Alzheimer's disease antibodies bind specifically to a neurofilament protein in Torpedo cholinergic neurons

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and neuritic plaques and by the degeneration of central cholinergic neurons. Recent studies indicated the presence of antibodies in the sera and cerebrospinal fluid of AD patients which react with neuronal tissue and which recognize cholinergic neurons. In order to identify the cholinergic antigens against which the AD antibodies are directed, we have recently used the purely cholinergic electromotor neurons of the electric fish Torpedo which are chemically homogenous and cross-react antigenically with mammalian cholinergic neurons. This study revealed that immunoglobulins (IgG) from sera of AD patients bind specifically to an antigen in Torpedo electromotor neurons with an apparent molecular weight of 200 kDa. In the present report we attempt to characterize this antigen. The similarity in size of this protein to that of the heavy neurofilament subunit (NF-H) and the association of neurofilaments with plaques and tangles prompted us to examine the possibility that it is a neurofilament protein. Our findings show that IgG from sera of AD patients bind to the NF-H protein of Torpedo cholinergic neurons. Comparison of the binding of AD and control IgG to Torpedo cholinergic NF-H revealed that AD IgG bind to this neurofilament protein more readily than do control IgG. In contrast, AD and control IgG bind similarly to NF-H obtained from the chemically heterogenous Torpedo spinal cord and rat brain. These findings suggest that AD sera contain a repertoire of anti-NF-H IgG and that a subpopulation of these antibodies whose levels are significantly elevated in AD binds to epitopes highly enriched in Torpedo cholinergic NF-H.(ABSTRACT TRUNCATED AT 250 WORDS)     

Family of human neuronal external surface epitopes defined by Torpedo monoclonal antibodies.

We are employing a library of monoclonal antibodies (MAbs) that were made to Torpedo cholinergic synaptosomes to identify conserved, physiologically vital epitopes of the neuronal surface. Our particular interest is in those epitopes that are present on some but not all neurons. In the present study we screened this library on different cell lines, the neuronal cell lines PC12, NG108, MC-IXC, and SY5Y, and the endocrine cell lines GH-3 and HIT. Of these cell lines, only SY5Y cells bind MAbs that define neuronal surface subsets. Utilizing its parent cell line, SK-N-SH, we verified that six MAbs, Tor 25, Tor 103, Tor 190, Tor 201, Tor 219, and Tor 233, bind the external neuronal surface. The cytolocalization of all six MAbs is very similar: the membrane of the cell body and its processes are finely outlined in a punctate distribution. Western blot analyses of Torpedo electric organ homogenates, a highly enriched source of antigenic material, revealed that each MAb identifies multiple polypeptides, two of which have the relative mobilities of 180 kD and 67 kD. In a screen of peripheral nerves from cases of amyotrophic lateral sclerosis (ALS), we found that all these MAbs revealed surface alterations; some displayed a decrease in binding, while others displayed an increase. The combined data provide evidence that these epitopes belong to an important, complex family of polypeptides of the external neuronal surface.     

Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.     

Dynamics of mammalian high-molecular-weight neurofilament subunit phosphorylation in cultured rat sympathetic neurons.

To better understand the function(s) of the multiphosphorylation repeat (MPR) of the high molecular weight neurofilament (NF) subunit (NF-H), we sought to determine how phosphorylation within this region is regulated in cultured rat sympathetic neurons. To do this, monoclonal antibodies specific to phosphorylated or nonphosphorylated tandem repeats of the amino acid sequence Lys-Ser-Pro-Ala-Glu-Ala found within the MPR were identified and used to determine the extent of phosphorylation, the time course of phosphorylation, and the rate of turnover of phosphate groups within the NF-H MPR in cultured sympathetic neurons. We showed that (1) the synthesis and phosphorylation of NF-H occurs in these neurons cultured for 1 or 4 weeks; (2) the conversion from poorly to more highly phosphorylated variants of NF-H occurs slowly in cultured neurons; and (3) the turnover of phosphate groups on both poorly and highly phosphorylated variants of NF-H occurs more rapidly than the turnover of NF-H itself. In addition, we showed that the 200-kD highly phosphorylated NF-H can contain at least five consecutive nonphosphorylated tandem repeats as well as phosphorylated tandem repeats, whereas we were unable to detect consecutively phosphorylated tandem repeats in the 160-kD form of NF-H. These findings allow us to propose a mechanism whereby NF-H is initially phosphorylated singly at sites distributed throughout the MPR. This "poorly phosphorylated NF-H," which is the predominant form in immature neurons, is then converted by phosphorylation at additional sites within the MPR to the more "highly phosphorylated" species of NF-H typically found in mature neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity of human anti-neurofilament autoantibodies

The specificities and isotypes of human antibodies that react with neurofilament (NF) proteins were examined by Western blot analysis. Two-thirds of the subjects tested had antibodies to the 200 kDa high molecular weight neurofilament protein (NF-H), and fewer had antibodies to the low and middle molecular weight neurofilament proteins (NF-L and NF-M respectively). Human autoantibodies bound to both native and dephosphorylated NF-H, but some antibodies bound to dephosphorylated NF-H only, indicating the presence of at least two target epitopes. They also recognized a fusion protein containing a segment of the NF-H protein produced by a cDNA clone in Escherichia coli, indicating that they bind to unmodified peptide epitopes. The anti-NF-H antibodies were mostly IgG, but were frequently complexed to IgA or IgM antibodies, possibly with rheumatoid factor or anti-idiotypic activity. These characteristics of anti-NF-H antibodies are most consistent with a secondary immune response that is antigen driven and T-cell dependent.     

Differential subcellular localization of phosphorylated neurofilament and tau proteins in degenerating neurons of the human entorhinal cortex

A panel of novel monoclonal antibodies was tested on the human entorhinal cortex for the recognition of age- and disease-related changes of neurofilament proteins (NF). Several antibodies identified phosphorylated NF-H subunit, which occurred preferentially in those aged between 60 and 80 years and were localized in degenerating neurons. Such neurons also contained neurofibrillary tangles, but neurofilament aggregates did not co-localize with tangles, nor did the quantity nor the number of NF-positive neurons correlate with the severity of Alzheimer's disease. This points to a susceptibility of NF in a subset of neurons for phosphorylation- and metabolically related morphological changes during neurodegeneration.     

Cyclin dependent kinase-5 (CDK-5) phosphorylates neurofilament heavy (NF-H) chain to generate epitopes for antibodies that label neurofilament accumulations in amyotrophic lateral sclerosis (ALS) and is present in affected motor neurones in ALS.

1. Accumulations of phosphorylated neurofilaments are seen in affected motor neurones in amyotrophic lateral sclerosis (ALS). 2. The authors demonstrate that cyclin dependent kinase-5 (cdk-5) will induce cellular phosphorylation of neurofilament heavy chain side-arms to generate epitopes for several antibodies that label these neurofilament accumulations. 3. By creating recombinant neurofilament fragments of NF-H side-arm domains, the authors also map the epitopes for several of these antibodies. 4. Finally, the authors demonstrate that cdk-5 is also present in affected motor neurones in ALS. These studies implicate cdk-5 in the pathogenesis of ALS.     

Phosphorylation-dependent epitopes on neurofilament proteins and neurofilament densities differ in axons in the corticospinal and primary sensory dorsal column tracts in the rat spinal cord.

The highest molecular weight neurofilament protein (NF-H) is multiply phosphorylated at epitopes which can be distinguished by specific monoclonal antibodies on Western blots. Eight characterized antibodies were used in immunocytochemistry to examine the tissue distributions of phosphorylated variants of NF-H in axons of the adult rat spinal cord. The most striking difference in staining was found between axons in the cuneate tract and those in the neighboring dorsal corticospinal tract. Axons in the cuneate tract reacted intensely with antibodies to phosphorylated epitopes of NF-H and poorly with antibodies to dephosphorylated epitopes of NF-H, whereas the reverse was the case for the axons of the dorsal corticospinal tract. These differences showed that systematic variations in the phosphorylation of NF-H in long-tract axons in the central nervous system occur as a function of cell type. When the cytoskeletons of these axons were compared by electron microscopy, the neurofilaments of the cuneate fibers were seen to be more abundant and formed a latticework, more compactly organized than the neurofilaments of the dorsal corticospinal axons. By comparison, the dorsal corticospinal axons were relatively richer in microtubules than the cuneate axons. Although the cuneate fiber tract contained many more large (greater than 2.0 microns 2 in cross section) axons than did the dorsal corticospinal tract, these differences in cytoskeletal organization were apparent even when myelinated axons of similar sizes (0.4 micron 2 to 2.0 microns 2) were compared. In addition, the number of neurofilaments in cuneate axons in the 0.4 to 2.0 microns 2 size range was significantly better correlated with axon size than was the case for this size range of dorsal corticospinal axons. Thus, the differences seen in the organization of the neurofilament latticework and the phosphorylation of NF-H between axons found in these two tracts both appeared to be correlated with cell type, and were independent of length or caliber of the axons.     

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)     

Normal neuronal cell bodies of the nucleus tractus mesencephalici nervi trigemini react with antibodies against phosphorylated epitopes on neurofilaments

Reactivity to antibodies directed against phosphorylated neurofilament epitopes is normally present in axons. Phosphorylated neurofilament immunoreactivity is not generally found in neuronal perikarya, except in abnormal states such as Alzheimer's disease. We found that cytoplasm of neurons of the nucleus tractus mesencephalici nervi trigemini in normal mice and rats reacts with monoclonal antibodies against phosphorylated epitopes on neurofilaments. This suggests either that phosphorylated epitopes on neurofilaments are localized in the perikarya of some normal neurons or that the antibodies that were used (SMI 31 and SMI 34) recognize more than phosphorylated epitopes.     

Neuroimmunology of myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disease in which the immune system attacks the nicotinic acetylcholine receptors (AChRs) of the neuromuscular junction. Anti-AChR antibodies are present in 85% of patients and bind to distinct epitopes on the surface of the AChR alpha subunits, as defined by competition with monoclonal anti-AChR antibodies. There are at least three types of the disease, defined by thymic histology, age of onset, and HLA associations, and anti-AChR antibodies show some differences in fine specificity between those with thymic hyperplasia and those with thymic tumors. Peripheral blood lymphocytes from MG patients contain T lymphocytes specifically sensitized to AChR. These are stimulated by purified Torpedo AChR and some human alpha subunit synthetic peptides. The T and B cell epitopes on the primary sequence of the alpha subunit are currently being mapped using recombinant human AChR subunit fragments.     

Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form

A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.     

Individual neurofilament subunits reassembled in vitro exhibit unique biochemical, morphological and immunological properties

Purified bovine neurofilament (NF) subunit proteins were reassembled in vitro to form either homopolymeric or heteropolymeric intermediate-sized filaments using single or paired combinations of NF triplet proteins. Using conditions established for the reassembly of bovine NF triplet proteins, we demonstrated that the low Mr NF subunit (NF-L) alone and in combination with the middle Mr NF subunit (NF-M) reassembled very efficiently, i.e. greater than 95% of these proteins formed filaments within 90 min from the start of reassembly. In contra-distinction, the high Mr NF subunit (NF-H) alone and in combination with NF-M or NF-L underwent reassembly to a lesser extent, i.e. 62-88% of these proteins reassembled within 90 min. Immunolabeling of the reassembled NF polymers revealed striking differences in the organization of rod domain determinants. Specifically, antibodies specific for epitopes in the rod domains of NF-H, NF-M and NF-L failed to bind heteropolymeric filaments but recognized rod domains in the homopolymers. In contrast, antibodies specific to head and tail domains of all NF proteins labeled the reassembled hetero- and homopolymeric NFs. Double-labeling of heteropolymers demonstrated that pairs of different NF subunits coassembled into intermediate-sized filaments. Our results also showed that only copolymeric filaments of NF-L and NF-M, but not NF-L/NF-H and NF-M/NF-H were able to form long and stable 10-nm wide filaments. These observations provide new insights into the requirements for stable filament formation from NF subunits. In particular, they support the notion that only NF-L/NF-M, but not NF-L/NF-H or NF-M/NF-H might assemble into a stable filamentous network in vivo.     

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)     

Deposition of detergent-resistant neurofilaments into Lewy body fibrils

To assess the contribution of neurofilaments (NF) to the detergent-resistant cortical Lewy body (LB) fibril we extracted LBs from Diffuse LB diseased brains and used monoclonal antibodies to probe Western transfers of solubilized LB-derived protein. Antibodies to epitopes located in the COOH-termini of the 200- and 170kDa NF subunits (NF-H and NF-M) labelled LB proteins corresponding to full length and partially truncated or variably phosphorylated NF-H and NF-M. LB-derived protein at approximately 7-kDa did not contain epitopes detected by monoclonal antibodies to NF-L, τ or the COOH-termini of the NF-H and NF-M. We conclude that NF-H and NF-M are incorporated as integral insoluble components of the cortical LB fibril.     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Triton-soluble phosphovariants of the heavy neurofilament subunit in developing and mature mouse central nervous system

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100–insoluble cytoskeleton. The dynamic nature of these subunit associations in vivo remains unresolved, and the applicability of this assembly model to NFs in other neuronal compartments or to developing neurons is unknown. Here, we report that a unique pool of Triton X-100–soluble, extensively phosphorylated, high molecular weight NF subunits (NF-H, or H-200) are abundantly expressed in the mouse CNS during early postnatal development and persist in the perikaryal compartment of some mature neurons. Triton-soluble H-200 subunits appeared at postnatal day 14 (P14) and remained high through P60, beyond which the percentage declined to marginal levels by P120. Medium and low molecular weight NF (NF-M and NF-L, respectively) were at all times only detectable within the cytoskeleton. Comparison of soluble and cytoskeleton-associated H-200 immunoreactivity indicated that certain phosphorylation-dependent epitopes were confined to the cytoskeleton. Pulse-chase radiolabeling analyses in optic pathway demonstrated that some Triton-soluble NF-H subunits are extensively phosphorylated within retinal perikarya before they are incorporated into Triton-insoluble structures. These findings indicate that the assembly behaviors of NF-H differ substantially from those of NF-M and NF-L, and that the interaction of NF-H with NFs may be more dynamic than is generally recognized, especially during brain development and within specific compartments of mature neurons. J. Neurosci. Res. 48:515–523, 1997. © 1997 Wiley-Liss Inc.     

Neurofilament antibodies and spiral ganglion neurons of the mammalian cochlea

The spiral ganglia of the cat, gerbil, mouse, rat, and human were immunohistochemically stained with various monoclonal neurofilament antibodies. Three antibodies to the 200-kD neurofilament protein (R-3, Dr?ger et al., '84; ICN anti-200, clone NE14, Debus et al., '83; RT-97, Wood and Anderton, '81) labeled the somata of type II spiral ganglion neurons but not those of type I ganglion neurons. In the extreme base of the cochlea of cats, mice and rats, there was intense labeling of a few (less than 0.5% of the total ganglion population) large neurons resembling type I ganglion neurons. Several other neurofilament antibodies (Amersham anti-68, Amersham and ICN anti-160, and SMI-32) did not specifically label type II ganglion neurons but instead labeled all neurons of the spiral ganglion. These two patterns of labeling prompted us to investigate the cause for this difference. Because antibodies against the 200-kD neurofilament protein preferentially labeled type II neurons and because 200-kD neurofilament is highly phosphorylated, we treated cochlear tissue with alkaline phosphatase in order to remove phosphate groups. This treatment eliminated the intense labeling of type II neurons with R-3, ICN anti-200, and RT-97, but had no effect on the intense labeling of ganglion cell bodies observed with the other neurofilament antibodies tested. This evidence suggests that labeling occurs because of the cytoplasmic presence of phosphorylated 200-kD neurofilament protein in type II ganglion neurons. Populations of neurons may thus differ in their neurofilament epitopes and monoclonal antibodies can be used to mark such differences.     

Epitopes located in spatially separate domains of each neurofilament subunit are present in Parkinson's disease Lewy bodies.

Subcortical Lewy bodies are the pathological hallmark of idiopathic Parkinson's disease. This study sought to determine the extent to which each neurofilament subunit [low (NF-L), mid (NF-M), or high (NF-H)] was present in Lewy bodies by using light, confocal, and electron microscopy. A battery of 37 antineurofilament antibodies, characterized as to subunit specificity, epitope domain, and phosphorylation status, was employed to probe substantia nigra Lewy bodies from 15 Parkinson's disease cases. All 37 antibodies labelled Lewy bodies. The epitopes recognized by these antibodies included those in the NF-L rod and tail domains; the NF-M head, rod, and tail domains, as well as epitopes within, and flanking, the multiphosphorylation repeat site; and the NF-H rod domain and multiphosphorylation repeat sites. With these probes, nearly the entire length of each subunit could be demonstrated in Lewy bodies. However, the staining pattern of the Lewy bodies suggested that the tail domains of NF-M and NF-H were present in the periphery of the Lewy body core and in the Lewy body corona, but they appeared to be altered or missing in the center of the Lewy body core. In contrast, the head domain of NF-M, the tail domain of NF-L, and the rod domains of all three subunits are present throughout the Lewy body. These results strongly suggest that the entire extent of each neurofilament subunit is found in Lewy bodies but that the neurofilament subunits may be altered during the processing of these filaments into Lewy bodies.     

The protein phosphatase inhibitor okadaic acid increases axonal neurofilaments and neurite caliber,and decreases axonal microtubules in NB2a/d1 Cells

When cells were treated with dbcAMP for 3 days to induce the outgrowth of axonal neurites, the addition of the phosphatase inhibitor okadaic acid (0A; 5 nM) for the last 24 hr markedly increased neurofilament subunit immunoreactivity including phosphate-dependent NF-H epitopes in axonal neurites, increased axonal neurite caliber by approximately 30 %, but did not increase neurite contour length. Ultrastructural analysis demonstrated a >2-fold increase in neurofilaments and indicated that neurofilaments were phosphorylated to a similar extent in the presence and absence of OA. Vimentin immunoreactivity, which undergoes down-regulation during dbcAMP-mediated differentiation, was not increased by OA. OA did not induce the precocious appearance of delayed phosphate-dependent neurofilament epitopes suggesting that it did not induce the activation of additional neurofilament kinases. NF-H subunits from cytoskeletons of OA-treated cells were less susceptible to degradation by an endogenous calcium-dependent protease, providing a possible mechanism for neuro filament accumulation during OA treatment. By contrast, OA decreased axonal neurite microtubules, and eliminated stabilized (acetylated) axonal microtubules. OA treatment at earlier times prevented and reversed neurite outgrowth. Despite increased deposition of phosphorylated neurofilaments, OA did not hasten the development of colchicine resistance to neurites, suggesting that stabilization of the axonal cytoskeletal lattice requires neurofilament-microtubule interaction. © 1993 Wiley-Liss, Inc.