The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction.

Ahnak, a 700 kDa protein, is expressed in a variety of cells and has been implicated in different cell-type-specific functions. In the human heart, we observed an endogenous carboxyl-terminal 72 kDa ahnak fragment that copurified with myofibrillar proteins. Immunocytochemistry combined with confocal microscopy localized this fragment to the intercalated discs and close to the Z-line of cardiomyocytes. No endogenous myofibrillar ahnak fragment was observed in the skeletal muscle. We elucidated the role of the recombinant carboxyl-terminal ahnak fragment (ahnak-C2) in actin filament organization and in the function of muscle fibers. Addition of ahnak-C2 to actin filaments induced filament bundling into paracrystalline-like structures as revealed by electron microscopy. Incubation of demembranated skeletal muscle fibers with ahnak-C2 attenuated the decline in isometric force development upon repeated contraction-relaxation cycles. Our results suggest that the carboxyl-terminal ahnak domain exerts a stabilizing effect on muscle contractility via its interaction with actin of thin filaments.

Polymerization of proteins into amyloid protofibrils shares common critical oligomeric states but differs in the mechanisms of their formation.

Amyloid protofibril formation of phosphoglycerate kinase (PGK) and Syrian hamster prion protein (SHaPrP(90-232)) were investigated by static and dynamic light scattering, size exclusion chromatography and electron microscopy. Changes in secondary structure were monitored by Fourier transform infrared spectroscopy and by circular dichroism. Protofibril formation of the two proteins is found to be a two-stage process. At the beginning, an ensemble of critical oligomers is built up. These critical oligomeric states possess a predominant beta-sheet structure and do not interact considerably with monomers. Initial oligomerization and transition to beta-sheet structure are coupled events differing in their details for both proteins. Intermediate oligomeric states (dimers, trimers, etc.) are populated in case of PGK, whereas SHaPrP(90-232) behaves according to an apparent two-state reaction between monomers and octamers rich in beta-structure with a reaction order varying between 2 and 4. All oligomers coalesce to PGK protofibrils in the second stage, while SHaPrP(90-232) protofibrils are only formed by a subpopulation. The rates of both growth stages can be tuned in case of PGK by different salts preserving the underlying generalized diffusion-collision mechanism. The different kinetics of the early misfolding and oligomerization events of the two proteins argue against a common mechanism of protofibril formation. A classification scheme for misassembly mechanisms of proteins based on energy landscapes is presented. It includes scenarios of downhill polymerization to which protofibril formation of PGK and SHaPrP(90-232) belong.

Local origin and activity-dependent generation of nestin-expressing protoplasmic astrocytes in CA1.

Since reports that precursor cells in the adult subventricular zone (SVZ) contribute to regenerative neuro- and gliogenesis in CA1, we wondered whether a similar route of migration might also exist under physiological conditions. Permanent labeling of SVZ precursor cells with a lentiviral vector for green fluorescent protein did not reveal any migration from the SVZ into CA1 in the intact murine brain. However, in a nestin-GFP reporter mouse we found proliferating cells within the corpus callosum/alveus region expressing nestin and glial fibrillary acidic protein similar to precursor cells in the neighboring neurogenic region of the adult dentate gyrus. Within 3 weeks of BrdU administration, BrdU-positive nestin-GFP-expressing protoplasmic astrocytes emerged in CA1. Similar to precursor cells isolated from the dentate gyrus and the SVZ, nestin-GFP-expressing cells from corpus callosum/alveus were self-renewing and multipotent in vitro, whereas cells isolated from CA1 were not. Nestin-GFP-expressing cells in CA1 differentiated into postmitotic astrocytes characterized by S100beta expression. No new neurons were found in CA1. The number of nestin-GFP-expressing astrocytes in CA1 was increased by environmental enrichment. We conclude that astrogenesis in CA1 is influenced by environmental conditions. However, SVZ precursor cells do not contribute to physiological cellular plasticity in CA1.

Interaction of huntingtin fragments with brain membranes--clues to early dysfunction in Huntington's disease.

Abstract Huntingtin is a large, multi-domain protein of unknown function in the brain. An abnormally elongated polyglutamine stretch in its N-terminus causes Huntington's disease (HD), a progressive neurodegenerative disorder. Huntingtin has been proposed to play a functional role in membrane trafficking via proteins involved in endo- and exocytosis. Here, we supply evidence for a direct association between huntingtin and membranes. In the brains of R6/2 mice with HD pathology, a 64 kDa N-terminal huntingtin fragment accumulated in postsynaptic membranes during the pre-symptomatic period of 4-8 weeks of age. In addition, an oligomeric fragment of approximately 200 kDa was detected at 8 weeks of age. Simultaneous progressive changes in distribution of amphiphysin, synaptojanin, and subunits of NMDA- and AMPA-receptors provide a strong indication of dysfunctional synaptic trafficking. Composition of the major phospholipids in the synaptic membranes was unaffected. In vitro, large unilamellar vesicles of brain lipids readily associated with soluble N-terminal huntingtin exon 1 fragments and stimulated fibrillogenesis of mutant huntingtin aggregates. Moreover, interaction of both mutant and wild-type huntingtin exon 1 fragments with brain lipids caused bilayer perturbation, mediated through a proline-rich region adjacent to the polyglutamines. This suggests that lipid interactions in vivo could influence misfolding of huntingtin and may play an early role in HD pathogenesis.

Ischemic acute renal failure induces differential expression of small heat shock proteins.

AlphaB-crystallin and heat shock protein (hsp) 25 are structurally and functionally related small stress proteins induced by a variety of insults, including heat and ischemia. Cytoprotection by these two hsp is thought to result from molecular chaperoning and/or cytoskeletal stabilization. Because renal ischemia is characterized by disruption of the renal tubular cell actin cytoskeleton, this study was conducted to determine the localization and quantify the expression and phosphorylation of both hsp in renal cortex, isolated glomeruli, outer medulla, and inner medulla of rats after bilateral renal ischemia. Sham-operated kidneys had similarly small amounts of hsp25 and alphaB-crystallin in cortex and glomeruli, with substantially greater amounts of alphaB-crystallin versus hsp25 in outer and inner medulla. Ischemia resulted in significantly increased hsp25 (and hsp70i) but variable alphaB-crystallin levels in cortex and outer medulla, and progressively decreased glomerular hsp25 phosphorylation. In sham-operated kidneys, hsp25 localized to glomeruli, vessels, and collecting ducts, with alphaB-crystallin primarily in medullary thin limbs and collecting ducts. After ischemia, hsp25 accumulated in proximal tubules in cortex and outer medulla, while alphaB-crystallin labeling became nonhomogeneous in outer medulla, and increased in Bowman's capsule. It is concluded that: (1) There is striking differential expression of hsp25 and alphaB-crystallin in various renal compartments; and (2) Renal ischemia results in differential accumulation of hsp25 and alphaB-crystallin, with hsp25 part of a generalized stress response in renal proximal tubular cells, which may play a role in recovery from ischemia-induced actin filament disruption.

Formation of critical oligomers is a key event during conformational transition of recombinant syrian hamster prion protein.

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90-232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90-232 showed an infrared absorbance spectrum typical of proteins with a predominant alpha-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3-2 m guanidine hydrochloride), the secondary structure of SHaPrP90-232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel beta-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in alpha-helical structures and the increase in beta-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No beta-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90-232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90-232. A new model for the structural transition and aggregation process of the prion protein is described.