COMMENTARY ON MORISHIMA-KAWASHIMA ET AL. 375

0197-4580(95)00033-X

COMMENTARY

ALZHEIMER ABNORMALLY PHOSPHORYLATED TAU IS MORE HYPERPHOSPHORYLATED THAN THE FETAL TAU

AND CAUSES THE DISRUPTION OF MICROTUBULES

K H A L I D I Q B A L A N D I N G E G R U N D K E - I Q B A L

New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, N Y 10314

ALZHEIMER'S disease (AD) probably has polyetiology, which include genetic, environmental, and metabolic factors. Indepen- dent of the etiologic agent, histopathologicaUy, AD is charac- terized by the presence of painted helical filaments (PHF) in the neurons and ~-amyloid in the extracellular space in the brain parenchyma and in the walls of the brain blood vessels. How- ever, there is growing evidence from a number of laboratories that dementia in AD patients is associated with neurofibrillary degeneration; ~-amyloid alone in the absence of Alzheimer neurofibrillary degeneration does not produce the disease clin- ically (7,13,34). Thus, it is critical to understand the molecular mechanism of Alzheimer neurofibrillary degeneration, and in turn, to devise a rational therapeutic intervention, the mapping of the phosphorylation sites of the Alzheimer abnormally phos- phorylated tau is required to identify the protein kinases and protein phosphatases involved in the abnormal phosphorylation.

Since the discoveries of tau as the major protein subunit of PHF (21) and the abnormal phosphorylation of tau in AD brain (22,27), several phosphorylation sites in PHF-tau have been identified (see refs. 9,12,20,23,26,33,40). Ihara's group, i.e., Morishima-Kawashima et al. and Hasegawa et al. (ref. 23), have made major contributions in mapping the phosphorylation sites of normal and Alzheimer abnormal taus (see M. Morishima- Kawashima et al., this issue). In addition to the 19 phosphory- lation sites reported by Morishima-Kawashima et al., there are two more sites (see Fig. 1), i.e., Ser 46 (26) and Thr 123 (12). Tau in PHF has been previously shown to be partially phosphor- ylated at these two sites (12,23,26).

Prior to Morishima-Kawashima's studies, some investigators had suggested (a) that the Alzheimer abnormal phosphorylation of tau was the same as that occurring in normal brain during development or even in adulthood and (b) that almost all sites were canonical sites for the-proline-directed protein kinases (PDPKs). Maps of the phosphorylation sites of PHF-tau and fetal tan provide three important findings: (a) that the PHF-tau is phosphorylated at almost twice more sites than the fetal tau; (b) that 7 of 10 additional phosphorylation sites in PHF-tau are non-PDPK sites; (c) that the extent of phosphorylation at given sties in fetal tau is lesser than in PHF-tau.

Recent studies on brain surgical tissue of epileptic patients have also revealed fetal brain-like phosphorylation (and these studies have been interpreted as hyperphosphorylation in nor- mal brain (see Morishima-Kawashima, this issue). However, (a) unlike in fetal brain, in the epileptic cases the tau hyperphos- phorylation is observed only when tissue is rapidly homogenized

in SDS-polyacrylamide gel electrophoresis sample buffer; (b) for brain surgery of the epileptic patients the blood flow to the tar- geted area is stopped and thus hypoxia might play a role in the observed hyperphosphorylation.

Soluble Abnormally Phosphorylated Tau in AD Brain

In addition to PHF, there is a significant pool (up to 40% of total abnormal tau) of soluble abnormally phosphorylated tan (AD P-tan) in AD brain (see ref. 38). The soluble abnormal tau binds to normal tan and becomes sedimentable at 200,000 x g, (1,38) and for this reason had escaped detection in previous stud- ies. Like PHF-tau, the soluble abnormal tau is hypophosphor- ylated (5-9 moles, phosphates/mole of tau) at all the sites that were seen in PHF-tau using phosphorylation-dependent antibod- ies (see refs. 19,38). Furthermore, immunocytochemical stud- ies have shown the presence of the abnormally phosphorylated tau as amorphous non-PHF deposits in pretangle neurons (3,4). Thus, these studies indicate that in AD brain the abnormal phos- phorylation of tau most likely precedes and is probably involved in its polymerization into PHF.

Mechanism of Alzheimer Neurofibrillary Degeneration

Ultrastructural studies of brain biopsies have revealed that in certain selected neurons in AD brain the microtubule system is disrupted and replaced by neurofibrillary tangles of PHF (15,55). Tau stimulates microtubule assembly by polymerizing with tubulin and maintains the microtubule structure (57). The levels of tau in homogenates of frontal cortex from AD patients are several-fold higher than in age-matched controls, and this increase is in the form of abnormally phosphorylated protein (35). Both immunocytochemical (3,4) and biochemical (27,38) studies have shown in AD brain the presence of a significant pool of the abnormally phosphorylated tan that is not polymer- ized into PHF and is not associated with ubiquitin. These stud- ies suggest that the abnormal phosphorylation of tau probably precedes both its polymerization into PHF and the incorpora- tion of ubiquitin.

Microtubule assembly is defective in AD brain. Microtubules can be assembled in vitro from the cytosol of normal fresh au- topsy brain obtained within 5 h postmortem but not from iden- tically treated brains of AD cases. The microtubule assembly from the Alzheimer brains, however, is induced by the addition of DEAE dextran, a polycation that mimics the effect of tau for microtubule assembly (27). Because tau in AD brain cytosol is

376 COMMENTARY ON MORISHIMA-KAWASHIMA ET AL.

Phosphorylation Sites of PHF-tau ((>) and Fetal tau (O)

181 198199 202 208210212214 217 231 235

TPP-- SGDRSGYSSPGSPGTPGSRSRTPSLPTPPTREPKKVAVVRTPPKSPSSAK

N_H 2 ~ / ~ O H

t ~ 6 ~ 1 0 0 / 2 3 ~ 200 262 \ 300 / 400

KESPLQ HVTQAR IGSTENLK ~

TDHGAEWYKSPVVSGDTSPKHLSNVSSTGSIDMVDSPQLATLADEVSA 386

FIG. 1. Phosphorylation sites of PHF-tau (O) and fetal (O) tau. In PHF-tau, the phosphorylation sites were determined both by phosphorylation-dependent antibodies: Ser 46, Thr 123, Ser 199/Ser 202, Ser 396, and Ser 404; and by mass spectrometry: Ser 198, Ser 202, Ser 208, Ser 210, Ser 214, Thr 212, Thr 217, Thr 231, Ser 235, Ser 262, Ser 396, Ser 400, Thr 403, Ser 404, Ser 409, Ser 412, Ser 413, and Set 422, The canonical sites for PDPKs are marked by arrows. Phosphorylation sites shared by PHF-tau and fetal tau are indicated by half solid/half clear circles. The position of the four tubulin binding repeat domains in the tau sequence are shown by open squares. The second repeat domain shown by (r-l) is absent in the three repeat tau isoforms. Figure adapted from Morishima-Kawashima et al. (see this volume).

abnormally phosphorylated (22,27,38) and phosphorylation of tau depresses tau's ability to promote microtubule assembly (41), it appears that this alteration of tan in the Alzheimer brain might have been the cause of the microtubule assembly defect. Both PHF-tau (28) and the non-PHF soluble abnormally phosphor- ylated tau (1) when dephosphorylated with alkaline phosphatase stimulate in vitro microtubule assembly. Whereas normal tan promotes the GTP binding to the exchangeable site in the 3- subunit of tubulin, the PHF-tau does it only after prior dephos- phorylation of the abnormal sites (36). Furthermore, the soluble abnormally phosphorylated tau isolated from AD brain binds to normal tau and not to tubulin, and inhibits the microtubule assembly (1). These findings suggest that the neurofibrillary de- generation is probably caused by the binding of the abnormally phosphorylated tan to normal tau and thereby disrupting the microtubules in the affected neurons (see Fig. 2).

For a neuron to function it must be able to transport mate- rials between its cell body and synapses, and integrity of the microtubule system is essential for this axonal transport. A breakdown of the microtubule system probably leads to a com- promised axonal transport and eventually a retrograde degen- eration of the affected neurons. The retrograde degeneration in AD brain has been confirmed both by Golgi stain studies, show- ing massive loss of the neuronal arborization (50), and by the presence of thickening of the distal ends of the neurites, an early stage of neuropil threads, in pretangle neurons stained for the abnormally phosphorylated tau (11).

Role o f Protein Kinases and Protein Phosphatases in the Abnormal Phosphorylation o f Tau

Protein phosphorylation is one of the major mechanisms for regulation of cellular function. The state of phosphorylation of

substrate proteins depends on the relative activities of protein kinases and phosphoprotein phosphatases. Eleven of twenty one abnormal phosphorylation sites of the Alzheimer hyperphos- phorylated tan identified to date are canonical sites for the proline-directed protein kinases (PDPKs), suggesting that more than one protein kinase are likely to be involved in the abnor- mal phosphorylation. At present, the nature of these protein kinases is not clearly understood. Several protein kinases, both PDPKs and non-PDPKs have been shown to phosphorylate tau (5,6,8,9,14,24,30,31,32,39,42,47,48,49,51,52,58). Two PDPKs, the mitogen activated protein (MAP) kinase and the glycogen synthase kinase-3 (GSK-3) have been shown to phosphorylate tau at several of the abnormal phosphorylation sites (e.g., refs. 14,32). However, the kinetics of phosphorylation at the abnor- mal site(s) are very slow, suggesting that tan (abnormal sites) is not a preferred substrate for these kinases. The abnormal phosphorylation of tau might involve certain site-site and/or protein-protein interactions. The nature of these interactions is not presently understood.

Studies (22,26,27) showing the dephosphorylation of the abnormally phosphorylated sites of tau after treatment with alkaline phosphatase in vitro suggested that the protein phos- phorylation/dephosphorylation defect might be the result, in part, of a deficiency in a protein phosphatase system or systems in the affected neurons in AD. The activities of the phospho- seryl/phosphothreonyl protein phosphatases (PP)-1 and -2A to- ward phosphorylase kinase, and phosphotyrosyl phosphatase toward poly (Glu,Tyr) 4:1 are decreased in brains of AD cases (18). Furthermore, by immunocytochemical studies it has been observed that PP-1, PP-2A, PP-2B, and cytosolic phosphoty- rosyl protein phosphatase PTP-IB are all present in pyramidal neurons in human brain (45). To date, all the tan abnormal phosphorylation sites identified are Set or Thr; no phosphory-

COMMENTARY ON MORISHIMA-KAWASHIMA ET AL. 377

[ - ' ~ Pretangles - - -~ ANT/PHF I - ' ~ ANT/PHF II " - ~ Ghost tangles

P-Tau-Tau ~ ' ] ~ ' ~ + Tubulin ~--'~ Microtubules ~¢

Protein Kinases ! Protein . Phosphatases~ ¢ |

FIG. 2. A hypothetical scheme showing the mechanism of neurofibrillary degeneration in Alzheimer disease. Tau is phosphorylated by several protein kinases. Because of a decrease (1.) in the activities of protein phosphatases that normally dephosphorylate tau, an imbal- ance of phosphorylation/dephosphorylation occurs, leading to the abnormal hyperphos- phorylation of tau U). The latter (1) does not bind to tubulin to promote assembly of microtubules, (2) binds to normal tan and inhibits the microtubule assembly, and (3) be- comes stabilized and polymerizes into PHE The affected neurons degenerate retrogradely both as a result of the breakdown of the microtubule system, and because of the accumu- lation of PHF as Alzheimer neurofibrillary tangles (ANT) filling the entire cell cytoplasm, leaving behind ghost tangles in the extracellular space. Alzheimer disease changes are indi- cated by hatched lines and arrows. The increase in protein kinase activity(s) is as yet not supported by any data and is therefore indicated by a broken arrow.

iation of Tyr has been observed. However, a decrease in phos- photyrosine phosphatase in AD brain might contribute to hyperphosphorylation of tau by keeping MAP kinase activated for extended periods; dephosphorylation of MAP kinase at either Ser/Thr or Tyr inactivates it (46). Furthermore, treatment of cultured neuroblastoma cells with inhibitors of PP-2A and PP-2B leads to increased phosphorylation of tau (Tanaka et al., in preparation).

Studies on site-specific dephosphorylation by protein phos- phatases have revealed that the soluble abnormally phosphory- lated tau isolated from AD brain is rapidly dephosphorylated at the abnormal sites Set 46, Set 199/Ser 202, Ser 235, and Ser 396/Ser 404 by protein phosphatase PP-2B, at all the above sites except Ser 235 by PP-2A, and at only Ser 199/Ser 202 and Set 396/Ser 404 by PP-1. The activities of all the three phospha- tases, i.e., PP-2B, PP-2A and PP-1 toward the abnormally phosphorylated tau are markedly increased by the presence of Mn 2+. Dephosphorylation of the abnormal tau by PP-2C at none of the above sites has been detected (16,17,19). Unlike the soluble abnormally phosphorylated tau, the PHF are less favor- able substrate for dephospho:rylation both by PP-2A and PP-2B. The two phosphatases, PP-2A and PP-2B, can dephosphorylate PHF at Set 199/Ser 202 but not Ser 396 using the same condi- tions as used for the dephosphorylation of the soluble abnor- mally phosphorylated tau (56).

In conclusion, microtubule associated protein tau is abnor-

mally hyperphosphorylated in the brain of patients with Alzhei- mer disease (AD) and the abnormal tau is the major protein subunit of paired helical filaments (PHF). The hyperphosphor- ylation of tau in AD brain is markedly greater than that seen in normal brain either during development or in the adulthood. One half of the phosphorylation sites in the Alzheimer tau are canonical for the proline-directed protein kinases (PDPK), the remaining half are the non-PDPK sites. In addition to PHF, there is a significant pool of the soluble abnormally phosphor- ylated tau in AD brain. The abnormal phosphorylation of tau probably precedes its polymerization into PHF. The abnormal tau does not bind to tubulin but competes with tubulin in bind- ing to normal tau and thereby inhibits the assembly of micro- tubules in the affected neurons. The abnormal tau can be dephosphorylated enzymatically and by this way its microtubule assembly promoting activity can be restored. The activities of protein phosphatases might be decreased in the affected neurons in AD brain, allowing the abnormal hyperphosphorylation of tall.

ACKNOWLEDGEMENTS

Secretarial support was provided by Joanne Lopez and Padmini Reginald. This work was supported in part by funds from the New York State Office of Mental Retardation and Developmental Disabilities, Na- tional Institutes of Health Grants AG05892, AG08076, NS18105, and Zenith Award from the Alzheimer's Association, USA.

378 C O M M E N T A R Y O N M O R I S H I M A - K A W A S H I M A ET AL.

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0197-4580(95)00032-1

AUTHORS' RESPONSE TO COMMENTARIES

M A H O M O R I S H I M A - K A W A S H I M A A N D Y A S U O I H A R A

Department of Neuropathology, Institute for Brain Research, Faculty of Medicine, ,University of Tokyo, 7-3-1 Hongo, Bunkyoku, Tokyo 113, Japan

AS two commentaries pointed out, Iqbal and his colleagues have shown that "soluble" hyperphosphorylated non -PHF tau is present in AD brain and that this species of tau (AD P-tau) binds to normal tau resulting in suppression of its microtubule assem- bly promoting activity (1,10). It is important to note that this tau species is indeed insolub]~e in the conventional buffer but be- came soluble in 8M urea (10). In fact, AD P-tau was isolated f rom the 27,000 to 200,000 x g fraction of AD brain homog- enates. This is similar to the condition established by Greenberg and Davies for the preparat ion o f nonbundled P H F and P H F - tau (5). PHF- tau can be solubilized with 8M urea (6) and once solubilized, it is soluble in conventional buffers. As Kopke et al. stated (10), at least some part of AD P-tau is derived from P H K The remaining may come from an aggregated (sedimentable)

form of tau which cannot be seen as P H F by EM and may rep- resent the early stage of P H F formation. In this context, it is reasonable to postulate real soluble hyperphosphorylated spe- cies of tau as a soluble precursor for P H E However , we have been unable to detect significant amounts of such soluble tau in the 100,000 x g supernatants of AD brain homogenates. Only a trace amount of such tau is immunochemical ly detectable in the cytosolic fraction, which can be explained by P H F disrup- tion during homogenizat ion. This observation has been really puzzling to us and lead us to consider two possibilities; (a) once normal soluble tau is hyperphosphorylated, it immediately aggregates or is incorporated into growing P H F and its cytosolic concentrations are kept vary low; (b) aggregation of normal sol- uble tau precedes hyperphosphorylat ion; as is known in the