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The mechanisms underpinning concussion, traumatic brain injury, and chronic traumatic encephalopathy, and the relationships between these disorders, are poorly understood. We examined post-mortem brains from teenage athletes in the acute-subacute period after mild closed-head impact injury and found astrocytosis, myelinated axonopathy, microvascular injury, perivascular neuroinflammation, and phosphorylated tau protein pathology. To investigate causal mechanisms, we developed a mouse model of lateral closed-head impact injury that uses momentum transfer to induce traumatic head acceleration. Unanaesthetized mice subjected to unilateral impact exhibited abrupt onset, transient course, and rapid resolution of a concussion-like syndrome characterized by altered arousal, contralateral hemiparesis, truncal ataxia, locomotor and balance impairments, and neurobehavioural deficits. Experimental impact injury was associated with axonopathy, blood-brain barrier disruption, astrocytosis, microgliosis (with activation of triggering receptor expressed on myeloid cells, TREM2), monocyte infiltration, and phosphorylated tauopathy in cerebral cortex ipsilateral and subjacent to impact. Phosphorylated tauopathy was detected in ipsilateral axons by 24 h, bilateral axons and soma by 2 weeks, and distant cortex bilaterally at 5.5 months post-injury. Impact pathologies co-localized with serum albumin extravasation in the brain that was diagnostically detectable in living mice by dynamic contrast-enhanced MRI. These pathologies were also accompanied by early, persistent, and bilateral impairment in axonal conduction velocity in the hippocampus and defective long-term potentiation of synaptic neurotransmission in the medial prefrontal cortex, brain regions distant from acute brain injury. Surprisingly, acute neurobehavioural deficits at the time of injury did not correlate with blood-brain barrier disruption, microgliosis, neuroinflammation, phosphorylated tauopathy, or electrophysiological dysfunction. Furthermore, concussion-like deficits were observed after impact injury, but not after blast exposure under experimental conditions matched for head kinematics. Computational modelling showed that impact injury generated focal point loading on the head and seven-fold greater peak shear stress in the brain compared to blast exposure. Moreover, intracerebral shear stress peaked before onset of gross head motion. By comparison, blast induced distributed force loading on the head and diffuse, lower magnitude shear stress in the brain. We conclude that force loading mechanics at the time of injury shape acute neurobehavioural responses, structural brain damage, and neuropathological sequelae triggered by neurotrauma. These results indicate that closed-head impact injuries, independent of concussive signs, can induce traumatic brain injury as well as early pathologies and functional sequelae associated with chronic traumatic encephalopathy. These results also shed light on the origins of concussion and relationship to traumatic brain injury and its aftermath.awx350media15713427811001., (© The Author(s) (2018). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Tau misprocessing to form aggregates and other toxic species has emerged as a major feature in our developing understanding of the etiology and pathogenesis of Alzheimer's disease (AD). The significance of tau misprocessing in AD has been further emphasized by recent studies showing that tau can be secreted from neurons via exosomes and may itself be an important agent in the spreading of neurofibrillary lesions within the brain. Tau secretion occurs most readily under disease-associated conditions in cellular models, suggesting that cellular changes responsible for secretion, possibly including tau oligomerization, could play a key role in the propagation of neurofibrillary lesions in neurodegenerative disease. Here we show that overexpression of 4R0N human tau in neuroblastoma cells recruits mitochondrial and axonogenesis-associated proteins relevant to neurodegeneration into the exosomal secretion pathway via distinct mechanisms. The recruitment of mitochondrial proteins appears to be linked to autophagy disruption (exophagy) in multiple neurodegenerative conditions but has few known direct links to AD and tau. By contrast, the involvement of synaptic plasticity and axonogenesis markers is highly specific to both tau and AD and may be relevant to the reactivation of developmental programs involving tau in AD and the recently demonstrated ability of secreted tau to establish tissue distribution gradients in CNS neuropil. We also found a highly significant correlation between genes that are significantly downregulated in multiple forms of AD and proteins that have been recruited to exosomes by tau, which we interpret as strong evidence for the central involvement of tau secretion in AD cytopathogenesis. Our results suggest that multiple cellular mechanisms may link tau secretion to both toxicity and neurofibrillary lesion spreading in AD and other tauopathies.

When the microtubule (MT)-associated protein tau is not bound to axonal MTs, it becomes hyperphosphorylated and vulnerable to proteolytic cleavage and other changes typically seen in the hallmark tau deposits (neurofibrillary tangles) of tau-associated neurodegenerative diseases (tauopathies). Neurofibrillary tangle formation is preceded by tau oligomerization and accompanied by covalent crosslinking and cytotoxicity, making tangle cytopathogenesis a natural central focus of studies directed at understanding the role of tau in neurodegenerative disease. Recent studies suggest that the formation of tau oligomers may be more closely related to tau neurotoxicity than the presence of the tangles themselves. It has also become increasingly clear that tau pathobiology involves a wide variety of other cellular abnormalities including a disruption of autophagy, vesicle trafficking mechanisms, axoplasmic transport, neuronal polarity, and even the secretion of tau, which is normally a cytosolic protein, to the extracellular space. In this review, we discuss tau misprocessing, toxicity and secretion in the context of normal tau functions in developing and mature neurons. We also compare tau cytopathology to that of other aggregation-prone proteins involved in neurodegeneration (alpha synuclein, prion protein, and APP). Finally, we consider potential mechanisms of intra- and interneuronal tau lesion spreading, an area of particular recent interest.

Our recent identification of an exosomal route for tau protein secretion(1) marks a key similarity between tau and other aggregation-prone proteins implicated in neurodegenerative disease pathogenesis and is to some extent congruent with the popular idea that tau pathology spreads between neurons via a "prionlike" template-mediated protein misfolding mechanism in AD and other tauopathies. However, the observation that much of the phosphotau in CSF samples from early AD patients is exosomal (and thus likely to have been secreted) calls into question a very widely held and plausible assumption - the idea that the elevated CSF-tau in AD is due to the passive release and accumulation of tau in the CSF as a consequence of widespread neuronal death. Here we examine this issue directly and explore some of the broader implications of this study for our understanding of AD pathogenesis and the prospects for improving its diagnosis and treatment.

Aggregation-prone proteins associated with neurodegenerative disease, such as α synuclein and β amyloid, now appear to share key prion-like features with mammalian prion protein, such as the ability to recruit normal proteins to aggregates and to translocate between neurons. These features may shed light on the genesis of stereotyped lesion development patterns in conditions such as Alzheimer disease and Lewy Body dementia. We discuss the qualifications of tau protein as a possible "prionoid" mediator of lesion spread based on recent characterizations of the secretion, uptake and transneuronal transfer of human tau isoforms in a variety of tauopathy models, and in human patients. In particular, we consider (1) the possibility that prionoid behavior of misprocessed tau in neurodegenerative disease may involve other aggregation-prone proteins, including PrP itself, and (2) whether "prionlike" tau lesion propagation might include mechanisms other than protein-protein templating.

Blast exposure is associated with traumatic brain injury (TBI), neuropsychiatric symptoms, and long-term cognitive disability. We examined a case series of postmortem brains from U.S. military veterans exposed to blast and/or concussive injury. We found evidence of chronic traumatic encephalopathy (CTE), a tau protein-linked neurodegenerative disease, that was similar to the CTE neuropathology observed in young amateur American football players and a professional wrestler with histories of concussive injuries. We developed a blast neurotrauma mouse model that recapitulated CTE-linked neuropathology in wild-type C57BL/6 mice 2 weeks after exposure to a single blast. Blast-exposed mice demonstrated phosphorylated tauopathy, myelinated axonopathy, microvasculopathy, chronic neuroinflammation, and neurodegeneration in the absence of macroscopic tissue damage or hemorrhage. Blast exposure induced persistent hippocampal-dependent learning and memory deficits that persisted for at least 1 month and correlated with impaired axonal conduction and defective activity-dependent long-term potentiation of synaptic transmission. Intracerebral pressure recordings demonstrated that shock waves traversed the mouse brain with minimal change and without thoracic contributions. Kinematic analysis revealed blast-induced head oscillation at accelerations sufficient to cause brain injury. Head immobilization during blast exposure prevented blast-induced learning and memory deficits. The contribution of blast wind to injurious head acceleration may be a primary injury mechanism leading to blast-related TBI and CTE. These results identify common pathogenic determinants leading to CTE in blast-exposed military veterans and head-injured athletes and additionally provide mechanistic evidence linking blast exposure to persistent impairments in neurophysiological function, learning, and memory.

Recent demonstrations that the secretion, uptake, and interneuronal transfer of tau can be modulated by disease-associated tau modifications suggest that secretion may be an important element in tau-induced neurodegeneration. Here, we show that much of the tau secreted by M1C cells occurs via exosomal release, a widely characterized mechanism that mediates unconventional secretion of other aggregation-prone proteins (α-synuclein, prion protein, and β-amyloid) in neurodegenerative disease. Exosome-associated tau is also present in human CSF samples and is phosphorylated at Thr-181 (AT270), an established phosphotau biomarker for Alzheimer disease (AD), in both M1C cells and in CSF samples from patients with mild (Braak stage 3) AD. A preliminary analysis of proteins co-purified with tau in secreted exosomes identified several that are known to be involved in disease-associated tau misprocessing. Our results suggest that exosome-mediated secretion of phosphorylated tau may play a significant role in the abnormal processing of tau and in the genesis of elevated CSF tau in early AD.

While the interneuronal propagation of neurofibrillary lesions in Alzheimer's disease and other tauopathies now appears to involve the spreading of tau-associated toxicity, little is known about its mechanism. We characterized the movement of human tau through the brain of a non-transgenic lower vertebrate tauopathy model in which full-length wild type and mutant human tau isoforms were expressed in identified neurons, thus permitting the identification and localization of EC tau sources. We describe two distinct patterns of tau spreading that correspond to tau species that lack (MTBR-) and contain (MTBR+) the tau microtubule-binding region. These patterns illustrate the production, migration and uptake of EC tau and resemble some of the extracellular tau deposits typically seen in human brain after repeated traumatic injury in cases of chronic traumatic encephalopathy (CTE). We propose that misprocessed human tau can spread between CNS neurons via a variety of non-synaptic mechanisms as well as synaptically mediated mechanisms.

We used a nontransgenic cellular tauopathy model in which individual giant neurons in the lamprey CNS (ABCs) overexpress human tau isoforms cell autonomously to characterize the still poorly understood consequences of disease-associated tau processing in situ. In this model, tau colocalizes with endogenous microtubules and is nontoxic when expressed at low levels, but is misprocessed by a toxicity-associated alternative pathway when expressed above levels that saturate dendritic microtubules, causing abnormally phosphorylated, vesicle-associated tau to accumulate in ABC distal dendrites. This causes localized microtubule loss and eventually dendritic degeneration, which is preceded by tau secretion to the extracellular space. This sequence is reiterated at successively more proximal dendritic locations over time, suggesting that tau-induced dendritic degeneration is driven by distal dendritic accumulation of hyperphosphorylated, vesicle-associated tau perpetuated by localized microtubule loss. The implications for the diagnosis and treatment of human disease are discussed.

Hyperphosphorylation of tau is closely associated with its aggregation by as yet undefined mechanisms. We attempted herein to further investigate the interrelationships between tau aggregation and phosphorylation by inhibition and activation of cdk5 and GSK3β in cells expressing normal tau and a mutant form of tau (3PO-tau), which generates intracellular aggregates while retaining microtubule-binding capacity). Aggregates were routinely observed in cells expressing 3PO-tau, but never in cells expressing normal tau, whether or not cdk5 or GSK3β was overexpressed. In addition, in cells expressing 3PO-tau, both the percentage of cells with aggregates, as well as the size of aggregates, was increased following overexpression of cdk5 or GSK3β, decreased following treatment with pharmacological agents (roscovitine and lithium) active against these kinases, and increased following treatment with the phosphatase inhibitor okadaic acid. These findings collectively indicate that phosphorylation potentiates aggregation in the presence of one or more key tau mutations. These findings confirm and extend prior studies in which overexpression of the cdk5 activator p35, or GSK3β, induced phosphorylation, mislocalization and/or aggregation of tau.

Abnormal tau cleavage is prominent in the neurofibrillary degeneration characteristic of Alzheimer's disease (AD) and related tauopathies. We recently showed that cleaved human tau is secreted by specific mechanisms when overexpressed. Here we examined the effect of expressing N-terminal and full length tau constructs in transiently and stably transfected neuronal lines. We show that secreted tau exhibits a cleavage pattern similar to CSF-tau from human AD patients and that tau secretion is specifically inhibited by the presence of the exon 2 insert. These results suggest that tau secretion may play a hitherto unsuspected role in AD and related tauopathies., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The mechanisms by which tau-containing lesions are propagated between adjacent and synaptically interconnected parts of the brain are a potentially important but poorly understood component of human tauopathies such as Alzheimer's disease, Pick's disease, and corticobasal degeneration. Since the utility of currently available transgenic models for studying intercellular aspects of tauopathy is limited by their broad patterns of tau expression in the central nervous system, we used an in situ tauopathy model that replicates tau-induced cytodegeneration in identified neurons on a tau-negative background to determine whether tau secretion or interneuronal transfer might play a role in lesion propagation. We found that the N-terminal half of tau is required for tau secretion and is efficiently exported to the extracellular space and adjacent neurons at relatively low levels of overexpression. By contrast, full-length tau is secreted by a separate mechanism that is correlated with phosphorylation of tau at tyrosine 18 and dendritic degeneration, is exacerbated by tauopathy mutations, and blocked by mutations that inhibit tau:tau interactions. Anterograde transneuronal tau movement occurred with the expression of tau containing the P301L tauopathy mutant, but not with wild type tau isoforms. Our results are consistent with recent studies suggesting a role for molecular "templating" in the propagation of neurofibrillary lesions and provide a novel conceptual and experimental basis for studying the mechanisms of interneuronal propagation and toxicity in human neurodegenerative disease.

Small-molecule inhibitors of neurofibrillary lesion formation may have utility for treatment of Alzheimer's disease and certain forms of frontotemporal lobar degeneration. These lesions are composed largely of tau protein, which aggregates to form intracellular fibrils in affected neurons. Previously it was shown that chronic overexpression of human tau protein within identified neurons (anterior bulbar cells) of the sea lamprey induced a phenotype-resembling tauopathic neurodegeneration, including the formation of tau filaments, fragmentation of dendritic arbors, and eventual cell death. Development of this neurodegenerative phenotype was blocked by chronic administration of a benzothiazole derivative termed N3 ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) to lamprey aquaria. Here we examined the mechanism of action of N3 and an alkene analog termed N4 ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole) in vitro and in the lamprey model. Results showed that although both compounds entered the lamprey central nervous system, only N3 arrested tauopathy. On the basis of in vitro aggregation assays, neither compound was capable of directly inhibiting tau filament formation. However, N3, but not N4, was capable of partially antagonizing the binding of Thioflavin S to synthetic tau filaments. The results suggest that occupancy of N3-binding sites on nascent tau filaments may significantly retard the progressive degeneration accompanying tau overexpression in lamprey.

Exonic mutations in the gene coding for human tau cause familial neurofibrillary degenerative diseases (tauopathies) which exhibit mutation-specific characteristics. It is thus unclear whether such mutations have similar effects on tau structure and function in vivo and if they act via similar cytopathological mechanisms in vulnerable neuron types. We have previously shown that overexpressing wild type human tau isoforms in identified giant neurons (ABCs) of the lamprey CNS results in characteristic, stereotyped cytopathological changes in these cells over several weeks. Here, we use this model to compare the cytopathological consequences of expressing wild type and exonic mutant tau isoforms (P301L, G272V, V337M, and R406W) at a high level of resolution. We show that each of the four exonic htau mutations tested accelerate degeneration in ABCs when compared to their WT parent isoforms, and that the patterns of human tau distribution, phosphorylation and cytopathology, while similar, vary characteristically from one another among both WT and mutant isoforms in a single identified neuron in situ. Our results therefore suggest that at least some of the differences between the effects of these mutations in humans are due to cell autonomous, mutation specific differences in the cytopathological mechanism of tau-induced neurodegeneration.

The carboxyl terminal "tail" domains of the heavy and middle molecular weight mammalian neurofilament (NF) proteins regulate inter-NF spacing and formation of organized networks. The C-terminal region of the larger of the two lamprey NF subunits (NF-180) resembles these mammalian proteins in that it consists of a proximal glutamate-rich region and a distal region containing multiple phosphorylation sites. To investigate the role of these two sidearm domains in the organization of lamprey NFs, we generated plasmids lacking the glutamate-rich domain, the domain containing multiple phosphorylation sites, or both, and examined the impact of the resultant mutant proteins on the endogenous NF network in differentiated NB21/d1 neuroblastoma cells. We present evidence that, like mammalian NFs, the glutamate-rich region of NF-180 sidearm plays a critical role in NF architecture.

Acute and chronic liver diseases are an assortment of disorders brought to the clinician's attention by abnormal liver function tests or specific signs and symptoms. The differential diagnosis includes disorders that have primary or secondary liver involvement. This paper will be limited to the epidemiology, clinical manifestations, diagnosis, treatment, and prevention of the different viral liver diseases: A, B, C, D, E and G.

The estrogen receptor is the master transcriptional regulator of breast cancer phenotype and the archetype of a molecular therapeutic target. We mapped all estrogen receptor and RNA polymerase II binding sites on a genome-wide scale, identifying the authentic cis binding sites and target genes, in breast cancer cells. Combining this unique resource with gene expression data demonstrates distinct temporal mechanisms of estrogen-mediated gene regulation, particularly in the case of estrogen-suppressed genes. Furthermore, this resource has allowed the identification of cis-regulatory sites in previously unexplored regions of the genome and the cooperating transcription factors underlying estrogen signaling in breast cancer.

The large group of neurodegenerative diseases which feature abnormal metabolism and accumulation of tau protein (tauopathies) characteristically produce a multiplicity of cellular and systemic abnormalities in human patients. Understanding the complex pathogenetic mechanisms by which abnormalities in tau lead to systemic neurofibrillary degenerative disease requires the construction and use of model experimental systems in which the behavior of human tau can be analyzed under controlled conditions. In this paper, we survey the ways in which in vitro, cellular and whole-animal models of human tauopathy are being used to add to our knowledge of the pathogenetic mechanisms underlying these conditions. In particular, we focus on the complementary advantages and limitations of various approaches to constructing tauopathy models presently in use with respect to those of murine transgenic tauopathy models.

Although tau filament formation is a central event in familial tauopathies and Alzheimer's disease (AD), the cellular consequences of neurofibrillary tangle (NFT) formation are poorly understood because of the unavailability of mammalian in vivo cellular models of neurofibrillary degeneration (NFD). We have shown that human tau forms filaments and is associated with cytodegeneration when overexpressed chronically in identified neurons (ABCs) in the lamprey central nervous system (CNS). In this model, degeneration occurs according to a stereotyped sequence that closely resembles the pattern seen in tangle-bearing neurons in AD, with both tau deposition and fragmentation beginning in distal dendrites and progressing proximally over time. This sequence has been divided into four stages ranging from (1) mild beading of terminal dendrites only through (4) extensive dendritic fragmentation and loss. Here, we show that lipid-soluble, low-molecular-weight (approx 300 Da) proprietary compounds that have been demonstrated to block tau filament formation in vitro can significantly retard the progressive degeneration of ABCs that express human tau23. Bath application of one of these compounds for periods of up to 50 d after plasmid injection prevented degeneration beyond stage 2 in 90% of all treated cells, whereas over half of control cells showed severe degeneration by this time. This provides the first in vivo experimental evidence directly supporting a causal role for tau filament formation in the pathogenesis of NFD and suggests that intensive effort toward developing therapeutic agents for AD and other NFDs targeted at blocking tau filament formation is warranted.

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

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.

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., (Copyright 2000 Wiley-Liss, Inc.)

Accumulation of abnormally modified tau protein (PHF-tau) is the principal intracellular lesion in a variety of neurodegenerative diseases, including Alzheimer's Disease (AD), but the cellular mechanisms underlying this accumulation are unknown. In this study, the cellular metabolism of PHF-tau purified from AD brain was investigated by microinjecting it into identified central neurons of the lamprey, a lower vertebrate. Dephosphorylation of 2 critical epitopes (the PHF-1 and TAU-1 sites), occurred within a few hours of PHF-tau microinjection, while proteolysis was complete by 2 days. These results constitute the first demonstration of the intracellular degradation of PHF-tau in an experimental in vivo system and suggest that the degradation of PHF-tau in situ is preceded by dephosphorylation. They also suggest that intracellular PHF-tau accumulation is primarily due to the failure of normal dephosphorylation and/or proteolytic mechanisms during neurofibrillary degenerative disease.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.