Biomedical subjects

M J Carden

Publications and source records attributed to M J Carden.

29 records · Page 2Linked to original sources

PubMed1987

Studies of neurofilaments that accumulate in proximal axons of rats intoxicated with beta,beta'-iminodipropionitrile (IDPN).

The paradigm of IDPN neuropathy was produced in rats in order to examine the neurofilaments (NFs) that accumulate in the proximal motor and sensory axons of intoxicated animals, and to compare the aggregated NFs with control NFs and with the depleted populations of NFs in the distal portions of the same experimental nerves. NFs were probed biochemically and histochemically, using a large and well-characterized library of monoclonal antibodies that included antibodies that are monospecific for each of the rat NF protein subunits (NF-H, NF-M, and NF-L) as well as antibodies that recognized differential phosphorylated states of rat NF-H and NF-M. All antibodies tested showed enhanced immunostaining of enlarged axons and of large spheroids in the spinal cord and dorsal root ganglia of experimental animals. Biochemical analyses of IDPN-treated animals revealed enrichment of NF-H, NF-M, and NF-L in homogenates of dorsal root ganglia and of proximal motor and sensory nerve roots as well as depletion of the three subunits in distal nerve roots and in sciatic nerves. Immunoblot revealed a uniform enrichment of NF-H, NF-M, and NF-L in NF aggregates as well as the same admixture of phosphorylated and dephosphorylated epitopes of NF-H and NF-M in experimental and in control tissues. The global increase of immunoreactivity in axonal swellings to antibodies that react with phosphorylated, nonphosphorylated, and phosphorylation-independent NF epitopes suggests that IDPN induces an accumulation of NFs in proximal axons without necessarily altering the state of NF phosphorylation.

Animals↗

PubMed1987

Phosphate dependent and independent neurofilament epitopes in the axonal swellings of patients with motor neuron disease and controls.

Axonal accumulations of neurofilaments (NFs) may result from abnormalities intrinsic to NF subunits (e.g., proteolytic cleavage, altered phosphorylation), or from abnormalities extrinsic to NFs that retard the transport of these neuron-specific intermediate filaments. To evaluate this hypothesis, we probed the NF-rich axonal swellings seen in normal spinal cords, "globules", and the larger ones, "spheroids", seen in the spinal cords of patients with motor neuron disease by using a library of monoclonal antibodies that recognize human NF proteins. These monoclonal antibodies discriminate different phosphorylation states of individual NF subunits. The NF protein determinants of globules and spheroids were similar to each other and to the determinants that predominate in normal spinal cord axons. NF protein-positive inclusion bodies were only seen in the anterior horn cells of one patient with motor neuron disease, and they contained NF protein determinants similar to those normally expressed in perikarya. Thus, the NFs in globules and spheroids appear to be derived from axonal NF proteins, and both kinds of axonal swellings may arise by similar mechanisms. Although our data do not exclude a structural defect in NF proteins to account for the accumulation of NFs in these axonal swellings, the hypotheses being advanced to explain the formation of NF-rich globules and spheroids based on intrinsic alterations of NF proteins must consider that the immunological integrity of disparate NF protein epitopes in different states of phosphorylation is retained in both globules and spheroids.

Adolescent↗

PubMed1987

Phosphorylation of neurofilament proteins and chromatolysis following transection of rat sciatic nerve.

States of phosphorylation of neurofilament proteins were examined in the perikarya of rat sensory and motor neurons between 3 and 28 d following either a distal transection [6-7 cm from the L4-L5 dorsal root ganglia (DRG)] or a proximal transection (1-2 cm from the L4-L5 DRG) of the sciatic nerve. Paraffin sections of the right (experimental) and left (control) L4 and L5 DRG from animals with unilateral transection of the right distal sciatic nerve were stained immunocytochemically with monoclonal antibodies to phosphorylation-dependent (NF-P), dephosphorylation-dependent (NF-dP), or phosphorylation-independent (NF-ind) epitopes on the largest (NF200), mid-sized (NF150), or smallest (NF68) neurofilament protein subunits. Increased immunoreactivity to NF-P on NF200 and NF150 was detected in experimental DRC at 10 d, peaking by 20 d, and declining to near control levels by 28 d. Conversely, immunoreactivity to NF-dP declined in experimental DRG beginning at 6 d, reaching a maximum decline at 10-16 d, and returning to near control levels by 28 d. Immunocytochemical changes were confirmed with biochemical studies on tissue homogenates that demonstrated an increase of immunoreactivity to NF-P and a decrease of reactivity to NF-dP in the experimental DRG. Changes in immunoreactivities to NF-P and NF-dP were observed only in the perikarya of large neurons and were closely associated with chromatolytic changes in these neurons. Marked enhancement of chromatolysis, as well as the immunoreactivities to NF-P and NF-dP, occurred following a proximal (left side) versus distal (right side) transection in the same animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats.

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns.

Monoclonal antibodies (mAbs) to rat neurofilament (NF) proteins NF-L, NF-M, and NF-H were used to examine the developmental programs of NF expression in rat embryos. The ability of these mAbs to recognize differentially phosphorylated states of NF-M and NF-H (Lee et al., 1987, the preceding paper) was exploited in order to examine the temporal and spatial patterns of NF phosphorylation during early neuronal development in vivo. NF proteins were first detected on the twelfth day postfertilization (E12) using NF-L- or NF-M-specific mAbs. By E13, the coexpression of NF-L and NF-M was widespread, reflecting dramatic increases of immunoreactivity to both subunits. Partial phosphorylation, denoted P[+], of NF-M was already present in perikarya and neurites of E12 neurons. Extensively phosphorylated, or P[+++], isoforms of NF-M appeared in E13 axons, thereby establishing a proximodistal gradient of NF phosphorylation during the earliest phase of NF expression. Immunoblots of tissue homogenates revealed that most NF-M of E13 embryos exists in a partially phosphorylated, or P[+], isoform. Unequivocal staining for NF-H first appeared at E15, a time at which NF-L and NF-M had already attained their adult patterns of immunocytochemical staining. Levels of NF-H were extremely low at E15 but could be detected in all of its differentially phosphorylated states, i.e., nonphosphorylated P[-], partly P[+], and highly P[+++] phosphorylated isoforms. P[+++] isoforms of NF-H were restricted to the distal portions of E15 axons, although staining of more proximal axons, like those in adult, was noted by E17. Immunoblots of E17 embryos revealed most NF-H as P[-] and P[+] isoforms. Quantities of immunoreactive NF-H increased very slowly and remained well below those of NF-M and NF-L for several weeks beyond birth. These results show that sequential forms of NFs are expressed by developing and maturing neurons throughout the nervous system. An "immature" form of NFs, composed of NF-M and NF-L, appears to function in establishing the neuronal phenotype and in initiating and maintaining neurite outgrowth. Addition of NF-H confers a "mature" state to the NF. This delayed expression of NF-H is a slow and graduated process that coincides in time with the stabilization of neuronal circuitries and may be important in modulating axonal events, such as the slowing of cytoskeletal transport and the growth of axonal caliber.

Animals↗

PubMed1986

Chemical cross-linking analyses of ox neurofilaments.

Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among 'rod-domain' fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.

Animals↗

PubMed1986

2,5-Hexanedione neuropathy is associated with the covalent crosslinking of neurofilament proteins.

Protein composition in different segments along nerves from rats intoxicated with 2,5-hexanedione (HD) was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by immunoblotting, using monoclonal antibodies specific for each of the three neurofilament polypeptide components (H, M, and L). Comparison with nerve protein extracts from normal (control) rats revealed a disappearance of the largest neurofilament polypeptide (H), accompanied by accumulation of higher-molecular-weight products that were immunoreactive with H-specific antibodies. Both the extent of this crosslinking and its localization in particular portions of peripheral nerves showed a correlation with HD dosage and with the known progression of ultrastructural features during HD-induced neuropathy. Similar changes were not detected for the M and L neurofilament components.

Animals↗

PubMed1986

Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit.

We have obtained five monoclonal antibodies to the Mr 200,000 neurofilament component (NF200) after immunization with polypeptides purified from enzymatically dephosphorylated bovine neurofilaments. In immunoblots of untreated neurofilament protein and protein from filaments exposed to phosphatase, these antibodies recognize nonphosphorylated or dephosphorylated, but not phosphorylated, forms of NF200. The epitopes recognized by these new monoclonal antibodies reside in the carboxyterminal domain of the NF200 polypeptide as defined by immunoreaction with limited chymotryptic fragments. Immunohistochemical studies of bovine cerebellum, spinal cord, trigeminal ganglion, and trigeminal nerve with these new monoclonal antibodies demonstrate immunoreactivity primarily in neuronal perikarya; axons and dendrites are weakly or infrequently immunostained. After enzymatic dephosphorylation of these tissues, a more extensive distribution of immunoreactivity is seen, especially in axons and dendrites. Immunostaining of cultured rat sympathetic neurons is restricted to cell bodies. These data provide evidence for the in situ existence of NF200 epitopes that are not phosphorylated in some classes of neurons or regions of a neuron, but are modified by phosphorylation in other neurons or neuronal domains. These new monoclonal antibodies are distinctly different from those in a large library (over 100) raised to, and specific for, phosphorylated neurofilament proteins. They are novel tools for probing neurofilament distribution, metabolism, structure, and possibly function.

Animals↗

PubMed1986

Structural similarities and differences between neurofilament proteins from five different species as revealed using monoclonal antibodies.

Neurofilament (NF) proteins isolated from human, rabbit, rat, and chicken spinal cord white matter were immunoblotted with monoclonal antibodies (MAbs) raised to bovine NF immunogens. The aim of these experiments was to test the degree of epitope conservation among NF proteins from different vertebrate species. In so doing, the validity of using antibodies raised to NF from one species (cow) for detecting NF proteins of other species was also tested. The MAbs used for these experiments were characterized using bovine NFs to define polypeptide specificity, the approximate location of epitopes within each NF polypeptide, and the effects on antibody recognition of the presence or absence of phosphorylated residues in these polypeptides (see Carden et al., 1985). Our findings indicate that epitopes located in the alpha-helical core domains of NF-H, NF-M, and NF-L are distinct from each other, yet are strongly conserved among the different species. Epitopes located in the noncore or peripheral domains of the NF polypeptides show variable degrees of cross-species preservation. For example, all epitopes in the peripheral domains of bovine NF-H that require the presence of phosphate groups for recognition (phosphorylation dependent) are widely expressed in all species studied. Many phosphorylation-dependent epitopes of bovine NF-H are also present in bovine NF-M, as well as in NF-H of non-bovine species. In addition, epitopes in the peripheral domain of NF-H that require dephosphorylation of NF polypeptides for recognition (dephosphorylation dependent) are also found in NF-H of other mammalian species but not in NF-H of chicken.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

The structure, biochemical properties, and immunogenicity of neurofilament peripheral regions are determined by phosphorylation state.

Treatment of freshly isolated, bovine neurofilaments with Escherichia coli alkaline phosphatase removes over 90% of the phosphate groups from serine residues of the Mr 200,000 and 150,000 polypeptide components (NF200 and NF150). Dephosphorylated NF200 and NF150 remain associated with filaments, but migrate in sodium dodecyl sulfate gels with reduced apparent molecular weights. Unusual migration appears to be due to modification at regions of these polypeptides that are peripheral to the neurofilament backbone as defined by limited chymotryptic digestion. Over 90 monoclonal antibodies recognizing epitopes located within the peripheral domain of native NF200 all show reduced affinity for dephosphorylated NF200. A single monoclonal antibody binds within the filament-associated domain of NF200 and its recognition of NF200 is unaffected upon treatment of neurofilaments with phosphatase. Around 50% of our monoclonal antibodies that bind NF150 monospecifically and at epitopes within its peripheral domain have reduced affinities for NF150 from phosphatase-treated filaments, while the remaining 50% bind native and dephosphorylated NF150 equally well. The smallest neurofilament component (NF70) contains few phosphate groups, most of which remain after treatment of neurofilaments with phosphatase. The resulting form of NF70 migrates normally in gels and its recognition by antibodies is unchanged. We conclude that phosphorylation modifies the structure of the two larger neurofilament polypeptides along domains that are peripheral to the filamentous backbone and that these effects are more pronounced for NF200 than for NF150.

Alkaline Phosphatase↗

PubMed1983

Neurofilaments from ox spinal nerves. Isolation, disassembly, reassembly and cross-linking properties.

An isolation procedure for neurofilaments from ox spinal nerves is described where the triplet polypeptides (which have molecular weights of 205 000, 158 000 and 72 000) constitute more than 80% of the preparation. Soon after purification, the neurofilaments form a gel that is stable for many weeks. The purified neurofilaments disassemble in low-salt buffers at pH greater than 7.0 into soluble particles that contain all of the triplet polypeptides. Greater than 90% of the protein can reassemble to form filaments. The thiol-containing residues in the filaments can be cross-linked. Analyses of the complexes formed show that in the filament the 205 000-mol.wt. components are arranged to that they can be cross-linked to themselves and to the 158 000-mol.wt. polypeptides, and that the 72 000-mol.wt. components are arranged so that their thiol groups can be cross-linked together.

Animals↗