Staging of neurofibrillary degeneration caused by human tau overexpression in a unique cellular model of human tauopathy.

The hyperphosphorylation of human tau and its aggregation into neurofibrillary tangles are central pathogenic events in familial tauopathies and Alzheimer's disease. However, the cellular consequences of neurofibrillary tangle formation in vivo have not been directly studied because cellular models of human neurofibrillary degeneration have been unavailable until recently. Incorporation of human tau into filaments in vivo and the association of filamentous tau with cytodegeneration were first demonstrated experimentally with the overexpression of human tau in identified neurons (anterior bulbar cells) in the lamprey central nervous system. In this system, filamentous tau deposits are associated with the loss of dendritic microtubules and synapses, plasma membrane degeneration, and eventually the formation of extracellular tau deposits and cell death. Here we show that human tau hyperphosphorylation in anterior bulbar cells is spatiotemporally correlated with a highly stereotyped sequence of degenerative stages closely resembling those seen in human neurofibrillary degeneration. Hyperphosphorylated tau deposits first appear in the distal dendrites and somata, together with degenerative changes that begin in distal dendrites and progress proximally over time. This sequence is independent of the tau isoform used, the presence of epitope tags and the method used to overexpress tau, and thus has important implications for the cytopathogenesis of human neurofibrillary disease.

The single neurofilament subunit of the lamprey forms filaments and regulates axonal caliber and neuronal size in vivo.

Neurofilaments (NFs) are composed of a heteropolymer of three related subunits in mammalian neurons, where they are a major component of the cytoskeleton in large neurons and are thought to regulate axonal diameter. NFs in the lamprey, while ultrastructurally and functionally indistinguishable from mammalian NFs, are polymers of a single subunit protein, NF180. In this study, we use the simplicity of lamprey NFs and the accessibility of the lamprey central nervous system (CNS) to examine the effects of overproducing NFs in an identified giant neuron in vivo, and thus to elucidate the role of NFs in regulating neuronal size and axonal caliber in the vertebrate CNS. We show that overexpression of NF180 tagged with a variant of Green Fluorescent Protein (EYFP) in identified lamprey neurons (ABCs) and in human neuroblastoma (NB2a) cells results in the assembly of exogenous NF180 into ultrastructurally normal NFs that are tightly packed and unphosphorylated. These accumulate in the somata of NB2a cells and produce somatic swelling by 3 days post-transfection. NF180 overexpression in lamprey ABCs in vivo causes exogenous NFs to accumulate in ABC axons, somata, and dendrites, and induces a significant increase in axonal diameter without increasing axonal NF packing density. Overexpression of EYFP alone has none of these effects. We conclude that NF180 normally plays a critical role in determining axonal caliber in ABCs and may influence neuronal size in situations where NFs accumulate in the soma, such as after axonal injury.

Human tau filaments induce microtubule and synapse loss in an in vivo model of neurofibrillary degenerative disease.

The intracellular accumulation of tau protein and its aggregation into filamentous deposits is the intracellular hallmark of neurofibrillary degenerative diseases such as Alzheimer's Disease and familial tauopathies in which tau is now thought to play a critical pathogenic role. Until very recently, the lack of a cellular model in which human tau filaments can be experimentally generated has prevented direct investigation of the causes and consequences of tau filament formation in vivo. In this study, we show that human tau filaments formed in lamprey central neurons (ABCs) that chronically overexpress human tau resemble the 'straight filaments' seen in Alzheimer's Disease and other neurofibrillary conditions, and are distinguishable from neurofilaments by their ultrastructure, distribution and intracellular behavior. We also show that tau filament formation in ABCs is associated with a distinctive pattern of dendritic degeneration that closely resembles the cytopathology of human neurofibrillary degenerative disease. This pattern includes localized cytoskeletal disruption and aggregation of membranous organelles, distal dendritic beading, and the progressive loss of dendritic microtubules and synapses. These results suggest that tau filament formation may be responsible for many key cytopathological features of neurofibrillary degeneration, possibly via the loss of microtubule based intracellular transport.

Neuronal morphology, axonal integrity, and axonal regeneration in situ are regulated by cytoskeletal phosphorylation in identified lamprey central neurons.

The CNS of the sea lamprey (Petromyzon marinus) contains giant, individually identifiable neurons that can be microinjected intracellularly in the living animal. We have used the unique accessibility of this system to investigate the role played by serine/threonine kinases and phosphatases in regulating cytoskeletal stability in identified reticulospinal neurons (ABCs) in situ. Injection of broad spectrum kinase and phosphatase inhibitors induce marked changes in ABC gross morphology and in the extent and morphology of sprouts induced by axotomy. The kinase inhibitor K-252a causes regenerating sprouts to be longer and narrower than those seen in control preparations, and significantly reduces the diameters of axon stumps; this latter effect is similar to the effect of microinjecting anti neurofilament (NF) antibodies. By contrast, the phosphatase inhibitor okadaic acid (OA) causes the selective disruption of axonal integrity, blocking axonal regeneration and causing axon stump retraction in axotomized ABCs. The microtubule (MT) disrupting drug colchicine has an effect similar but less marked than OA on ABC axonal morphology. Both OA and colchicine also induce the formation of large somatodendritic swellings in axotomized (but not intact) ABCs by 1-3 weeks post-injection. Immunocytochemical analyses indicate that both colchicine and OA treatments result in the destabilization of MTs and the phosphorylation of NFs, while OA induces the accumulation of phosphorylated tau protein in some dendritic swellings. Control injections of inactive substances have none of these effects. These results suggest that OA does not have its primary effect on NF assembly at the doses used, but may block axonal regeneration by inducing a prolonged disruption of axonal MTs, possibly via an indirect mechanism involving the hyperphosphorylation of tau and other MAPs. K-252a, on the other hand, may interfere with NF assembly and sidearm phosphorylation, thereby reducing NF transport into both axon stumps and sprouts and in turn reducing sprout diameter. The implications of these results for the respective roles of MTs, MAPs, and NFs in axonal regeneration in the vertebrate CNS are discussed.

Human tau becomes phosphorylated and forms filamentous deposits when overexpressed in lamprey central neurons in situ.

Microinjection of plasmids encoding human tau (htau) protein into identified lamprey reticulospinal neurons (anterior bulbar cells, or ABCs) in situ induces chronic htau expression. htau protein is transported to both the axon and dendrites of expressing ABCs by mechanisms that require the C-terminal domain of htau protein but do not require directed htau mRNA transport. htau becomes phosphorylated at the PHF-1 (Ser-396/404) and TAU-1/AT8 (Ser-199/202) epitopes throughout ABCs with heavy htau accumulations; many such cells also exhibit degenerative changes, which include the development of extracellular htau deposits. Finally, expression of htau protein fused to green fluorescent protein induced the somatodendritic accumulation of filaments containing htau when examined by immunoelectron microscopy. These results suggest that chronic expression of htau in lamprey ABCs may be useful for studying cellular mechanisms underlying tau hyperphosphorylation and filament formation in vertebrate central neurons in situ.

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons.

Axotomy within 500 microm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompanied by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.

Neurofilament spacing, phosphorylation, and axon diameter in regenerating and uninjured lamprey axons.

It has been postulated that phosphorylation of the carboxy terminus sidearms of neurofilaments (NFs) increases axon diameter through repulsive electrostatic forces that increase sidearm extension and interfilament spacing. To evaluate this hypothesis, the relationships among NF phosphorylation, NF spacing, and axon diameter were examined in uninjured and spinal cord-transected larval sea lampreys (Petromyzon marinus). In untransected animals, axon diameters in the spinal cord varied from 0.5 to 50 microns. Antibodies specific for highly phosphorylated NFs labeled only large axons (> 10 microns), whereas antibodies for lightly phosphorylated NFs labeled medium-sized and small axons more darkly than large axons. For most axons in untransected animals, diameter was inversely related to NF packing density, but the interfilament distances of the largest axons were only 1.5 times those of the smallest axons. In addition, the lightly phosphorylated NFs of the small axons in the dorsal columns were widely spaced, suggesting that phosphorylation of NFs does not rigidly determine their spacing and that NF spacing does not rigidly determine axon diameter. Regenerating neurites of giant reticulospinal axons (GRAs) have diameters only 5-10% of those of their parent axons. If axon caliber is controlled by NF phosphorylation via mutual electrostatic repulsion, then NFs in the slender regenerating neurites should be lightly phosphorylated and densely packed (similar to NFs in uninjured small caliber axons), whereas NFs in the parent GRAs should be highly phosphorylated and loosely packed. However, although linear density of NFs (the number of NFs per micrometer) in these slender regenerating neurites was twice that in their parent axons, they were highly phosphorylated. Following sectioning of these same axons close to the cell body, axon-like neurites regenerated ectopically from dendritic tips. These ectopically regenerating neurites had NF linear densities 2.5 times those of uncut GRAs but were also highly phosphorylated. Thus, in the lamprey, NF phosphorylation may not control axon diameter directly through electrorepulsive charges that increase NF sidearm extension and NF spacing. It is possible that phosphorylation of NFs normally influences axon diameter through indirect mechanisms, such as the slowing of NF transport and the formation of a stationary cytoskeletal lattice, as has been proposed by others. Such a mechanism could be overridden during regeneration, when a more compact, phosphorylated NF backbone might add mechanical stiffness that promotes the advance of the neurite tip within a restricted central nervous system environment.

Early cytoskeletal changes following injury of giant spinal axons in the lamprey.

The spinal cord of the larval sea lamprey contains identified giant axons that readily regenerate following spinal transection. In this study, we used serial light and electron microscopy to analyze the early ultrastructural consequences of axotomy in the proximal stumps of these axons near the lesion site. Axotomy results in two types of striking ultrastructural changes: 1) changes associated with the degeneration of axoplasm and subsequent retraction of the cut axon from the lesion and 2) changes associated with the early stages of axonal regeneration. Degenerative changes include the disruption of mitochondria to form large vacuoles, the collapse of neurofilaments into closely packed masses (condensed filamentous cores; CFCs), and the appearance of amorphous electron-dense bodies (dense granular masses; DGMs). Events associated with regeneration include the disappearance of vacuoles, DGMs, and CFCs and the appearance of small, sprout-like projections from the axon stump. Thus, we show that degenerative and regenerative events can be clearly separated from one another in identified axons, unlike the situation in the central nervous systems of amniote vertebrates such as mammals.

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons.

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the "rod" domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament "sidearm" domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rod-specific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1-2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons.

Axotomy-induced neurofilament phosphorylation is inhibited in situ by microinjection of PKA and PKC inhibitors into identified lamprey neurons.

Close axotomy of identified lamprey neurons induces phosphorylation of somatodendritic neurofilaments (NFs), followed by ectopic regeneration of neurofilamentous sprouts from the dendrites. We used in situ intracellular microinjection to study the mechanism of axotomy-induced NF phosphorylation. We found that inhibitors of protein kinase C (PKC) and protein kinase A (PKA) block somatodendritic NF phosphorylation for up to 15 days when injected at the time of axotomy. Injection of PKA catalytic subunit, diacylglycerol, or okadaic acid induces somatodendritic NF phosphorylation in intact neurons with the same time course as close axotomy. These results suggest that transient activation of PKC, PKA, and/or serine phosphatase inhibition by axotomy triggers persistent intracellular changes that may be related to polarity loss in these neurons.

Microtubule destabilization and neurofilament phosphorylation precede dendritic sprouting after close axotomy of lamprey central neurons.

Axotomy of giant lamprey (Petromyzon marinus) central neurons (anterior bulbar cells) close to their somata results in ectopic axon-like sprouting from the dendritic tips. Such sprouts first appear as swellings at the tips of a small subset of dendrites 2-3 weeks after "close" axotomy. We report here that immunocytochemical examination of these swellings reveals a structure and composition that differs from that of conventional growth cones; incipient sprouts contain many highly phosphorylated neurofilaments (NFs), little tubulin, and virtually no stable (acetylated) microtubules (MTs). The dendrites of anterior bulbar cells after close axotomy also show pronounced changes in NF protein and tubulin staining patterns prior to the emergence of sprouts from the dendrites. The amount of tyrosinated tubulin increases greatly; this rise is tightly coupled to the appearance of highly phosphorylated NFs and the loss of nonphosphorylated NFs in the dendrites. Acetylated tubulin is generally reduced after close axotomy and is selectively lost from dendrites that gave rise to sprouts. These changes indicate that an invasion of the dendrites by phosphorylated NFs may be linked to the destabilization of dendritic MTs, and in some dendrites this may lead to a marked loss of stable MTs, which is correlated with the emergence of NF-filled sprouts from the dendritic tips.

Aryl acylamidase from Rhodococcus erythropolis NCIB 12273.

A Rhodococcus erythropolis strain was isolated from soil on the basis of its ability to use acetaminophen as the sole source of both carbon and energy for growth. When grown in a complex medium containing an anilide inducer compound, the bacterium exhibited aryl acylamidase (EC 3.5.1.13) activity. This activity was not subject to carbon or nitrogen repression by the growth medium constituents as the enzyme was present throughout the exponential growth phase. The anilide was converted to the corresponding aniline, which was not further degraded. The enzyme was partially purified by a variety of methods including a batch ion exchange procedure, column ion exchange chromatography and hydrophobic interaction chromatography. The enzyme had a maximum activity at around pH 8.0 and had a Km for acetaminophen of 0.11 mM. Electrochemical assays of aryl acylamidase activity are described. The enzyme is suitable for use as a reagent in the clinical diagnostic measurement of acetaminophen.

Sprouts emerging from the dendrites of axotomized lamprey central neurons have axonlike ultrastructure.

We have examined the dendritic and axonal ultrastructure of intact anterior bulbar reticulospinal neurons (ABCs) in the CNS of the larval sea lamprey and compared it with that of the dendrites and neuritic sprouts from ABCs examined 2 months following axotomy. Dendrites and axons of intact ABCs are distinguishable from one another by several ultrastructural criteria: (1) the predominance of microtubules in the dendritic cytoskeleton and neurofilaments in that of the axon, (2) the exclusively postsynaptic status of the dendrites versus the presynaptic status of the axon, and (3) the presence of polyribosomes and large numbers of mitochondria in the dendrites and their respective absence and scarcity in the axon. The ultrastructure of axonal sprouts evoked by axotomy of ABCs 1-1.5 mm from their somata ("intermediate axotomy") in many ways resembled that of intact axons. Axonal sprouts were presynaptic to other neurons, and their cytoskeletons consisted mainly of neurofilaments. They also exhibited some features not seen in either axons or dendrites, such as numerous clusters of small vesicles that were not associated with synapses and, in some cases, close associations with glial elements. We also examined sprouts emerging from the dendrites of ABCs following axotomy within 500 microns of their somata ("close axotomy") and found that such "dendritic" sprouts closely resembled axonal sprouts; they possessed neurofilament-dominated cytoskeletons, were presynaptic to other neurons, and were often associated with glial elements. The dendrites of ABCs undergoing dendritic sprouting retained their normal gross morphology but possessed a mixture of "axonal" and "dendritic" ultrastructural characteristics, exhibiting neurofilament-dominated cytoskeletons while remaining entirely postsynaptic to other neurons. However, there were significantly fewer synapses on the dendrites of axotomized cells than were found on the dendrites of intact ABCs. We conclude that sprouts evoked by axotomy are intrinsically axonal in character whether they originate from the axon stump or from the dendritic tree. Our results also suggest that the materials necessary for axonal regeneration may displace elements of the dendritic cytoskeleton as they are transported through the dendrites to the emerging "dendritic" sprouts following close axotomy.

Dendritic amputation redistributes sprouting evoked by axotomy in lamprey central neurons.

In the previous paper (Hall and Cohen, 1988), we showed that axotomy of anterior bulbar cells (ABCs) in the hindbrain of the larval lamprey results in the sprouting of axonlike neurites from either the end of the proximal axon stump, the dendritic tips, or both, depending on the site of axotomy. Here we show that, unlike axotomy, dendritic amputation (dendrotomy) does not by itself induce sprouting from ABCs. However, dendrotomy does induce sprouting from dendrites in the immediate vicinity of the dendritic lesion in cells that have been previously axotomized. We found that dendrotomy acts primarily to rearrange the distribution of sprouts induced by axotomy rather than serving as an additional stimulus to neurite outgrowth. We propose that (1) dendritic sprouting in ABCs occurs because the dendritic tips become attractive sites for sprout initiation when they are either directly injured (as with dendrotomy) or are situated relatively close to the site of injury (as with axotomy close to the soma), and (2) the axon stump, dendritic stumps, and uninjured dendritic tips of the cell compete to initiate a limited total amount of sprouting induced by axotomy. The probability that a given locus will support sprouting is determined both by its proximity to the nearest lesion site and by whether there are other attractive potential sprouting sites in the cell.

The pattern of dendritic sprouting and retraction induced by axotomy of lamprey central neurons.

We have investigated some of the factors controlling the distribution of axonal and dendritic sprouting following axotomy of a subset of Muller giant interneurons (anterior bulbar cells or ABCs) in the hindbrain of the larval sea lamprey (Petromyzon marinus). Sprouts originated from different sites in the cell depending on the distance of the axonal lesion from the soma. When the axon was cut close to the soma (within 500 microns), the dendritic tips sprouted profusely, whereas the proximal axon stump showed few sprouts and frequently disappeared entirely. Axotomy further from the soma (1000-1400 microns) resulted in less sprouting from the dendrites and more from the axon stump, with the total amount of dendritic plus axonal sprouting remaining constant. Axotomy at sites distant from the soma (1 cm or more) did not result in dendritic sprouting. No sprouts were ever observed emerging from the soma proper or from the axon stump except at the lesion site. Neuritic sprouts from dendrites and axon were similar in their gross morphology. Sprouts resembled axons rather than dendrites whatever their sites of origin; they followed linear, rostrocaudally oriented paths in the "basal plate" region of the hindbrain. Dendritic and axonal sprouts grew both rostrally and caudally within the brain. Either "close" or "distant" axotomy resulted in the retraction of the dendritic tree and of both dendritic and axonal sprouts by several months postaxotomy. Reaxotomy close to the soma 30 d after a distant axotomy accelerated the onset of this evoked dendritic retraction. Reaxotomy close to the soma also induced sprouting significantly sooner than did close axotomy alone. These results suggest that axotomy close to the soma causes axonal regeneration to be shunted into ectopic locations at the dendritic tips. The emerging sprouts then follow guidance cues appropriate for regenerating ABC axons.

Control of neuron shape during development and regeneration.

We have examined the ability of Mueller reticulospinal neurons in the CNS of the larval sea lamprey to sprout following axonal and dendritic injury. Axotomy induces regenerative sprouting exclusively from the axon stump if it occurs at a site distant from the soma in the spinal cord. However, axotomy within the hindbrain at a site close to the soma results in profuse neuritic sprouting from the dendrites. The gross morphology and trajectories of these "dendritic" sprouts resemble those of regenerating axons. Amputation of Mueller cell dendrites (dendrotomy) without axotomy does not result in neuritic sprouting from either the axon or dendrites, indicating that axotomy is specifically required for sprouting to occur. However, dendrotomy is capable of altering the distribution of sprouting in a previously axotomized Mueller cell by inducing sprouting at the site of the dendrotomy lesion. Sprouts of both dendritic and axonal origin tend to follow linear, rostrocaudally oriented paths along or near the ventral surface of the hindbrain. Some sprouts form very large, palmate growth cones on the marginal surface, which in turn give rise to many branches that continue to grow either rostrally or caudally along the surface of the brain. We discuss the possibility that both dendritic and axonal sprouts evoked by axotomy of Mueller neurons are recapitulating initial axonal development during embryogenesis, and that their trajectories are determined by developmental guidance cues persisting in the ventral hindbrain.

Extensive dendritic sprouting induced by close axotomy of central neurons in the lamprey.

Massive dendritic sprouting was induced in identified giant reticulospinal neurons of the lamprey by axotomy close to the soma. An axonal lesion slightly farther from the cell body induced new growth from both dendrites and axon. The amount of new growth per cell was the same whether it originated from the dendrites alone or from axonal and dendritic compartments. The location of the axonal lesion therefore determines where, in the neuron, membrane is inserted to produce the new neurites. The dendritic tree of a differentiated vertebrate central neuron was shown to have sufficient plasticity to extend new growth for several millimeters beyond the normal dendritic domain.