Carolyn A. Rankin

Tau phosphorylation by GSK-3β promotes tangle-like filament morphology

BackgroundNeurofibrillary tangles (NFTs) are intraneuronal aggregates associated with several neurodegenerative diseases including Alzheimers disease. These abnormal accumulations are primarily comprised of fibrils of the microtubule-associated protein tau. During the progression of NFT formation, disperse and non-interacting tau fibrils become stable aggregates of tightly packed and intertwined filaments. Although the molecular mechanisms responsible for the conversion of disperse tau filaments into tangles of filaments are not known, it is believed that some of the associated changes in tau observed in Alzheimers disease, such as phosphorylation, truncation, ubiquitination, glycosylation or nitration, may play a role.ResultsWe have investigated the effects of tau phosphorylation by glycogen synthase kinase-3β (GSK-3β) on tau filaments in an in vitro model system. We have found that phosphorylation by GSK-3β is sufficient to cause tau filaments to coalesce into tangle-like aggregates similar to those isolated from Alzheimers disease brain.ConclusionThese results suggest that phosphorylation of tau by GSK-3β promotes formation of tangle-like filament morphology. The in vitro cell-free experiments described here provide a new model system to study mechanisms of NFT development. Although the severity of dementia has been found to correlate with the presence of NFTs, there is some question as to the identity of the neurotoxic agents involved. This model system will be beneficial in identifying intermediates or side reaction products that might be neurotoxic.

Renal expression of a transforming growth factor-α transgene accelerates the progression of inherited, slowly progressive polycystic kidney disease in the mouse

Polycystic kidney disease (PKD) is a prevalent inherited disease in human beings. The pathogenesis of PKD is as yet unclear. The epidermal growth factor family of proteins has been implicated in PKD based largely on in vitro data. To determine whether these growth factors contribute to the progression of inherited PKD in vivo, we crossed mice with a transgene for human transforming growth factor-alpha (TGF-alpha, a member of the epidermal growth factor (EGF) family) and mice with the pcy gene (which causes a slowly progressive form of PKD very similar to human autosomal dominant PKD). Renal expression of the TGF-alpha transgene in cystic mice (homozygous for the pcy gene) accelerated the development of PKD as shown by an increased kidney weight as a percent of body weight and an increased volume density of renal cysts at 8.5 weeks of age. However, renal expression of the TGF-alpha transgene did not appear to precociously initiate cyst development (at 6.5 weeks), nor did it cause an increase in the final degree of renal enlargement (at 29 weeks). Thus TGF-alpha accelerated the enlargement of cysts once initiated. At 8.5 weeks of age, renal expression of the TGF-alpha mRNA correlated positively with the amount of renal enlargement. At all time points studied, cystic kidneys exhibited increased expression of c-myc mRNA as compared with phenotypic normal kidneys, consistent with PKD being a hyperplastic disease of renal tubules. However, the renal expression of c-myc in 8.5 week cystic kidneys, with or without the transgene, did not correlate with the degree of renal enlargement. The results of this study suggest that EGF-like proteins may accelerate the progression of inherited renal cystic disease. However, the final degree of cystic change is dictated by the primary disease process rather than by the continued presence of growth factor.

E3 Ubiquitin-Protein Ligase Activity of Parkin Is Dependent on Cooperative Interaction of RING Finger (TRIAD) Elements

The parkin gene codes for a 465-amino acid protein which, when mutated, results in autosomal recessive juvenile parkinsonism (AR-JP). Symptoms of AR-JP are similar to those of idiopathic Parkinsons disease, with the notable exception being the early onset of AR-JP. We have cloned and expressed human Parkin in Escherichia coli and have examined Parkin-mediated ubiquitination in an in vitro ubiquitination assay using purified recombinant proteins. We found that Parkin has E3 ubiquitin ligase activity in this system, demonstrating for the first time that the E3 activity is an intrinsic function of the Parkin protein and does not require posttranslational modification or association with cellular proteins other than an E2 (human Ubc4 E2 was utilized in this ubiquitination assay). Mutagenesis of individual elements of the conserved RING TRIAD domain indicated that at least two elements were required for ubiquitin ligase activity and suggested a functional cooperation between the RING finger elements. Since the activity assays were conducted with recombinant proteins purified from E. coli, this is the first time TRIAD element interaction has been demonstrated as an intrinsic feature of Parkin E3 activity.

Assessing the Toxicity of Tau Aggregation

Abnormally phosphorylated and aggregated tau protein is the primary component of pathological structures that are closely associated with neurodegeneration in Alzheimers disease, Pick disease, corticobasal degeneration, progressive supranuclear palsy and many other neurodegenerative tauopathies, leading to the hypothesis that these structures are toxic mediators of disease progression. Results from animal models designed to test this hypothesis have yielded evidence that can suggest either a pathogenic, beneficial, or incidental role for tau aggregation. This review summarizes the differences in construction of recent model systems and assay methods that examine tau pathology and toxicity. We have found that the expression levels of tau and the modifications of tau used to enhance its aggregation have a large impact on the results. It is clear from the data that tau aggregation is toxic, but it is less clear which form of tau aggregate is the toxic species.

Parkin, A top level manager in the cell's sanitation department

Parkin belongs to a class of multiple RING domain proteins designated as RBR (RING, in between RING, RING) proteins. In this review we examine what is known regarding the structure/function relationship of the Parkin protein. Parkin contains three RING domains plus a ubiquitin-like domain and an in-between-RING (IBR) domain. RING domains are rich in cysteine amino acids that act as ligands to bind zinc ions. RING domains may interact with DNA or with other proteins and perform a wide range of functions. Some function as E3 ubiquitin ligases, participating in attachment of ubiquitin chains to signal proteasome degradation; however, ubiquitin may be attached for purposes other than proteasome degradation. It was determined that the C-terminal most RING, RING2, is essential for Parkin to function as an E3 ubiquitin ligase and a number of substrates have been identified. However, Parkin also participates in a number of other fiunctions, such as DNA repair, microtubule stabilization, and formation of aggresomes. Some functions, such as participation in a multi-protein complex implicated in NMDA activity at the post synaptic density, do not require ubiquitination of substrate molecules. Recent observations of RING proteins suggest their function may be regulated by zinc ion binding. We have modeled the three RING domains of Parkin and have identified a new set of RING2 ligands. This set allows for binding of two rather than just one zinc ion, opening the possibility that the number of zinc ions bound acts as a molecular switch to modulate Parkin function.

Pre-assembled tau filaments phosphorylated by GSK-3b form large tangle-like structures.

Hyperphosphorylated tau protein is a major component of neurofibrillary tangles, a prominent intracellular hallmark of Alzheimers disease. Both hyperphosphorylated tau and neurofibrillary tangles have been shown to correlate with dementia in Alzheimers disease, but the relationship between hyperphosphorylation and tangle formation is not clear. Using a cell-free in vitro model system, in which tau polymerization is induced by arachidonic acid, we show that GSK-3beta phosphorylation of pre-assembled tau filaments makes those filaments prone to coalesce into large neurofibrillary tangle-like structures. Five phosphorylation sites, S199, T205, T231, S396 and S404, were identified in the phosphorylated filaments; many of the five are within epitopes recognized by Alzheimers disease-associated antibodies. These tangle-like structures are optically visible and are similar to those formed by polymerization of GSK-3beta phosphorylated tau monomer and to those isolated from Alzheimers disease tissue. We conclude that the phosphorylation of tau by GSK-3beta either prior to or following polymerization promotes polymer/polymer interactions that result in stable clusters of tau filaments.

Isolated RING2 domain of parkin is sufficient for E2-dependent E3 ligase activity.

The E3 ubiquitin ligase activity of the parkin protein is implicated in playing a protective role against neurodegenerative disorders including Parkinsons, Huntingtons, and Alzheimers diseases. Parkin has four zinc-containing domains: RING0, RING1, IBR (in-between ring), and RING2. Mutational analysis of full-length parkin suggests that the C-terminal RING2 domain contains the catalytic core. Here, a catalytically competent recombinant RING2 containing an N-terminal GB1 solubility peptide is described. In cell-free in vitro ubiqitination reactions, the RING2 construct catalyzes the transfer of ubiquitin from the E2 enzyme UbcH7 to the attached GB1 tag. This intramolecular autoubiquitination reaction indicates that (a) ubiquitination by RING2 can occur in the absence of other parkin domains and (b) UbcH7 can interact directly with RING2 to transfer its bound ubiquitin. Mass spectrometry identified sites of mono- and diubiquitin attachment to two surface-exposed lysine residues (Lys24 and Lys39) on the GB1 peptide. The sites of diubiquitination involved Lys11 and Lys48 linkages, which have been identified as general signals for proteasome degradation. Cleaving the linker between the GB1 tag and RING2 resulted in loss of ubiquitination activity, indicating that the substrate must be tethered to RING2 for proper presentation to the active site. Atomic absorption spectrometry and selective mutation of zinc ligands indicated that only one of the two zinc binding sites on RING2, the N-terminal site, needs to be occupied by zinc for expression of ubiquitination activity. This is consistent with the hypothesis that the second, C-terminal, zinc binding site on RING2 has a regulatory rather than a catalytic function.

A one-carbon modification of protein lysine associated with elevated oxidative stress in human substantia nigra.

We describe for the first time a naturally occurring lysine modification that is converted to methyllysine by reduction with sodium borohydride. This modification is ∼1.7 times as abundant in soluble proteins from human substantia nigra pars compacta as in proteins from other brain regions, possibly as a result of elevated oxidative stress in the nigra. Proteins from cultured PC12 cells exposed to oxidative stress conditions also contain elevated levels of this lysine modification. The abundance of the naturally occurring modification is roughly 0.08 nmoles/mg protein in either unstressed brain or PC12 cells. Modification levels remain stable in isolated proteins incubated for 2 h at 37°C in pH 7 buffer. We propose that the endogenous modification is the lysine Schiff base, ε‐N‐methylenelysine, and that lysine modifications may result from a reaction with formaldehyde in vivo. Rat brain contains ∼60 nmoles/g wet weight of formaldehyde, which probably includes both free and reversibly bound forms. Adding ∼35 µm HCHO to PC12 cell growth medium introduces methylenelysine modifications in cell proteins and impairs cell viability. The existence of this post‐translational modification suggests new mechanisms of oxidative stress that may contribute to tissue degeneration, including loss of nigral dopamine neurons during normal aging and in Parkinsons disease.