Payam Dibaj

Nanoscopy in a living mouse brain.

Super high-resolution microscopy resolves neuron dynamics in the cerebral cortex of a living mouse. We demonstrated superresolution optical microscopy in a living higher animal. Stimulated emission depletion (STED) fluorescence nanoscopy reveals neurons in the cerebral cortex of a mouse with <70-nanometer resolution. Dendritic spines and their subtle changes can be observed at their relevant scales over extended periods of time.

NO mediates microglial response to acute spinal cord injury under ATP control in vivo

To understand the pathomechanisms of spinal cord injuries will be a prerequisite to develop efficient therapies. By investigating acute lesions of spinal cord white matter in anesthetized mice with fluorescently labeled microglia and axons using in vivo two‐photon laser‐scanning microscopy (2P‐LSM), we identified the messenger nitric oxide (NO) as a modulator of injury‐activated microglia. Local tissue damages evoked by high‐power laser pulses provoked an immediate attraction of microglial processes. Spinal superfusion with NO synthase and guanylate cyclase inhibitors blocked these extensions. Furthermore, local injection of the NO‐donor spermine NONOate (SPNO) or the NO‐dependent second messenger cGMP induced efficient migration of microglial cells toward the injection site. High‐tissue levels of NO, achieved by uniform superfusion with SPNO and mimicking extended tissue damage, resulted in a fast conversion of the microglial shape from ramified to ameboid indicating cellular activation. When the spinal white matter was preconditioned by increased, ambient ATP (known as a microglial chemoattractant) levels, the attraction of microglial processes to local NO release was augmented, whereas it was abolished at low levels of tissue ATP. Because both signaling molecules, NO and ATP, mediate acute microglial reactions, coordinated pharmacological targeting of NO and purinergic pathways will be an effective mean to influence the innate immune processes after spinal cord injury.

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.

In Vivo Imaging Reveals Distinct Inflammatory Activity of CNS Microglia versus PNS Macrophages in a Mouse Model for ALS

Mutations in the enzyme superoxide dismutase-1 (SOD1) cause hereditary variants of the fatal motor neuronal disease Amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous: neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring non-neuronal cells. In vivo imaging by two-photon laser-scanning microscopy was used to compare the role of microglia/macrophage-related neuroinflammation in the CNS and PNS using ALS-linked transgenic SOD1G93A mice. These mice contained labeled projection neurons and labeled microglia/macrophages. In the affected lateral spinal cord (in contrast to non-affected dorsal columns), different phases of microglia-mediated inflammation were observed: highly reactive microglial cells in preclinical stages (in 60-day-old mice the reaction to axonal transection was ∼180% of control) and morphologically transformed microglia that have lost their function of tissue surveillance and injury-directed response in clinical stages (reaction to axonal transection was lower than 50% of control). Furthermore, unlike CNS microglia, macrophages of the PNS lack any substantial morphological reaction while preclinical degeneration of peripheral motor axons and neuromuscular junctions was observed. We present in vivo evidence for a different inflammatory activity of microglia and macrophages: an aberrant neuroinflammatory response of microglia in the CNS and an apparently mainly neurodegenerative process in the PNS.

Kir4.1 channels regulate swelling of astroglial processes in experimental spinal cord edema

In glial cells, inwardly rectifying K+ channels (Kir) control extracellular [K+]o homeostasis by uptake of K+ from the extracellular space and release of K+ into the microvasculature. Kir channels were also recently implicated in K+‐associated water influx and cell swelling. We studied the time‐dependent expression and functional implication of the glial Kir4.1 channel for astroglial swelling in a spinal cord edema model. In this CNS region, Kir4.1 is expressed on astrocytes from the second postnatal week on and co‐localizes with aquaporin 4 (AQP4). Swelling of individual astrocytes in response to osmotic stress and to pharmacological Kir blockade were analyzed by time‐lapse‐two‐photon laser‐scanning microscopy in situ. Application of 30% hypotonic solution induced astroglial soma swelling whereas no swelling was observed on astroglial processes or endfeet. Co‐application of hypotonic solution and Ba2+, a Kir channel blocker, induced prominent swelling of astroglial processes. In Kir4.1−/− mice, however, somatic as well as process swelling was observed upon application of 30% hypotonic solutions. No additional effect was provoked upon co‐application with Ba2+. Our experiments show that Kir channels prevent glial process swelling under osmotic stress. The underlying Kir channel subunit that controls glial process swelling is Kir4.1, whereas changes of the glial soma are not substantially related to Kir4.1.

Influence of methylene blue on microglia-induced inflammation and motor neuron degeneration in the SOD1(G93A) model for ALS.

Mutations in SOD1 cause hereditary variants of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Pathophysiology of the disease is non-cell-autonomous, with toxicity deriving also from glia. In particular, microglia contribute to disease progression. Methylene blue (MB) inhibits the effect of nitric oxide, which mediates microglial responses to injury. In vivo 2P-LSM imaging was performed in ALS-linked transgenic SOD1G93A mice to investigate the effect of MB on microglia-mediated inflammation in the spinal cord. Local superfusion of the lateral spinal cord with MB inhibited the microglial reaction directed at a laser-induced axon transection in control and SOD1G93A mice. In vitro, MB at high concentrations inhibited cytokine and chemokine release from microglia of control and advanced clinical SOD1G93A mice. Systemic MB-treatment of SOD1G93A mice at early preclinical stages significantly delayed disease onset and motor dysfunction. However, an increase of MB dose had no additional effect on disease progression; this was unexpected in view of the local anti-inflammatory effects. Furthermore, in vivo imaging of systemically MB-treated mice also showed no alterations of microglia activity in response to local lesions. Thus although systemic MB treatment had no effect on microgliosis, instead, its use revealed an important influence on motor neuron survival as indicated by an increased number of lumbar anterior horn neurons present at the time of disease onset. Thus, potentially beneficial effects of locally applied MB on inflammatory events contributing to disease progression could not be reproduced in SOD1G93A mice via systemic administration, whereas systemic MB application delayed disease onset via neuroprotection.

Long-lasting post-mortem activity of spinal microglia in situ in mice.

As CNS macrophages, microglia show a high spontaneous motility of their processes, continuously surveying their microenvironment. Upon CNS injury, microglia react by immediate cellular polarization and process extension toward the lesion site as well as by subsequent amoeboid lesion‐directed migration and phagocytosis. To determine the ability of microglia to fulfill their role within distinctively lesioned tissue in the absence of life support, we investigated microglial activity and responsiveness to laser‐induced axonal injuries in the spinal dorsal columns in situ after cardiac and respiratory arrest, i.e., post‐mortem, in the progressively degrading nervous tissue. For this purpose, we used time‐lapse two‐photon laser scanning microscopy in double transgenic mice expressing enhanced green fluorescent protein in microglia and enhanced yellow fluorescent protein in projection neurons. Depending on the premortal condition of the animal, microglial activity and responsiveness remain for up to5–10 hr post‐mortem. Thereby, the continuously decreasing glial reaction is independent of oxygen and glucose supply but requires residual ATP, suggesting a parasitic form of energy, such as a transmembrane uptake of ATP released from injured nervous tissue. Even though initially microglia are able to detect axonal injury after disruption of the blood supply, the later aspects of glial reaction, for example amoeboid conversion and migration, are absent post‐ mortem, corresponding to the failure of microglia to prevent secondary damage after injury of nervous tissue.

Septin/anillin filaments scaffold central nervous system myelin to accelerate nerve conduction

Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity. DOI: http://dx.doi.org/10.7554/eLife.17119.001

In Vivo Imaging and Noninvasive Ablation of Pyramidal Neurons in Adult NEX-CreERT2 Mice

To study the function of individual neurons that are embedded in a complex neural network is difficult in mice. Conditional mutagenesis permits the spatiotemporal control of gene expression including the ablation of cells by toxins. To direct expression of a tamoxifen-inducible variant of Cre recombinase (CreERT2) selectively to cortical neurons, we replaced the coding region of the murine Nex1 gene by CreERT2 cDNA via homologous recombination in embryonic stem cells. When injected with tamoxifen, adult NEX-CreERT2 mice induced reporter gene expression exclusively in projection neurons of the neocortex and hippocampus. By titrating the tamoxifen dosage, we achieved recombination in single cells, which allowed multiphoton imaging of neocortical neurons in live mice. When hippocampal projection neurons were genetically ablated by induced expression of diphteria toxin, within 20 days the inflammatory response included the infiltration of CD3+ T cells. This marks a striking difference from similar studies, in which dying oligodendrocytes failed to recruit cells of the adaptive immune system.

In vivo imaging reveals rapid morphological reactions of astrocytes towards focal lesions in an ALS mouse model

Pathophysiology of the motoneuron disease amyotrophic lateral sclerosis (ALS) is non-cell-autonomous. In mouse models of familiar ALS, neurotoxicity is derived not only from mutant motor neurons but also from mutant neighbouring glial cells. In vivo imaging by two-photon laser-scanning microscopy was used to study rapid morphological reactions of astroglial cells towards laser-induced axonal transection in ALS-linked transgenic SOD1(G93A) mice. In the affected lateral spinal cord, mutated astroglial cells extended branches towards injured axons within a time frame of minutes to hours post lesion while in control animals astrocytes lack any rapid morphological alteration within the studied time frame. This suggests that astrocytes partially contribute to the rapid response of non-neuronal cells to acute axonal lesions in ALS mice.