Tau-dependent microtubule disassembly initiated by prefibrillar beta-amyloid.

Alzheimer's Disease (AD) is defined histopathologically by extracellular beta-amyloid (Abeta) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Abeta lies upstream of tau, but the steps that connect Abeta to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Abeta in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Abeta42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Abeta40, and microtubules were insensitive to fibrillar Abeta. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Abeta.

Axon cytoskeleton ultrastructure in chronic inflammatory demyelinating polyneuropathy.

INTRODUCTION: To detail the extent and pattern of axon cytoskeleton alterations in chronic inflammatory demyelinating polyneuropathy (CIDP). METHODS: Nerve biopsies from 7 cases of CIDP, including 4 cases with severe fiber loss, were compared with 5 controls by morphometric transmission electron microscopy (TEM). RESULTS: Despite demyelination of single fibers, myelin ultrastructure was otherwise normal. Contrary to immunolabeling, TEM revealed a decrease in neurofilament (NF) density in every case, although there were pronounced variations among fibers even in the same sample. The NF decrease reached the same extent in large- and small-diameter fibers. It was observed in normally myelinated fibers, suggesting they were demyelinated at a distance from the section. Minimal inter-NF distance increased roughly inversely to NF density. Microtubules increased in 3 cases previously characterized by increased growth-associated protein (GAP-43) immunolabeling. CONCLUSION: These data demonstrate the severity and constancy of axonal lesions, and especially of NF, in residual fibers in our cases of CIDP.

Histomorphological study of myelinated nerve fibres in the periodontal ligament of human canine.

OBJECTIVE: This study aims to describe the human periodontal ligament (PDL) using serial sections, with a focus on mechanoreceptor distribution and morphology. MATERIALS AND METHODS: One permanent lower canine with surrounding PDL and alveolar bone tissues was retrieved from a human cadaver. After being embedded into paraffin block, the canine was horizontally cut in 6 µm thick serial sections. At root levels of 0.3, 1.5, 3, 4.5 and 6 mm from the apex, five slices each level were evaluated. Immunocytochemisty was performed on the same serial sections, enabling a more reliable description of neural structures. RESULTS: The distribution of myelinated fibres varied from apical to coronal level, with a total number of 38 at 0.3 mm from the apex, 25 at 1.5 mm, 25 at 3 mm, 31 at 4.5 mm and 32 at 6 mm. At all times, mesial and buccal regions were typically more densely innervated (p < 0.01) except at the 3 mm level. The average density of myelinated nerve fibres increased by arriving closer to the apex. However, the average diameter did not show any significant differences amongst quadrants or root levels (p > 0.05). The average diameter of myelinated fibres varied between 5.3-7.8 µm. Grouped myelinated axons were twice as common as isolated ones, with the innervation being rather close to the alveolar bone. Isolated myelinated axons showed a tendency to group around large blood vessels. CONCLUSION: The present results add to the understanding of human PDL innervation, indicating dense innervations by myelinated nerve fibres in close proximity to collagen fibres and alveolar bone. It also reveals that apical as well as mesial and buccal sites of the human canine are more densely innervated.

Cajal's second great battle for the neuron doctrine: the nature and function of neurofibrils.

One hundred years ago, a novel kind of reticularism threatened to displace the neuron doctrine as the established model of functional organization of the nervous system. The challenging paradigm, championed by Stephan von Apáthy and Albrecht Bethe, held that nerve impulses propagate along neurofibrils connected in a continuous network throughout all nerve cells. Santiago Ramón y Cajal, a leading figure in the conception of the neuron doctrine, headed again the battle against this return of reticularism. Dissatisfied with the available staining techniques, he devised the "reduced silver nitrate method" that even Camillo Golgi recognized as the best at the time for revealing the neurofibrils. In 1904 Cajal already published over a dozen papers in three languages describing neurofibril distributions in the nervous systems of diverse vertebrates and invertebrates, under both normal and experimental conditions. Next he investigated the involvement of neurofibrils in the process of nerve regeneration. This unprecedented survey led him to the conclusion that the neurofibrils are linear "colonies" of particles constituting a semi-solid, dynamic internal skeleton of the nerve cell. Apáthy reacted with a long invective paper that Cajal had no choice but acknowledging. His comprehensive reply, published in 1908, meant the effective end of the renewed reticularist campaign against the neuron doctrine. Along the way, a visionary and today almost forgotten chapter in the history of the cytoskeleton had also been written.

Subunit structure of paired helical filaments in Alzheimer's disease.

The neurofibrillary tangles that occur in the brain in cases of senile dementia of the Alzheimer type contain a distinctive type of filament, the paired helical filament (PHF). We have developed a method for isolating the tangles postmortem in sufficient yield for structural study of PHFs by electron microscopy of negatively stained and shadowed preparations. This material shows the characteristic helical structure seen in sectioned embedded material. In addition, two striking fragmentation patterns are observed. (a) Some filaments show sharp transverse breaks at apparently random positions along the filament. (b) In a few PHFs one strand is missing for a variable length, whereas the other appears to maintain its structural integrity. The shadowed specimens show the PHF to be wound in a left-handed manner. These observations indicate that the PHF consists of subunits of very limited axial extent arranged along two left-handed helical strands. The visualization of the substructure within the PHFs is rather variable and a model building approach has therefore been adopted, which has allowed the main features seen in the images to be interpreted. The subunit appears to have at least two domains in a radial direction and an axial extent of less than 5 nm. The whole structure can best be described as a twisted ribbon and indeed alkali treatment does untwist PHFs to give flat ribbons. The nature of the proposed model makes it most unlikely that the PHF is formed by a simple collapse of normal cytoskeletal elements, such as neurofilaments.

Neurofilament proteins of rat peripheral nerve and spinal cord.

Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.

Immunological and ultrastructural studies of neurofilaments isolated from rat peripheral nerve.

Neurofilaments were isolated from desheathed and minced segments of rat peripheral nerve by osmotic shock into 0.01 M Tris-HCI buffer, pH 7.2. Freshly isolated neurofilaments were observed to undergo disassembly by progressive fragmentation upon exposure of dilute tissue extracts to this buffer. Low- and high-speed centrifugations of these tissue extracts separated membranous and particulate constituents and produced a progressive enrichment of 68,000-dalton polypeptide band in successive supernates, as determined by analyses of soluble proteins by SDS-polyacrylamide electrophoresis. The final high-speed supernatant fractions (S3) of nerve extracts, which were predominantly composed of 68,000-dalton polypeptide, were used to raise a specific experimental antisera in rabbits. Utilizing techniques of immune electron microscopy, experimental rabbit antisear was shown to contain antibodies against neurofilaments. Intact neurofilaments isolated from rat nerves and attached to carbon-coated grids became decorated when exposed to experimental rabbit antisera or purified gamma globulin (IgG) derivatives. The decoration of neurofilaments closely resembled the IgG coating seen in immune electron microscopy. Antibody absorption techniques were used to identify the biochemical constituency of neurofilamentous antigenic determinants. The decoration of neurofilament by experimental IgG was not altered by additions of tubulin or bovine serum albumin, but was prevented by additions of S3 fractions as well as the 68,000-dalton polypeptide of this fraction which was eluted and recovered from polyacrylamide gels. These findings are indicative that a 68,000-dalton polypeptide is a constituent subunit of rat peripheral nerve neurofilaments.

Helical substructure of neurofilaments isolated from Myxicola and squid giant axons.

Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope. The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea. When the ropelike structure comprising the neurofilaments untwists, two strands 4--5.5nm in diameter can be resolved. Upon further denaturation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures. Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments. The strands appear to consist of protofilaments 2--2.5 nm in diameter. These observations suggest that the neurofilament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they su-port the filamentous rather than the golbular model of intermediate filament structure.

Chemical and structural changes of neurofilaments in transected rat sciatic nerve.

The sequence of changes occurring in transected rat sciatic nerve was examined by electron microscopy and by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. Representative segments of transected nerves were processed for ultrastructural examinations between 0 and 34 days after the transection of sciatic nerves immediately below the sacro-sciatic notch. The remainder of the transected nerves and the intact portions of sciatic nerves were desheathed and immediately homogenized in 1 percent SDS containing 8 M urea and 50 mM dithioerythritol. Solubilized proteins were analyzed on 12 percent gels at pH 8.3 in a discontinuous electrophoretic system. Initial changes were limited to the axons of transected nerve fibers and were characterized by the loss of microtubules and neurofilaments and their replacement by an amorphous floccular material. These changes became widespread between 24 and 48 h after transection. The disruption of neurofilaments during this interval occurred in parallel with a selective loss of 69,000, 150,000 and 200,000 mol wt proteins from nerve homogenates, thus corroborating the view that these proteins represent component subunits of mammalian neurofilaments. Furthermore, the selective changes of neurofilament proteins in transected nerves indicate their inherent lability and suggest their susceptibility to calcium-mediated alterations. Electrophoretic profiles of nerve proteins during the 4-34-day interval after nerve transection reflected the breakdown and removal of myelin, the proliferation of Schwann cells and the deposition of endoneurial collagen. A marked increase of intermediate-sized filaments within proliferating Schwann cell processes was not accompanied by the appearance of neurofilamentlike proteins in gels of nerve homogenates.

Growth of neurites without filopodial or lamellipodial activity in the presence of cytochalasin B.

To examine the role in neurite growth of actin-mediated tensions within growth cones, we cultured chick embryo dorsal root ganglion cells on various substrata in the presence of cytochalasin B. Time-lapse video recording was used to monitor behaviors of living cells, and cytoskeletal arrangements in neurites were assessed via immunofluorescence and electron microscopic observations of thin sections and whole, detergent-extracted cells decorated with the S1 fragment of myosin. On highly adhesive substrata, nerve cells were observed to extend numerous (though peculiarly oriented) neurites in the presence of cytochalasin, despite their lack of both filopodia and lamellipodia or the orderly actin networks characteristic of typical growth cones. We concluded that growth cone activity is not necessary for neurite elongation, although actin arrays seem important in mediating characteristics of substratum selectivity and neurite shape.

Slow axonal transport of neurofilament proteins: impairment of beta,beta'-iminodipropionitrile administration.

beta,beta'-Iminodipropionitrile (IDPN) administration prevented normal slow axonal transport of [35S]methionine- or [3H]leucine-labeled proteins in rat sciatic motor axons. Ultrastructural and electrophoretic studies showed that the neurofilament triplet proteins in particular were retained within the initial 5 millimeters of the axons, resulting in neurofilament-filled axonal swellings. Fast anterograde and retrograde axonal transport were not affected. The IDPN thus selectively impaired slow axonal transport. The neurofibrillary pathology in this model is the result of the defective slow transport of neurofilaments.

Alzheimer's disease: X-ray spectrometric evidence of aluminum accumulation in neurofibrillary tangle-bearing neurons.

The elemental content of neurons of the hippocampus was studied by a combination of scanning electron microscopy and x-ray spectrometry in autopsy-derived brain tissue from three cases of senile dementia (Alzheimer type) and three nondemented elderly controls. Foci of aluminum were detected within the nuclear region of a high percentage of neurons containing neurofibrillary tangles from the cases of senile dementia as well as the elderly controls. The adjacent normal-appearing neurons from both groups of patients were virtually free of detectable aluminum. These findings suggest that the association of aluminum to Alzheimer's disease extends to the neuronal level.

Deposits of amyloid beta protein in the central nervous system of transgenic mice.

Alzheimer's disease is characterized by widespread deposition of amyloid in the central nervous system. The 4-kilodalton amyloid beta protein is derived from a larger amyloid precursor protein and forms amyloid deposits in the brain by an unknown pathological mechanism. Except for aged nonhuman primates, there is no animal model for Alzheimer's disease. Transgenic mice expressing amyloid beta protein in the brain could provide such a model. To investigate this possibility, the 4-kilodalton human amyloid beta protein was expressed under the control of the promoter of the human amyloid precursor protein in two lines of transgenic mice. Amyloid beta protein accumulated in the dendrites of some but not all hippocampal neurons in 1-year-old transgenic mice. Aggregates of the amyloid beta protein formed amyloid-like fibrils that are similar in appearance to those in the brains of patients with Alzheimer's disease.

Acetylcholinesterase accelerates assembly of amyloid-beta-peptides into Alzheimer's fibrils: possible role of the peripheral site of the enzyme.

Acetylcholinesterase (AChE), an important component of cholinergic synapses, colocalizes with amyloid-beta peptide (A beta) deposits of Alzheimer's brain. We report here that bovine brain AChE, as well as the human and mouse recombinant enzyme, accelerates amyloid formation from wild-type A beta and a mutant A beta peptide, which alone produces few amyloid-like fibrils. The action of AChE was independent of the subunit array of the enzyme, was not affected by edrophonium, an active site inhibitor, but it was affected by propidium, a peripheral anionic binding site ligand. Butyrylcholinesterase, an enzyme that lacks the peripheral site, did not affect amyloid formation. Furthermore, AChE is a potent amyloid-promoting factor when compared with other A beta-associated proteins. Thus, in addition to its role in cholinergic synapses, AChE may function by accelerating A beta formation and could play a role during amyloid deposition in Alzheimer's brain.

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein.

We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.

Acute and delayed implantation of positively charged 2-hydroxyethyl methacrylate scaffolds in spinal cord injury in the rat.

OBJECT: Hydrogels are nontoxic, chemically inert synthetic polymers with a high water content and large surface area that provide mechanical support for cells and axons when implanted into spinal cord tissue. METHODS: Macroporous hydrogels based on 2-hydroxyethyl methacrylate (HEMA) were prepared by radical copolymerization of monomers in the presence of fractionated NaCl particles. Male Wistar rats underwent complete spinal cord transection at the T-9 level. To bridge the lesion, positively charged HEMA hydrogels were implanted either immediately or 1 week after spinal cord transection; control animals were left untreated. Histological evaluation was performed 3 months after spinal cord transection to measure the volume of the pseudocyst cavities and the ingrowth of tissue elements into the hydrogels. RESULTS: The hydrogel implants adhered well to the spinal cord tissue. Histological evaluation showed ingrowth of connective tissue elements, blood vessels, neurofilaments, and Schwann cells into the hydrogels. Morphometric analysis of lesions showed a statistically significant reduction in pseudocyst volume in the treated animals compared with controls and in the delayed treatment group compared with the immediate treatment group (p < 0.001 and p < 0.05, respectively). CONCLUSIONS: Positively charged HEMA hydrogels can bridge a posttraumatic spinal cord cavity and provide a scaffold for the ingrowth of regenerating axons. The results indicate that delayed implantation can be more effective than immediate reconstructive surgery.

Neurofilaments consist of distinct populations that can be distinguished by C-terminal phosphorylation, bundling, and axonal transport rate in growing axonal neurites.

We examined the steady-state distribution and axonal transport of neurofilament (NF) subunits within growing axonal neurites of NB2a/d1 cells. Ultrastructural analyses demonstrated a longitudinally oriented "bundle" of closely apposed NFs that was surrounded by more widely spaced individual NFs. NF bundles were recovered during fractionation and could be isolated from individual NFs by sedimentation through sucrose. Immunoreactivity toward the restrictive C-terminal phospho-dependent antibody RT97 was significantly more prominent on bundled than on individual NFs. Microinjected biotinylated NF subunits, GFP-tagged NF subunits expressed after transfection, and radiolabeled endogenous subunits all associated with individual NFs before they associated with bundled NFs. Biotinylated and GFP-tagged NF subunits did not accumulate uniformly along bundled NFs; they initially appeared within the proximal portion of the NF bundle and only subsequently were observed along the entire length of bundled NFs. These findings demonstrate that axonal NFs are not homogeneous but, rather, consist of distinct populations. One of these is characterized by less extensive C-terminal phosphorylation and a relative lack of NF-NF interactions. The other is characterized by more extensive C-terminal NF phosphorylation and increased NF-NF interactions and either undergoes markedly slower axonal transport or does not transport and undergoes turnover via subunit and/or filament exchange with individual NFs. Inhibition of phosphatase activities increased NF-NF interactions within living cells. These findings collectively suggest that C-terminal phosphorylation and NF-NF interactions are responsible for slowing NF axonal transport.

Structural aspects of pathology in Alzheimer's disease.

The characteristic lesions of Alzheimer's disease, neurofibrillary tangles and neuritic plaques, are the sites of accumulation of abnormal fibrillar material. The structure of the paired helical filament from tangles has been analysed by electron microscopy and biochemical studies have shown that it contains microtubule associated protein tau as a component. Fibrils of beta-amyloid in the neuritic plaque arise by polymerization of a small proteolytic fragment of a much larger precursor protein. It is not yet clear what triggers the events that lead to assembly of the abnormal structures nor why the structures once formed are so resistant to turnover.

Non-steroidal anti-inflammatory drugs have anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.

The pathogenesis of Alzheimer's disease (AD) is characterized by cerebral deposits of amyloid beta-peptides (A beta) and neurofibrillary tangles which are surrounded by inflammatory cells. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of NSAIDs such as ibuprofen, aspirin, meclofenamic acid sodium salt, diclofenac sodium salt, ketoprofen, flurbiprofen, naproxen, sulindac sulfide and indomethacin on the formation, extension, and destabilization of beta-amyloid fibrils (fA beta) at pH 7.5 at 37 degrees C in vitro. All examined NSAIDs dose-dependently inhibited formation of fA beta from fresh A beta(1-40) and A beta(1-42), as well as their extension. Moreover, these NSAIDs dose-dependently destabilized preformed fA betas. The overall activity of the molecules examined was in the following order: ibuprofen approximately sulindac sulfide >or= meclofenamic acid sodium salt>aspirin approximately ketoprofen >or= flurbiprofen approximately diclofenac sodium salt>naproxen approximately indomethacin. Although the mechanisms by which these NSAIDs inhibit fA beta formation from A beta, and destabilize preformed fA beta in vitro are still unclear, NSAIDs may be promising for the prevention and treatment of AD.

Excitable neurofibrils and the problem of identifying the structure of central excitatory synapses in the nineteenth century.

Neurofibrils, identified after staining with Cajal's reduced silver nitrate, for example, were thought by many senior histologists in the nineteenth and early-twentieth centuries to conduct action potentials. There was no basis for this popular idea, although it was the impetus for intense study of the "neurofibrillar network" within neurons by Golgi, Cajal, Freud, and many others. Here, I trace the way in which this "excitable neurofibrillary" hypothesis led to major problems in the attempt by histologists to identify the central excitatory synapse, postulated by Sherrington on functional grounds and eventually described by Berkley.