Aggregation of Expanded Huntingtin in the Brains of Patients With Huntington Disease

Huntingtin containing an expanded polyglutamine causes neuronal death and Huntington disease. Although expanded huntingtin is found in virtually every cell type, its toxicity is limited to neurons of certain areas of the brain, such as cortex and caudate/putamen. In affected areas of the brain, expanded huntingtin is not found in its intact monomeric form. It is found instead in the form of N-terminal fragments, oligomers and polymers, all of which accumulate in the cortex. Whereas the oligomer is mostly soluble, the polymers and the fragments associate with each other and with other proteins to form the insoluble inclusions characteristic of the disease. It is likely that the aggregates containing expanded huntingtin are toxic to neurons, but it remains to be determined whether the oligomer or the inclusion is the toxic species.Key Words: huntingtin, polyglutamine, aggregation, oligomer, polymer, N-terminal fragments, transglutaminase     

Molecular cloning and characterization of human trabeculin-alpha, a giant protein defining a new family of actin-binding proteins

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-alpha (M(r) = 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of alpha-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrin-like motif in the central region of the protein; two potential Ca(2+)-binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-alpha appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9-486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-alpha in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.     

HLM1, an essential signaling component in the hypersensitive response,is a member of the cyclic nucleotide-gated channel ion channel family

The hypersensitive response (HR) in plants is a programmed cell death that is commonly associated with disease resistance. A novel mutation in Arabidopsis, hlm1, which causes aberrant regulation of cell death, manifested by a lesion-mimic phenotype and an altered HR, segregated as a single recessive allele. Broad-spectrum defense mechanisms remained functional or were constitutive in the mutant plants, which also exhibited increased resistance to a virulent strain of Pseudomonas syringae pv tomato. In response to avirulent strains of the same pathogen, the hlm1 mutant showed differential abilities to restrict bacterial growth, depending on the avirulence gene expressed by the pathogen. The HLM1 gene encodes a cyclic nucleotide-gated channel, CNGC4. Preliminary study of the HLM1/CNGC4 gene pro-duct in Xenopus oocytes (inside-out patch-clamp technique) showed that CNGC4 is permeable to both K(+) and Na(+) and is activated by both cGMP and cAMP. HLM1 gene expression is induced in response to pathogen infection and some pathogen-related signals. Thus, HLM1 might constitute a common downstream component of the signaling pathways leading to HR/resistance.     

A biomimetic motility assay provides insight into the mechanism of actin-based motility

Abiomimetic motility assay is used to analyze the mechanism of force production by site-directed polymerization of actin. Polystyrene microspheres, functionalized in a controlled fashion by the N-WASP protein, the ubiquitous activator of Arp2/3 complex, undergo actin-based propulsion in a medium that consists of five pure proteins. We have analyzed the dependence of velocity on N-WASP surface density, on the concentration of capping protein, and on external force. Movement was not slowed down by increasing the diameter of the beads (0.2 to 3 microm) nor by increasing the viscosity of the medium by 10(5)-fold. This important result shows that forces due to actin polymerization are balanced by internal forces due to transient attachment of filament ends at the surface. These forces are greater than the viscous drag. Using Alexa488-labeled Arp2/3, we show that Arp2/3 is incorporated in the actin tail like G-actin by barbed end branching of filaments at the bead surface, not by side branching, and that filaments are more densely branched upon increasing gelsolin concentration. These data support models in which the rates of filament branching and capping control velocity, and autocatalytic branching of filament ends, rather than filament nucleation, occurs at the particle surface.     

Preservation of ischemia and isoflurane-induced preconditioning after brain death in rabbit hearts

We sought to determine whether brain death-induced catecholamine release preconditions the heart, and if not, whether it precludes further protection by repetitive ischemia or isoflurane. Anesthetized rabbits underwent 30 min of coronary occlusion and 4 h of reperfusion. The effect on infarct size of either no intervention (controls), ischemic preconditioning (IPC), or isoflurane inhalation (Iso) was evaluated with or without previous brain death (BD) induced by subdural balloon inflation. Plasma catecholamine levels were measured at several time points. Although it dramatically increase plasma catecholamine levels, BD failed to reduce infarct size that averaged 0.49 +/- 0.34 without BD versus 0.45 +/- 0.27 g with BD. IPC and Iso, alone as well as after BD, significantly reduced infarct size that averaged 0.11 +/- 0.04, 0.21 +/- 0.15, 0.10 +/- 0.09, and 0.22 +/- 0.10 g in IPC, Iso, BD + IPC, and BD + Iso groups, respectively (means +/- SD, P < 0.05 vs. controls). BD-induced catecholamines "storm" does not precondition the rabbit heart that however retains the ability to be protected by repetition of brief ischemia or isoflurane inhalation.     

Cholesterol gallstone formation in overweight mice establishes that obesity per se is not linked directly to cholelithiasis risk

The relationship between obesity and cholesterol cholelithiasis is not well understood at physiologic or genetic levels. To clarify whether obesity per se leads to increased prevalence of cholelithiasis, we examined cholesterol gallstone susceptibility in three polygenic (KK/H1J, NON/LtJ, NOD/LtJ) and five monogenic [carboxypeptidase E (Cpe (fat)), agouti yellow (A(y)), tubby (tub), leptin (Lep(ob)), leptin receptor (Lepr (db))] murine models of obesity during ingestion of a lithogenic diet containing dairy fat, cholesterol, and cholic acid. At 8 weeks on the diet, one strain of polygenic obese mice was resistant whereas the others revealed low or intermediate prevalence rates of cholelithiasis. Monogenic obese mice showed distinct patterns with either high or low gallstone prevalence rates depending upon the mutation. Dysfunction of the leptin axis, as evidenced by the Lep(ob) and the Lepr (db) mutations, markedly reduced gallstone formation in a genetically susceptible background strain, indicating that in mice with this genetic background, physiologic leptin homeostasis is a requisite for cholesterol cholelithogenesis. In contrast, the Cpe (fat) mutation enhanced the prevalence of cholelithiasis markedly when compared with the background strain. Since CPE converts many prohormones to hormones, a deficiency of biologically active cholecystokinin is a likely contributor to enhanced susceptibility to cholelithiasis through compromising gallbladder contractility and small intestinal motility. Because some murine models of obesity increased, whereas others decreased cholesterol gallstone susceptibility, we establish that cholesterol cholelithiasis in mice is not simply a secondary consequence of obesity per se. Rather, specific genes and distinct pathophysiological pathways are responsible for the shared susceptibility to both of these common diseases.     

Salt Stress Induced Changes in the Exoproteome of the Halotolerant Bacterium Tistlia consotensis Deciphered by Proteogenomics

The ability of bacteria to adapt to external osmotic changes is fundamental for their survival. Halotolerant microorganisms, such as Tistlia consotensis, have to cope with continuous fluctuations in the salinity of their natural environments which require effective adaptation strategies against salt stress. Changes of extracellular protein profiles from Tistlia consotensis in conditions of low and high salinities were monitored by proteogenomics using a bacterial draft genome. At low salinity, we detected greater amounts of the HpnM protein which is involved in the biosynthesis of hopanoids. This may represent a novel, and previously unreported, strategy by halotolerant microorganisms to prevent the entry of water into the cell under conditions of low salinity. At high salinity, proteins associated with osmosensing, exclusion of Na⁺ and transport of compatible solutes, such as glycine betaine or proline are abundant. We also found that, probably in response to the high salt concentration, T. consotensis activated the synthesis of flagella and triggered a chemotactic response neither of which were observed at the salt concentration which is optimal for growth. Our study demonstrates that the exoproteome is an appropriate indicator of adaptive response of T. consotensis to changes in salinity because it allowed the identification of key proteins within its osmoadaptive mechanism that had not previously been detected in its cell proteome.     

Mutations in CSPP1 Cause Primary Cilia Abnormalities and Joubert Syndrome with or without Jeune Asphyxiating Thoracic Dystrophy

Joubert syndrome (JBTS) is a recessive ciliopathy in which a subset of affected individuals also have the skeletal dysplasia Jeune asphyxiating thoracic dystrophy (JATD). Here, we have identified biallelic truncating CSPP1 (centrosome and spindle pole associated protein 1) mutations in 19 JBTS-affected individuals, four of whom also have features of JATD. CSPP1 mutations explain ∼5% of JBTS in our cohort, and despite truncating mutations in all affected individuals, the range of phenotypic severity is broad. Morpholino knockdown of cspp1 in zebrafish caused phenotypes reported in other zebrafish models of JBTS (curved body shape, pronephric cysts, and cerebellar abnormalities) and reduced ciliary localization of Arl13b, further supporting loss of CSPP1 function as a cause of JBTS. Fibroblasts from affected individuals with CSPP1 mutations showed reduced numbers of primary cilia and/or short primary cilia, as well as reduced axonemal localization of ciliary proteins ARL13B and adenylyl cyclase III. In summary, CSPP1 mutations are a major cause of the Joubert-Jeune phenotype in humans; however, the mechanism by which these mutations lead to both JBTS and JATD remains unknown.     

Herpes simplex virus dances with amyloid precursor protein while exiting the cell

Herpes simplex type 1 (HSV1) replicates in epithelial cells and secondarily enters local sensory neuronal processes, traveling retrograde to the neuronal nucleus to enter latency. Upon reawakening newly synthesized viral particles travel anterograde back to the epithelial cells of the lip, causing the recurrent cold sore. HSV1 co-purifies with amyloid precursor protein (APP), a cellular transmembrane glycoprotein and receptor for anterograde transport machinery that when proteolyzed produces A-beta, the major component of senile plaques. Here we focus on transport inside epithelial cells of newly synthesized virus during its transit to the cell surface. We hypothesize that HSV1 recruits cellular APP during transport. We explore this with quantitative immuno-fluorescence, immuno-gold electron-microscopy and live cell confocal imaging. After synchronous infection most nascent VP26-GFP-labeled viral particles in the cytoplasm co-localize with APP (72.8+/-6.7%) and travel together with APP inside living cells (81.1+/-28.9%). This interaction has functional consequences: HSV1 infection decreases the average velocity of APP particles (from 1.1+/-0.2 to 0.3+/-0.1 μm/s) and results in APP mal-distribution in infected cells, while interplay with APP-particles increases the frequency (from 10% to 81% motile) and velocity (from 0.3+/-0.1 to 0.4+/-0.1 μm/s) of VP26-GFP transport. In cells infected with HSV1 lacking the viral Fc receptor, gE, an envelope glycoprotein also involved in viral axonal transport, APP-capsid interactions are preserved while the distribution and dynamics of dual-label particles differ from wild-type by both immuno-fluorescence and live imaging. Knock-down of APP with siRNA eliminates APP staining, confirming specificity. Our results indicate that most intracellular HSV1 particles undergo frequent dynamic interplay with APP in a manner that facilitates viral transport and interferes with normal APP transport and distribution. Such dynamic interactions between APP and HSV1 suggest a mechanistic basis for the observed clinical relationship between HSV1 seropositivity and risk of Alzheimer's disease.