Abstract
The hippocampus is a very heterogeneous brain structure with different mechanical properties reflecting its functional variety. In particular, adult neurogenesis in rodent hippocampus has ...been associated with specific viscoelastic properties in vivo and ex vivo. Here, we study the microscopic mechanical properties of hippocampal subregions using ex vivo atomic force microscopy (AFM) in correlation with the expression of GFP in presence of the nestin promoter, providing a marker of neurogenic activity. We further use magnetic resonance elastography (MRE) to investigate whether in vivo mechanical properties reveal similar spatial patterns, however, on a much coarser scale. AFM showed that tissue stiffness increases with increasing distance from the subgranular zone (p = 0.0069), and that stiffness is 39% lower in GFP than non-GFP regions (p = 0.0004). Consistently, MRE showed that dentate gyrus is, on average, softer than Ammon´s horn (shear wave speed = 3.2 ± 0.2 m/s versus 4.4 ± 0.3 m/s, p = 0.01) with another 3.4% decrease towards the subgranular zone (p = 0.0001). The marked reduction in stiffness measured by AFM in areas of high neurogenic activity is consistent with softer MRE values, indicating the sensitivity of macroscopic mechanical properties in vivo to micromechanical structures as formed by the neurogenic niche of the hippocampus.
Owing to the small size of mitochondria and the complexity of their motility patterns, mitochondrial tracking is technically challenging. Mitochondria are often tracked manually; however, this is ...time‐consuming and prone to measurement error. Here, we examined the suitability of four commercial and open‐source software alternatives for automated mitochondrial tracking in neurons compared with manual measurements. We show that all the automated tracking tools dramatically underestimated track length, mitochondrial displacement and movement duration, with reductions ranging from 45 to 77% of the values obtained manually. In contrast, mitochondrial velocity was generally overestimated. Only the number of motile mitochondria and their directionality were similar between strategies. Despite these discrepancies, we show that automated tools successfully detected transport alterations after applying an oxidant agent. Thus, automated methods appear to be suitable for assessing relative transport differences between experimental groups, but not for absolute quantification of mitochondrial dynamics. Although useful for objective and time‐efficient measurements of mitochondrial movements, results provided by automated methods should be interpreted with caution.
We examined the suitability of automated tools for tracking mitochondria within neurons, compared with manual tracking (lines indicating mitochondrial movements are marked on each micrograph). The results from automated programs differed significantly from those obtained manually, and both correlation and agreement between methods were poor. However, automated tools could detect relative transport alterations induced by experimental interventions. Thus, automated mitochondrial tracking may be suitable for assessing relative differences in transport, but not for providing absolute quantification of mitochondrial dynamics.
Fractalkine receptor (CX3CR1)‐deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise ...implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild‐type and CX3CR1‐deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1‐deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1‐deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b+ NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4+ T cells and kill these autoreactive cells more efficiently than the immature CD11b− fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.
Fractalkine receptor‐mediated recruitment of mature CD11b+ NK cells into the CNS contributes to control neuroinflammation in the EAE. Modulation of autoreactive CD4+ T‐cell response by mature NK cells inside the CNS may represent a mechanism of EAE regulation.
Abstract Mechanically, the brain is characterized by both solid and fluid properties. The resulting unique material behavior fosters proliferation, differentiation, and repair of cellular and ...vascular networks, and optimally protects them from damaging shear forces. Magnetic resonance elastography (MRE) is a noninvasive imaging technique that maps the mechanical properties of the brain in vivo. MRE studies have shown that abnormal processes such as neuronal degeneration, demyelination, inflammation, and vascular leakage lead to tissue softening. In contrast, neuronal proliferation, cellular network formation, and higher vascular pressure result in brain stiffening. In addition, brain viscosity has been reported to change with normal blood perfusion variability and brain maturation as well as disease conditions such as tumor invasion. In this article, the contributions of the neuronal, glial, extracellular, and vascular networks are discussed to the coarse‐grained parameters determined by MRE. This reductionist multi‐network model of brain mechanics helps to explain many MRE observations in terms of microanatomical changes and suggests that cerebral viscoelasticity is a suitable imaging marker for brain disease.
In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary ...progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from
mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.
Summary We previously demonstrated a correlation between the frequency of CX3CR1‐expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1high NK ...cells were more cytotoxic than their CX3CR1neg/low counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1high, CX3CR1neg/low NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin‐2 and promoted a strong up‐regulation of the key co‐stimulatory molecule CD40 on monocytes. Co‐expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1neg CD56bright, CX3CR1neg CD56dim and CX3CR1high CD56dim fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56dim discriminated between an intermediary CX3CR1neg CD56dim and fully mature CX3CR1high CD56dim NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.
In demyelinating diseases including multiple sclerosis (MS), neural stem cells (NSCs) can replace damaged oligodendrocytes if the local microenvironment supports the required differentiation process. ...Although chitinase-like proteins (CLPs) form part of this microenvironment, their function in this differentiation process is unknown. Here, we demonstrate that murine Chitinase 3-like-3 (Chi3l3/Ym1), human Chi3L1 and Chit1 induce oligodendrogenesis. In mice, Chi3l3 is highly expressed in the subventricular zone, a stem cell niche of the adult brain, and in inflammatory brain lesions during experimental autoimmune encephalomyelitis (EAE). We find that silencing Chi3l3 increases severity of EAE. We present evidence that in NSCs Chi3l3 activates the epidermal growth factor receptor (EGFR), thereby inducing Pyk2-and Erk1/2- dependent expression of a pro-oligodendrogenic transcription factor signature. Our results implicate CLP-EGFR-Pyk2-MEK-ERK as a key intrinsic pathway controlling oligodendrogenesis.
During neuroinflammation, monocytes that infiltrate the central nervous system (CNS) may contribute to regenerative processes depending on their activation status. However, the extent and mechanisms ...of monocyte-induced CNS repair in patients with neuroinflammatory diseases remain largely unknown, partly due to the lack of a fully human assay platform that can recapitulate monocyte-neural stem cell interactions within the CNS microenvironment. We therefore developed a human model system to assess the impact of monocytic factors on neural stem cells, establishing a high-content compatible assay for screening monocyte-induced neural stem cell proliferation and differentiation. The model combined monocytes isolated from healthy donors and human embryonic stem cell derived neural stem cells and integrated both cell-intrinsic and -extrinsic properties. We identified CNS-mimicking culture media options that induced a monocytic phenotype resembling CNS infiltrating monocytes, while allowing adequate monocyte survival. Monocyte-induced proliferation, gliogenic fate and neurogenic fate of neural stem cells were affected by the conditions of monocytic priming and basal neural stem cell culture as extrinsic factors as well as the neural stem cell passage number as an intrinsic neural stem cell property. We developed a high-content compatible human in vitro assay for the integrated analysis of monocyte-derived factors on CNS repair.
Oxidative stress and mitochondrial dysfunction appear to contribute to axon degeneration in numerous neurological disorders. However, how these two processes interact to cause axonal damage—and how ...this damage is initiated—remains unclear. In this study we used transected motor axons from murine peripheral roots to investigate whether oxidative stress alters mitochondrial dynamics in myelinated axons. We show that the nodes of Ranvier are the initial sites of mitochondrial damage induced by oxidative stress. There, mitochondria became depolarized, followed by alterations of the external morphology and disruption of the cristae, along with reduced mitochondrial transport. These mitochondrial changes expanded from the nodes of Ranvier bidirectionally towards both internodes and eventually affected the entire mitochondrial population in the axon. Supplementing axonal bioenergetics by applying nicotinamide adenine dinucleotide and methyl pyruvate, rendered the mitochondria at the nodes of Ranvier resistant to these oxidative stress-induced changes. Importantly, this inhibition of mitochondrial damage protected the axons from degeneration.
In conclusion, we present a novel ex vivo approach for monitoring mitochondrial dynamics within axons, which proved suitable for detecting mitochondrial changes upon exogenous application of oxidative stress. Our results indicate that the nodes of Ranvier are the site of initial mitochondrial damage in peripheral axons, and suggest that dysregulation of axonal bioenergetics plays a critical role in oxidative stress-triggered mitochondrial alterations and subsequent axonal injury. These novel insights into the mechanisms underlying axon degeneration may have implications for neurological disorders with a degenerative component.
•We imaged axonal mitochondria in peripheral root explants exposed to oxidative stress.•Oxidative stress altered mitochondrial dynamics and function in myelinated axons.•Oxidative damage to mitochondria was initiated at the nodes of Ranvier.•NAD+ and pyruvate prevented oxidative damage to mitochondria.•NAD+ and pyruvate protected axons from oxidative stress-induced degeneration.
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