Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aß) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aß, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.

Serum-induced neurite retraction in CAD cells--involvement of an ATP-actin retractile system and the lack of microtubule-associated proteins.

Cultured catecholamine-differentiated cells [which lack the microtubule-associated proteins (MAPs): MAP1B, MAP2, Tau, STOP, and Doublecortin] proliferate in the presence of fetal bovine serum, and, in its absence, cease dividing and generate processes similar to the neurites of normal neurons. The reintroduction of serum induces neurite retraction, and proliferation resumes. The neurite retraction process in catecholamine-differentiated cells was partially characterized in this study. Microtubules in the cells were found to be in a highly dynamic state, and tubulin in the microtubules consisted primarily of the tyrosinated and deacetylated isotypes. Increased levels of acetylated or Δ2-tubulin (which are normally absent) did not prevent serum-induced neurite retraction. Treatment of differentiated cells with lysophosphatidic acid or adenosine deaminase induced neurite retraction. Inhibition of Rho-associated protein kinase, ATP depletion and microfilament disruption each (individually) blocked serum-induced neurite retraction, suggesting that an ATP-dependent actomyosin system underlies the mechanism of neurite retraction. Nocodazole treatment induced neurite retraction, but this effect was blocked by pretreatment with the microtubule-stabilizing drug paclitaxel (Taxol). Paclitaxel did not prevent serum-induced or lysophosphatidic acid-induced retraction, suggesting that integrity of microtubules (despite their dynamic state) is necessary to maintain neurite elongation, and that paclitaxel-induced stabilization alone is not sufficient to resist the retraction force induced by serum. Transfection with green fluorescent protein-Tau conferred resistance to retraction caused by serum. We hypothesize that, in normal neurons (cultured or in vivo), MAPs are necessary not only to stabilize microtubules, but also to establish interactions with other cytoskeletal or membrane components to form a stable structure capable of resisting the retraction force.

Quantification of acetylated tubulin.

The acetylation/deacetylation of Lys40 of the α-subunit is an important posttranslational modification undergone by tubulin during the life of a cell. Many previous studies have addressed the physiological role of this acetylation process using various approaches based on changes of acetylated tubulin (AcTubulin) content. In most of these studies, however, the actual amounts of AcTubulin were not known and it was difficult to draw conclusions. We present here a simple method to estimate the percentage of AcTubulin relative to total tubulin. The method is based on acetylation of the tubulin sample with acetic anhydride, Western blotting stained by antiAcTubulin antibody, and comparison of the optical density of the AcTubulin band with that of a corresponding sample that was not chemically acetylated.

A novel method for purification of polymerizable tubulin with a high content of the acetylated isotype.

Tubulin can be acetylated/deacetylated on Lys40 of the α-subunit. Studies of the post-translational acetylation/deacetylation of tubulin using biochemical techniques require tubulin preparations that are enriched in AcTubulin (acetylated tubulin) and (for comparison) preparations lacking AcTubulin. Assembly-disassembly cycling of microtubules gives tubulin preparations that contain little or no AcTubulin. In the present study we demonstrated that this result is owing to the presence of high deacetylating activity in the extracts. This deacetylating activity in rat brain homogenates was inhibited by TSA (Trichostatin A) and tubacin, but not by nicotinamide, indicating that HDAC6 (histone deacetylase 6) is involved. TSA showed no effect on microtubule polymerization or depolymerization. We utilized these properties of TSA to prevent deacetylation during the assembly-disassembly procedure. The effective inhibitory concentration of TSA was 3 µM in the homogenate and 1 µM in the subsequent cycling steps. By comparison with immunopurified AcTubulin, we estimated that ~64% of the tubulin molecules in the three cycled preparations were acetylated. The protein profiles of these tubulin preparations, as assessed by SDS/PAGE and Coomassie Blue staining, were identical to that of a preparation completely lacking AcTubulin obtained by assembly-disassembly cycles in the absence of TSA. The tyrosination state and in vitro assembly-disassembly kinetics were the same regardless of the degree of acetylation.

Lack of stabilized microtubules as a result of the absence of major maps in CAD cells does not preclude neurite formation.

In many laboratories, the requirement of microtubule-associated proteins (MAPs) and the stabilization of microtubules for the elongation of neurites has been intensively investigated, with controversial results being obtained. We have observed that the neurite microtubules of Cath.a-differentiated (CAD) cells, a mouse brain derived cell, are highly dynamic structures, and so we analyzed several aspects of the cytoskeleton to investigate the molecular causes of this phenomenon. Microtubules and microfilaments were present in proportions similar to those found in brain tissue and were distributed similarly to those in normal neurons in culture. Neurofilaments were also present. Analysis of tubulin isospecies originating from post-translational modifications revealed an increased amount of tyrosinated tubulin, a diminished amount of the detyrosinated form and a lack of the Delta2 form. This tyrosination pattern is in agreement with highly dynamic microtubules. Using western blot analyses with specific antibodies, we found that CAD cells do not express several MAPs such as MAP1b, MAP2, Tau, doublecortin, and stable-tubule-only-peptide. The presence of the genes corresponding to these MAPs was verified. The absence of the corresponding mRNAs confirmed the lack of expression of these proteins. The exception was Tau, whose mRNA was present. Among the several MAPs investigated, LIS1 was the only one to be expressed in CAD cells. In addition, we determined that neurites of CAD cells form and elongate at the same rate as processes in a primary culture of hippocampal neurons. Treatment with nocodazol precluded the formation of neurites, and induced the retraction of previously formed neurites. We conclude that the formation and elongation of neurites, at least in CAD cells, are dependent on microtubule integrity but not on their stabilization or the presence of MAPs.

Acetylated tubulin associates with the fifth cytoplasmic domain of Na(+)/K(+)-ATPase: possible anchorage site of microtubules to the plasma membrane.

We showed previously that NKA (Na(+)/K(+)-ATPase) interacts with acetylated tubulin resulting in inhibition of its catalytic activity. In the present work we determined that membrane-acetylated tubulin, in the presence of detergent, behaves as an entity of discrete molecular mass (320-400 kDa) during molecular exclusion chromatography. We also found that microtubules assembled in vitro are able to bind to NKA when incubated with a detergent-solubilized membrane preparation, and that isolated native microtubules have associated NKA. Furthermore, we determined that CD5 (cytoplasmic domain 5 of NKA) is capable of interacting with acetylated tubulin. Taken together, our results are consistent with the idea that NKA may act as a microtubule-plasma membrane anchorage site through an interaction between acetylated tubulin and CD5.