Neurofibrillary tangle-associated alteration of stathmin in Alzheimer's disease.

Stathmin (p19), a 19-kDa cytosolic phosphorotein, plays a key role in converting extracellular signals into intracellular biochemical changes. Antibodies and cDNA specific for stathmin were used to study its levels and localization in normal and Alzheimer's disease (AD) brain tissue. The stathmin protein concentration was reduced in AD neocortex as assessed by Western blotting, whereas the concentration of its mRNA detected by both in situ hybridization and slot blot were increased in AD. The alteration of the stathmin protein concentration was negatively correlated with neurofibrillary tangle numbers but not with plaque numbers. Immunoreactivity was evenly localized to the cytoplasm of neurons in control cortical sections, whereas in AD it was preferentially localized to some of the neurofibrillary tangle-bearing neurons. Numbers of stathmin-positive neurons were inversely correlated with tangle numbers but not with plaque numbers in the frontal cortex of AD patients.

Casein kinase II is associated with neurofibrillary tangles but is not an intrinsic component of paired helical filaments.

Neurofibrillary tangles (NFT) are pathological cytoskeletal structures composed of paired helical filaments (PHF), and are found in neurons of patients afflicted with many neurodegenerative disorders, including Alzheimer's disease (AD). We previously found that an antiserum against casein kinase II (CK-II) stained NFT intensely in the brain tissue of AD patients. In the current study, we found that the anti-CK-II antiserum stains NFT and neuronal inclusions in many other neurodegenerative diseases as well, including Guam-Parkinson dementia complex, chromosome 18 deletion syndrome, progressive supranuclear palsy, Kufs' disease, and Pick's disease. This antiserum reacted, in crude brain homogenates, with both a doublet of Mr 43,000 and a Mr 27,000 Da protein which could correspond to the alpha, alpha', and beta chains of CK-II. The staining of these bands was adsorbed by preincubating anti-CK-II antiserum with purified CK-II. Preincubation of brain sections with purified CK-II strongly intensified the immunostaining of NFT with anti-CK-II, suggesting that NFT may bind CK-II. In the AD brain homogenates, the particulate CK-II levels are increased whereas the cytosolic levels are decreased without a change in total CK-II levels, consistent with the idea that CK-II binds to the particulate PHF, a major constituent of NFT. In accord with these findings, purified PHF bound CK-II, but purified PHF did not contain CK-II as its component. These results suggest that CK-II might be an extraneously deposited component of NFT. Thus, the altered CK-II compartmentalization might have significant consequences in the pathogenesis of AD.

Casein kinase II alteration precedes tau accumulation in tangle formation.

Previous studies have shown altered casein kinase II (CK-II) in Alzheimer's disease (AD). For the present study, the authors analyzed CK-II immunoreactivity at various stages of tangle formation using quantitative laser confocal microscopy and immunoelectron microscopy. AD hippocampal pyramidal cells without neurofibrillary tangles (NFTs) displayed 15% more anti-tau immunoreactivity (P less than 0.01) and 43% more anti-CKII immunolabeling than controls (P less than 0.001). In AD, tangle-bearing hippocampal neurons with strong anti-tau immunoreactivity (threefold increase from controls) showed a significant 22% increase in anti-CKII immunolabeling (P less than 0.01), compared with those without NFTs. Neurons with early neurofibrillary changes showed diffuse anti-CKII immunostaining in their cytoplasm and cell processes. In tangle-bearing neurons, in which a higher level of tau immunoreactivity was detected, anti-CKII immunolabeling was distributed along a fibrillar meshwork in cell bodies and processes. Linear regression analysis of anti-CKII and anti-tau immunoreactivity in AD showed a positive correlation (r = 0.53, P less than 0.001). At the ultrastructural level, anti-CKII was immunolocalized to the paired helical filaments (PHF) of the tangle-bearing neurons, as well as to PHF in neuropil threads and some dystrophic neurites in plaques. These results suggest a possible role for CK-II in tangle formation.

Increased immunoreactivity of brain spectrin in Alzheimer disease: a marker for synapse loss?

Alzheimer disease (AD) is characterized, among other pathological alterations, by an extensive synapse loss. Brain spectrin is a membrane skeleton protein found in synapses, and its immunoreactivity has been shown to increase in the rat model of denervation. In order to test the hypothesis that there is an increase in brain spectrin immunoreactivity in relation to the synapse pathology in AD, we studied brain sections and homogenates from AD and control cases and found increased anti-brain spectrin immunostaining of neurons, fibers, and plaques, with a relative decrease in the granular pattern of neuropil immunoreactivity. Western blot analysis showed a 25% increase in the 150 kDa bands (degradation products) in the cytosolic fraction and a decrease in the 240 kDa band (intact brain spectrin) in the particulate fraction. Altered immunostaining of brain sections and Western blot was not observed with an antibody against red blood cell spectrin demonstrating the specific change of brain spectrin. These results support the contention that increased brain spectrin immunoreactivity is a marker of synapse or neuronal loss and further supports the concept of synapse pathology in AD.

Aberrant casein kinase II in Alzheimer's disease.

Abnormal protein phosphorylation has been identified in Alzheimer's disease (AD) for several proteins including a Mr 60,000 protein, a Mr 86,000 protein and a microtubule-associated protein tau. The Mr 86,000 protein is phosphorylated by protein kinase C, whereas protein kinases responsible for other aberrant phosphorylation reactions are not known. In addition to protein kinase C, another kinase, casein kinase II (CK-II), has now been shown to be aberrant in AD. The spermine-dependent CK-II activity is reduced by 84% in AD and the amount of CK-II as determined by its immunoreactivity on a Western blot is reduced by 63%. Furthermore, the distribution of CK-II in AD is altered. Although the neuronal cell body reacts well with CK-II antisera in the normal cortex, the non-tangle-bearing neurons in the AD cortex showed a 15-30% decrease in anti-CK-II immunoreactivity. The neurofibrillary tangles, on the other hand, stain very strongly with rabbit anti-CK-II and indicates that CK-II may be involved in the pathology of AD. The study of CK-II immunoreactivity for dementing diseases other than AD revealed a similar reduction, suggesting the CK-II involvement in the common process of neurodegeneration.

Aberrant protein phosphorylation and cytoarchitecture in Alzheimer's disease.

The neurofibrillary tangle (NFT) is a pathological cytoskeletal structure found in diseased neurons of patients with Alzheimer's disease (AD). Immunocytochemical studies have revealed that the NFT contains cytoskeletal components which are excessively phosphorylated. It is not known if the overphosphorylation of cytoskeletal components is causally involved in the formation of NFT or if it is a simple reflection of abnormal cytoskeletal structure. A major pathological feature of AD is selective neuronal degeneration. Because neuronal survival is supported by the combined function of many growth factors which trigger various protein kinase reactions, some of the cascade reactions necessary for the survival of neurons may be aberrant in AD neurons. In fact, the concentration of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PK-C) is lower in AD cortex than in control cortex. Furthermore, the endogenous phosphorylation reaction catalyzed by this kinase revealed diminished levels of phosphate incorporation into a major PK-C substrate, Mr 86,000 protein (P86). Is this aberrant PK-C system pertinent to NFT formation? The number of tangles does not correlate well with the degree of PK-C abnormality. Therefore, some other protein kinases may be involved in NFT formation. Under conditions where cAMP-dependent protein kinase (PK-A) and PK-C were not stimulated, the levels of in vitro phosphorylation of a Mr 60,000 protein (P60) by an endogenous kinase were found to be increased in AD cortex as compared to control. This increased P60 phosphorylation was not detected in AD patients without NFT, suggesting the involvement of P60 phosphorylation in the process of tangle formation. In fact, the degree of the P60 phosphorylation showed a positive correlation with tangle numbers. P60 phosphorylation was induced in neurons under various conditions where PK-C activity was impaired. Thus, it is possible that the aberrant P60 phosphorylation is consequential to aberrant PK-C. The other protein kinase relevant to NFT formation is casein kinase II (CK-II). CK-II concentration is reduced in AD cortex but its specific activity is increased. Immunohistochemical study of CK-II revealed a general decrease in its staining in AD neurons and a strong staining of NFT. Conversely, anti-paired helical filament antibody cross-reacted with partially purified CK-II. Thus, many protein kinases are affected in AD and some of them are associated with aberrant cytoarchitecture in AD neurons. Further study of protein phosphorylation in AD neurons should prove useful in elucidating the formation of pathological cytoarchitecture and eventual cell death in this disease.

Increase in cross-linking of type I and type III collagens associated with volume-overload hypertrophy.

Types I, III, IV, and V collagen were isolated and characterized from eight normal dog hearts and seven with volume-overload hypertrophy. Animals with volume-overload hypertrophy were killed at a time when left ventricular end-diastolic pressure and stiffness were increased. The collagens were characterized by solubility properties, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, and enzyme-linked immunosorbent assay. The percentage of collagen obtained from canine left ventricles was decreased from 32.4% in normal hearts to 15.0% in hypertrophied hearts. We attribute this to a diminution in the extractability of types I and III collagen, which fell from 199.5 mg type I/g collagen and 76.4 mg type III/g collagen in normal hearts to 83.5 mg type I/g collagen and 26.4 mg type III/g collagen in hypertrophied hearts. The amount of types IV and V collagen isolated remained constant in both the control and arteriovenous shunt hearts averaging 15.7 mg type IV/g collagen and 32.1 mg type V/g collagen in control hearts and 12.5 mg type IV/g collagen and 28.9 mg type V/g collagen in hypertrophied hearts. The reduction in quantity of types I and III collagen probably reflects a greater degree of cross-linking in these two types of collagen. Cyanogen bromide peptide analysis confirmed that there was an increase of high molecular weight cross-linked peptides from 3.96% in normal samples to 8.88% in hypertrophied samples. We conclude that cross-linking of types I and III collagen increases in volume-overload hypertrophy and that this is associated with a rise in diastolic stiffness.

Precorneal factors influencing the ocular distribution of topically applied liposomal inulin.

The primary objective of this study was to investigate the effect of several precorneal factors on the retention of liposomes in tears and their interaction with the corneal and conjunctival surfaces. It was found that adsorption of liposomes onto these surfaces was requisite to the ocular absorption of inulin. Over a range of 10 to 50 microliter, the availability of binding sites at the corneal and conjunctival surfaces rather than the size of the instilled volume controlled the extent of liposomal adsorption and ultimately availability of inulin to the intraocular tissues. The inulin liposomes were in facile association with the adsorptive surfaces, as evidenced by their low resistance to removal by rinsing the eye with saline and by the lack of sustained inulin concentrations in any of the ocular tissues studied. This property of liposomes, coupled with the slow rate (1% per hour) at which they released inulin, was responsible for the absence of inulin in the aqueous humor as late as 240 minutes post-dosing. It was concluded that, for liposomes to be effective in ocular drug delivery, they must show affinity for and be bound to the corneal surface and, in addition, must release their contents at optimal rates.

Subcellular distribution of esterases in the bovine eye.

The distribution of esterases within a cell can influence the rate and extent at which drugs containing ester linkages would be hydrolyzed. The objective of this study was to evaluate the subcellular distribution of esterases in the bovine eye. Ocular tissue homogenates were subjected to differential centrifugation to yield mitochondrial, microsomal and cytoplasmic fractions. Each fraction was then incubated with 1- or 2-naphthyl esters and its esterase activity determined. No esterase activity was detected in the mitochondrial fraction. For the corneal epithelium and iris-ciliary body about 80% of the esterase activity in the tissue homogenates, on a per milligram protein basis, was associated with the microsomes, the remainder was associated with the cytoplasm. Of the tissues studied, the iris-ciliary body had the highest esterase activity, 15 and 7 times that in the corneal epithelium and corneal stroma, respectively. The corneal endothelium was devoid of esterase activity. In comparison with the rabbit eye, the specific esterase activity in the bovine eye was lower. Surprisingly, unlike the rabbit, the corneal epithelium of the bovine eye was enzymatically less active than the stroma. Based on these preliminary data, it was concluded that the esterase activity varies with the tissue and that within a cell the esterases were distributed between the cytoplasm and the microsomes according to a ratio specific for a tissue.