Grzegorz Wicher

c-Jun Reprograms Schwann Cells of Injured Nerves to Generate a Repair Cell Essential for Regeneration

Summary The radical response of peripheral nerves to injury (Wallerian degeneration) is the cornerstone of nerve repair. We show that activation of the transcription factor c-Jun in Schwann cells is a global regulator of Wallerian degeneration. c-Jun governs major aspects of the injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-Jun is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. We show that absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death. We conclude that a single glial transcription factor is essential for restoration of damaged nerves, acting to control the transdifferentiation of myelin and Remak Schwann cells to dedicated repair cells in damaged tissue.

MALDI mass spectrometry based molecular phenotyping of CNS glial cells for prediction in mammalian brain tissue

The development of powerful analytical techniques for specific molecular characterization of neural cell types is of central relevance in neuroscience research for elucidating cellular functions in the central nervous system (CNS). This study examines the use of differential protein expression profiling of mammalian neural cells using direct analysis by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). MALDI-MS analysis is rapid, sensitive, robust, and specific for large biomolecules in complex matrices. Here, we describe a newly developed and straightforward methodology for direct characterization of rodent CNS glial cells using MALDI-MS-based intact cell mass spectrometry (ICMS). This molecular phenotyping approach enables monitoring of cell growth stages, (stem) cell differentiation, as well as probing cellular responses towards different stimulations. Glial cells were separated into pure astroglial, microglial, and oligodendroglial cell cultures. The intact cell suspensions were then analyzed directly by MALDI-TOF-MS, resulting in characteristic mass spectra profiles that discriminated glial cell types using principal component analysis. Complementary proteomic experiments revealed the identity of these signature proteins that were predominantly expressed in the different glial cell types, including histone H4 for oligodendrocytes and S100-A10 for astrocytes. MALDI imaging MS was performed, and signature masses were employed as molecular tracers for prediction of oligodendroglial and astroglial localization in brain tissue. The different cell type specific protein distributions in tissue were validated using immunohistochemistry. ICMS of intact neuroglia is a simple and straightforward approach for characterization and discrimination of different cell types with molecular specificity.

Metastasis-associated S100A4 (Mts1) protein is expressed in subpopulations of sensory and autonomic neurons and in Schwann cells of the adult rat

S100A4 (Mts1) is a member of a family of calcium‐binding proteins of the EF‐hand type, which are widely expressed in the nervous system, where they appear to be involved in the regulation of neuron survival, plasticity, and response to injury or disease. S100A4 has previously been demonstrated in astrocytes of the white matter and rostral migratory stream of the adult rat. After injury, S100A4 is markedly up‐regulated in affected central nervous white matter areas as well as in the periventricular area and rostral migratory stream. Here, we show that S100A4 is expressed in a subpopulation of dorsal root, trigeminal, geniculate, and nodose ganglion cells; in a subpopulation of postganglionic sympathetic and parasympathetic neurons; in chromaffin cells of the adrenal medulla; and in satellite and Schwann cells. In dorsal root ganglia, S100A4‐positive cells appear to constitute a subpopulation of small ganglion neurons, a few of which coexpressed calcitonin gene‐related peptide (CGRP) and Griffonia simplicifolia agglutinin (GSA) isolectin B4 (B4). S100A4 protein appears to be transported from dorsal root ganglia to the spinal cord, where it is deposited in the tract of Lissauer. After peripheral nerve or dorsal root injury, a few S100A4‐positive cells coexpress CGRP, GSA, or galanin. Peripheral nerve or dorsal root injury induces a marked up‐regulation of S100A4 expression in satellite cells in the ganglion and in Schwann cells at the injury site and in the distal stump. This pattern of distribution partially overlaps that of the previously studied S100B and S100A6 proteins, indicating a possible functional cooperation between these proteins. The presence of S100A4 in sensory neurons, including their processes in the central nervous system, suggests that S100A4 is involved in propagation of sensory impulses in specific fiber types. J. Comp. Neurol. 473:233–243, 2004.

Low density lipoprotein receptor-related protein-2/megalin is expressed in oligodendrocytes in the mouse spinal cord white matter.

Lipoprotein receptor‐related protein‐2 (LRP2)/megalin is a member of the low density lipoprotein receptor (LDLR) family, and is essential in absorptive epithelia for endocytosis of lipoproteins, low molecular weight proteins, cholesterol and vitamins, as well as in cellular signaling. Previous studies have shown megalin expression in ependymal cells and choroid plexus. We have investigated megalin expression in the spinal cord of postnatal mice with immunohistochemistry and immunoblot. Antibodies recognizing either the cytoplasmic tail (MM6) or the extracellular domain (E11) of megalin labeled oligodendrocytes in the spinal cord white matter, in parallel with myelination. MM6 antibodies, predominantly labeled the nuclei, whereas E11 antibodies labeled the cytoplasm of these cells. MM6 antibodies labeled also nuclei of oligodendrocytes cultured from embryonic mouse spinal cord. Immunoblots of spinal cord showed intact megalin, as well as its carboxyterminal fragment, the part remaining after shedding of the extracellular domain of megalin. Megalin‐immunoreactive oligodendrocytes also expressed presenilin 1, an enzyme responsible for γ‐secretase mediated endodomain cleavage. These findings show that spinal cord oligodendrocytes are phenotypically different from those in the brain, and indicate that megalin translocates signals from the cell membrane to the nucleus of oligodendrocytes during the formation and maintenance of myelin of long spinal cord pathways.

Low-density lipoprotein receptor-related protein (LRP)-2/megalin is transiently expressed in a subpopulation of neural progenitors in the embryonic mouse spinal cord.

The lipoprotein receptor LRP2/megalin is expressed by absorptive epithelia and involved in receptor‐mediated endocytosis of a wide range of ligands. Megalin is expressed in the neuroepithelium during central nervous system (CNS) development. Mice with homozygous deletions of the megalin gene show severe forebrain abnormalities. The possible role of megalin in the developing spinal cord, however, is unknown. Here we examined the spatial and temporal expression pattern of megalin in the embryonic mouse spinal cord using an antibody that specifically recognizes the cytoplasmic part of the megalin molecule. In line with published data, we show expression of megalin in ependymal cells of the central canal from embryonic day (E)11 until birth. In addition, from E11 until E15 a population of cells was found in the dorsal part of the developing spinal cord strongly immunoreactive against megalin. Double labeling showed that most of these cells express vimentin, a marker for immature astrocytes and radial glia, but not brain lipid binding protein (BLBP), a marker for radial glial cells, or glial fibrillary acidic protein (GFAP), a marker for mature astrocytes. These findings indicate that the majority of the megalin‐positive cells are astroglial precursors. Megalin immunoreactivity was mainly localized in the nuclei of these cells, suggesting that the cytoplasmic part of the megalin molecule can be cleaved following ligand binding and translocated to the nucleus to act as a transcription factor or regulate other transcription factors. These findings suggest that megalin has a crucial role in the development of astrocytes of the spinal cord. J. Comp. Neurol. 492:123–131, 2005.

Intracellular calcium-binding protein S100A4 influences injury-induced migration of white matter astrocytes

Astrocytes play a crucial role in central nervous system (CNS) pathophysiology. White and gray matter astrocytes are regionally specialized, and likely to respond differently to CNS injury and in CNS disease. We previously showed that the calcium-binding protein S100A4 is exclusively expressed in white matter astrocytes and markedly up-regulated after injury. Furthermore, down-regulation of S100A4 in vitro significantly increases the migration capacity of white matter astrocytes, a property, which might influence their function in CNS tissue repair. Here, we performed a localized injury (scratch) in confluent cultures of white matter astrocytes, which strongly express S100A4, and in cultures of white matter astrocytes, in which S100A4 was down-regulated by transfection with short interference (si) S100A4 RNA. We found that S100A4-silenced astrocytes rapidly migrated into the injury gap, whereas S100A4-expressing astrocytes extended hypertrophied processes toward the gap, but without closing it. To explore the involvement of S100A4 in migration of astrocytes in vivo, we induced focal demyelination and transient glial cell elimination in the spinal cord white matter by ethidium bromide injection in S100A4 (−/−) and (+/+) mice. The results show that astrocyte migration into the demyelinated area is promoted in S100A4 (−/−) compared to (+/+) mice, in which a pronounced glial scar was formed. These data indicate that S100A4 reduces the migratory capacity of reactive white matter astrocytes in the injured CNS and is involved in glial scar formation after injury.

Adult motor neurons show increased susceptibility to axotomy-induced death in mice lacking clusterin

Clusterin is a highly conserved, amphiphatic glycoprotein present in most tissues. It has been shown to be involved in the regulation of lipid transportation, clearance of cellular debris from the extracellular space and intracellular signal transduction. Clusterin is markedly up‐regulated after neural injury but the functional significance of this response is unclear. Here, we show that clusterin up‐regulation is substantially greater in hypoglossal motor neurons after hypoglossal nerve avulsion compared with nerve transection. Quantitative analyses of motor neuron numbers after the same lesions in clusterin(–/–) and clusterin(+/+) mice showed significantly larger numbers of surviving motor neurons in clusterin(+/+) mice. These results suggest that clusterin has a neuroprotective role after axotomy.

Megalin deficiency induces critical changes in mouse spinal cord development.

Low density lipoprotein receptor-related protein, megalin, is a multifunctional lipoproptein receptor expressed by absorptive epithelia for endocytosis of numerous ligands. Megalin is widely expressed during embryonic life and is essential for development of the nervous system as evidenced by severe forebrain abnormalities in megalin (−/−). Here, we investigated the influence of megalin deficiency on prenatal spinal cord development in mice. In contrast to wild-type mice, cells expressing Olig2 and NG2, that is, oligodendroglial precursor cells, are absent from embryonic stage E16 in megalin (−/−) mice. At the end of prenatal development, there is a failure in vertebral development, and the number of astrocytes are markedly reduced in megalin (−/−) mice. These findings indicate that megalin is essential in astro–oligodendroglial interactions during development of the spinal cord.

Extracellular clusterin promotes neuronal network complexity in vitro

Clusterin (apolipoprotein J), a highly conserved amphiphatic glycoprotein and chaperone, has been implicated in a wide range of physiological and pathological processes. As a secreted protein, clusterin has been shown to act extracellularly where it is involved in lipid transportation and clearance of cellular debris. Intracellularly, clusterin may regulate signal transduction and is upregulated after cell stress. After neural injury, clusterin may be involved in nerve cell survival and postinjury neuroplasticity. In this study, we investigated the role of extracelullar clusterin on neuronal network complexity in vitro. Quantitative analysis of clustrin-treated neuronal cultures showed significantly higher network complexity. These findings suggest that in addition to previously demonstrated neuroprotective roles, clusterin may also be involved in neuronal process formation, elongation, and plasticity.

Interleukin-33 Promotes Recruitment of Microglia/Macrophages in Response to Traumatic Brain Injury

Traumatic brain injury (TBI) is a devastating condition, often leading to life-long consequences for patients. Even though modern neurointensive care has improved functional and cognitive outcomes, efficient pharmacological therapies are still lacking. Targeting peripherally derived, or resident inflammatory, cells that are rapid responders to brain injury is promising, but complex, given that the contribution of inflammation to exacerbation versus improved recovery varies with time post-injury. The injury-induced inflammatory response is triggered by release of alarmins, and in the present study we asked whether interleukin-33 (IL-33), an injury-associated nuclear alarmin, is involved in TBI. Here, we used samples from human TBI microdialysate, tissue sections from human TBI, and mouse models of central nervous system injury and found that expression of IL-33 in the brain was elevated from nondetectable levels, reaching a maximum after 72 h in both human samples and mouse models. Astrocytes and oligodendrocytes were the main producers of IL-33. Post-TBI, brains of mice deficient in the IL-33 receptor, ST2, contained fewer microglia/macrophages in the injured region than wild-type mice and had an altered cytokine/chemokine profile in response to injury. These observations indicate that IL-33 plays a role in neuroinflammation with microglia/macrophages being cellular targets for this interleukin post-TBI.