I. Grundke-Iqbal

EVIDENCE THAT ALZHEIMER NEUROFIBRILLARY TANGLES ORIGINATE FROM NEUROTUBULES

Antiserum against normal human neurotubules purified by in-vitro assembly precipitated both neurotubules and a polypeptide isolated from Alzheimer neurofibrillary tangles in Ouchterlony double-diffusion tests. The antiserum specifically labelled neurofibrillary tangles, in isolated neurons by immunofluorescence and in tissue sections by the peroxidase-antiperoxidase technique. These results indicate that neurofibrillary tangles in Alzheimers disease probably originate from neurotubules.

Mechanism of neurofibrillary degeneration and pharmacologic therapeutic approach

Neurofibrillary degeneration is a key histopathological brain lesion of Alzheimer disease (AD) and related neurodegenerative disorders such as frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), commonly referred to as tauopathies. Microtubule associated protein (MAP) tau, which is a major MAP of a normal mature neuron is abnormally hyperphosphorylated in tauopathies and is the major protein subunit of paired helical filaments (PHF)/straight filaments (SF) which accumulate in the soma (as neurofibrillary tangles) and dystrophic neurites (as neuropil threads and as dystrophic neurites surrounding the beta-amyloid core in neuritic plaques in AD) of the affected neurons. Unlike normal tau which stimulates assembly and stabilizes microtubules, the abnormally hyperphosphorylated tau inhibits assembly and disrupts microtubules. The abnormally hyperphosphorylated tau competes with tubulin/microtubules in associating with normal tau, MAP1 and MAP2. This sequestration of normal MAPs by the abnormal tau results in the breakdown of the microtubules. The association of the abnormal tau with normal tau and not with MAP1 or MAP2 results in the formation of tangles of tau filaments. All these toxic properties of the abnormally hyperphosphorylated tau are eliminated by its enzymatic dephosphorylation. Activities of phosphoseryl/phosphothreonyl protein phosphatases (PP)-2A and PP-1 which can dephosphorylate the abnormal tau to a normal-like state are compromised in AD brain. Dephosphorylation by PP-2A and PP-2B and to a lesser extent by PP-1 restores the normal microtubule assembly promoting activity in AD P-tau in vitro. Neurofibrillary tangles of PHF isolated from AD brain are also dissociated on in vitro dephosphorylation with PP-2A, and the tau released by this treatment can stimulate microtubule assembly. Thus, it appears that the abnormal hyperphosphorylation of tau leads to neurodegeneration through breakdown of the microtubule network and that the abnormal tau on association with normal tau forms neurofibrillary tangles of tau filaments i.e. PHF/SF. Increase in tau phosphatase activity is a promising approach to inhibit neurofibrillary degeneration and thereby the diseases characterized by this lesion.

OLIGODENDROGLIA FROM HUMAN AUTOPSIED BRAIN: BULK ISOLATION AND SOME CHEMICAL PROPERTIES1

Abstract— Oligodendroglia were isolated from fresh and frozen human autopsied brains by a modification of our technique for isolation of neuronal perikarya and astroglia (Iqbal & Tellez‐Nagel, 1972). Cerebral white matter was minced in a hypertonic hexose‐Ficoll buffered medium, passed through successive screens of decreasing pore size, and the cells were then separated on a discontinuous sucrose density gradient using low speed centrifugation. Cells could also be isolated by substituting sucrose for hexoses in the cell isolation medium. About 95% of the isolated cells had morphology characteristic of oligodendroglia. The usual contaminants in this fraction were capillary fragments, red blood cells, and a few astrocytes. Free myelin was only sparsely seen. About 5–10% of the isolated oligodendroglia had one or more loops of loose membranes extending from the cell plasma membrane. These membraneous loops resembled myelin. The ultrastructural preservation of the cells was poor. The average yield per gram of wet tissue was 52 million cells amounting to 2.4 mg protein, 283 μg DNA, and 94 μg RNA. The protein, DNA, and RNA contents per average cell were 47, 5.3, and 1.8 pg respectively. About 50% of the tissue DNA was recovered as isolated cells, suggesting a larger proportion of oligodendroglia to astroglia, the other principal cell type in human white matter.

Chemical relationship of the paired helical filaments of Alzheimer's dementia to normal human neurofilaments and neurotubules.

Intraneuronal fibrillary tangles are prominent features of several neurological diseases, including especially Alzheimer presenile and senile dementia, and to a much lesser degree in the normal aged human brain. These tangles are made up of abnormal fibrillar elements each about 22 nm at its widest, periodically reduced to 10 nm at about every 80 nm. Each abnormal fiber seems to be a pair of 10 nm filaments helically wound around each other. In this study the protein subunit of these paired helical filaments isolated from cases of Alzheimers dementia was compared with the major protein subunits of normal neurofilaments and neurotubules by two-dimensional peptide maps of the tryptic or chymotryptic digests of these proteins labelled with 125I. The paired helical filament protein is very similar in its peptide maps to identically treated major neurofilament protein and to the beta monomer of neurotubule, while it is not so similar to the alpha tubulin. These data suggest that the paired helical filament protein subunit is closely related chemically to the normal neurofilament protein subunit, and the beta tubulin.

On neurofilament and neurotubule proteins from human autopsy tissue.

Abstract— Neurofilaments and neurotubules are the principal fibers of the mature normal neuron. In this study the protein subunits of these neurofibrils were isolated from human autopsy tissue, and compared by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, and by two dimensional peptide maps of the tryptic digest of these proteins labelled with 125I. The α and the β monomers of neurotubule are related but distinct in their peptide maps, while the major neurofilament protein subunit (molecular weight, 50,000) is remarkably similar to β tubulin. The neurofilament fraction binds colchicine, but the specificity is not yet determined. Neurotubule and neurofilament are also similar in having minor proteins which coelectrophorese on the gels. These results suggest that neurofilament and neurotubule may share one or more protein subunits.

Regulation of expression, phosphorylation and biological activity of tau during differentiation in SY5Y cells.

Tau, one of the best characterized microtubule-associated proteins (MAPs), is a phosphoprotein, the biological activity of which is regulated by its degree of phosphorylation. The objective of the present study was to evaluate the regulation, phosphorylation and the biological activity of tau during differentiation. On differentiation, the tau/tubulin ratio increased about 3-fold regardless whether cells were optimally differentiated with retinoic acid and aphidicolin or with retinoic acid alone which does not inhibit proliferation. The phosphorylation at the Tau-1 (Ser-195/Ser-198/Ser-199/Ser-202) and PHF-1 (Ser-396/Ser-404) sites was increased, mostly in the retinoic acid treated cells, whereas phosphorylation of tau at the 12E8 (Ser-262/Ser-356) epitope was decreased in both groups by approximately 60%. Phosphorylation at the 12E8 site is thought to be one of the most prominent factors affecting the biological activity of tau. However, the microtubule binding activity of tau increased only slightly upon differentiation. Furthermore, a large part of the tau that bound to taxol-stabilized microtubules was phosphorylated at all three sites indicating that these sites are not major sites determining the biological activity of tau. These data show that differentiation of SY5Y cells results in increased tau levels rather than dephosphorylation of tau to meet the additional need in taus biological activity.

Neurofibrillary Pathology: An Update

Neurofibril is the term coined by light microscopists to refer to the fibrillary structures in the neuron. These neurofibers correspond to a variety of linear structures as seen with the electron microscope. The principal fibrils of the normal mature neuron are the neurotubules and the neurofilaments (Fig. 1).

Pharmacological targets to inhibit alzheimer neurofibrillary degeneration

Neurofibrillary degeneration appears to be required for the clinical expression of Alzheimer disease (AD) and related tauopathies. Given the polyetiology of these diseases and the pivotal involvement of neurofibrillary degeneration in their pathogenesis, inhibition of this lesion offers a promising therapeutic target. Studies from our laboratories have shown that there is a protein phosphorylation/dephosphorylation imbalance and that the microtubule associated protein tau is abnormally hyperphosphorylated in the brain of patients with AD and in this form it is the major protein subunit of paired helical filaments/neurofibrillary tangles (PHF/NFT). The abnormal tau which is polymerized into PHF/NFT neither promotes or inhibits in vitro microtubule assembly. In contrast the cytosolic abnormally hyperphosphorylated tau from AD brain, the AD P-tau neither associates with tubulin nor promotes in vitro microtubule assembly but instead it sequesters normal tau, MAP1 and MAP2 and inhibits microtubule assembly. The AD P-tau readily self-assembles in vitro into tangles of PHF/straight filaments under physiological conditions of protein concentration, pH, ionic strength and reducing conditions and this self assembly requires the abnormal hyperphosphorylation of this protein. The activity of phosphoseryl/phosphothreonyl protein phosphatase (PP)-2A which regulates the phosphorylation of tau, is compromised in AD brain. Thus, modulation of the activities of protein phosphatase-2A and tau kinases and inhibition of the sequestration of normal MAPs by AD P-tau offer promising therapeutic opportunities to inhibit neurofibrillary degeneration and the diseases characterized by this lesion.