Immunosuppression-induced leukoencephalopathy from tacrolimus (FK506)

Tacrolimus (FK506) has recently been approved for immunosuppression in organ transplantation, although its use is accompanied by a wide spectrum of neurotoxic side effects. We describe the clinical, radiological, and pathological features of 3 cases of tacrolimus-related leukoencephalopathy. The syndrome of immunosuppression-related leukoencephalopathy is proposed as an uncommon neurological syndrome occurring in patients with organ transplants involving demyelination, in particular in the parieto-occipital region and centrum semiovale. Although the syndrome is not associated with a particular (absolute) serum level of tacrolimus, it resolves spontaneously upon decreasing the dose. The tacrolimus-related syndrome has a similar radiographic and pathologic appearance as the analogous syndrome that occurs in patients taking cyclosporine.

Partial purification of a novel mitogen for oligodendroglia.

A protein with a MWapp of 50-70 kDa isolated from the salt extract of crude membranes from neonatal rat brain increases the numbers of oligodendroglia in mixed glial cultures prepared from neonatal rat cerebral white matter. After partial purification by ion exchange and gel exclusion chromatography, and elution from an SDS-polyacrylamide gel, this protein ("oligodendroglial trophic factor," OTF) elicited half-maximal oligodendroglial recruitment at a concentration of 5 ng/mL. OTF is a mitogen for oligodendroglia, and to a lesser extent, for oligodendroglial progenitor (O2A) cells, but does not stimulate proliferation of astroglia, Schwann cells, or endoneurial fibroblasts. OTF, unlike platelet-derived growth factor (PDGF), is not an oligodendroglial survival factor. Antibodies against PDGF and basic fibroblast growth factor (bFGF) do not interfere with the accumulation of oligodendroglia induced by OTF. When OTF is given simultaneously with either PDGF or bFGF, there is an additive increase in the numbers of cells of the oligodendroglial lineage.

Alzheimer disease A68 proteins injected into rat brain induce codeposits of beta-amyloid, ubiquitin, and alpha 1-antichymotrypsin.

Aberrantly phosphorylated tau proteins (i.e., A68 or PHF-tau) and beta-amyloid or A4 (beta A4) peptides are major components of pathologic lesions in Alzheimer disease (AD). Although A68 and beta A4 colocalize in AD neurofibrillary tangles (NFTs) and amyloid-rich senile plaques (SPs), the mechanisms leading to the convergence of A68, beta A4, and other proteins in the same AD lesions are unknown. To probe the biological properties of A68 in vivo, and to assess interactions of A68 with endogenous proteins in the rodent brain, we injected A68, dephosphorylated A68 (DEP-A68), and normal adult human tau protein into the hippocampus and neocortex of rats. In marked contrast to DEP-A68 and tau, A68 resisted rapid proteolysis and induced codeposits of three rodent proteins--i.e., beta A4, ubiquitin, and alpha 1-antichymotrypsin (ACT)--that accumulate in AD NFTs and SPs together with A68. These findings suggest that A68 may interact with beta A4, ubiquitin, and ACT in neuronal perikarya as well as in the extracellular space after release of A68 from degenerating neurons. The model system described here will facilitate efforts to elucidate mechanisms leading to the convergence of A68, beta A4, ubiquitin, and ACT in hallmark lesions of AD.

Abnormal tau phosphorylation at Ser396 in Alzheimer's disease recapitulates development and contributes to reduced microtubule binding.

Abnormally phosphorylated tau proteins (A68) are the building blocks of Alzheimer's disease (AD) paired helical filaments. The biological consequences of the conversion of normal adult tau to A68 remain unknown. Here we demonstrate that native A68 does not bind to microtubules (MTs), yet dephosphorylated A68 regains the ability to bind to MTs. Ser396 is phosphorylated in A68, but not in normal adult tau, whereas fetal tau is phosphorylated transiently at this site. Phosphorylation of tau at Ser396 by protein kinases in CHO cells and rat brain produces an electrophoretic mobility similar to that of A68. Using CHO cells transfected with an Ala396 mutant, we show that the phosphorylation of tau at Ser396 reduces its affinity for MTs and its ability to stabilize MTs against nocodazole-induced depolymerization. Our results demonstrate that the abnormal phosphorylation of tau in AD involves Ser396, and we suggest that this may be mediated by the inappropriate activation of fetal kinases or the reduced activity of tau protein phosphatases. Thus, phosphorylation of Ser396 may destabilize MTs in AD, resulting in the degeneration of affected cells.

Altered tau and neurofilament proteins in neuro-degenerative diseases: diagnostic implications for Alzheimer's disease and Lewy body dementias.

The neuronal cytoskeleton is one of the most profoundly altered organelles in late life neuro-degenerative disorders that are characterized by progressive impairments in cognitive abilities. The elucidation of the protein building blocks of these organelles as well as advances in understanding how these proteins become altered in Alzheimer's disease (AD) and other less common dementing illnesses, i.e., diffuse Lewy body disease (DLBD) or the Lewy body variant of AD (LBVAD), will provide insights into the molecular basis of these disorders. Within, we review evidence that normal adult human tau is abnormally phosphorylated and converted into the subunits of AD paired helical filaments (PHFs), and that Lewy bodies (LBs) represent accumulation of altered neurofilament (NF) triplet subunits. Although the precise biological consequences of PHF and LB formation in neurons is unknown, growing evidence suggests that the formation of PHFs and LBs from normal neuronal cytoskeletal proteins could have deleterious effects on neuronal function and survival. Finally, insights into the composition of PHFs and LBs could lead to the development of novel strategies for the timely and accurate diagnosis of AD, DLBD and the LBVAD.

Regions with abundant neurofibrillary pathology in human brain exhibit a selective reduction in levels of binding-competent tau and accumulation of abnormal tau-isoforms (A68 proteins).

Paired helical filaments, the dominant filamentous components of Alzheimer's disease (AD), neurofibrillary tangles (NFT), neuropil threads, and the dystrophic neurites associated with amyloid rich senile plaques, are composed of abnormally phosphorylated derivatives of tau known as A68 proteins. Indeed the inappropriate phosphorylation of Ser396, which is adjacent to the microtuble binding domain in tau, may contribute to the transformation of tau into A68 and prevent A68 from efficiently binding to microtubules. The reduced levels of normal soluble tau proteins in AD brains may be the consequence of a multi-step process whereby normal tau is converted into A68 and sequestered in paired helical filaments. To elucidate the events involved in this process, we compared the relative levels of binding-competent (BC) and binding-incompetent (BI) tau with the level of A68 in six different regions (hippocampus, fornix, frontal grey and white matter, and cerebellar grey and white matter) of fresh AD and control brains. When the AD brains were compared as a group with neurologically normal and diseased non-AD controls, quantitative immunoblot analysis demonstrated a selective reduction of BC tau in regions of the AD brains with abundant neurofibrillary lesions (NFTs, neuropil threads, and senile plaque neurites) and in their associated white matter areas. The level of BI tau was similar in both AD and control brains. In contrast, A68 was present only in the AD brains, but it was confined to those brain regions with abundant NFTs, neuropil threads, and senile plaques. We view the reductions in BC tau in fornix and frontal white matter to be a consequence of the reductions in their associated grey matter regions i.e., hippocampus and frontal grey matter. Although there is no strict relationship between the reduction of BC tau and the level of A68 within an individual brain, the comparison of the AD group with the control group suggests that the grey matter of the affected regions may be the site for the conversion of BC tau into A68. Further, this process may occur rapidly or via pathways that do not involve BI tau since the levels of BI tau were similar in AD and control brains. Although the complete sequence of events leading to the transformation of tau into A68 and paired helical filaments remains to be elucidated, our data provide compelling evidence that A68 proteins are generated from tau-proteins in selected regions of the AD brain where neurofibrillary lesions comprised of paired helical filaments accumulate.

Immunocytochemical studies with antibodies to three proteins prominent in the isolated microtubule cytoskeleton of a trypanosomatid.

The cytoskeleton of Crithidia fasciculata consists of a corset of parallel microtubules enclosing the cell body and closely underlying the plasma membrane. Distinct sets of crosslinks appear to connect tubules to each other and to membrane. Our objective is to determine the composition of these crosslinks and to elucidate the basis of this spectacular example of membrane-microtubule interaction. We purified three proteins (designated COP-33, -41, and -61 by their subunit Mr), which were consistently abundant in highly purified cytoskeletons. All three bound strongly to microtubules in vitro, and the first two induced bundles through periodic crosslinking. Polyclonal antibodies against each have been used to try to localize these proteins in thin sections of cells or whole mounts of cytoskeletons. Antibodies to COP-41 bound specifically to glycosomes, organelles that encapsulate many glycolytic enzymes in these protozoa, and COP-41 has been identified as glyceraldehyde 3-P dehydrogenase.

Periodic crosslinking of microtubules by cytoplasmic microtubule-associated and microtubule-corset proteins from a trypanosomatid.

The dominant element in the cytoskeleton of Crithidia fasciculata is a peripheral corset of microtubules enclosing the cell body and closely underlying the plasma membrane. A lateral spacing of 50 nm is maintained by crosslinks, and microtubules may also be linked to the plasma membrane. We have characterized groups of polypeptides that associate with microtubules polymerized in vitro from the cytoplasm, or that are associated with the corset complex. They differ except for one of Mr 33,000 present in both groups. The corresponding native corset protein appears to be a dimer of Mr 66,000. These protein(s) copolymerize with brain tubulin, and the resultant polymer consists of pairs or small parallel bundles of microtubules, joined by periodic crosslinks spaced about 8.5 nm apart.

Calcium/phospholipid-dependent kinase recognizes sites in microtubule-associated protein 2 which are phosphorylated in living brain and are not accessible to other kinases.

Microtubule-associated protein 2 (MAP-2) purified after microtubule assembly cycles from bovine brain had been shown to contain about 10 esterified phosphates (mol/mol), which were relatively phosphatase resistant and essentially confined to the projection domain which contributes to the visible arms on microtubules. The kinase responsible for phosphorylating these sites had not been identified. We have approached this question by using a phosphatase that releases the bulk of these residues and then determining which kinase can now add additional residues corresponding to those released. Three kinases were chosen because of their abundance in brain and/or proximity to microtubules. Of these only Ca/phospholipid-dependent kinase was able to recognize the previously occupied sites. We also found that MAP-2 isolated from rat brain without assembly cycles contained more phosphate than previously recognized, greater than 30 mol/mol, suggesting that 20 of these had been inadvertently released by phosphatase during assembly cycles. All 3 kinases (Ca/phospholipid-dependent, cAMP-dependent, and Ca/calmodulin-dependent kinase II) recognized more sites in the bovine than in the rat MAP-2.

The sites at which brain microtubule-associated protein 2 is phosphorylated in vivo differ from those accessible to cAMP-dependent kinase in vitro.

The most conspicuous brain microtubule-associated protein, MAP-2, has been shown to contain 8-10 mol of covalently bound phosphate/mol, as isolated. The MAP-2-associated cAMP-dependent protein kinase can add 10-12 more phosphates, using cycled microtubule preparations, but it does not catalyze exchange between ATP and the pre-existing protein phosphate. We now show that the phosphates that turn over in vivo, after intracerebral injection of 32Pi, are primarily in the projection domain of MAP-2, whereas the sites phosphorylated in vitro are more concentrated in the binding domain. Phosphoserine and phosphothreonine were recovered in a 6:1 ratio from partial acid hydrolysates of MAP-2 phosphorylated either in vivo or in vitro. A protein phosphatase, purified from brain, released residues from in vitro sites in both domains. The enzyme did not release appreciable phosphate that had turned over in vivo, and similar specificity was shown by three other purified protein phosphatases: calcineurin (also from brain) and smooth muscle phosphatases I and II. Thus, even in the projection domain, different sites may be involved.