Environmental enrichment during the chronic phase after experimental stroke promotes functional recovery without synergistic effects of EphA4 targeted therapy.

Worldwide, stroke is the main cause of long-term adult disability. After the initial insult, most patients undergo a subacute period with intense plasticity and rapid functional improvements. This period is followed by a chronic phase where recovery reaches a plateau that is only partially modifiable by rehabilitation. After experimental stroke, various subacute rehabilitation paradigms improve recovery. However, in order to reach the best possible outcome, a combination of plasticity-promoting strategies and rehabilitation might be necessary. EphA4 is a negative axonal guidance regulator during development. After experimental stroke, reduced EphA4 levels improve functional outcome with similar beneficial effects upon the inhibition of EphA4 downstream targets. In this study, we assessed the effectiveness of a basic enriched environment in the chronic phase after photothrombotic stroke in mice as well as the therapeutic potential of EphA4 targeted therapy followed by rehabilitation. Our findings show that environmental enrichment in the chronic phase improves functional outcome up to 2 months post-stroke. Although EphA4 levels increase after experimental stroke, subacute EphA4 inhibition followed by environmental enrichment does not further increase recovery. In conclusion, we show that environmental enrichment during the chronic phase of stroke improves functional outcome in mice with no synergistic effects of the used EphA4 targeted therapy.

Controversies and prospects about microglia in maternal immune activation models for neurodevelopmental disorders.

Activation of the maternal immune system during pregnancy is a well-established risk factor for neuropsychiatric disease in the offspring, yet, the underlying mechanisms leading to altered brain function remain largely undefined. Microglia, the resident immune cells of the brain, are key to adequate development of the central nervous system (CNS), and are prime candidates to mediate maternal immune activation (MIA)-induced brain abnormalities. As such, the effects of MIA on the immunological phenotype of microglia has been widely investigated. However, contradicting results due to differences in read-out and methodological approaches impede final conclusions on MIA-induced microglial alterations. The aim of this review is to critically discuss the evidence for an activated microglial phenotype upon MIA.

Age-specific function of α5ß1 integrin in microglial migration during early colonization of the developing mouse cortex.

Microglia, the immune cells of the central nervous system, take part in brain development and homeostasis. They derive from primitive myeloid progenitors that originate in the yolk sac and colonize the brain mainly through intensive migration. During development, microglial migration speed declines which suggests that their interaction with the microenvironment changes. However, the matrix-cell interactions allowing dispersion within the parenchyma are unknown. Therefore, we aimed to better characterize the migration behavior and to assess the role of matrix-integrin interactions during microglial migration in the embryonic brain ex vivo. We focused on microglia-fibronectin interactions mediated through the fibronectin receptor α5ß1 integrin because in vitro work indirectly suggested a role for this ligand-receptor pair. Using 2-photon time-lapse microscopy on acute ex vivo embryonic brain slices, we found that migration occurs in a saltatory pattern and is developmentally regulated. Most importantly, there is an age-specific function of the α5ß1 integrin during microglial cortex colonization. At embryonic day (E) 13.5, α5ß1 facilitates migration while from E15.5, it inhibits migration. These results indicate a developmentally regulated function of α5ß1 integrin in microglial migration during colonization of the embryonic brain.

Reducing EphA4 before disease onset does not affect survival in a mouse model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons resulting in severe neurological symptoms. Previous findings of our lab suggested that the axonal guidance tyrosine-kinase receptor EphA4 is an ALS disease-modifying gene. Reduction of EphA4 from developmental stages onwards rescued a motor neuron phenotype in zebrafish, and heterozygous deletion before birth in the SOD1G93A mouse model of ALS resulted in improved survival. Here, we aimed to gain more insights in the cell-specific role of decreasing EphA4 expression in addition to timing and amount of EphA4 reduction. To evaluate the therapeutic potential of lowering EphA4 later in life, we ubiquitously reduced EphA4 levels to 50% in SOD1G93A mice at 60 days of age, which did not modify disease parameters. Even further lowering EphA4 levels ubiquitously or in neurons, did not improve disease onset or survival. These findings suggest that lowering EphA4 as target in ALS may suffer from a complex therapeutic time window. In addition, the complexity of the Eph-ephrin signalling system may also possibly limit the therapeutic potential of such an approach in ALS. We suggest here that a specific EphA4 knockdown in adulthood may have a limited therapeutic potential for ALS.

Lowering EphA4 Does Not Ameliorate Disease in a Mouse Model for Severe Spinal Muscular Atrophy.

EphA4 is a receptor of the Eph-ephrin system, which plays an important role in axon guidance during development. Previously, we identified EphA4 as a genetic modifier of amyotrophic lateral sclerosis (ALS) in both zebrafish and rodent models, via modulation of the intrinsic vulnerability, and re-sprouting capacity of motor neurons. Moreover, loss of EphA4 rescued the motor axon phenotype in a zebrafish model of spinal muscular atrophy (SMA). Similar to ALS, SMA is a neurodegenerative disorder affecting spinal motor neurons resulting in neuromuscular junction (NMJ) denervation, muscle atrophy and paralysis. In this study, we investigated the disease modifying potential of reduced EphA4 protein levels in the SMNΔ7 mouse model for severe SMA. Reduction of EphA4 did not improve motor function, survival, motor neuron survival or NMJ innervation. Our data suggest that either lowering EphA4 has limited therapeutic potential in SMA or that the clinical severity hampers the potential beneficial role of EphA4 reduction in this mouse model for SMA.

EphA4 loss improves social memory performance and alters dendritic spine morphology without changes in amyloid pathology in a mouse model of Alzheimer's disease.

BACKGROUND: EphA4 is a receptor of the ephrin system regulating spine morphology and plasticity in the brain. These processes are pivotal in the pathophysiology of Alzheimer's disease (AD), characterized by synapse dysfunction and loss, and the progressive loss of memory and other cognitive functions. Reduced EphA4 signaling has been shown to rescue beta-amyloid-induced dendritic spine loss and long-term potentiation (LTP) deficits in cultured hippocampal slices and primary hippocampal cultures. In this study, we investigated whether EphA4 ablation might preserve synapse function and ameliorate cognitive performance in the APPPS1 transgenic mouse model of AD. METHODS: A postnatal genetic ablation of EphA4 in the forebrain was established in the APPPS1 mouse model of AD, followed by a battery of cognitive tests at 9 months of age to investigate cognitive function upon EphA4 loss. A Golgi-Cox staining was used to explore alterations in dendritic spine density and morphology in the CA1 region of the hippocampus. RESULTS: Upon EphA4 loss in APPPS1 mice, we observed improved social memory in the preference for social novelty test without affecting other cognitive functions. Dendritic spine analysis revealed altered synapse morphology as characterized by increased dendritic spine length and head width. These modifications were independent of hippocampal plaque load and beta-amyloid peptide levels since these were similar in mice with normal versus reduced levels of EphA4. CONCLUSION: Loss of EphA4 improved social memory in a mouse model of Alzheimer's disease in association with alterations in spine morphology.

Reduction of ephrin-A5 aggravates disease progression in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the brainstem, spinal cord and motor cortex. ALS is characterized by genetic and clinical heterogeneity, suggesting the existence of genetic factors that modify the phenotypic expression of the disease. We previously identified the axonal guidance EphA4 receptor, member of the Eph-ephrin system, as an ALS disease-modifying factor. EphA4 genetic inhibition rescued the motor neuron phenotype in zebrafish and a rodent model of ALS. Preventing ligands from binding to the EphA4 receptor also successfully improved disease, suggesting a role for EphA4 ligands in ALS. One particular ligand, ephrin-A5, is upregulated in reactive astrocytes after acute neuronal injury and inhibits axonal regeneration. Moreover, it plays a role during development in the correct pathfinding of motor axons towards their target limb muscles. We hypothesized that a constitutive reduction of ephrin-A5 signalling would benefit disease progression in a rodent model for ALS. We discovered that in the spinal cord of control and symptomatic ALS mice ephrin-A5 was predominantly expressed in neurons. Surprisingly, reduction of ephrin-A5 levels in SOD1G93A mice accelerated disease progression and reduced survival without affecting disease onset, motor neuron numbers or innervated neuromuscular junctions in symptomatic mice. These findings suggest ephrin-A5 as a modifier of disease progression that might play a role in the later stages of the disease. Similarly, we identified a more aggressive disease progression in patients with lower ephrin-A5 protein levels in the cerebrospinal fluid without modifying disease onset. In summary, we identified reduced expression of ephrin-A5 to accelerate disease progression in a mouse model of ALS as well as in humans. Combined with our previous findings on the role of EphA4 in ALS our current data suggests different contribution for various members of the Eph-ephrin system in the pathophysiology of a motor neuron disease.

Magnetofection is superior to other chemical transfection methods in a microglial cell line.

BACKGROUND: Microglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection. NEW METHOD: Magnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines. RESULTS: Magnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24h after transfection. COMPARISON WITH EXISTING METHODS: Here we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipication (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%). CONCLUSION: Transfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines.

Maternal immune activation evoked by polyinosinic:polycytidylic acid does not evoke microglial cell activation in the embryo.

Several studies have indicated that inflammation during pregnancy increases the risk for the development of neuropsychiatric disorders in the offspring. Morphological brain abnormalities combined with deviations in the inflammatory status of the brain can be observed in patients of both autism and schizophrenia. It was shown that acute infection can induce changes in maternal cytokine levels which in turn are suggested to affect fetal brain development and increase the risk on the development of neuropsychiatric disorders in the offspring. Animal models of maternal immune activation reproduce the etiology of neurodevelopmental disorders such as schizophrenia and autism. In this study the poly (I:C) model was used to mimic viral immune activation in pregnant mice in order to assess the activation status of fetal microglia in these developmental disorders. Because microglia are the resident immune cells of the brain they were expected to be activated due to the inflammatory stimulus. Microglial cell density and activation level in the fetal cortex and hippocampus were determined. Despite the presence of a systemic inflammation in the pregnant mice, there was no significant difference in fetal microglial cell density or immunohistochemically determined activation level between the control and inflammation group. These data indicate that activation of the fetal microglial cells is not likely to be responsible for the inflammation induced deficits in the offspring in this model.