William W. Schlaepfer

The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF‐M. Analysis of the sequence has allowed us to investigate the structure of the carboxy‐terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy‐terminal region of the protein contains a 13 amino acid proline‐ and serine‐rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys‐Ser‐Pro‐Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled‐coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.

Calcium-induced degeneration of axoplasm in isolated segments of rat peripheral nerve

Abstract Comparative studies were conducted on the pattern of degenerative changes in transected rat saphenous nerves and in excised segments of the same nerve incubated at 37°C in dextrose-enriched oxygenated Ringers solution with varying calcium ion concentrations. Identical features of Wallerian degeneration were observed in the transected nerve and in nerve segments incubated in Ringers solution. A characteristic initial alteration of this process was the granular disintegration of axoplasmic microtubules and neurofilaments, a phenomenon which was shown to be calcium-dependent in the isolated nerve segments. This primary axonal alteration as well as the associated linear fragmentation of myelin sheaths could be prevented by substitution of equimolar concentrations of magnesium for calcium (Ca-free media) or by the addition of 10 m M EGTA (Ca-chelated media) to Ringers solution. Furthermore, the granular disintegrative axoplasmic change could be accelerated by the addition of 10 or 50 m M CaCl 2 to Ringers incubation media or by subjecting the nerve preparations to chemical (detergent) or physical (freeze-thawing) membrane disrupting procedures. These findings support a Ca-influx hypothesis of Wallerian degeneration, whereby degradative axonal membrane alterations and a spontaneous influx of calcium ion into the axoplasm are critical phenomena in the degeneration of isolated axons.

Characterization of the calcium-induced disruption of neurofilaments in rat peripheral nerve.

Transverse frozen sections of desheathed rat peripheral nerve were incubated in media of different composition prior to fixation and processing for electron microscopic examination. Neurofilaments remained intact when these tissues were incubated in calcium-free media. A loss of neurofilaments and their replacement by granular debris occurred in myelinated and unmyelinated fibers following incubation in media containing 2 mM calcium. The calcium-mediated disruption of neurofilaments was inhibited by preincubation or incubation with 1 mM p-chloromercuribenzoate (PCMB). The inhibition by preincubation with PCMB could be partially reversed by subsequent preincubation with 10 mM dithioerythritol (DTE). Calcium-mediated breakdown of neurofilaments did not occur after prolonged preincubation in calcium-free media, a finding which suggested that neurofilament disruption was dependent upon a tissue factor which could be lost or inactivated in frozen-sectioned nerve tissues. The findings of the present study provide morphological evidence that neurofilament disruption in mammalian peripheral nerve is mediated by a calcium-activated, PCMB-sensitive enzyme in the axoplasm of myelinated and unmyelinated nerve fibers.

PyK2 and FAK connections to p190Rho guanine nucleotide exchange factor regulate RhoA activity, focal adhesion formation, and cell motility

Integrin binding to matrix proteins such as fibronectin (FN) leads to formation of focal adhesion (FA) cellular contact sites that regulate migration. RhoA GTPases facilitate FA formation, yet FA-associated RhoA-specific guanine nucleotide exchange factors (GEFs) remain unknown. Here, we show that proline-rich kinase-2 (Pyk2) levels increase upon loss of focal adhesion kinase (FAK) in mouse embryonic fibroblasts (MEFs). Additionally, we demonstrate that Pyk2 facilitates deregulated RhoA activation, elevated FA formation, and enhanced cell proliferation by promoting p190RhoGEF expression. In normal MEFs, p190RhoGEF knockdown inhibits FN-associated RhoA activation, FA formation, and cell migration. Knockdown of p190RhoGEF-related GEFH1 does not affect FA formation in FAK−/− or normal MEFs. p190RhoGEF overexpression enhances RhoA activation and FA formation in MEFs dependent on FAK binding and associated with p190RhoGEF FA recruitment and tyrosine phosphorylation. These studies elucidate a compensatory function for Pyk2 upon FAK loss and identify the FAK–p190RhoGEF complex as an important integrin-proximal regulator of FA formation during FN-stimulated cell motility.

Monoclonal Antibodies to Gel‐Excised Glial Filament Protein and Their Reactivities with Other Intermediate Filament Proteins

Abstract: A series of 14 monoclonal antibodies (MAs) has been obtained from a single rat‐mouse fusion using gel‐excised bovine glial filament (GF) proteins as immunogens. These MAs were characterized by two separate immunochemical assays and by two different immunohistochemical methods. Nine MAs demonstrated specificity for GF proteins. One MA also recognized an epitope shared by intermediate filaments (IF) of the vimentin class (VF). Using the enzyme‐linked immunosorbent assay, four of the MAs recognized 200,000, 150,000, and 51,000 dalton proteins, suggesting that these MAs were specific for GF proteins (the 51,000 dalton protein) and neurofilament (NF) proteins (the two high‐molecular‐weight proteins). However, in both of the immunohistochemical assay systems, these MAs stained neurons and their processes but not astroglial cells. These observations strongly suggest that the 51,000–dalton protein recognized by these four MAs was not derived from GF proteins but instead represents derivatives of NF protein subunits comigrating in gels with GF proteins. These data provide additional information concerning the unique and shared antigenic determinants of the three classes of IF (NF, GF, and VF) of the CNS. In addition, they draw attention to the fact that proteins of certain IF may undergo degradation and comigrate in gels with the proteins of unrelated IF. This emphasizes the need for the use of independent immunochemical and immunohistochemical assays in the characterization of the specificity of MAs.

Structural alterations of peripheral nerve induced by the calcium ionophore A23187.

Desheathed segments of rat peripheral nerve were incubated at 37 degrees C in oxygenated Ringers solution with and without the addition of calcium ionophore, A23187, 10 microgram/ml. Nerve fibers incubated in the presence of both ionophore and calcium revealed extensive granular disintegration of their axonal microtubules and neurofilaments after 30 and 60 min incubation intervals. These changes were not seen following control incubations in Ringers solution without ionophore or in calcium-free Ringers solution containing ionophore and EGTA, 1 mmole/1. Ionophore-induced alterations were also noted in Schwann cell cytoplasm. The granular degradative alteration of axoplasm caused by exposure of nerve fibers to ionophore and calcium were believed to be due to an ionophore-mediated influx of calcium into the axoplasm with resultant elevation of intra-axoplasmic calcium concentration. These axoplasmic changes were indistinguishable from the axoplasmic alteration occurring in the distal portions of transpected neurites during Wallerian degeneration. The findings support the view that abnormal calcium influxes are determinants in the degeneration of peripheral nerve.

An Immunoblot Study of Neurofilament Degradation In Situ and During Calcium-Activated Proteolysis

Abstract: The degradation of neurofilament (NF) proteins was examined by immunoblot methods to identify, characterize, and monitor the appearance of immunoreactive breakdown products during the loss of NF triplet proteins. Individual NF proteins and their breakdown products were identified using polyclonal and monoclonal antibodies to NF proteins. NF degradation was compared during calcium‐activated proteolysis of isolated rat NF, during an experimental influx of calcium into excised rat spinal nerve roots, and during NF breakdown in transected rat peripheral nerve. These different experimental conditions produced similar patterns of NF fragmentation, including the transient appearance of NF immunobands between Mr 150,000–200,000 and 110,000–120,000 as well as the appearance and accumulation of NF immunobands between Mr 45,000 and 65,000. Most immunoreactive NF fragments remained Triton‐insoluble. Low levels of the same immunoreactive fragments were present in control neural tissues, suggesting that calciumactivated proteolysis may be operative in the turnover and/or processing of NF proteins in vivo. Very similar patterns of NF degradation during experimental calcium influxes into different CNS and PNS tissues are indicative of the widespread distribution of calcium‐activated NF protease in neural tissues.

Uptake and binding of calcium by axoplasm isolated from giant axons of Loligo and Myxicola.

1. Axoplasm isolated from giant axons of the squid Loligo and of the polychaete worm Myxicola continues to bind Ca and maintain an ionized Ca concentration close to 0.1 microgram which is similar to that seen in intact axons. 2. Injection of Ca into isolated axoplasm only produces a transient rise in ionized Ca showing that axoplasm can buffer a Ca challenge. 3. In order to characterize the Ca‐binding systems isolated axoplasm was placed in small dialysis tubes and exposed to a variety of artificial axoplasms containing 45Ca. 4. In the presence of ATP, orthophosphate and succinate, Ca uptake appreciable and after 4 hr exposure of Loligo axoplasm to 0.1 microgram‐Ca, approximately 100 mumole Ca/kg axoplasm was bound. Binding could be divided operationally into two distinct processes, one that requires ATP or succinate togeth with orthophosphate and is blocked by cyanide and oligomyocin, and one that is unaffected by these reagents. 5. Energy‐dependent binding has a large capacity, but a rather low affinity for Ca, being half‐maximal between 20 and 60 microgram‐Ca. In Loligo, its properties closely parallel those of a crude mitochondrial preparation isolated from axoplasm; but there are some interesting differences in Myxicola. Energy‐independent binding is half‐maximal at ionized Ca concentrations between 80 and 160 nM but is readily saturated and has a capacity of 6‐60 mumole/kg axoplasm. 6. Ca binding by Loligo is greatest in media containing roughly physiological concentrations of K and is reduced by isosmotic replacement of K by Na. This effect seems to be confined to the energy‐dependent, presumed mitochondrial, component of binding. 7. Ca binding by Loligo axoplasm is reduced by both La and Mn ions.

An immunohistochemical study of human central and peripheral nervous system tumors, using monoclonal antibodies against neurofilaments and glial filaments

Monoclonal antibodies that recognize either neurofilaments or glial filaments were used with the peroxidase-antiperoxidase (PAP) method to retrospectively study 100 tumors of the central and peripheral nervous systems in paraffin-embedded sections. Only neoplasms of putative neuronal origin or with presumed neuronal differentiation (paraganglioma, ganglioglioma, ganglioneuroblastoma, ganglioneuroma, neuroblastoma, ovarian teratoma and pheochromocytoma) contained tumor cells with immunoreactive neurofilament, but such cells were more common in the more differentiated or benign neoplasms in this category. Glial filament immunoreactivity was observed in tumor cells of glial origin and in tumor cells with foci of glial differentiation arising within the central nervous system, consistent with findings from previous studies using anti-glial-filament antisera. With the exception of a benign cystic teratoma, no glial filament immunoreactivity was observed outside the central nervous system. Some immunoreactive neurofilaments, but not glial filaments, were arranged in presumably abnormal balls, cords, or clumps within tumor cells, possibly reflecting cytoskeletal alterations related to neoplastic transformation. These findings indicate that monoclonal antibodies against intermediate filament proteins such as neurofilaments and glial filaments retain their specificity and sensitivity when employed in paraffin sections in conjunction with the peroxidase-antiperoxidase method. They suggest that such reagents are useful probes for the evaluation of the histogenesis or degree of differentiation in human nervous system tumors. Finally, they permit the speculation that the analysis of the intermediate filaments of tumor cells, as contrasted with those in normal cells, may provide new insights into the biology of neoplasms.

An Immunoperoxidase Study of Senile Cerebral Amyloidosis with Pathogenetic Considerations

Samples of human cerebral cortex were obtained from twelve autopsied patients with Alzheimers disease or “normal” aging. Rabbit or goat anti-human antisera to the following plasma proteins: IgG, F(ab‘)2, Fc, kappa and lambda light chains, IgM, IgA, fibrinogen, albumin, C3, lysozyme, haptoglobin, macroglobulin, and microglobulin; antibodies to the following intracellular proteins: glial fibrillary acidic (GFA) protein, filamin, actin, non-muscle myosin, tubulin, cholinergic vesicle proteins, and neurofilament (NF) proteins were utilized in the immunoglobulin peroxidasc bridge. Amyloid cores of classical or perivascular plaques and dyshoric angiopathy exhibited a strong reaction for intact IgG and for both of its light chains, moderate reactions for lysozyme, fibrinogen, albumin and IgA, and weak reactions for IgM, C3, Fc, F(ab’)2, haptoglobin, macroglobulin and microglobulin. Antibodies to all three NF proteins, individually and pooled, stained dyshoric and plaque amyloid, while antibodies to other intracellular proteins did not. The coronae of classical plaques and many primitive plaques stained for GFA, but inconsistently for IgG, both light chains, lysozyme, actin, tubulin, and NF proteins. Affected vessels of three patients with Congophilic angiopathy were reactive for all plasma proteins (especially IgG, fibrinogen, and albumin) and for NF proteins. NF staining in Congophilic blood vessels, although variable, revealed a peripheral or adventitial distribution, whereas plasma proteins tended to be localized in the media of the vessel wall. The distributions of Congo red and NF positivity were often identical. Both NF and Congo red staining was sensitive to oxidation. Isolated NF proteins were Congophilic and capable of displaying apple-green birefringence. A hypothesis concerning the role of NF proteins in senile cerebral amyloid is presented.