Raya Eilam

The Runx3 transcription factor regulates development and survival of TrkC dorsal root ganglia neurons

The RUNX transcription factors are important regulators of linage‐specific gene expression in major developmental pathways. Recently, we demonstrated that Runx3 is highly expressed in developing cranial and dorsal root ganglia (DRGs). Here we report that within the DRGs, Runx3 is specifically expressed in a subset of neurons, the tyrosine kinase receptor C (TrkC) proprioceptive neurons. We show that Runx3‐deficient mice develop severe limb ataxia due to disruption of monosynaptic connectivity between intra spinal afferents and motoneurons. We demonstrate that the underlying cause of the defect is a loss of DRG proprioceptive neurons, reflected by a decreased number of TrkC‐, parvalbumin‐ and β‐galactosidase‐positive cells. Thus, Runx3 is a neurogenic TrkC neuron‐specific transcription factor. In its absence, TrkC neurons in the DRG do not survive long enough to extend their axons toward target cells, resulting in lack of connectivity and ataxia. The data provide new genetic insights into the neurogenesis of DRGs and may help elucidate the molecular mechanisms underlying somatosensory‐related ataxia in humans.

Glatiramer acetate-specific T cells in the brain express T helper 2/3 cytokines and brain-derived neurotrophic factor in situ

The ability of a remedy to modulate the pathological process in the target organ is crucial for its therapeutic activity. Glatiramer acetate (GA, Copaxone, Copolymer 1), a drug approved for the treatment of multiple sclerosis, induces regulatory T helper 2/3 cells that penetrate the CNS. Here we investigated whether these GA-specific T cells can function as suppressor cells with therapeutic potential in the target organ by in situ expression of T helper 2/3 cytokines and neurotrophic factors. GA-specific cells and their in situ expression were detected on the level of whole-brain tissue by using a two-stage double-labeling system: (i) labeling of the GA-specific T cells, followed by their adoptive transfer, and (ii) detection of the secreted factors in the brain by immunohistological methods. GA-specific T cells in the CNS demonstrated intense expression of the brain-derived neurotrophic factor and of two antiinflammatory cytokines, IL-10 and transforming growth factor β. No expression of the inflammatory cytokine IFN-γ was observed. This pattern of expression was manifested in brains of normal and experimental autoimmune encephalomyelitis-induced mice to which GA-specific cells were adoptively transferred, but not in control mice. Furthermore, infiltration of GA-induced cells to the brain resulted in bystander expression of IL-10 and transforming growth factor β by resident astrocytes and microglia. The ability of infiltrating GA-specific cells to express antiinflammatory cytokines and neurotrophic factor in the organ in which the pathological processes occur correlates directly with the therapeutic activity of GA in experimental autoimmune encephalomyelitis/multiple sclerosis.

miRNA malfunction causes spinal motor neuron disease

Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9–based regulatory mechanisms in adult neurons and neurodegenerative states.

Neurogenesis and Neuroprotection Induced by Peripheral Immunomodulatory Treatment of Experimental Autoimmune Encephalomyelitis

Brain insults such as the autoimmune inflammatory process in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) induce a measure of neurogenesis, but its regenerative therapeutic consequence is limited, because it fails to regenerate functional neurons and compensate the damage. Here, we investigated whether peripheral immunomodulatory treatment for MS/EAE, glatiramer acetate (GA), can enhance neurogenesis and generate neuroprotection in the CNS of EAE-inflicted mice. EAE was induced by myelin oligodendrocyte glycoprotein peptide, either in yellow fluorescent protein (YFP) 2.2 transgenic mice, which selectively express YFP on their neuronal population, or in C57BL/6 mice. The in situ effect of GA was studied in various brain regions; neuroprotection and neurogeneration were evaluated and quantified by measuring the expression of different neuronal antigens and in vivo proliferation markers. The results demonstrated that in EAE-inflicted mice, neuroproliferation was initially elevated after disease appearance but subsequently declined below that of naive mice. In contrast, GA treatment in various stages of the disease led to sustained reduction in the neuronal/axonal damage typical to the neurodegenerative disease course. Moreover, three processes characteristic of neurogenesis, namely cell proliferation, migration, and differentiation, were augmented and extended by GA treatment in EAE mice compared with EAE-untreated mice and naive controls. The newborn neuroprogenitors manifested massive migration through exciting and dormant migration pathways, into injury sites in brain regions, which do not normally undergo neurogenesis, and differentiated to mature neuronal phenotype. This suggests a direct linkage between immunomodulation, neurogenesis, and an in situ therapeutic consequence in the CNS.

Differential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal differentiation

Two receptor tyrosine kinases, ErB-3 and ErbB-4, mediate signaling by Neu differentiation factors (NDFs, also called neuregulins), while ErbB-1 and ErbB-2 serve as co-receptors. We show that the two NDF/neuregulin receptors differ in spatial and temporal expression patterns: The kinase-defective receptor, ErbB-3, is expressed primarily in epithelial layers of various organs, in the peripheral nervous system, and in adult brain, whereas ErbB-4 is restricted to the developing central nervous system and to the embryonic heart. An example of alternating expression of the two receptors is provided by the developing cerebellum: During postnatal cerebellar development, ErbB-4 expression slightly decreases along with a decline in NDF transcription, whereas ErbB-3 expression commences after the peak of neurogenesis. To study functional differences, we established primary brain cultures and found that ErbB-3 was expressed only in oligodendrocytes, whereas ErbB-4 expression was shared by oligodendrocytes, astrocytes and neurons. Blocking the action of endogenous NDF in vitro, by using a soluble form of ErbB-4, accelerated neurite outgrowth in both primary cultures and in neuronal-type cultures of the P19 teratocarcinoma, suggesting an inhibitory effect of NDF on neural differentiation. Apparently, ErbB-3 is associated with proliferation of P19 cells, whereas ErbB-4 correlates with a differentiated phenotype. We conclude that the two NDF receptors play distinct, rather than redundant, developmental and physiological roles.

Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis

The interplay between demyelination and remyelination is critical in the progress of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In the present study, we explored the capacity of glatiramer acetate (GA, Copaxone) to affect the demyelination process and/or lead to remyelination in mice inflicted by chronic EAE, using both scanning electron microscopy and immunohistological methods. Spinal cords of untreated EAE mice revealed substantial demyelination accompanied by tissue destruction and axonal loss. In contrast, in spinal cords of GA-treated mice, in which treatment started concomitantly with disease induction (prevention), no pathology was observed. Moreover, when treatment was initiated after the appearance of clinical symptoms (suppression) or even in the chronic disease phase (delayed suppression) when substantial demyelination was already manifested, it resulted in a significant decrease in the pathological damage. Detection of oligodendrocyte progenitor cells (OPCs) expressing the NG2 or O4 markers via colocalization with the proliferation marker BrdU indicated their elevated levels in spinal cords of GA-treated mice. The mode of action of GA in this system is attributed to increased proliferation, differentiation, and survival of OPCs along the oligodendroglial maturation cascade and their recruitment into injury sites, thus enhancing repair processes in situ.

Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice

BACKGROUND AND PURPOSE Cannabis extracts and several cannabinoids have been shown to exert broad anti‐inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration.

In vivo up‐regulation of brain‐derived neurotrophic factor in specific brain areas by chronic exposure to Δ9‐tetrahydrocannabinol

Cannabinoids are widely abused drugs. Here we show that chronic administration of Δ9‐tetrahydrocannabinol (Δ9‐THC), the active psychotropic agent in marijuana and hashish, at 1.5 mg per kg per day intraperitoneally for 7 days, increases the expression, at both mRNA and protein levels, of brain‐derived neurotrophic factor (BDNF), in specific rat brain areas, notably in those involved in reward and addiction. Real‐time PCR revealed a 10‐fold up‐regulation of BDNF mRNA in the nucleus accumbens (NAc) upon chronic Δ9‐THC treatment, but there was no change at 3 or 24 h after a single injection. Smaller increases in mRNA levels were found in the ventral tegmental area (VTA), medial prefrontal cortex and paraventricular nucleus (PVN). Immunohistochemistry showed large increases in BDNF‐stained cells in the NAc (5.5‐fold), posterior VTA (4‐fold) and PVN (1.7‐fold), but no change was observed in the anterior VTA, hippocampus or dorsal striatum. Altogether, our study indicates that chronic exposure to Δ9‐THC up‐regulates BDNF in specific brain areas involved with reward, and provides evidence for different BDNF expression in the anterior and posterior VTA. Moreover, BDNF is known to modulate synaptic plasticity and adaptive processes underlying learning and memory, leading to long‐term functional and structural modification of synaptic connections. We suggest that Δ9‐THC up‐regulation of BDNF expression has an important role in inducing the neuroadaptive processes taking place upon exposure to cannabinoids.

Contribution of brain inflammation to neuronal cell death in neuronopathic forms of Gaucher's disease

Gauchers disease, the most common lysosomal storage disorder, is caused by the defective activity of glucocerebrosidase, the lysosomal hydrolase that degrades glucosylceramide. The neuronopathic forms of Gauchers disease are characterized by severe neuronal loss, astrocytosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. In the current study, we delineate the role of neuroinflammation in the pathogenesis of neuronopathic Gauchers disease and show significant changes in levels of inflammatory mediators in the brain of a neuronopathic Gauchers disease mouse model. Levels of messenger RNA expression of interleukin -1β, tumour necrosis factor-α, tumour necrosis factor-α receptor, macrophage colony-stimulating factor and transforming growth factor-β were elevated by up to ∼30-fold, with the time-course of the increase correlating with the progression of disease severity. The most significant elevation was detected for the chemokines CCL2, CCL3 and CCL5. Blood-brain barrier disruption was also evident in mice with neuronopathic Gauchers disease. Finally, extensive elevation of nitrotyrosine, a hallmark of peroxynitrite (ONOO(-)) formation, was observed, consistent with oxidative damage caused by macrophage/microglia activation. Together, our results suggest a cytotoxic role for activated microglia in neuronopathic Gauchers disease. We suggest that once a critical threshold of glucosylceramide storage is reached in neurons, a signalling cascade is triggered that activates microglia, which in turn releases inflammatory cytokines that amplify the inflammatory response, contributing to neuronal death.

Noninvasive Magnetic Resonance Imaging of Transport and Interstitial Fluid Pressure in Ectopic Human Lung Tumors

Tumor response to blood borne drugs is critically dependent on the efficiency of vascular delivery and transcapillary transfer. However, increased tumor interstitial fluid pressure (IFP) forms a barrier to transcapillary transfer, leading to resistance to drug delivery. We present here a new, noninvasive method which estimates IFP and its spatial distribution in vivo using contrast-enhanced magnetic resonance imaging (MRI). This method was tested in ectopic human non-small-cell lung cancer which exhibited a high IFP of approximately 28 mm Hg and, for comparison, in orthotopic MCF7 human breast tumors which exhibited a lower IFP of approximately 14 mm Hg, both implanted in nude mice. The MRI protocol consisted of slow infusion of the contrast agent [gadolinium-diethylenetriaminepentaacetic acid (GdDTPA)] into the blood for approximately 2 hours, sequential acquisition of images before and during the infusion, and measurements of T1 relaxation rates before infusion and after blood and tumor GdDTPA concentration reached a steady state. Image analysis yielded parametric images of steady-state tissue GdDTPA concentration with high values of this concentration outside the tumor boundaries, approximately 1 mmol/L, declining in the tumor periphery to approximately 0.5 mmol/L, and then steeply decreasing to low or null values. The distribution of steady-state tissue GdDTPA concentration reflected the distribution of IFP, showing an increase from the rim inward, with a high IFP plateau inside the tumor. The changes outside the borders of the tumors with high IFP were indicative of convective transport through the interstitium. This work presents a noninvasive method for assessing the spatial distribution of tumor IFP and mapping barriers to drug delivery and transport.