Evidence that the rab5 effector APPL1 mediates APP-ßCTF-induced dysfunction of endosomes in Down syndrome and Alzheimer's disease.

ß-Amyloid precursor protein (APP) and its cleaved products are strongly implicated in Alzheimer's disease (AD). Endosomes are highly active APP processing sites, and endosome anomalies associated with upregulated expression of early endosomal regulator, rab5, are the earliest known disease-specific neuronal response in AD. Here, we show that the rab5 effector APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif) mediates rab5 overactivation in Down syndrome (DS) and AD, which is caused by elevated levels of the ß-cleaved carboxy-terminal fragment of APP (ßCTF). ßCTF recruits APPL1 to rab5 endosomes, where it stabilizes active GTP-rab5, leading to pathologically accelerated endocytosis, endosome swelling and selectively impaired axonal transport of rab5 endosomes. In DS fibroblasts, APPL1 knockdown corrects these endosomal anomalies. ßCTF levels are also elevated in AD brain, which is accompanied by abnormally high recruitment of APPL1 to rab5 endosomes as seen in DS fibroblasts. These studies indicate that persistent rab5 overactivation through ßCTF-APPL1 interactions constitutes a novel APP-dependent pathogenic pathway in AD.

Neurofilament subunits are integral components of synapses and modulate neurotransmission and behavior in vivo.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (α-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

Slow axonal transport.

New studies provide further evidence that the neuronal cytoskeleton is the product of a dynamic interplay between axonal transport processes and locally regulated assembly mechanisms. These data confirm that the axonal cytoskeleton in mammalian systems is largely stationary and is maintained by a smaller pool of moving subunits or polymers. Slow axonal transport in certain lower species, however, may exhibit quite different features.

The slow axonal transport of cytoskeletal proteins.

Once presumed to be relatively uniform, the axonal cytoskeleton can vary markedly in size and composition along its length. New studies emphasize the interactiveness of neurofilaments and identify a family of cytoskeletal proteins that may cross-link the various cytoskeletal polymers of the axon, and anchor this network to the membrane skeleton. These and other findings support a model of the axonal cytoskeleton as a stationary but dynamic structure. Current evidence continues to support the possibility that axonally transported polymers/oligomers and/or monomers may serve as precursors to the cytoskeleton in different situations. Although the motors for slow transport of cytoskeletal proteins remain elusive, possible candidates are emerging.

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments.

The 200-kD subunit of neurofilaments (NF-H) functions as a cross-bridge between neurofilaments and the neuronal cytoskeleton. In this study, four phosphorylated NF-H variants were identified as major constituents of axons from a single neuron type, the retinal ganglion cell, and were shown to have characteristics with different functional implications. We resolved four major Coomassie Blue-stained proteins with apparent molecular masses of 197, 200, 205, and 210 kD on high resolution one-dimensional SDS-polyacrylamide gels of mouse optic axons (optic nerve and optic tract). Proteins with the same electrophoretic mobilities were radiolabeled within retinal ganglion cells in vivo after injecting mice intravitreally with [35S]methionine or [3H]proline. Extraction of the radiolabeled protein fraction with 1% Triton X-100 distinguished four insoluble polypeptides (P197, P200, P205, P210) with expected characteristics of NF-H from two soluble neuronal polypeptides (S197, S200) with few properties of neurofilament proteins. The four Triton-insoluble polypeptides displayed greater than 90% structural homology by two-dimensional alpha-chymotryptic iodopeptide map analysis and cross-reacted with four different monoclonal and polyclonal antibodies to NF-H by immunoblot analysis. Each of these four polypeptides advanced along axons primarily in the Group V (SCa) phase of axoplasmic transport. By contrast, the two Triton-soluble polypeptides displayed only a minor degree of alpha-chymotryptic peptide homology with the Triton-insoluble NF-H forms, did not cross-react with NF-H antibodies, and moved primarily in the Group IV (SCb) wave of axoplasmic transport. The four NF-H variants were generated by phosphorylation of a single polypeptide. Each of these polypeptides incorporated 32P when retinal ganglion cells were radiolabeled in vivo with [32P]orthophosphate and each cross-reacted with monoclonal antibodies specifically directed against phosphorylated epitopes on NF-H. When dephosphorylated in vitro with alkaline phosphatase, the four variants disappeared, giving rise to a single polypeptide with the same apparent molecular mass (160 kD) as newly synthesized, unmodified NF-H. The NF-H variants distributed differently along optic axons. P197 predominated at proximal axonal levels; P200 displayed a relatively uniform distribution; and P205 and P210 became increasingly prominent at more distal axonal levels, paralleling the distribution of the stationary neurofilament network.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons.

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthesis, axonal transport, and turnover of the high molecular weight microtubule-associated protein MAP 1A in mouse retinal ganglion cells: tubulin and MAP 1A display distinct transport kinetics.

Microtubule-associated proteins (MAPs) in neurons establish functional associations with microtubules, sometimes at considerable distances from their site of synthesis. In this study we identified MAP 1A in mouse retinal ganglion cells and characterized for the first time its in vivo dynamics in relation to axonally transported tubulin. A soluble 340-kD polypeptide was strongly radiolabeled in ganglion cells after intravitreal injection of [35S]methionine or [3H]proline. This polypeptide was identified as MAP 1A on the basis of its co-migration on SDS gels with MAP 1A from brain microtubules; its co-assembly with microtubules in the presence of taxol or during cycles of assembly-disassembly; and its cross-reaction with well-characterized antibodies against MAP 1A in immunoblotting and immunoprecipitation assays. Glial cells of the optic nerve synthesized considerably less MAP 1A than neurons. The axoplasmic transport of MAP 1A differed from that of tubulin. Using two separate methods, we observed that MAP 1A advanced along optic axons at a rate of 1.0-1.2 mm/d, a rate typical of the Group IV (SCb) phase of transport, while tubulin moved 0.1-0.2 mm/d, a group V (SCa) transport rate. At least 13% of the newly synthesized MAP 1A entering optic axons was incorporated uniformly along axons into stationary axonal structures. The half-residence time of stationary MAP 1A in axons (55-60 d) was 4.6 times longer than that of MAP 1A moving in Group IV, indicating that at least 44% of the total MAP 1A in axons is stationary. These results demonstrate that cytoskeletal proteins that become functionally associated with each other in axons may be delivered to these sites at different transport rates. Stable associations between axonal constituents moving at different velocities could develop when these elements leave the transport vector and incorporate into the stationary cytoskeleton.

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons.

The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the " 140,000" neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The "140,000" proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The "140,000" NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the "140,000" mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the "140,000" component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of "140,000" NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.

Posttranslational processing of alpha-tubulin during axoplasmic transport in CNS axons.

Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta-tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha-tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.

Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber.

The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation-dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150-microns level and further increased a total of threefold by the 1,200-microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl-terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network.

Local control of neurofilament accumulation during radial growth of myelinating axons in vivo. Selective role of site-specific phosphorylation.

The accumulation of neurofilaments required for postnatal radial growth of myelinated axons is controlled regionally along axons by oligodendroglia. Developmentally regulated processes previously suspected of modulating neurofilament number, including heavy neurofilament subunit (NFH) expression, attainment of mature neurofilament subunit stoichiometry, and expansion of interneurofilament spacing cannot be primary determinants of regional accumulation as we show each of these factors precede accumulation by days or weeks. Rather, we find that regional neurofilament accumulation is selectively associated with phosphorylation of a subset of Lys-Ser-Pro (KSP) motifs on heavy neurofilament subunits and medium-size neurofilament subunits (NFMs), rising >50-fold selectively in the expanding portions of optic axons. In mice deleted in NFH, substantial preservation of regional neurofilament accumulation was accompanied by increased levels of the same phosphorylated KSP epitope on NFM. Interruption of oligodendroglial signaling to axons in Shiverer mutant mice, which selectively inhibited this site-specific phosphorylation, reduced regional neurofilament accumulation without affecting other neurofilament properties or aspects of NFH phosphorylation. We conclude that phosphorylation of a specific KSP motif triggered by glia is a key aspect of the regulation of neurofilament number in axons during axonal radial growth.

Increased axonal proteolysis in myelin-deficient mutant mice.

Protein degradation within retinal ganglion cell axons in vitro is 50 to 110 percent faster than normal in mutant mice exhibiting deficiencies of myelin in the central nervous system. Proteolysis is increased proximally and distally within retinal ganglion cell axons of mice carrying the jumpy mutation or its allele, myelin synthesis deficiency, and is increased distally within those axons of quaking mice. The proteolytic defect is axon (neuron)-specific since the rate of protein degradation within glial cells is normal. Increased axonal proteolysis does not bear a simple relation to hypomyelination since shiverer, another mouse mutant deficient in central myelin, displayed normal rates of axonal protein degradation under the same conditions. These observations suggest an abnormal axon-glial interaction in mice with primary glial defects and raise the possibility that the functioning of histologically normal axons (neurons) may be altered in dysmyelinating diseases.

Gene expression and cellular content of cathepsin D in Alzheimer's disease brain: evidence for early up-regulation of the endosomal-lysosomal system.

In Alzheimer's disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

Neurofilament phosphorylation: a new look at regulation and function.

Dynamic remodeling of cytoskeleton architecture is necessary for axonal growth and guidance, signal transduction and other fundamental aspects of neuron function. Protein phosphorylation plays a key part in these remodeling processes. Since neurofilaments are major cytoskeletal constituents and are among the most highly phosphorylated neuronal proteins, the control of their behavior serves as a possible model for understanding how phosphorylation regulates the many other phosphoproteins in the cytoskeleton. Recent studies show that neurofilament protein subunits are phosphorylated on both their amino-terminal head domains and carboxy-terminal tails by different protein kinases. This review considers the implications of this complex regulation for neurofilament function in normal neurons and in disease states characterized by neurofibrillary pathology.

The endosomal-lysosomal system of neurons: new roles.

The repertoire of the lysosomal system extends beyond its function in degrading biologic macromolecules for energy and recycling purposes. Controlled shifts in lysosomal activity help neurons to regulate their cytoplasmic volume and to remodel local cellular domains. Newly identified regulatory controls over targeting to lysosomes and the limited proteolytic actions of 'lysosomal' hydrolases, together with other recent findings, are suggesting potential roles for the endosomal-lysosomal system in modifying functions of specific proteins, acquiring nutrients essential for growth and repair, influencing the output of secretory products, and helping neurons to modulate trophic signals. The prominent involvement of the endosomal-lysosomal system in Alzheimer's disease and other major pathologies has redoubled interest in how this system serves neurons.

Calpain inhibition mediates autophagy-dependent protection against polyglutamine toxicity.

Over recent years, accumulated evidence suggests that autophagy induction is protective in animal models of a number of neurodegenerative diseases. Intense research in the field has elucidated different pathways through which autophagy can be upregulated and it is important to establish how modulation of these pathways impacts upon disease progression in vivo and therefore which, if any, may have further therapeutic relevance. In addition, it is important to understand how alterations in these target pathways may affect normal physiology when constitutively modulated over a long time period, as would be required for treatment of neurodegenerative diseases. Here we evaluate the potential protective effect of downregulation of calpains. We demonstrate, in Drosophila, that calpain knockdown protects against the aggregation and toxicity of proteins, like mutant huntingtin, in an autophagy-dependent fashion. Furthermore, we demonstrate that, overexpression of the calpain inhibitor, calpastatin, increases autophagosome levels and is protective in a mouse model of Huntington's disease, improving motor signs and delaying the onset of tremors. Importantly, long-term inhibition of calpains did not result in any overt deleterious phenotypes in mice. Thus, calpain inhibition, or activation of autophagy pathways downstream of calpains, may be suitable therapeutic targets for diseases like Huntington's disease.

Colocalization of lysosomal hydrolase and beta-amyloid in diffuse plaques of the cerebellum and striatum in Alzheimer's disease and Down's syndrome.

The lysosomal hydrolases, cathepsin D (Cat D) and beta-hexosaminidase A (HEX), which are normally intracellular enzymes, colocalize with beta-amyloid in a subgroup of diffuse plaques in the cerebellum and striatum of individuals with Alzheimer's disease or Down's syndrome. Using specific antisera in combination with single- and double-label immunocytochemical techniques, extracellular hydrolase was detected in 30 to 40% of the diffuse plaques in the cerebellar molecular layer and nearly all of the diffuse plaques in the striatum. In both Alzheimer's disease and Down's syndrome, about 5 to 10% of the cerebellar Purkinje cells contained abnormally increased numbers of hydrolase-positive lysosomes despite their normal appearance by conventional histologic stains. Occasional atrophic Purkinje cells identified by Nissl stain were intensely immunostained. By confocal imaging analysis, abnormal hydrolase-laden Purkinje cell dendrites were seen coursing through some hydrolase-positive plaques and were continuous with dendritic branches that terminated within deposits of extracellular hydrolase and beta-amyloid. In the striatum, intensely immunostained abnormal-appearing neurons were commonly associated with extracellular deposits of hydrolase immunoreactivity and beta-amyloid within diffuse plaques and in the less commonly seen classical plaques. In both brain regions, other hydrolase-negative beta-amyloid deposits were seen, these being associated with blood vessels. The presence of HEX immunoreactivity in neurons, but not in glia, and its abundance in plaques support earlier studies, suggesting that neurons are the principal source of plaque hydrolase. An endosomal-lysosomal system upregulation, with increased hydrolase expression and extracellular enzyme deposition in plaques, is, like beta-amyloid deposition, an early marker of metabolic dysfunction potentially related to primary etiologic events in Alzheimer's disease and Down's syndrome.

Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease.

In Alzheimer's disease (AD), the neuropathologic hallmarks of beta-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal-lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to A beta peptide that forms beta-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, beta CTF - the beta-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating beta-cleavage of APP. beta-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of gamma-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of gamma-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the gamma-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human A beta 40 and A beta 42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for A beta generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to beta-amyloid deposition.

The regulation of neurofilament protein dynamics by phosphorylation: clues to neurofibrillary pathobiology.

Neurofilament proteins are continuously modified during their lifetime by a succession of protein kinases and phosphatases. Site-specific phosphorylation or dephosphorylation within different polypeptide domains of each neurofilament subunit is now believed to regulate such behaviors of neurofilaments as subunit polymerization and exchange, axonal transport, interactions with other cytoskeletal proteins and degradation. Local regulation of phosphorylation events could account for variations in the size, morphology and dynamics of the neurofilament network in different regions of the neuron. The apparent greater plasticity of the neurofilament network in regions like the perikaryon, initial segment and nodes along the axon may provide some insight into the vulnerability of these regions in neurofibrillary disease.