Claes-Henric Berthold

Absence of Glial Fibrillary Acidic Protein and Vimentin Prevents Hypertrophy of Astrocytic Processes and Improves Post-Traumatic Regeneration

The regenerative capacity of the CNS is extremely limited. The reason for this is unclear, but glial cell involvement has been suspected, and oligodendrocytes have been implicated as inhibitors of neuroregeneration (Chen et al., 2000, GrandPre et al., 2000; Fournier et al., 2001). The role of astrocytes in this process was proposed but remains incompletely understood (Silver and Miller, 2004). Astrocyte activation (reactive gliosis) accompanies neurotrauma, stroke, neurodegenerative diseases, or tumors. Two prominent hallmarks of reactive gliosis are hypertrophy of astrocytic processes and upregulation of intermediate filaments. Using the entorhinal cortex lesion model in mice, we found that reactive astrocytes devoid of the intermediate filament proteins glial fibrillary acidic protein and vimentin (GFAP-/-Vim-/-), and consequently lacking intermediate filaments (Colucci-Guyon et al., 1994; Pekny et al., 1995; Eliasson et al., 1999), showed only a limited hypertrophy of cell processes. Instead, many processes were shorter and not straight, albeit the volume of neuropil reached by a single astrocyte was the same as in wild-type mice. This was accompanied by remarkable synaptic regeneration in the hippocampus. On a molecular level, GFAP-/-Vim-/- reactive astrocytes could not upregulate endothelin B receptors, suggesting that the upregulation is intermediate filament dependent. These findings show a novel role for intermediate filaments in astrocytes and implicate reactive astrocytes as potent inhibitors of neuroregeneration.

Intermediate Filament Protein Partnership in Astrocytes

Intermediate filaments are general constituents of the cytoskeleton. The function of these structures and the requirement for different types of intermediate filament proteins by individual cells are only partly understood. Here we have addressed the role of specific intermediate filament protein partnerships in the formation of intermediate filaments in astrocytes. Astrocytes may express three types of intermediate filament proteins: glial fibrillary acidic protein (GFAP), vimentin, and nestin. We used mice with targeted mutations in the GFAP or vimentin genes, or both, to study the impact of loss of either or both of these proteins on intermediate filament formation in cultured astrocytes and in normal or reactive astrocytesin vivo. We report that nestin cannot form intermediate filaments on its own, that vimentin may form intermediate filaments with either nestin or GFAP as obligatory partners, and that GFAP is the only intermediate filament protein of the three that may form filaments on its own. However, such filaments show abnormal organization. Aberrant intermediate filament formation is linked to diseases affecting epithelial, neuronal, and muscle cells. Here we present models by which the normal and pathogenic functions of intermediate filaments may be elucidated in astrocytes.

Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.

Glial fibrillary acidic protein (GFAP) is the main component of the intermediate filaments in cells of astroglial lineage, including astrocytes in the CNS, nonmyelin forming Schwann cells and enteric glia. To address the function of GFAP in vivo, we have disrupted the GFAP gene in mice via targeted mutation in embryonic stem cells. Mice lacking GFAP developed normally, reached adulthood and reproduced. We did not find any abnormalities in the histological architecture of the CNS, in their behavior, motility, memory, blood‐brain barrier function, myenteric plexi histology or intestinal peristaltic movement. Comparisons between GFAP and S‐100 immunohistochemical staining patterns in the hippocampus of wild‐type and mutant mice suggested a normal abundance of astrocytes in GFAP‐negative mice, however, in contrast to wild‐types, GFAP‐negative astrocytes of the hippocampus and in the white matter of the spinal cord were completely lacking intermediate filaments. This shows that the loss of GFAP intermediate filaments is not compensated for by the up‐regulation of other intermediate filament proteins, such as vimentin. The GFAP‐negative mice displayed post‐traumatic reactive gliosis, which suggests that GFAP up‐regulation, a hallmark of reactive gliosis, is not an obligatory requirement for this process.

Intermediate filaments regulate astrocyte motility.

Intermediate filaments (IFs) compose, together with actin filaments and microtubules, the cytoskeleton and they exhibit a remarkable but still enigmatic cell‐type specificity. In a number of cell types, IFs seem to be instrumental in the maintenance of the mechanical integrity of cells and tissues. The function of IFs in astrocytes has so far remained elusive. We have recently reported that glial scar formation following brain or spinal cord injury is impaired in mice deficient in glial fibrillary acidic protein and vimentin. These mice lack IFs in reactive astrocytes that are normally pivotal in the wound repair process. Here we show that reactive astrocytes devoid of IFs exhibit clear morphological changes and profound defects in cell motility thereby revealing a novel function for IFs.

Axoplasmic organelles at nodes of Ranvier. I: Occurrence and distribution in large myelinated spinal root axons of the adult cat

SummaryUsing light microscopy (LM) and electron microscopy (EM) we have examined the occurrence and distribution of axoplasmic organelles in large myelinated nerve fibres of the L7 ventral and dorsal spinal roots of the cat with special reference to the paranode-node-paranode (pnp)-regions. Ninety-eight percent of the 550 Toluidine Blue-stained paranode-node-paranode-regions examined in the light microscope contained dark-blue bodies accumulated distal to the midlevel of the paranode-node-paranode-region. Further, a veil of Toluidine Blue positive material was observed in about 50% of the paranode-node-paranode-regions. In about 25% of these paranode-node-paranode-regions the veil lay distal to the midlevel of the paranode-node-paranode-region and in the. remainder it lay proximally. Electron microscopy suggested that the ultrastructural equivalents of the dark-blue bodies and of the veil were dense lamellar bodies and a diffuse granular material, respectively. Our calculations indicate that from 70% to more than 90% of some organelles (dense lamellar bodies, multivesicular bodies and vesiculo-tubular membranous organelles) present in an axon are accumulated in the paranode-node-paranode-regions. The occurrence of these organelles in the individual paranode-node-paranode-regions varied within wide limits also in adjacent fibres. The dense lamellar and multivesicular bodies dominated the distal part of the paranode-node-paranode-regions while the vesiculo-tubular membranous organelles dominated the proximal part, i.e. the organelles showed a mutual proximo-distal segregation with reference to the midlevel of the paranode-node-paranode-region. of seventeen paranode-node-paranode-regions analyzed ultrastructurally, seven were classified as ‘fully segregated’, that is 67% or more of the lamellar and multivesicular bodies, present in the whole paranode-node-paranode-region, lay distal to the mid-level, and 67% or more of the vesiculo-tubular membranous organelles lay proximal to it.

Electron microscopic serial section analysis of nodes of Ranvier in lumbosacral spinal roots of the cat: ultrastructural organization of nodal compartments in fibres of different sizes

SummaryThe general ultrastructural organization of nodes of Ranvier in peripheral nerve fibres from 2 to 20 μm in diameter (D) was investigated in the adult cat using serially sectioned ventral and dorsal spinal roots. The study was performed in order to collect and systematize information considered necessary for a morphometric analysis of the node of Ranvier. In all cases a node of Ranvier could be divided into a central nodal axon segment and a surrounding nodal Schwann cell compartment. The latter included a nodal gap matrix substance, more or less overlapping nodal Schwann cell collars and, as a rule, also a Schwann cell brush-border emanating from the nodal Schwann cell collars and occupying the nodal gap. The relative size and the organization level of the nodal Schwann cell compartment increased with increasing fibre size up to a fibre diameter of 8–10 μm. At this fibre size the nodal gap was of a fairly even height (1 μm) all around the nodal axon and contained a thick brush-border of densely packed, more or less radially arranged Schwann cell microvilli. In very small fibres (D < 3 μm) the nodal gap was low (<0.1 μm) and contained no or few microvilli. In fibres >10 μm in diameter the relative size and the degree of structural order of the nodal Schwann cell compartment decreased with increasing fibre size. Drastic sectorial variations in nodal gap height and local thinning-out of the brush-border became prominent features in the largest fibres. The possiblein vivo organization of the nodal Schwann cell compartment is discussed. Preliminary calculations indicate that the extracellular space directly surrounding the nodal axon might be quite small and that the area open for free communication between this extracellular space and the endoneurial space might be very much restricted, measuring as little as 2% of the area of the nodal axolemma. Algorithms for calculating various nodal structural parameters are discussed.

Electron microscopic serial section analysis of nodes of Ranvier in lumbar spinal roots of the cat: A morphometric study of nodal compartments in fibres of different sizes

SummarySerially sectioned nodes of Ranvier from nerve fibres 2–20 μm in diameter of feline ventral and dorsal spinal roots were examined electron microscopically, reconstructed to scale and analysed morphometrically. The assumed ‘fresh-state’ value ot several structural variables, considered to be of functional significance, were calculated by the use of compensation factors. The compensated data were plotted against fibre and axon diameters. It was calculated that the membranous area of the ‘fresh-state’ nodal axon segment increased more or less exponentially from less than 5 μm2 to 30 μm2 with increasing fibre diameter (D). Most variables associated with the nodal gap and the Schwann cell initially increased rapidly withD and then levelled out or even decreased in fibres with aD value greater than 8–12 μm. The area open for communication between the nodal axolemma and the endoneurial space was 30–100 times smaller than the membrane area of the nodal axolemma. The volume of the extracellular space in the nodal gap, outside the nodal axolemma, increased linearly from less than 0.1 μm3 to about 0.6 μm3 with increasing fibre size. The Schwann cell membrane area facing the nodal gap outnumbered the membrane area of the nodal axon by 10–15 times in nerve fibres with aD value between 5 and μm. Some functional implications of the ‘fresh-state’ nodal model are discussed.

Expression of the myelin and oligodendrocyte progenitor marker sulfatide in neurons and astrocytes of adult rat brain

Sulfatide is a myelin component of the central (CNS) and peripheral nervous system (PNS) and is used extensively to identify oligodendrocyte progenitor cells. We have explored sulfatide expression in CNS gray matter (cerebellum, cerebral cortex, and hippocampus) and the PNS in adult rats using an anti‐sulfatide antibody (Sulph I) and confocal microscopy. Biochemical analyses revealed two Sulph I antigens, sulfatide and seminolipid; sulfatide was present at about five times higher concentration, and the affinity of Sulph I for sulfatide was 2.5 times higher than that for seminolipid. Thus sulfatide was considered the dominant antigen. We found Sulph I immunostaining, in addition to that in myelinated areas in subpopulations of astrocytes and neurons. Astrocyte Sulph I staining was localized to the cell bodies and in some cases also to the processes. In the cerebellum, some Sulph I‐positive astrocytes corresponded to Golgi epithelial cell bodies. We also found Sulph I staining in neuronal cell bodies, which in some neurons was clearly localized to the cytoplasm and in others to the nuclear membrane. Sulph I immunostaining in the PNS was located in the myelin sheath and paranodal end segments. These results demonstrate the expression of sulfatide in cell types other than oligodendrocytes and Schwann cells, showing that sulfatide is not a selective marker for adult oligodendrocyte progenitor cells. Moreover, these findings show that sulfatide is localized also to intracellular compartments and indicate that other roles of sulfatide in astrocytes and neurons, compared to myelin, might be considered.

Lysosomal activity at nodes of Ranvier during retrograde axonal transport of horseradish peroxidase in alpha-motor neurons of the cat

SummaryLysosomal activity at nodes of Ranvier of feline hindlimb alpha-motor neurons was examined by light and electron microscopical acid phosphatase (AcPase) histochemistry during retrograde axonal transport of intramuscularly injected horseradish peroxidase (HRP). Several nodes along the PNS parts of the alpha-motor axons of the HRP-injected side showed accumulations of AcPase-positive bodies in the constricted nodal axon segment and the adjacent paranodal axoplasm. Such lysosomal accumulations were most prominent in the ventral root and differed in number and intensity depending on survival time after the HRP injection. At nodes showing high AcPase activity the axoplasm proximal to the nodal midlevel was occupied by many small, AcPase-positive, vesiculotubular profiles. Larger AcPase-positive bodies were mainly situated distal to the nodal midlevel. Double incubation for demonstration of both HRP and AcPase activity showed similar accumulations of AcPase-positive bodies at some of the HRP-transporting nodes. The AcPase activity differed considerably between nodes exhibiting comparable levels of HRP-positivity. Many of the AcPase-positive bodies also contained HRP reaction product. At some HRP-positive nodes the number of AcPase-positive bodies situated in the paranodal axon-Schwann cell network was elevated when compared to nodes of the contralateral, control side. In contrast to the PNS nodes, the nodal occurrence and distribution of lysosomes in the CNS part of alpha-motor axons seemed not to be affected by HRP transport.These observations support our previous proposal that nodes of Ranvier in the PNS parts of alpha-motor axons, in contrast to their CNS nodes, possess an ability to control passage of and initiate lysosomal degradation of axonally transported substances. Such an ability may provide a protective function to the motor neuron by restricting the intraneuronal transport of materials imbibed by the axon terminals outside the CNS.

Changes in shape and size of cat spinal root myelinated nerve fibers during fixation and Vestopal-w embedding for electron microscopy.

The influence of glutaraldehyde fixation, osmication, acetone dehydration, and Vestopal-W embedding on shape and dimensions of cat myelinated lumbar spinal root nerve fibers was investigated using light and electron microscopy. Glutaraldehyde fixation gave no clear changes in nerve fiber shape or dimensions. The subsequent preparative steps introduced a decrease in the whole fiber “diameter” of about 10% irrespective of the original fiber “diameter”. Myelin sheath thickness decreased by about 30–35%. With this background it was calculated that axon “diameter” should change with the g value of the fiber; viz., Y = (X − Z)/g + Z, where X, Y, and Z are terms expressing the change (%) in values for fiber “diameter”, axon “diameter”, and myelin sheath thickness, respectively. It was noted that the axon “diameter” of fibers with g values of 0.4, 0.5, 0.6, and 0.7, changed by +20, +15, +5 and 0%, respectively. Experimental data confirmed these calculations. The most striking preparatory change at the nodes of Ranvier was widening of the node gaps.