus midazolam sedation in our cardiogenic shock registry. MATERIALS AND METHODS: Mechanically ventilated patients suffering from cardiogenic shock were retrospectively enrolled from the cardiogenic shock registry of the university hospital of Munich. 174 patients treated predominantly with propofol were matched by propensity-score to 174 patients treated predominantly with midazolam. RESULTS: Catecholamine doses were similar on admission but significantly lower in the propofol group on days 1-4 of ICU stay. Mortality rate was 38% in the propofol and 52% in the midazolam group after 30 days (p = 0.002). Rate of ≥BARC3 bleeding was significantly lower in the propofol group compared to the midazolam group (p = 0.008). Sedation with midazolam was significantly associated with ICU mortality. CONCLUSION: In this observational cohort study, sedation with propofol in comparison to midazolam was linked to a reduced dose of catecholamines, decreased mortality and bleeding rates for patients with cardiogenic shock. Based on this study and in contrast to current recommendations, propofol should be given consideration for sedation in cardiogenic shock patients.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation. Under steady-state conditions, CNS cell types prefer distinct modes of energy metabolism. Unexpectedly, the comparison with KD revealed distinct cell type-specific strategies to manage the altered availability of energy metabolites. Astrocytes and neurons but not oligodendrocytes demonstrated metabolic plasticity. Moreover, inflammatory demyelinating disease changed the neuronal metabolic signature in a similar direction as KD. Together, these findings highlight the importance of the metabolic cross-talk between CNS cells and between the periphery and the brain to manage altered nutrition and neurological disease.

Inducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis.

Up to one person in a population of 10,000 is diagnosed once in lifetime with an encephalitis, in 50-70% of unknown origin. Recognized causes amount to 20-50% viral infections. Approximately one third of affected subjects develops moderate and severe subsequent damage. Several neurotropic viruses can directly infect pyramidal neurons and induce neuronal death in cortex and hippocampus. The resulting encephalitic syndromes are frequently associated with cognitive deterioration and dementia, but involve numerous parallel and downstream cellular and molecular events that make the interpretation of direct consequences of sudden pyramidal neuronal loss difficult. This, however, would be pivotal for understanding how neuroinflammatory processes initiate the development of neurodegeneration, and thus for targeted prophylactic and therapeutic interventions. Here we utilized adult male NexCreERT2xRosa26-eGFP-DTA (= 'DTA') mice for the induction of a sterile encephalitis by diphtheria toxin-mediated ablation of cortical and hippocampal pyramidal neurons which also recruits immune cells into gray matter. We report multifaceted aftereffects of this defined process, including the expected pathology of classical hippocampal behaviors, evaluated in Morris water maze, but also of (pre)frontal circuit function, assessed by prepulse inhibition. Importantly, we modelled in encephalitis mice novel translationally relevant sequelae, namely altered social interaction/cognition, accompanied by compromised thermoreaction to social stimuli as convenient readout of parallel autonomic nervous system (dys)function. High resolution magnetic resonance imaging disclosed distinct abnormalities in brain dimensions, including cortical and hippocampal layering, as well as of cerebral blood flow and volume. Fluorescent tracer injection, immunohistochemistry and brain flow cytometry revealed persistent blood-brain-barrier perturbance and chronic brain inflammation. Surprisingly, blood flow cytometry showed no abnormalities in circulating major immune cell subsets and plasma high-mobility group box 1 (HMGB1) as proinflammatory marker remained unchanged. The present experimental work, analyzing multidimensional outcomes of direct pyramidal neuronal loss, will open new avenues for urgently needed encephalitis research.

Neuronal cholesterol synthesis is essential for repair of chronically demyelinated lesions in mice.

Astrocyte-derived cholesterol supports brain cells under physiological conditions. However, in demyelinating lesions, astrocytes downregulate cholesterol synthesis, and the cholesterol that is essential for remyelination has to originate from other cellular sources. Here, we show that repair following acute versus chronic demyelination involves distinct processes. In particular, in chronic myelin disease, when recycling of lipids is often defective, de novo neuronal cholesterol synthesis is critical for regeneration. By gene expression profiling, genetic loss-of-function experiments, and comprehensive phenotyping, we provide evidence that neurons increase cholesterol synthesis in chronic myelin disease models and in patients with multiple sclerosis (MS). In mouse models, neuronal cholesterol facilitates remyelination specifically by triggering oligodendrocyte precursor cell proliferation. Our data contribute to the understanding of disease progression and have implications for therapeutic strategies in patients with MS.

Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport.

BACKGROUND: Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. METHODS: Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. RESULTS: Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. CONCLUSIONS: Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

Microglia facilitate repair of demyelinated lesions via post-squalene sterol synthesis.

The repair of inflamed, demyelinated lesions as in multiple sclerosis (MS) necessitates the clearance of cholesterol-rich myelin debris by microglia/macrophages and the switch from a pro-inflammatory to an anti-inflammatory lesion environment. Subsequently, oligodendrocytes increase cholesterol levels as a prerequisite for synthesizing new myelin membranes. We hypothesized that lesion resolution is regulated by the fate of cholesterol from damaged myelin and oligodendroglial sterol synthesis. By integrating gene expression profiling, genetics and comprehensive phenotyping, we found that, paradoxically, sterol synthesis in myelin-phagocytosing microglia/macrophages determines the repair of acutely demyelinated lesions. Rather than producing cholesterol, microglia/macrophages synthesized desmosterol, the immediate cholesterol precursor. Desmosterol activated liver X receptor (LXR) signaling to resolve inflammation, creating a permissive environment for oligodendrocyte differentiation. Moreover, LXR target gene products facilitated the efflux of lipid and cholesterol from lipid-laden microglia/macrophages to support remyelination by oligodendrocytes. Consequently, pharmacological stimulation of sterol synthesis boosted the repair of demyelinated lesions, suggesting novel therapeutic strategies for myelin repair in MS.

Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.

Dietary cholesterol promotes repair of demyelinated lesions in the adult brain.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.

Inducible targeting of CNS astrocytes in Aldh1l1-CreERT2 BAC transgenic mice.

Background: Studying astrocytes in higher brain functions has been hampered by the lack of genetic tools for the efficient expression of inducible Cre recombinase throughout the CNS, including the neocortex. Methods: Therefore, we generated BAC transgenic mice, in which CreERT2 is expressed under control of the Aldh1l1 regulatory region. Results: When crossbred to Cre reporter mice, adult Aldh1l1-CreERT2 mice show efficient gene targeting in astrocytes. No such Cre-mediated recombination was detectable in CNS neurons, oligodendrocytes, and microglia. As expected, Aldh1l1-CreERT2 expression was evident in several peripheral organs, including liver and kidney. Conclusions: Taken together, Aldh1l1-CreERT2 mice are a useful tool for studying astrocytes in neurovascular coupling, brain metabolism, synaptic plasticity and other aspects of neuron-glia interactions.