Donatella D'Urso

Inhibition of collagen IV deposition promotes regeneration of injured CNS axons

Scarring impedes axon regrowth across the lesion site and is one major extrinsic constraint to effective regeneration in the adult mammalian central nervous system. In the present study we determined whether specific biochemical or immunochemical modulation of one major component of the scar, the basal membrane (BM), would provide a means to stimulate axon regeneration in the mechanically transected postcommissural fornix of the adult rat. Basal membrane developed within the first 2 weeks after transection in spatiotemporal coincidence with the abrupt growth arrest of spontaneously regrowing axons. Local injection of anticollagen IV antibodies or α, α′‐dipyridyl, an inhibitor of collagen triple helix formation and synthesis, significantly reduced lesion‐induced BM deposition. This treatment allowed massive axon elongation across the lesion site. Anterograde tracing provided unequivocal evidence that regenerating axons follow their original pathway, reinnervate the appropriate target, the mammillary body, and become remyelinated with compact myelin. Presynaptic electrophysiological recordings of regenerated fibre tracts showed recovery to nearly normal conduction properties. Our results indicate that lesion‐induced BM is an impediment for successful axonal regeneration and its reduction is a prerequisite and sufficient condition for regrowing axons to cross the lesion site.

Reversible Inhibitory Effects of Interferon‐γ and Tumour Necrosis Factor‐α on Oligodendroglial Lineage Cell Proliferation and Differentiation In Vitro

We have investigated the effects of the two prominent inflammatory cytokines, interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α), on oligodendroglial lineage cell development and survival. Purified oligodendrocytes and oligodendrocyte precursors obtained from neonatal rat brain primary cultures were subcultured in a defined, serum‐free medium and exposed to IFN‐γ (1‐100 U/ml), TNF‐α (25‐100 ng/ml) or both (100 U/ml and 50 ng/ml respectively) from day 1 to day 3 or from day 3 to day 6. While cell survival was not affected in any of the conditions tested, IFN‐γ dose‐dependently inhibited [3H]thymidine or bromodeoxyuridine incorporation (by up to 50%) and the reduction of the tetrazolium salt 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT; by up to 33%). TNF‐α synergized with IFN‐γ, but was ineffective by itself. Moreover, IFN‐γ totally antagonized the induction by basic fibroblast growth factor and platelet‐derived growth factor of the proliferation of the oligodendroglial lineage cell population under study. IFN‐γ also blocked the differentiation of oligodendrocyte precursors, as evidenced by cell morphology, immunostaining for early and late differentiation markers (galactocerebroside and myelin basic protein respectively) and activity of ceramide galactosyl transferase. Again, the effect of IFN‐γ was potentiated by TNF‐α, which was ineffective when tested alone. The inhibitory activity of IFN‐γ was rapidly reversible: 3 days after removal of the cytokine, administered from day 1 to day 3, complete recovery of cell proliferation and differentiation could be documented. The cytokine‐induced arrest in the expression of differentiation antigens was accompanied by perturbations in the expression of the corresponding mRNAs, revealed by a semiquantitative reverse transcription‐polymerase chain reaction method. In particular, the message for myelin basic protein (and, in the case of treatment from days 3 to 6, also that for myelin associated glycoprotein) was decreased in cultures exposed to IFN‐γ, and further depressed in cultures treated with IFN‐γ and TNF‐α, while TNF‐α alone was ineffective. The above observations may help explain the role of IFN‐γ and TNF‐α in the pathogenesis of inflammatory demyelinating diseases, in which increases in the levels of these substances have been described. In particular, in the case of multiple sclerosis, our results may bear on the problem of defective remyelination and are consistent with the frequent relapsing‐remitting course of the disease.

Peripheral Myelin Protein-22 is Expressed in Rat and Mouse Brain and Spinal Cord Motoneurons

Peripheral myelin protein PMP‐22 is expressed by Schwann cells and is a constituent of peripheral nervous system (PNS) myelin. Two PMP‐22 transcripts, SR73 and CD25, differing in their 5’non‐coding sequences have been described. SR73 is present both in the PNS and in non‐neural tissue, whereas CD25 mRNA is almost exclusively expressed in Schwann cells. PMP‐22 mRNA is also present in the central nervous system (CNS), but at much lower levels than in the PNS. We have investigated the regional distribution of PMP‐22 mRNA in the rat and mouse CNS by the reverse transcriptase‐polymerase chain reaction method, using oligonucleotide primers specific for the SR73 or CD25 transcripts. SR73 mRNA was detected in all the CNS regions analysed, whereas the CD25 message was present only in the brainstem and the spinal cord. Localization of the PMP‐22 transcripts, determined by in situ hybridization in 21 day‐old animals, showed selective expression in the motor nuclei. The PMP‐22 signal was very weak in the nuclei of the oculomotor and trochlear nerves and absent in the nucleus of the abducens nerve. A strong PMP‐22 signal was observed in the motor nuclei of the trigeminal, facial, ambigus, vagus, hypoglossal and accessory spinal nerves and in the ventral horn of the spinal cord. The PMP‐22‐positive cells were identified as motoneurons on the basis of topographic and morphological criteria, as well as immunolabelling with neuron‐specific antibodies. Immunostaining with specific anti‐PMP‐22 antibodies demonstrated that neurons expressing the PMP‐22 message also expressed the corresponding protein in their cell bodies and axons. In contrast, neurons in the sensory nuclei of cranial nerves and dorsal root ganglion neurons were PMP‐22‐negative. In the trembler mouse, a PNS dysmyelinating mutant with a missense mutation in PMP‐22, the motoneuronal expression of PMP‐22 mRNA was either normal or increased.

Basal membrane-depleted scar in lesioned CNS: characteristics and relationships with regenerating axons

The lesion scar formed after CNS injury is an impediment to axonal regeneration and leads to growth arrest or misrouting of sprouting axons. Our previous study showed that pharmacological reduction of basal membrane formation within the scar can overcome this scar impermeability [Stichel C. C. et al. (1999) Eur. J. Neurosci. 11, 632-646]. The aim of the present study was to characterize the basal membrane-depleted scar and to analyse its relationships with penetrating axons. The experiments comprised two groups of animals in which the left postcommissural fornix was transected; in addition, one group received a local immediate injection of the collagen IV-reducing agent dipyridyl, while the other group received an injection of phosphate-buffered saline. Immunohistochemical methods were used to characterize scar formation and scar-axon relationships. Animals receiving dipyridyl showed reduction of collagen IV-immunopositive basal membrane in the lesion center, which was accompanied by: (i) a decrease in laminin, as well as chondroitin and heparan sulfate proteoglycan, deposition in the lesion center; (ii) an increase in chondroitin and keratan sulfate proteoglycan expression in the perilesional area; (iii) a typical activation pattern of astrocytes and microglia/macrophages; (iv) axons regenerating through this modified scar were closely associated with various glial cell types and crossed a prominent chondroitin/keratan sulfate proteoglycan matrix. Our results suggest that neither the formation of a reactive astroglial network nor the accumulation of microglia/macrophages or the deposition of chondroitin and keratan sulfate proteoglycans in the perilesional area represent a barrier to regrowing axons. The present approach demonstrates that the lesion-induced basal membrane itself is the primary determinant of scar impermeability.

Focal ischaemia of the rat brain elicits an unusual inflammatory response: early appearance of CD8+ macrophages/microglia

Cerebral ischaemia leads to profound glial activation and leukocyte infiltration into the infarct area. In this study, we provide evidence for a dual macrophage response in focal ischaemic lesions of the rat brain. We show that a considerable proportion of macrophages in the ischaemic lesions express the CD8αβ heterodimer to date only described on CD8+ T cells. As known from other lesion paradigms, CD4+ macrophages were also present. Interestingly, CD8‐ and CD4‐expressing macrophages formed two non‐overlapping subpopulations. CD8+ macrophages reached their maximum during the first week with pronounced downregulation thereafter whereas CD4+ cells persisted at high levels into the second week. In contrast to cerebral ischaemia, macrophages in the spleen and in Wallerian degeneration after optic nerve axotomy expressed CD4, but not CD8. In experimental autoimmune encephalomyelitis, CD8 was mainly associated with T cells and very weakly detectable on some ramified cells resembling activated microglia. In conclusion, we show that cerebral ischaemia triggers an unusual inflammatory response characterized by the appearance of CD8+/CD4– macrophages that might exert specific functions in the pathogenesis of ischaemic brain damage.

Influence of elevated expression of rat wild-type PMP22 and its mutant PMP22Trembler on cell growth of NIH3T3 fibroblasts

Abstract.The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth-arrest-specific gene gas3. The biological function of PMP22 is unknown, but recent progress in the analysis of rat Schwann cells expressing altered levels of PMP22 revealed that one role of PMP22 is as a negative growth modulator. We have investigated the influence of rat PMP22 (rPMP22) and a mutant of PMP22 (rPMP22Tr) resembling the murine trembler mutation on cell growth of retrovirus-vector-infected mouse NIH3T3 cells. Transduced cells carrying the two different sense constructs expressed rPMP22 and rPMP22Tr mRNAs and proteins. Elevated levels of rPMP22 and rPMP22Tr significantly reduced fibroblast growth as judged by proliferation assays. Despite a negative modulatory influence of rPMP22 and rPMP22Tr on cell proliferation, cell cycle analyses by flow cytometry did not reveal an influence of rPMP22 or rPMP22Tr on the synchronous progression of resting NIH3T3 cells from G0 into S phase. However, cell cycle analyses by flow cytometry of asynchronously dividing cultures demonstrated that the expression of rPMP22 and rPMP22Tr increased the fraction of cells in the G1 phase of the cell cycle. Furthermore, cell death analyses revealed that, in contrast to control cells and cells carrying the rPMP22Tr construct, a significantly increased fraction of NIH3T3 cells expressing rPMP22 exit the proliferation compartment showing hallmarks of programmed cell death. These results indicate that (i) rPMP22 and rPMP22Tr act as negative modulators of proliferation in murine fibroblasts probably through extension of the G1 phase of the cell cycle and (ii) rPMP22 but not rPMP22Tr promotes programmed death of these cells.

Studies on the effects of altered PMP22 expression during myelination in vitro

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot‐Marie‐Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over‐ or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT‐PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation. J. Neurosci. Res. 48:31–42, 1997.

In vitro differentiation of neural progenitor cells from prenatal rat brain: Common cell surface glycoprotein on three glial cell subsets

Glial progenitor cell differentiation and cell lineage relationships during brain development are complex hierarchical processes depending on genetic programming, cell‐cell interactions, and microenvironmental factors. The identification of precursor cell‐specific antigens provides a tool for the study of both normal development and deviations from lineage‐specific differentiation associated with malignant transformation. Monoclonal antibody (mAb) RB13‐6 recognizes a 130‐kDa cell surface glycoprotein (gp130RB13‐6) expressed by a subset of 9OAcGD3‐positive glial precursor cells scattered in the rat neuroepithelium on prenatal day (PRD) 13. During prenatal development the fraction of gp130RB13‐6‐positive fetal brain cells (FBC) decreased from about 18% (PRD 14) to about 1.5% (PRD 22), coinciding with increasing fractions of more mature cell types, as indicated by the elevated expression of p24RB21‐15, another cell surface determinant specified by mAb RB21‐15 (Kindler‐Röhrborn et al.; Differentiation 30:53–60, 1985) and other neural cell type‐specific markers. Accordingly, gp130RB13‐6‐positive precursor cells were localized in the ventricular zones throughout brain development. Concomitant with their formation and in the adult rat brain, ependymal layers lining the ventricular surface, choroid plexus, and the leptomeninges were intensely labeled by anti‐gp130RB13‐6 mAb. As visualized by confocal laser scanning microscopy of FBC cultures from PRD 13, gp130RB13‐6 was coexpressed with the RC1 antigen by progenitor cells morphologically resembling radial glia cells. In addition, a very small subpopulation of astrocytes coexpressing gp130RB13‐6 and glial fibrillary acidic protein (GFAP; <5%) occurred 3 days after seeding. Primary FBC cultures from PRD 18 contained an increased subset of astrocytes coexpressing gp130RB13‐6 and GFAP (∼25% of all gp130RB13‐6 expressing cells), apparently generated from gp130RB13‐6‐positive precursors. Corresponding to in vivo conditions, ciliated ependymal cells but also microglial cells/macrophages and leptomeningeal cells showed strong expression of gp130RB13‐6 in culture. We thus present a new glycoprotein on the cell surfaces of a glial progenitor cell subset for further studies of cell lineage relationships between radial glia cells, astrocytes, and ependymal cells. J. Neurosci. Res. 48:95–111, 1997.

Ins and outs of peripheral myelin protein-22: Mapping transmembrane topology and intracellular sorting

Molecular genetic studies have shown that the peripheral myelin protein 22 (PMP22) is a key gene in hereditary peripheral neuropathies and appears to be essential for the formation and maintenance of myelin in the PNS. Based on the amino acid sequence the predicted structure of PMP22 protein contains two major distinct hydrophilic regions and four transmembrane domains. To analyze the cellular localization and membrane topology of PMP22 we inserted an octapeptide tag‐sequence at the amino or at the carboxyl terminus of the PMP22 open reading frame and generated different chimeric constructs which were expressed in HeLa cells. The expression of the tagged PMP22 protein and its orientation with respect to the plasma membrane were analyzed using antibodies raised against specific PMP22 epitopes and the tag sequence. Combined indirect, double‐immunofluorescence labeling and confocal microscopy showed that PMP22 is synthesized in the rough endoplasmic reticulum of transfected cells and passes through the Golgi apparatus to the cell surface. We determined the transmembrane organization of PMP22 providing the first experimental evidence that confirms the cytoplasmic disposition of its N and C termini and the extracellular localization of the two hydrophilic domains containing amino acids 28–40 and 118–131. This study provides the basis for further analysis aimed to identify functional domains of wild‐type PMP22 and the cellular sorting of mutant forms of PMP22. J. Neurosci. Res. 49:551–562, 1997.

Loss of vessel wall viability in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzeheimers disease and an important cause of cerebral haemorrhage in the elderly. CAA is characterized by the deposition of Alzheimer amyloid β protein (Aβ) in cerebral and leptomeningeal vessel walls. In order to study the effect of cerebrovascular Aβ deposits in vivo, living canine leptomeninges obtained from old dogs affected by CAA were analysed by confocal laser scanning microscopy after immunofluorescence staining for Aβ and viability staining with fluorescein diacetate (FDA). Simultaneous detection of the two signals showed a segmental loss of leptomeningeal vessel wall viability at some site of Aβ deposition. Many of the non-viable vessels segments were also dilated, suggesting that Aβ-induced vascular cell death creates the loci minores resistentiae for the development of cerebral haemorrhage in CAA.