Bela Kosaras

T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive, motor neuron degenerative disease, in which the role of inflammation is not well established. Innate and adaptive immunity were investigated in the CNS of the Superoxide Dismutase 1 (SOD1)G93A transgenic mouse model of ALS. CD4+ and CD8+ T cells infiltrated SOD1G93A spinal cords during disease progression. Cell-specific flow cytometry and gene expression profiling showed significant phenotypic changes in microglia, including dendritic cell receptor acquisition, and expression of genes linked to neuroprotection, cholesterol metabolism and tissue remodeling. Microglia dramatically up-regulated IGF-1 and down-regulated IL-6 expression. When mutant SOD1 mice were bred onto a TCRβ deficient background, disease progression was significantly accelerated at the symptomatic stage. In addition, microglia reactivity and IGF-1 levels were reduced in spinal cords of SOD1G93A (TCRβ−/−) mice. These results indicate that T cells play an endogenous neuroprotective role in ALS by modulating a beneficial inflammatory response to neuronal injury.

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR–NF-κB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

Toll-like receptor 3 is a potent negative regulator of axonal growth in mammals.

Toll is a cell surface receptor with well described roles in the developmental patterning of invertebrates and innate immunity in adult Drosophila. Mammalian toll-like receptors represent a family of Toll orthologs that function in innate immunity by recognizing molecular motifs unique to pathogens or injured tissue. One member in this family of pattern recognition receptors, toll-like receptor 3 (TLR3), recognizes viral double-stranded RNA and host mRNA. We examined the expression and function of TLRs in the nervous system and found that TLR3 is expressed in the mouse central and peripheral nervous systems and is concentrated in the growth cones of neurons. Activation of TLR3 by the synthetic ligand polyinosine:polycytidylic acid (poly I:C) or by mRNA rapidly causes growth cone collapse and irreversibly inhibits neurite extension independent of nuclear factor κB. Mice lacking functional TLR3 were resistant to the neurodegenerative effects of poly I:C. Neonatal mice injected with poly I:C were found to have fewer axons exiting dorsal root ganglia and displayed related sensorimotor deficits. No effect of poly I:C was observed in mice lacking functional TLR3. Together, these findings provide evidence that an innate immune pattern recognition receptor functions autonomously in neurons to regulate axonal growth and advances a novel hypothesis that this class of receptors may contribute to injury and limited CNS regeneration.

Sensory innervation of the calvarial bones of the mouse.

Migraine sufferers frequently testify that their headache feels as if the calvarial bones are deformed, crushed, or broken (Jakubowski et al. [2006] Pain 125:286–295). This has lead us to postulate that the calvarial bones are supplied by sensory fibers. We studied sensory innervation of the calvaria in coronal and horizontal sections of whole‐head preparations of postnatal and adult mice, via immunostaining of peripherin (a marker of thinly myelinated and unmyelinated fibers) or calcitonin gene‐related peptide (CGRP; a marker more typical of unmyelinated nerve fibers). In pups, we observed nerve bundles coursing between the galea aponeurotica and the periosteum, between the periosteum and the bone, and between the bone and the meninges; as well as fibers that run inside the diploë in different orientations. Some dural fibers issued collateral branches to the pia at the frontal part of the brain. In the adult calvaria, the highest concentration of peripherin‐ and CGRP‐labeled fibers was found in sutures, where they appeared to emerge from the dura. Labeled fibers were also observed in emissary canals, bone marrow, and periosteum. In contrast to the case in pups, no labeled fibers were found in the diploë of the adult calvaria. Meningeal nerves that infiltrate the periosteum through the calvarial sutures may be positioned to mediate migraine headache triggered by pathophysiology of extracranial tissues, such as muscle tenderness and mild trauma to the skull. In view of the concentration of sensory fibers in the sutures, it may be useful to avoid drilling the sutures in patients undergoing craniotomies for a variety of neurosurgical procedures. J. Comp. Neurol. 515:331–348, 2009.

Development of later life spontaneous seizures in a rodent model of hypoxia induced neonatal seizures

Purpose:  To study the development of epilepsy following hypoxia‐induced neonatal seizures in Long‐Evans rats and to establish the presence of spontaneous seizures in this model of early life seizures.

Sensitization of central trigeminovascular neurons: blockade by intravenous naproxen infusion

We have previously observed that migraine attacks impervious to triptan therapy were readily terminated by subsequent i.v. administration of the non-steroidal anti-inflammatory drug (NSAID) ketorolac. Since such attacks were associated with periorbital allodynia--a symptom of central sensitization--we examined whether infusion of the NSAID naproxen can block sensitization of central trigeminovascular neurons in the medullary dorsal horn, using in vivo single-unit recording in the rat. Topical exposure of the cerebral dura to inflammatory soup (IS) for 5 min resulted in a short-term burst of activity (<8 min) and a long-lasting (>120 min) neuronal hyper-responsiveness to stimulation of the dura and periorbital skin (group 1). Infusion of naproxen (1 mg/kg) 2 h after IS (group 1) brought all measures of neuronal responsiveness back to the baseline values recorded prior to IS, and depressed ongoing spontaneous activity well below baseline. When given preemptively 1 h before IS (group 2), naproxen blocked the short-term burst of activity and every long-term measure of neuronal hyper-responsiveness that was studied in the central neurons. The same preemptive treatment, however, failed to block IS-induced short-term bursts of activity in C-unit meningeal nociceptors (group 3). The results suggest that parenteral administration of naproxen, unlike triptan therapy, can exert direct inhibition over central trigeminovascular neurons in the dorsal horn. Though impractical as a routine migraine therapy, parenteral NSAID administration should be useful as a non-narcotic rescue therapy for migraine in the setting of the emergency department.

Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia

The most common neurological symptom of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) is early life refractory epilepsy. As previous studies have shown enhanced excitatory glutamatergic neurotransmission in TSC and FCD brains, we hypothesized that neurons associated with these lesions may also express altered γ‐aminobutyric acid (GABA)A receptor (GABAAR)‐mediated inhibition.

WAVE1 Is Required for Oligodendrocyte Morphogenesis and Normal CNS Myelination

Myelin formation involves the outgrowth of an oligodendrocyte cell process that can be regarded as a giant lamellipodium because it is an actively growing structure with extruded cytoplasm. The actin cytoskeleton is critical to morphogenesis, but little is known about regulation of actin dynamics in oligodendrocytes. Wiskott-Aldrich syndrome protein family verprolin homologous (WAVE) proteins mediate lamellipodia formation; thus, we asked whether these proteins function in oligodendrocyte process formation and myelination. Here, we show that WAVE1 is expressed by oligodendrocytes and localizes to the lamella leading edge where actin polymerization is actively regulated. CNS WAVE1 expression increases at the onset of myelination. Expression of dominant-negative WAVE1 impaired process outgrowth and lamellipodia formation in cultured oligodendrocytes. Similarly, oligodendrocytes isolated from mice lacking WAVE1 had fewer processes compared with controls, whereas neurons and astrocytes exhibited normal morphology. In white matter of WAVE1−/− mice, we found regional hypomyelination in the corpus callosum and to a lesser extent in the optic nerve. In optic nerve from WAVE1−/− mice, there were fewer nodes of Ranvier but nodal morphology was normal, implicating a defect in myelin formation. Our in vitro findings support a developmentally dynamic and cell-autonomous role for WAVE1 in regulating process formation in oligodendrocytes. Additionally, WAVE1 function during CNS myelination appears to be linked to regional cues. Although its loss can be compensated for in many CNS regions, WAVE1 is clearly required for normal amounts of myelin to form in corpus callosum and optic nerve. Together, these data demonstrate a role for WAVE1 in oligodendrocyte morphogenesis and myelination.

Modulation of lipid peroxidation and mitochondrial function improves neuropathology in Huntington’s disease mice

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder. Oxidative damage has been associated with pathological neuronal loss in HD. The therapeutic modulation of oxidative stress and mitochondrial function using low molecular weight compounds may be an important strategy for delaying the onset and slowing the progression of HD. In the present study, we found a marked increase of 4-hydroxy-2-nonenal (4-HNE) adducts, a lipid peroxidation marker, in the caudate and putamen of HD brains and in the striatum of HD mice. Notably, 4-HNE immunoreactivity was colocalized with mutant huntingtin inclusions in the striatal neurons of R6/2 HD mice. Administration of nordihydroguaiaretic acid (NDGA), an antioxidant that functions by inhibiting lipid peroxidation, markedly reduced 4-HNE adduct formation in the nuclear inclusions of R6/2 striatal neurons. NDGA also protected cultured neurons against oxidative stress-induced cell death by improving ATP generation and mitochondrial morphology and function. In addition, NDGA restored mitochondrial membrane potential, mitochondrial structure, and synapse structure in the striatum of R6/2 mice and increased their lifespan. The present findings suggest that further therapeutic studies using NDGA are warranted in HD and other neurodegenerative diseases characterized by increased oxidative stress and altered mitochondrial function.

Role of cyclooxygenase-2 induction by transcription factor Sp1 and Sp3 in neuronal oxidative and DNA damage response

Cyclooxygenase‐2 (COX‐2) has been implicated in neuronal survival and death. However, the precise regulatory mechanisms involved in COX‐2 function are unclear. In the present study we found that COX‐2 is induced in response to glutathione depletion‐induced oxidative stress in primary cortical neurons. Two proximal specific Sp1 and Sp3 binding sites are responsible for the COX‐2 promoter activity under normal as well as oxidative stress conditions through enhanced Sp1 and Sp3 DNA binding activity. Site‐directed mutagenesis confirmed that −268/–267 positions serve as specific Sp1 and Sp3 recognition sites under oxidative stress. Enforced expression of Sp1 and Sp3 using HSV vectors increased the promoter activity, transcription, and protein level of COX‐2 in cortical neurons. The dominant negative form of Sp1 abrogated the oxidative stress‐induced promoter activity and expression of COX‐2. We also demonstrated that adenovirus‐mediated COX‐2 gene delivery protected neurons from DNA damage induced by oxidative, genotoxic, and excitotoxic stresses and by ischemic injury. Moreover, COX‐2−/− cortical neurons were more susceptible to DNA damage‐induced cell death. These results indicate that in primary neurons Sp1 and Sp3 play an essential role in the modulation of COX‐2 transcription, which mediates neuronal homeostasis and survival by preventing DNA damage in response to neuronal stress.—Lee, J., Kosaras, B., Aleyasin, H., Han, J. A., Park, D. S., Ratan, R. R., Kowall, N. W., Ferrante, R. J., Lee, S. W., Ryu, H. Role of cyclooxygenase‐2 induction by transcription factor Sp1 and Sp3 in neuronal oxidative and DNA damage response. FASEB J. 20, E1657–E1669 (2006)