Infiltration of the brain by pathogens causes Alzheimer's disease.

Despite very numerous studies on Alzheimer's disease (AD), especially on amyloid plaques and neurofibrillary tangles, little information has been obtained thus on the causes of the disease. Evidence is described here that implicates firstly herpes simplex virus type 1 (HSV1) as a strong risk factor when it is present in brain of carriers of the type 4 allele of the gene for apolipoprotein E (APOE-4). Indirect support comes from studies indicating the role of APOE in several diverse diseases of known pathogen cause. A second putative risk factor is the bacterium, Chlamydia pneumoniae. This pathogen has been identified and localized in AD brain. Current studies aimed at "proof of principle" address the entry of the organism into the CNS, the neuroinflammatory response to the organism, and the role that the organism plays in triggering AD pathology. An infection-based animal model demonstrates that following intranasal inoculation of BALB/c mice with C. pneumoniae, amyloid plaques/deposits consistent with those observed in the AD brain develop, thus implicating this infection in the etiology of AD.

Functional characterization of Na(+)/H(+) exchangers in primary cultures of prairie dog gallbladder.

Gallbladder Na(+) absorption is linked to gallstone formation in prairie dogs. We previously reported Na(+)/H(+) exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I(sc), (11.6 +/- 0.5 microA. cm(-2)), potential differences, V(t) (2.1 +/- 0.2 mV), and resistance, R(t) (169 +/- 12 omega. cm(2)). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable (22)Na(+) uptake under a H(+) gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of (22)Na(+) uptake was 21.4 +/- 1.3 nmol x mg prot(-1) x min(-1), of which 9.5 +/- 0.7 (approximately 45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for approximately 6%, approximately 66% and approximately 28% of GBECs' total NHE activity, respectively. GBECs exhibited saturable NHE kinetics ( V(max) 9.2 +/- 0.3 nmol x mg prot(-1) x min(-1); K(m) 11.4 +/- 1.4 m M Na(+)). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na(+) transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.

Chlamydia pneumoniae infection promotes the transmigration of monocytes through human brain endothelial cells.

We have investigated the effects of Chlamydia pneumoniae on human brain endothelial cells (HBMECs) and human monocytes as a mechanism for breaching the blood-brain barrier (BBB) in Alzheimer's disease (AD). HBMECs and peripheral blood monocytes may be key components in controlling the entry of C. pneumoniae into the human brain. Our results indicate that C. pneumoniae infects blood vessels and monocytes in AD brain tissues compared with normal brain tissue. C. pneumoniae infection stimulates transendothelial entry of monocytes through HBMECs. This entry is facilitated by the up-regulation of VCAM-1 and ICAM-1 on HBMECs and a corresponding increase of LFA-1, VLA-4, and MAC-1 on monocytes. C. pneumoniae infection in HBMECs and THP-1 monocytes up-regulates monocyte transmigration threefold in an in vitro brain endothelial monolayer. In this way, C. pneumoniae infection in these cell types may contribute to increased monocyte migration and promote inflammation within the CNS resulting from infection at the level of the vasculature. Thus, infection at the level of the vasculature may be a key initiating factor in the pathogenesis of neurodegenerative diseases such as sporadic AD.

Association between Sézary T cell-activating factor, Chlamydia pneumoniae, and cutaneous T cell lymphoma.

Sézary T cell-activating factor (SAF) was originally defined as an inducer of functional interleukin-2 (IL-2) receptors on normal and malignant T cells in patients suffering from Sézary syndrome. In fact, a combination of SAF and IL-2 stimulated the propagation of T cell lines from the peripheral blood mononuclear cells (PBMC) of those patients, with approximately one third of those cell lines containing the predominant malignant clone as determined via cytogenetic and/or T cell receptor gene rearrangement analysis. Although the primary source of SAF was mitogen-stimulated PBMC of a patient with Sézary syndrome, we were unable to isolate the gene encoding SAF from eukaryotic libraries. However, we observed SAF activity in the cytoplasm of one of the malignant cell lines in a complex containing RNA and DNA. This observation led us to consider the possibility that SAF is not of eukaryotic origin. Intracellular pathogens replicate in the cytoplasm of host cells and contain proteins, DNA, and RNA. Using a panel of antichlamydial antibodies with confirmation from polymerase chain reaction primers, we found that most patients with mycosis fungoides were positive for these determinants. Immunoelectron microscopy and protein blotting further confirmed antibody reactivity. We showed that Chlamydia pneumoniae were capable of infecting normal human keratinocytes in culture. We also demonstrated that C. pneumoniae antigen expression was associated with active disease because these determinants were not expressed after psoralen and ultraviolet A therapy. We hypothesize that chronic infection by C. pneumoniae leads to expansion of C. pneumoniae-specific T cells, thereby potentiating the development of cutaneous T cell lymphoma.

Role of infection in Alzheimer's disease.

Alzheimer's disease (AD) is a chronic condition in which inflammation has been shown to contribute to neurodegeneration. Current thinking suggests that deposition of beta-amyloid in the brain promotes inflammation resulting in neuronal damage/death. Alternatively, our data suggest that chronic inflammation observed in late-onset sporadic AD may be stimulated by infection with the obligate, intracellular bacterium, Chlamydia pneumoniae. Our results indicate that C. pneumoniae is found in high frequency in glial cells in areas of neuropathology within the brains of patients with AD. Based on our evidence, nervous system infection with C. pneumoniae should be considered a risk factor for sporadic AD.

Transglutaminase-catalyzed formation of Alzheimer-like insoluble complexes from recombinant tau.

Alzheimer's disease (AD) is a progressive neurodegenerative disease in which abnormal filamentous inclusions accumulate in dystrophic and dying nerve cells. These inclusions have been described as neurofibrillary tangles (NFTs) of which paired helical filaments (PHFs) are the primary constituents (1-3). The PHFs primarily are composed of the microtubule-associated protein tau, which has undergone posttranslational modification such as phosphorylation (4,5), glycation (6-9), and crosslinking by transglutaminase (TGase) (10-16). Crosslinking of proteins catalyzed by TGase results in the deposition of these proteins into insoluble matrices that are resistant to proteolytic digestion and chaotropic denaturation (for review see ref. 17). In this regard, TGase has been demonstrated to be associated with NFTs from the Alzheimer brain (13,14) and to exhibit elevated activity in the AD brain as compared with normal aged-matched control subjects (16). Here we discuss important aspects of TGase and in vitro experimental approaches that address its ability to catalyze the tau protein into insoluble complexes exhibiting biophysical and immuno-logical properties similar to those of the Alzheimer PHFs and NFTs.

Sézary T-cell activating factor is a Chlamydia pneumoniae-associated protein.

We previously identified a protein that was stimulatory for malignant Sézary T cells, termed Sézary T-cell activating factor (SAF). However, the identity of this protein has not been fully elucidated, nor has it's role been determined in the pathogenesis of cutaneous T-cell lymphoma (CTCL). The basis for epidermotropism and proliferation of malignant cells in the skin of patients with CTCL is unknown. Using a monoclonal antibody inhibitory for SAF activity, we demonstrated that SAF is present in the skin of 16 of 27 samples from patients with mycosis fungoides, the predominant form of CTCL. In this report, the SAF determinant is demonstrated to be associated with Chlamydia pneumoniae bacteria by immunohistochemistry, immunoelectron microscopy, and culture analysis. Reactivity of antibodies against an outer membrane protein of C. pneumoniae or against the lipopolysaccharide of Chlamydiae spp. demonstrated that these determinants are coexpressed in 90% of the SAF-positive samples. We confirmed the presence of C. pneumoniae DNA and RNA in the skin by PCR and reverse transcription-PCR and by sequence analysis of the PCR products. The expression of the C. pneumoniae antigens and SAF appears to be associated with active disease in that C. pneumoniae antigens were absent or greatly diminished in the skin of three patients examined after Psoralen and long-wave UVA radiation treatment. Our results suggest that SAF is a Chlamydia-associated protein and that further investigation is warranted to determine whether SAF and C. pneumoniae play a role in the pathogenesis of CTCL.

Frequency of apolipoprotein E (APOE) allele types in patients with Chlamydia-associated arthritis and other arthritides.

Genetic background is important in determining whether certain infecting bacteria disseminate to the joint and cause arthritis. We assessed whether APOE genotype is associated with the presence of DNA from Chlamydia or other bacteria in synovial tissues of patients with various arthritides. Nucleic acids from synovial tissues of 135 patients were screened by PCR for DNA from Chlamydia trachomatis, C. pneumoniae and other bacteria (pan-bacteria). APOE genotype was determined by a PCR-based method for all patients in each of four resulting groups comprised of about 35 individuals each, positive for C. trachomatis only, C. pneumoniae only, other bacteria, or no bacteria. RT-PCR was used to assess synovial APOE expression. The latter assays confirmed that APOE mRNA is present in synovial tissue. Determination of APOE genotype showed that patients PCR-negative in all assays, and those positive in the C. trachomatis - and pan-bacteria- (excluding Chlamydia) directed assays, had distributions of the APOE epsilon2, epsilon3 and epsilon4 alleles mirroring those of the general population (i.e. about 8%, 79% and 13%, respectively). In contrast, 68% of patients with C. pneumoniae DNA in synovium possessed a copy of the epsilon4 allele. These results indicate that no association exists between APOE genotype and synovial presence of C. trachomatis or other bacteria. However, individuals bearing at least one copy of the APOE epsilon4 allele may be at increased risk for synovial infection by C. pneumoniae.

Identification and localization of Chlamydia pneumoniae in the Alzheimer's brain.

We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.

The neuropathology of a chromosome 17-linked autosomal dominant parkinsonism and dementia ("pallido-ponto-nigral degeneration").

A group of similar autosomal dominant hereditary neurodegenerative disorders have been linked to chromosome 17 in thirteen kindreds. One of these disorders, known as pallido-ponto-nigral degeneration (PPND), is characterized by extensive degeneration of the globus pallidus and substantia nigra as well as accumulation of abnormally phosphorylated tau proteins. The authors now present comprehensive data on the cellular and molecular pathology of PPND, allowing its classification among chromosome 17-linked neurodegenerative disorders as well as its classification among sporadic and other familial tauopathies. First, we showed that PPND is characterized by abundant ballooned neurons in neocortical and subcortical regions as well as by tau-rich inclusions in the cytoplasm of neurons and oligodendroglia morphologically similar to those seen in corticobasal degeneration (CBD), but in a distribution pattern resembling progressive supranuclear palsy (PSP). Second, we demonstrated that antibodies to phosphorylation-independent (Alz50, 133, 304, Tau-2, T-46) as well as phosphorylation-dependent (AT8, PHF-6, 12E8, PHF-1, T3P, pS422) epitopes in human tau proteins stain these glial and neuronal inclusions as intensely as they stain CBD or PSP inclusions. Third, we probed PPND brain by Western blots using some of the same anti-tau antibodies to reveal 2 tau immunobands with molecular weights of 69 kD and 64 kD in gray and white matter extracts, as reported for both PSP and CBD. Finally, electron microscopy showed that these abnormal tau proteins formed flat twisted ribbons with a maximum diameter of 20 nanometers (nm) and a periodicity of about 200 nm, resembling those reported in CBD. Based on this, we conclude that PPND is a hereditary neurodegenerative disorder characterized by neuronal and glial tau-rich inclusions formed from aggregated filaments and hyperphosphorylated tau proteins and, hence, can be subcategorized into the tauopathy group of chromosome 17-linked neurodegenerative disorders. Further, since the morphologic and biochemical lesions of PPND overlap with those seen in sporadic CBD and PSP, we speculate that these disorders share common pathogenetic mechanisms.

Cognitive, neuroimaging, and pathological studies in a patient with Pick's disease.

We conducted cognitive, imaging, and neuropathological studies on a patient with Pick's disease. The patient was impaired at interpreting sentences with complex grammatical constructions, differing significantly from control subjects and patients with Alzheimer's disease (AD). Evaluation of regional brain functioning at rest, with positron emission tomography, revealed reduced left frontal activity compared with control subjects and AD patients. Autopsy demonstrated the classic pathology of Pick's disease, including massive neuron loss and gliosis in the frontal and cingulate cortex as well as numerous tau-positive hippocampal Pick bodies. The abnormal tau proteins were phosphorylated at the same amino acid residues as AD paired helical filament tau (PHFtau), but they exhibited a unique migration profile on western blot. Our observations support the hypothesis that a distinct variety of hyperphosphorylated tau in Pick's disease compromises the long-term viability of selectively vulnerable populations of neurons in frontal cortices that contribute to sentence processing.

Class III beta-tubulin isotype (beta III) in the adrenal medulla: III. Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial-like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a Gelfoam matrix system.

BACKGROUND: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation. To our knowledge, glial differentiation has not been reported in this cell line. METHODS: We have studied, by immunohistochemistry and immunoblotting, the presence of neuronal cytoskeletal antigens [class III beta-tubulin isotype (beta III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components], and synaptophysin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line. In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP. Immunohistochemistry was performed on explants ranging from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers. Additional immunoblot experiments using anti-GFAP and anti-beta III monoclonal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins. RESULTS: Beta III and MAP2 were demonstrated by immunohistochemistry and immunoblotting of PC12 explants maintained for up to 32 days in Gelfoam matrices with and without treatment with dcAMP. Intense filamentous and granular beta III staining of PC12 cells was observed in dcAMP-treated cultures concomitant with neuronal morphologic alterations (neuritogenesis and ganglionic phenotype). In untreated cultures, beta III staining was present in less differentiated cells, as well in cells undergoing neuritic development. The neuronal phenotype of PC12 cells was confirmed by staining for MAP2, tau, and NF proteins, as well as for synaptophysin. The presence of beta III, MAP2, MAP1B, tau, and NF proteins was confirmed by immunoblotting. Clusters of GFAP-positive and S-100 protein-positive spindle cells, phenotypically distinct from the chromaffin-like or neuronal cells, were demonstrated in Gelfoam explants at 5-30 days in vitro. In 30-day-old cultures treated with dcAMP, there was strong filamentous GFAP and diffuse S-100 protein staining in an increased number of sustentacular-like PC12 cells. GFAP staining was corroborated by immunoblotting of explants maintained under identical conditions in vitro. In contrast, immunoblots performed on homogenates from PC12 suspension and monolayer cultures were GFAP-negative. CONCLUSIONS: Neuronal and glial-like, presumed sustentacular, phenotypes were demonstrated in PC12 cells grown in Gelfoam matrices with and without treatment with dcAMP for up to 34 days. To our knowledge, the occurrence of glial differentiation in the PC12 line is a hitherto unreported finding. Adult rat medullary sustentacular cells are known to express S-100 and GFA proteins (Suzuki and Kachi, Kaibogaku Zasshi-Anat 70(2): 130-139, 1995), and the organ culture system employed in our study may well have favored this direction of differentiation.

The association of tissue transglutaminase with human recombinant tau results in the formation of insoluble filamentous structures.

To determine possible mechanisms by which NFTs are formed in Alzheimer's disease (AD), we investigated the ability of tissue transglutaminase (TGase) to convert human recombinant tau proteins into insoluble filamentous structures. TGase derived from guinea pig liver was activated by calcium to catalyze the in vitro cross-linking of the largest soluble recombinant tau isoform (htau40) into insoluble complexes as determined by electrophoresis following incubation in 4 M urea and SDS. The TGase-catalyzed formation of these insoluble complexes occurred within 15 min to 24 h and the decreased migration of the insoluble material correlated with increased calcium concentrations ranging from 2 mM to 50 mM when analyzed electrophoretically. TGase-treated human recombinant tau formed filamentous structures in vitro that were immunoreactive with antibodies to tau and TGase. These structures retained the insoluble characteristics typical of AD PHF/NFTs. Immunolabeling with the TGase antibody revealed that TGase is associated with the filaments formed from human recombinant tau in vitro as well as with PHFs isolated from NFTs from AD brains. These novel findings support an in vitro model for investigating the biophysical changes that occur in converting soluble tau proteins into an insoluble matrix consistent with the insoluble PHFs/NFTs which may contribute to neuronal degeneration and cell death in the AD brain.

Localization of transglutaminase in hippocampal neurons: implications for Alzheimer's disease.

The purpose of this investigation was to identify and localize tissue transglutaminase (TGase) within neurons from the hippocampi of normal aged individuals and of those with confirmed Alzheimer's disease (AD). This enzyme may be a factor in the molecular mechanisms of neurodegeneration and formation of insoluble macromolecular complexes found in the neurons of normal aged and AD brain tissue. An antibody made to the extracellular TGase, coagulation factor XIIIa, was found to be specific for purified intracellular guinea pig liver tissue TGase. The specificity for liver tissue TGase has enabled us to identify tissue TGase(s) within rat hippocampal neurons and within neurons from normal aged and AD hippocampal tissues. Degenerating neurons from the AD hippocampus, compared to neurons from the normal aged hippocampus, exhibited increased immunoreactivity for TGase and demonstrated co-labeling for PHF1 and anti-TGase. Our results suggest that TGase may be associated with the neurofibrillary degeneration observed in AD, thereby implicating TGase as a potential factor in the pathogenesis of Alzheimer's disease.

Reassembly of the 66 kD neurofilament protein in vitro following isolation and purification from bovine spinal cord.

NF-66, also known as alpha-internexin, has been characterized as a 66 kD mammalian neurofilament (NF) protein whose expression in developing rat brain precedes that of the low molecular weight NF protein (NF-L). NF-66 is thought to assemble into 10 nm diameter intermediate filaments in vitro, although the precise nature of the assembly process remains obscure. Likewise, the ability of NF-66 to polymerize with the low (NF-L), middle (NF-M), and high (NF-H) M(r)NF proteins has not been defined. This investigation describes the reassembly of bovine NF-66 regarding its formation into 10 nm diameter filaments as well as its potential for polymerization with other type IV intermediate filaments. NF-66 and the NF triplet proteins were isolated from bovine spinal cord using established biochemical extraction and isolation procedures (Balin et al., Brain Res 556:181-195, 1991), and purified by a combination of high performance liquid chromatography (HPLC) (DEAE anion exchange and hydroxylapatite column chromatography) and gel elution strategies. In vitro reassembly experiments revealed that NF-66 formed approximately 10 nm diameter filaments of varying length; immunoelectron microscopy demonstrated labeling of these filaments by a monoclonal antibody to intermediate filament antigen (IFA), a polyclonal antibody against rat NF-66 and by a monoclonal antibody generated against the core region of NF-M but cross-reactive with NF-66. This report is the first investigation to look at the in vitro interaction between NF-66 and other type IV intermediate filament proteins (NF-H, -M, and -L) and establishes that NF-66 forms heteropolymeric filaments with these other neurofilament proteins, as confirmed by double immunolabeling. These studies suggest that NF-66 could provide a nucleation site for the polymerization of later-expressed proteins during neuronal development.

Analysis of paired helical filaments (PHFs) found in Alzheimer's disease using freeze-drying/rotary shadowing.

Paired helical filaments (PHFs) isolated from Alzheimer's disease (AD) brains were analyzed using freeze-drying/rotary shadowing and immunoelectron microscopy. These filaments are the major contributors to the formation of neurofibrillary tangles (NFTs) found in AD, and are composed primarily of the microtubule-associated protein tau. We have focused on the identification of PHF-tau protein within isolated PHFs using anti-tau antibodies (tau 14, 46, 60). These PHFs consisted of both helically twisted and "straight" paired filaments. With freeze-drying/rotary shadowing, we are able to demonstrate in 2-D and 3-D subtle twists within the straight filaments as well as immunolabeling of the individual filamentous strands composing the PHFs. Additionally, projections emanating from numerous filaments and bridges between PHFs often were immunolabeled with anti-tau antibodies. Our results suggest that tau proteins are present in discrete, nonconfluent patterns within the PHFs and are components of both strands composing the PHFs. Tau proteins are present in the bridges between individual PHFs and may contribute to interconnecting PHFs into a complex macromolecular network such as the NFTs found in AD.

Neurofilament reassembly in vitro: biochemical, morphological and immuno-electron microscopic studies employing monoclonal antibodies to defined epitopes.

The reassembly process of purified native (phosphorylated) and enzymatically dephosphorylated bovine neurofilament (NF) subunits was studied to delineate how NF triplet proteins assemble together into intermediate-size filaments in vitro. We determined the time course for reassembly, the ultrastructural characteristics of reassembled NFs, and the topographical disposition of NF protein subdomains within reassembled NFs using quantitative biochemical techniques, negative staining and immunoelectron microscopy. Our data indicate that: (1) approximately 50% of the purified NF subunit proteins assembled within 30 min from the start of reassembly into 10- to 12-nm filaments, and by 90 min approximately 85-90% of the NF proteins reassembled, (2) low concentrations (0.15-0.5 mg/ml) of purified NF proteins were able to reassemble into long filaments, (3) the rate and ability of native phosphorylated and dephosphorylated NF proteins to assemble into NFs were comparable, (4) negative staining revealed a periodicity of approximately 18-22 nm and a protofilamentous substructure in reassembled NFs, (5) immunoelectron microscopy using domain specific anti-NF monoclonal antibodies (mAbs) to all 3 NF proteins demonstrated specific labeling patterns corresponding to the spatial relationships of subdomains within reassembled NFs, and (6) negative staining and immunolabeling revealed that reassembled NFs are very similar to isolated native NFs. We conclude that purified mammalian axonal NF triplet proteins, independent of their phosphorylation state, rapidly and efficiently reassemble in vitro to generate characteristic 10-nm filaments. Furthermore, immunological analysis reveals that the rod domains of NF-H, NF-M and NF-L are buried within the reassembled NF, whereas the head domain of NF-M and the tail domains of all 3 NF proteins remain exposed following reassembly.

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.