Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease (AD), and their presence correlates with the presence of dementia. A major constituent of NFT is the insoluble paired helical filament which shares some antigenic relationships with normal cytoskeletal elements, particularly neurofilaments. If neurofilament proteins (200, 145-160, and 68 kilodaltons [kd]) participate in the formation of NFT, the distribution of these constituents might be expected to be abnormal. To examine this issue, we used immunocytochemical methods to localize phosphorylated and nonphosphorylated epitopes of neurofilament proteins in hippocampal neurons of controls and patients with AD. Normally, the 200-kd neurofilament protein is not phosphorylated in the perikarya of neurons. However, in AD, many pyramidal neurons contained immunoreactive phosphorylated neurofilaments. Patterns of immunoreactivity (linear, flame-shaped, or skein-like within perikarya) greatly resembled the appearance of silver-stained NFT. This pattern of immunoreactivity was not present in hippocampal pyramidal neurons in controls, except in one aged patient in whom adjacent silver-stained sections revealed a few NFT. Patterns of immunoreactivity with antibodies for nonphosphorylated neurofilament proteins were similar in control and AD neurons. Our results indicate that some NFT are associated with abnormal distributions of high molecular weight phosphorylated neurofilament proteins. One domain of the 200-kd protein is believed to be a component of the side arms which link neurofilaments and interact with microtubules. Abnormal interactions of perikaryal neurofilaments could play a role in the genesis of NFT, and this abnormality of the cytoskeleton could contribute to the dysfunction of neurons at risk in AD.     

Immunocytochemical and ultrastructural evidence of dendritic degeneration in motor neurons of aluminum-intoxicated rabbits

Summary Using immunocytochemical and ultrastructural methods, we observed extensive and characteristic dendritic changes in motor neurons of rabbits inoculated intracisternally with aluminum phosphate. Anti-microtubule-associated protein 2 immunostaining revealed markedly reduced immunoreactivity in motor neuron dendrites and a reduced number of dendritic trees in aluminum phosphate-intoxicated rabbits. These dendritic changes were confirmed at the ultrastructural level; neurofilamentous accumulations, membranous inclusions and disrupted microtubules were common features of motor neuron dendrites, but less prominent in motor neuron axons. These observations suggest that dendrites are characteristically involved in aluminum intoxication in addition to the widely reported accumulation of phosphorylated neurofilament in perikarya and axons.     

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.     

Aluminum intoxication: a disorder of neurofilament transport in motor neurons

In the rabbit, intrathecal administration of aluminum salts (AlCl₃) induces accumulation of neurofilaments in nerve cells of the central nervous system. In motor neurons, the spatial pattern of neurofilamentous accumulation following aluminum intoxication suggests a defect in the axonal transport of neurofilament proteins. To test this hypothesis, we examined the distribution of radioactive cytoskeletal proteins in sciatic nerves of intoxicated and control animals. In the nerves of aluminum-injected animals, there was a 40% reduction in the relative amount of radioactive neurofilament proteins compared to tubulin. These results suggest that an abnormality in neurofilament transport may be important in the pathogenesis of the neurofibrillary pathology induced by aluminum intoxication.     

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.     

Comparative immunocytochemical characterization of neurofibrillary tangles in experimental maytansine and aluminum encephalopathies

The neurofibrillary tangles induced by maytansine and aluminum are stained by the peroxidase-anti-peroxidase method, using antibodies raised against the 68, 150 and 200 kdaltons polypeptide subunits of normal neurofilaments. The immunoreaction with each of the 3 antisera occurs regardless of the location of the neurofilament accumulation, whether in perikarya, dendrites or axons. It is very likely that the filaments of the neurofibrillary tangles induced by both aluminum and maytansine contain all the 3 neurofilament subunits.     

Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.     

Neurofibrillary axonal pathology in aluminum intoxication

Aluminum chloride injected into the cisterna magna of 3-to 4-week-old rabbits is known to produce neurofibrillary changes in the perikarya and dendrites of neurons of the spinal cord and brainstem. This report calls attention to the early appearance and prominence of neurofibrillary swellings in proximal axons of affected neurons. One and 2 days after intoxication, large axonal swellings containing maloriented neurofilaments were present in the ventral horns. Over the next days and weeks, neurofilaments accumulated in the neuronal perikarya and dendrites. Two months after injection, abnormalities in the cell bodies were less apparent but occasional giant axonal swellings persisted in the ventral horn. This sequence of pathological changes, reflecting disorganization of the neuronal cytoskeleton, may result from an increase in synthesis of neurofilament polypeptides, and impairment in integration of these constituents into the neuronal cytoskeleton, or a defect in the intracellular transport of neurofilaments.     

Role of proteasomes in the formation of neurofilamentous inclusions in spinal motor neurons of aluminum‐treated rabbits

We examined the role of the 20S proteasome in pathologic changes, including abnormal aggregation of phosphorylated neurofilaments, of spinal motor nerve cells from aluminum‐treated rabbits. Immunohistochemistry for the 20S proteasome revealed that many lumbar spinal motor neurons without intracytoplasmic neurofilamentous inclusions or with small inclusions were more intensely stained in aluminum‐treated rabbits than in controls, whereas the immunoreactivity was greatly decreased in some enlarged neurons containing large neurofilamentous inclusions. Proteasome activity in whole spinal cord extracts was significantly increased in aluminum‐treated rabbits compared with controls. Furthermore, Western blot analysis indicated that the 20S proteasome degraded non‐phosphorylated high molecular weight neurofilament (neurofilament‐H) protein in vitro. These results suggest that aluminum does not inhibit 20S proteasome activity, and the 20S proteasome degrades neurofilament‐H protein. We propose that abnormal aggregation of phosphorylated neurofilaments is induced directly by aluminum, and is not induced by the proteasome inhibition in the aluminum‐treated rabbits. Proteasome activation might be involved in intracellular proteolysis, especially in the earlier stages of motor neuron degeneration in aluminum‐treated rabbits.     

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.     

Neurofilament reorganisation and neurofilament antigen redistribution in spinal motoneurones following retrograde axonal transport of diphtheria toxin

Single unilateral injections of diphtheria toxin (DTX) into the external anal sphincter muscle or internal intercostal nerve of cat induced characteristic ultrastructural lesions in corresponding ipsilateral spinal motoneurones 6–8 days later. The chief neuronal lesion was a progressive disruption of Nissl body composition and organisation, which between days 8–19 post injection was accompanied by a progressive accumulation of neurofilaments in motoneuronal perikarya and dendrites. Some axons in the ipsilateral ventral horn became hypertrophied due to neurofilamentous accumulation. Related immunocytochemical investigations 6–35 days after injection of DTX revealed abnormal immunoreactivity intoxicated motoneurones for 200-kDa and 160-kDa phosphorylated neurofilament proteins, but not in contralateral motoneurones. By day 35 abnormal neurofilament immunostaining also occurred in ipsilateral and some contralateral interneurones but not contralateral motoneurones. Abnormalities of Nissl body endoplasmic reticulum, neurofilament organisation, and neurofilament protein immunostaining were identical after either intraneural and intramuscular injections of DTX, indicating abnormalities were attributable to toxicity and not injection-related axonal damage. Since DTX acts specifically in the soma to inhibit protein synthesis, neurofilament abnormalities are secondary to cytotoxicity and probably result from deficits in transference of existing partially phosphorylated neurofilaments to the axonal transport system, or axonal transport per se.Supported by the Wellcome Trust and Motor Neurone Disease Association (UK)     

Immunoreactive phosphorylated epitopes on neurofilaments in neuronal perikarya may be obscured by tissue preprocessing

Monoclonal antibodies (SMI 31 and SMI 34) against phosphorylated epitopes on neurofilaments react almost exclusively with neuronal axons in paraffin sections of perfused animals. Using fresh frozen (unfixed) rodent brain sections the immunoreactivity with these antibodies was also present in numerous neuronal cell bodies. Both antibodies also recognized intracellular antigens in the soma of living cells of a human neuroblastoma clone. These data suggest that phosphorylation of neurofilaments in neuronal perikarya is common in vivo. The phosphorylated neurofilament epitopes in neuronal perikarya may become hidden during tissue preprocessing and fixation.     

Neurofilamentous abnormalities in motor neurons in spontaneously occurring animal disorders

Cytoskeletal proteins have a characteristic distribution within neurons when immunocytochemical techniques are used on conventional paraffin sections. For example, phosphorylated neurofilaments are located within axons but are not normally present in the majority of perikarya of the central nervous system. This pattern can be altered in disease, and neurofilaments that accumulate within perikarya can be phosphorylated inappropriately. To determine whether retained neurofilaments were phosphorylated inappropriately, we used immunocytochemical techniques to examine several diseases in animals in which neurofilaments accumulate within neuronal perikarya. Our investigations of diseases with disparate etiologies show that, whenever neurofilaments are retained within the neuronal perikarya, they are phosphorylated. These results suggest that phosphorylation of neurofilaments in an inappropriate location, i.e. perikarya, may be a nonspecific disease-related response of neurons that can be initiated by a variety of cellular injuries.     

Activation of NMDA receptors and Ca2+/calmodulin-dependent protein kinase participate in phosphorylation of neurofilaments induced by protein kinase C

Aberrant phosphorylation of neurofilaments, similar to that occurring in various motor neuron diseases, is produced in cultured motor neurons by activation of protein kinase C (PKC). Following exposure to synthetic diacylglycerol, persistent change in the phosphorylation state of C-terminal domains of neurofilament proteins was detected by increased perikaryal immunoreactivity with the antibody SMI34; this antibody recognizes NF-M/NF-H when C-terminal KSP repeat domains are highly phosphorylated. SMI34 labeling of perikarya and dendrites was prevented by pretreatment with either the NMDA receptor antagonist APV or by the Ca²⁺/calmodulin-dependent protein kinase (CaMK) inhibitor KN-62, but not by antagonists of AMPA/kainate or metabotropic glutamate receptors or by inhibitors of arachidonic acid metabolic pathways. Thus, activation of PKC may induce neurofilament phosphorylation in motor neurons by acting in cooperation with stimulation of NMDA receptors and activation of CaMK. These mechanisms may be relevant to motor neuron disease and other neuronal injuries in which increased PKC activity has been measured. J. Neurosci. Res. 50:514–521, 1997. © 1997 Wiley-Liss, Inc.     

Perikaryal neurofilament phosphorylation in axotomized and 6-OH-dopamine-lesioned CNS neurons

The axon reaction in the central nervous system was studied using a monoclonal antibody to phosphorylated neurofilaments. Axotomy was performed by cutting the nigrostriatal pathway. We were able to show that phosphorylated epitopes of neurofilaments, that are usually restricted to axons, could be detected in the perikarya and dendrites of axotomized neurons as early as 3 days postlesion. These neurons remained labelled up to 17 days after axotomy and in some cases even up to 6 weeks. The cytoplasmic changes appearing in the lesioned neurons 8 days after axotomy seem to indicate that these neurons will probably degenerate or survive only in an atrophied, non-functional state as they are unable to regenerate their sectioned axon. Neurochemical lesions, using the neurotoxin 6-OH-dopamine, were performed to establish whether this reaction of perikaryal neurofilament phosphorylation may be a non-specific phenomenon accompanying neuronal degeneration or injury. Although cell loss was important, no labelled neurons could be observed following 6-OH-dopamine treatment. These results indicate that the induction of perikaryal neurofilament phosphorylation is a response to selective types of neuronal injury and concerns selective neuronal populations.     

Demonstration of hyperphosphorylated neurofilaments in neuronal perikarya in vivo by microinjection of antibodies into cultured spinal neurons

With conventional immunocytochemical techniques on fixed tissue, antibodies which recognize highly phosphorylated neurofilament proteins strongly label axons, but often react poorly with perikaryal neurofilaments. The reactivity of one such antibody, SMI31, with neurofilaments in vivo has been investigated by microinjecting purified SMI31 into large neurons in living cultures of embryonic mouse spinal cord. Microinjected SMI31 (SMI31I) labeled perikarya and dendrites in a fibrillar pattern indistinguishable from that of microinjected SMI32 (SMI32I), which labels hypophosphorylated neurofilaments of perikarya and dendrites in fixed tissue. SMI31 also labeled perikarya and dendrites when applied to whole unfixed cultures after extraction with 1% Triton X-100 or to cultures fixed in acetone after Triton-extraction, but prior to exposure to primary antibody. SMI31 labeled mainly axons when applied after fixation with acetone without Triton-extraction. Positive labeling of neurofilaments and various inclusions in neuronal somata with antibodies against highly phosphorylated neurofilaments has been described in a number of neurotoxic and neurodegenerative diseases and after neuronal injury. The results of this study indicate that explanations other than alterations in phosphorylation could account for these observations.     

The effect of axotomy and deafferentation on phosphorylation dependent antigenicity of neurofilaments in rat superior cervical ganglion neurons

Previous immunocytochemical studies have shown immunological differences between neurofilaments in axons and those in dendrites and perikarya of many mature neuron types: it is now known that non-phosphorylated epitopes are normally seen in cell bodies and dendrites of mature neurons, whereas phosphorylated epitopes are observed in axons. Further studies on a variety of cell types have shown that phosphorylated epitopes are induced in the cell-body associated neurofilaments following axotomy, and we show here that comparable changes are observed in axotomized rat superior cervical ganglion neurons (SCG). We have also observed that preganglionic section induces similar changes in the levels of neurofilament phosphorylation, so that phosphorylated epitopes become visible in the cell bodies and dendrites of denervated neurons. The changes in this case appear more slowly and are accompanied by the appearance of punctate nuclear staining detectable with phosphorylation sensitive neurofilament antibodies. The acquisition of these staining patterns by both the axotomized and denervated SCG cells proved to be reversible, so that after 10–12 days no neurons in either experimental group exhibited perikaryal phosphorylated epitopes. These results indicate that changes in the level of neurofilament phosphorylation are not only associated with neuronal damage, but can also be induced by deafferentation.     

Phosphorylation-related immunoreactivity and the rate of transport of neurofilaments in chronic 2,5-hexanedione intoxication

Axonal transport of neurofilaments and the phosphorylation of epitopes on neurofilament proteins was studied in rats chronically intoxicated with 2,5-hexanedione. Sensory axons arising from the L₅ dorsal root ganglion exhibited accelerated transport and a reduced abundance of neurofilament proteins. The binding of an antibody to phosphorylated neurofilament epitopes was compared to the binding of an antibody to non-phosphorylated epitopes by quantitative ELISA. This immunochemical index of neurofilament phosphorylation was reduced in dorsal roots, proximal peripheral sensory axons and ventral roots, but not in a distal nerve (the nerve to the soleus). Axotomy produced a reduction in neurofilament protein abundance comparable to hexanedione without any change in the immunochemical phosphorulation index. These results are consistent with the hypothesis that the state of phosphorylation of neurofilaments in proximal axons is correlated with the rate of neurofilament transport.     

Ultrastructural evidence of neurofilament involvement in immune-mediated motor neuron injury

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder. A pathologic hallmark of ALS is accumulation of neurofilaments in proximal axons of affected motor neurones. As the neurofilaments involved in immune-mediated spinal cord ventral horn motor neuron degeneration and loss, we developed immune-mediated motor neuron injury animal model by inoculating Lewis rats with swine spinal cord homogenate and investigated the ultrastructural features of neurofilament accumulation using transmission electron microscopy. Our results showed that there was aberrant accumulation of neurofilaments in perikarya and processes of remaining motor neurons in recipient animals, which is similar to those observed in ALS patients. These findings suggest that immune-mediated motor neuron injury may share a common pathogenesis with ALS.     

An antibody against hyperphosphorylated neurofilament proteins collapses the neurofilament network in motor neurons but not in dorsal root ganglion cells.

The carboxyl-terminal region of both the medium and high molecular weight neurofilament proteins contains repeated sequences that are sites for phosphorylation. The monoclonal antibody SMI31 specifically recognizes the conformation of these multiphosphorylated domains in an intermediate state of phosphorylation. Microinjection of SMI31 into living spinal motor neurons in culture resulted in a gradual collapse of the arrays of neurofilaments in perikarya and dendrites. In some cells, antibody-decorated filaments penetrated the axon and accumulated in proximal axonal segments causing their swelling. In dorsal root ganglion neurons, microinjected SMI31 bound to neurofilaments but did not induce collapse of the network or proximal axonal swelling. This study supports a role for phosphorylation of neurofilament sidearms in control of neurofilament transport and illustrates that interference with these sites has different consequences on neurofilament organization and morphology in different cell types.