Monoclonal antibodies to the heavy neurofilament subunit (NF-H) of Torpedo cholinergic neurons.

Previous studies from our laboratory suggest that Alzheimer's disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF-H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF-H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme-linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF-H protein of Torpedo cholinergic neurons than in NF-H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF-H and one (TC15) that reacts with both NF-H and the medium-size neurofilament subunit NF-M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF-H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF-H contain phosphorylated as well as non phosphorylated epitopes. All the MAbs cross-react with rat brain NF-H.     

A monoclonal antibody to non-phosphorylated neurofilament protein marks the vulnerable cortical neurons in Alzheimer's disease

Various cytoskeletal proteins have been implicated in the formation of neurofibrillary tangles in Alzheimer's disease. A monoclonal antibody to non-phosphorylated neurofilament protein labels a distinct subset of pyramidal cells in the normal human cortex which have a distribution very similar to that of neurofibrillary tangles in brains from patients with Alzheimer's disease. In addition, regions and layers that normally contain a high density of such cells, in Alzheimer's disease, have large numbers of neurofibrillary tangles and few remaining immunoreactive cells.     

Evidence for the presence of antibodies to cholinergic neurons in the serum of patients with Alzheimer's disease

Summary A blind study showing that serum from patients with Alzheimer's disease causes immunolysis of mammalian brain synaptosomes is reported. Control, aged-matched, sera were largely without effect. The immunolysis was directed mainly against cholinergic synaptosomes. The data presented support the hypothesis that autoimmune mechanisms may operate in the pathogenesis of Alzheimer's disease.     

Neurofibrillary tangles in cholinergic pedunculopontine neurons in Alzheimer's disease

The cholinergic neurons located within the pedunculopontine nucleus (Ch5) of patients with Alzheimer's disease (AD; n = 15), Parkinson's disease (PD; n = 2), and neurologically normal (n = 6) subjects were visualized immunohistochemically using choline acetyltransferase, pharmacohistochemically using acetylcholinesterase, or by reduced histochemical methods using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d). Each histochemical procedure localized a well-delineated, compact lateral group and a more diffuse medial group of neurons within the pedunculopontine nucleus. Co-localization experiments revealed that all three enzymes marked the same population of cholinergic neurons. The extent of pathological alterations associated with the cholinergic neurons within the compact lateral sector of the pedunculopontine nucleus was examined in sections that reacted for NADPH-d, counterstained with thioflavin-S. The average number of neurofibrillary tangles within this portion of the pedunculopontine nucleus was 25.4 (range 0-70) in patients with AD, 1.5 (range 1-2) in those with PD, and 1.2 (range 0-4) in aged control subjects. Of the total number of neurofibrillary tangles counted in AD cases, 72.7% were end-stage ghosts and 27.3% were tangle-bearing neurons. The pathological alteration of cholinergic neurons of the compact lateral aspect of the pedunculopontine nucleus may play a role in some of the behavioral features characteristic of AD.     

Apoptotic signals within the basal forebrain cholinergic neurons in Alzheimer's disease

A relatively early and substantial loss of basal forebrain cholinergic neurons is a constant feature of Alzheimer's disease (AD). However, the mechanisms that contribute to the selective vulnerability of these neurons are not fully delineated. In the present series of experiments, we determined the possible contribution of apoptotic processes and other pathologic cascades to the degeneration of the cholinergic neurons of the nucleus basalis of Meynert (NBM) in AD. In contrast to neurons in the frontal cortex which showed prominent DNA fragmentation as detected by the TUNEL method, no DNA fragmentation was observed within the NBM in any of the AD or normal brains. Similarly, immunoreactivity for the apoptotic signals Fas, Fas-ligand, Bax, Bcl-x, caspase-8, caspase-9 and caspase-3 was absent from the NBM of AD and control brains. In contrast, a substantial subpopulation of cholinergic neurons within the NBM in AD displayed prominent immunoreactivity for the apoptotic signal Fas-associated death domain (FADD) in the form of tangles. FADD immunoreactivity was also present in dystrophic neurites. FADD-positive tangle-like structures were localized in neurons which contained immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT) and the low affinity neurotrophin receptor p75NTR. While many of the NBM cholinergic neurons in control brains contained immunoreactivity for the calcium binding protein calbindin-D28K (CB), the NBM neurons in AD displayed a substantial loss of CB immunoreactivity. Importantly, most of FADD-immunoreactive cholinergic neurons were devoid of CB immunoreactivity, and, conversely, most CB-positive cholinergic neurons had no FADD immunoreactivity. FADD immunoreactivity within the basal forebrain was colocalized with phosphorylated tau immunoreactive tangles and dystrophic neurites. In contrast, FADD immunoreactivity did not appear to be related to the primarily diffuse amyloid-beta deposits intermingled between cholinergic neurons in AD NBM. Finally, many CD68-positive microglia were observed surrounding the NBM cholinergic neurons in AD. In conclusion, the findings of the present study indicate that, while the FADD apoptotic signaling pathway may be triggered within the basal forebrain cholinergic neurons in AD, the apoptotic cascade is most likely aborted as no DNA fragmentation was detected and the executioner caspase-3 was not up-regulated within these neurons. The findings also suggest possible relationships between loss of CB, FADD expression and phosphorylation of tau within the basal forebrain cholinergic neurons in AD.     

Anti-tubulin monoclonal antibodies that bind to specific neurons in Caenorhabditis elegans

We have identified 2 anti-tubulin monoclonal antibodies that bind to 2 different subpopulations of identified neurons in the simple nervous system of the nematode Caenorhabditis elegans. The antibodies also recognize specific tubulin isotypes from C. elegans that were separated by isoelectric focusing. Antibody 6-1 1B-1 intensely stained the mechano-sensory neurons ALML, ALMR, PLML, PLMR, PVM, and AVM, plus neuron PVR by indirect immunofluorescence, and it bound to 1 of 2 major alpha-tubulin isotypes separated by iso-electric focusing. The epitope for antibody (Ab) 6-1 1B-1 is acetylated alpha-tubulin (Piperno and Fuller, 1985). Antibody 2-28-33 stained a set of neurons that contain the neurotransmitter GABA. It bound to the 2 major and 1 of the minor beta-tubulin isotypes of C. elegans. These results suggest that specific alpha- and beta-tubulin isotypes are greatly enriched in the subsets of neurons recognized by these antibodies. Most (possibly all) of the neurons in each subset are functionally related. The antibodies should allow us to examine whether specific tubulin isotypes have specific functions in these neurons. They may also be useful for examining neuron morphologies in mutants of C. elegans which affect nerve development.     

Interactions between beta-amyloid and central cholinergic neurons: implications for Alzheimer's disease

Alzheimer's disease is an age-related neurodegenerative disorder that is characterized by a progressive loss of memory and deterioration of higher cognitive functions. The brain of an individual with Alzheimer's disease exhibits extracellular plaques of aggregated beta-amyloid protein (Abeta), intracellular neurofibrillary tangles that contain hyperphosphorylated tau protein and a profound loss of basal forebrain cholinergic neurons that innervate the hippocampus and the neocortex. Abeta accumulation may trigger or contribute to the process of neurodegeneration. However, the mechanisms whereby Abeta induces basal forebrain cholinergic cell loss and cognitive impairment remain obscure. Physiologically relevant concentrations of Abeta-related peptides have acute, negative effects on multiple aspects of acetylcholine (ACh) synthesis and release, without inducing toxicity. These data suggest a neuromodulatory influence of the peptides on central cholinergic functions. Long-term exposure to micromolar Abeta induces cholinergic cell toxicity, possibly via hyperphosphorylation of tau protein. Conversely, activation of selected cholinergic receptors has been shown to alter the processing of the amyloid precursor protein as well as phosphorylation of tau protein. A direct interaction between Abeta and nicotinic ACh receptors has also been demonstrated. This review addresses the role of Abeta-related peptides in regulating the function and survival of central cholinergic neurons and the relevance of these effects to cholinergic deficits in Alzheimer's disease. Understanding the functional interrelations between Abeta peptides, cholinergic neurons and tau phosphorylation will unravel the biologic events that precede neurodegeneration and may lead to the development of more effective pharmacotherapies for Alzheimer's disease.     

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.     

Neurofibrillary degeneration of cholinergic and noncholinergic neurons of the basal forebrain in Alzheimer's disease

Two principal features of Alzheimer's disease (AD) are (1) the occurrence of neurofibrillary tangles (NFTs) and senile plaques, and (2) the loss of cortical cholinergic activity because of dysfunction of neurons in the basal forebrain cholinergic system. The relationship of these two abnormalities is an unresolved issue in the pathology of AD. We used polyclonal antibodies specific for paired helical filaments (PHFs), combined with acetylcholinesterase (AChE) histochemistry, to assess the cytoskeletal changes of cholinergic and noncholinergic neurons in the basal forebrain in AD. In both sporadic and familial AD, the nucleus basalis of Meynert (nbM) showed a marked decrease in AChE-positive (AChE+) perikarya and abundant immunoreactive NFTs. In double-labeling studies of the nbM, PHF reactivity was found both in surviving AChE+ neurons and in many AChE- NFTs that were not associated with microscopically recognizable cell structures. Some surviving AChE+ perikarya did not contain NFTs. Numerous NFTs and senile plaques were identified by PHF immunoreactivity in other basal forebrain areas, including subnuclei of the amygdala that showed low or absent AChE activity. We conclude that the dysfunction and death of cholinergic neurons in the nbM is associated with extensive NFT formation, including apparently residual NFTs in loci where nbM neurons once existed; and many noncholinergic neurons and neurites in the basal forebrain show NFT and senile plaque formation. The cytopathology of AD involves neurons of varying transmitter specificities, including cholinergic neurons in the basal forebrain.     

Medium-sized neurofilament protein related to maturation of a subset of cortical neurons.

Neurofilaments are typical structures of the neuronal cytoskeleton and participate in the formation and stabilization of the axonal and dendritic architecture. In this study, we have characterized a murine monoclonal antibody, FNP7, that is directed against the medium-sized neurofilament subunit NF-M. This antibody identifies a subset of neurons in the cerebral cortex of various species including human and in organotypic cultures of rat cortex. In the neocortex of all species examined, the antibody labels pyramidal cells in layers III, V, and VI, with a distinctive laminar distribution between architectonic boundaries. In comparison with other antibodies directed against NF-M, the FNP7 antibody identifies on blots two forms of NF-M that appear relatively late during development, at the time when dynamic growth of processes changes to the stabilization of the formed processes. Dephosphorylation with alkaline phosphatase unmasks the site, making it detectable for the FNP7 antibody. The late appearance suggests that the site is present during early development in phosphorylated form and with increasing maturation becomes dephosphorylated, mainly in dendrites. This event may relate to changes in cytoskeleton stability in a late phase of dendritic maturation. Furthermore, mainly corticofugal projections and only few callosal axons are stained, suggesting a differential phosphorylation in a subset of axons. The antibody provides a useful marker to study subsets of pyramidal cells in vivo, in vitro, and under experimental conditions.     

Galantamine: a novel cholinergic agent for Alzheimer's disease

Galantamine has been recently approved for the symptomatic treatment of Alzheimer's disease (AD). Apart from inhibiting acetylcholinesterase, galantamine modulates the nicotinic receptors, although the clinical significance of this action remains uncertain. Through a broad research program, it has been shown that galantamine produces a cognitive, functional and behavioral benefit in patients with either mild or moderate AD. Initially, the maintenance dose must be 16 mg a day. Later on, 24 mg dose attempts are justified on an individual patient basis. A clinical stabilization for almost one year is observed. After that time, treated patients deteriorate at a similar pace than the non-treated ones, but they remain above the non-treated during at least one more year. Additional data suggest that positive effects of galantamine spread both caregiver burden and pharmacoeconomic areas. Tolerability is good, provided that titration is made slowly. The only contraindications of this drug are atrioventricular blockade and uncontrolled bronchospasm. Galantamine has also shown efficacy in mixed dementia. New possible indications are mild cognitive impairment and vascular dementia.     

Swollen cortical neurons in Creutzfeldt-Jakob disease contain a phosphorylated neurofilament epitope.

The distribution of swollen neurons and the presence of a phosphorylated neurofilament protein (NFP) epitope in these cells were studied in six cases of Creutzfeldt-Jakob disease (CJD). Swollen neurons are widely distributed in the cerebral cortex and are most abundant in the cingulate and parahippocampal gyri. They are more numerous in the panencephalopathic type of CJD than in the subacute spongiform encephalopathic type. A phosphorylated epitope of NFP was detected in the perikarya of swollen neurons by an immunocytochemical method using a series of monoclonal antibodies that distinguish phosphorylated and nonphosphorylated epitopes of NFP. This abnormal distribution of phosphorylated NFP epitopes indicates that the process of NFP phosphorylation is altered in neurons affected by CJD. This investigation, in accordance with previous studies, suggests that the abnormal post-translational modification of the neurofilament may play an important role in the pathogenesis of several neurodegenerative disorders.     

Developmentally regulated epitopes on a neurofilament protein visualized by monoclonal antibodies

Two monoclonal antibodies (mabs) which recognized the 68 kDa subunit of the rat neurofilament triplet were isolated. In immunoblots with SDS-solubilized and reduced proteins, these mabs recognized their epitopes equally well in embryonic and in adult tissue. However, these epitopes were developmentally regulated in paraformaldehyde-fixed rat brain sections. They were abundant in all compartments of differentiating neurons, whereas in mature neurons their presence was markedly attenuated, with a moderate abundance in perikarya and larger dendrites and low concentrations in axons. Thus, a differential developmental modification, possibly involving the masking of an epitope, is demonstrated for the small neurofilament polypeptide in rat and monkey brain tissue.     

Prospective strategies for cholinergic interventions in Alzheimer's disease

The cholinergic hypothesis of memory dysfunction has guided most of the recent proposals for treating the primary symptoms of AD. The efficacy of these treatments has been severely limited. This review examines two major lines of evidence which suggest that the cholinergic hypothesis may have to be expanded and revised. The cholinergic hypothesis focuses on pre-synaptic defects. It assumes cholinoceptive neurons would function normally with adequate stimulation. Evidence is not sufficient to support this assumption. In addition, dissociations have been demonstrated between muscarinic receptor number and functional response of cholinoceptive neurons. Various measures are proposed to investigate the functional integrity of muscarinic receptors in AD patients. AD often has been characterized as a disorder produced by generalized cholinergic hypoactivity. Evidence for cortisol hypersecretion, abnormal dexamethasone suppression, and the occurrence of depressive symptoms, motoric dysfunction and sleep abnormalities in AD patients is more consistent with regional cholinergic hyperactivity than generalized hypoactivity. Resolution of these discrepancies could shed new light on the pathophysiology and treatment strategies for AD. Cholinoceptive neurons could be hypersensitive, subsensitive or have unaltered responsivity. These options would have very different treatment implications. New developments in outcome assessment which are capable of discriminating varieties of differential response to treatment can spur treatment development and improve quality of care for patients with complex disorders such as AD.     

Extraneural cholinergic markers in Alzheimer's and Parkinson's disease

Muscarinic and nicotinic binding sites were analysed in lymphocytes from patients with Alzheimer's disease, Multi-infarct dementia, Parkinson's disease and age- matched controls. A significant decrease in the number of both muscarinic and nicotinic binding sites was obtained in lymphocytes from Alzheimer patients while in Parkinson patients a significant decrease was found only in the nicotinic binding sites. Using butyrylthiocholine as substrate, no change was observed in cholinesterase activity in plasma from Alzheimer patients, whereas a significant decrease in plasma cholinesterase activity was found in Parkinson patients.     

PET studies and cholinergic therapy in Alzheimer's disease

Alzheimer's disease (AD) is one of the most devastating brain disorders of elderly humans. The last decade has witnessed a steadily increasing effort directed at discovery of the etiology of the disease and development of pharmacological treatment stategies. Symptomatic treatment mainly focussing on cholinergic therapy has been clinical evaluated by randomized, double-blind, placebo controlled, parallel group studies measuring performance based tests of cognitive function, activity of daily living and behavior. Significant progress has been made in recent years to develop and apply functional brain imaging techniques allowing early diagnosis of dementia and evaluation of treatment efficacy. Positron emission tomography (PET) is a suitable method for functional studies of pathological changes in brain which as a clinical instrument not solely reveal dysfunctional changes early in the course of the disease but also may provide a deep insight into the functional mechanisms of new potential drug treatment strategies. The advantage with PET is the capacity not only to measure changes in glucose metabolism, cerebral blood flow but also to obtain further insight into neuronal communicative processes (transmitter/receptor interactions) in brain and pharmacokinetic events and drug mechanisms. PET studies have so far revealed disturbances in some neuroreceptor systems in brain of AD patients. A significant correlation can be observed between the impairment of nicotinic receptors in the temporal cortex and the cognitive impairment of AD patients. Cholinergic drugs including cholinesterase inhibitors such as physostigmine, tacrine, velnacrine as well as the acetylcholine releaser linopiridine have been reported to increase the cerebral blood flow in AD patients both after acute and fairly short period of treatment. Increase in cerebral glucose metabolism has also been measured following fairly long periods of treatment with cholinesterase inhibitors (months). The cholinergic nicotinic and muscarinic receptors do also respond to treatment with cholinesterase inhibitors in AD patients. An improvement of the nicotinic receptors has been found in cortical regions following treatment with cholinesterase inhibitors and nerve growth factors (NGF) to AD patients. Functional PET activation studies performed simultaneously with memory tasks will provide further valuable insight into the mechanisms of action of new drug, how they interact and can improve the efficacy of memory processes in AD brains.     

Presence of neurofilament protein in Alzheimer's neurofibrillary tangles (ANT)

Summary Localization of neurofilament (NF) protein in the neurons of rat's brain, spinal cord, and peripheral nerves was studied by the indirect immunofluorescent method using goat-antirabbit plus rabbit anti-NF antibody. The NF protein was purified from the calf brain by the method of Yen et al. (1976). Control sections were treated with nonspecific rabbit serum and with the experimental rabbit serum absorbed with the NF protein. Fluorescence in the axons of the neurons, such as the motor neurons of the spinal cord, peripheral nerves, pontine nuclei, or those of the cerebellar cortex including Purkinje cells, was strong, while that of the dendrites and perikarya was weaker. Glial processes also showed intense fluorescence. Fluorescence in glia cells was interpreted as representing an insufficient separation of NF protein as the antigen from the glial fibrillary acidic protein (GFA). Localization of NF protein in the Alzheimer's neurofibrillary tangles (ANT) in the hippocampus of the brain of a patient with Alzheimer's disease, was examined by the same way as described above. ANT showed the whole gamut of the color of fluorescence from completely positive green to mixed green with increasingly bluer tints. Astroglial processes also showed intense green fluorescence. The results suggest that ANT comprises NF protein as the structural component and that the increasingly blue fluorescence may represent fewer antigenic sites due to increasing degrees of structural insolubilization or aging of ANT. Possibility of crosslinkage between NF protein and contribution of pathological factors as the cause of ANT formation were discussed.     

Cortical load of PHF-tau in Alzheimer's disease is correlated to cholinergic dysfunction

Summary. To assess a potential relationship between cortical neurofibrillary degeneration and cortical cholinergic deafferentation, the load of PHF-tau was analysed in eight cortical regions and in the basal nucleus of Meynert in 12 cases with Alzheimer's disease by means of a sensitive ELISA employing the monoclonal antibody B5-2. The activity of choline acetyltransferase was determined on identical tissue samples. The results demonstrate a highly correlative relationship between the cortical distribution of the amount of PHF-tau, mainly present in neuropil threads, and cholinergic depletion early during the course of the disease. This relationship was less strong in more advanced stages. The results support the suggestion that the formation of PHF-tau in cholinergic axon terminals which might result in a loss of cholinergic synapses and a cholinergic dysconnection of the cortex, is an early event in AD. During the progression of the disease, formation of PHF-tau appears to spread over the cortex which results in a more even distribution of neuropil threads and a progressive involvement of non-cholinergic neurons. Received September 16, 1998; accepted December 10, 1998