Genistein improves neuropathology and corrects behaviour in a mouse model of neurodegenerative metabolic disease

Background

Neurodegenerative metabolic disorders such as mucopolysaccharidosis IIIB (MPSIIIB or Sanfilippo disease) accumulate undegraded substrates in the brain and are often unresponsive to enzyme replacement treatments due to the impermeability of the blood brain barrier to enzyme. MPSIIIB is characterised by behavioural difficulties, cognitive and later motor decline, with death in the second decade of life. Most of these neurodegenerative lysosomal storage diseases lack effective treatments. We recently described significant reductions of accumulated heparan sulphate substrate in liver of a mouse model of MPSIIIB using the tyrosine kinase inhibitor genistein.

Methodology/Principal Findings

We report here that high doses of genistein aglycone, given continuously over a 9 month period to MPSIIIB mice, significantly reduce lysosomal storage, heparan sulphate substrate and neuroinflammation in the cerebral cortex and hippocampus, resulting in correction of the behavioural defects observed. Improvements in synaptic vesicle protein expression and secondary storage in the cerebral cortex were also observed.

Conclusions/Significance

Genistein may prove useful as a substrate reduction agent to delay clinical onset of MPSIIIB and, due to its multimodal action, may provide a treatment adjunct for several other neurodegenerative metabolic diseases.     

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.     

Functional dissection of Escherichia coli trigger factor: unraveling the function of individual domains

In Escherichia coli, the ribosome-associated chaperone Trigger Factor (TF) promotes the folding of newly synthesized cytosolic proteins. TF is composed of three domains: an N-terminal domain (N), which mediates ribosome binding; a central domain (P), which has peptidyl-prolyl cis/trans isomerase activity and is involved in substrate binding in vitro; and a C-terminal domain (C) with unknown function. We investigated the contributions of individual domains (N, P, and C) or domain combinations (NP, PC, and NC) to the chaperone activity of TF in vivo and in vitro. All fragments comprising the N domain (N, NP, NC) complemented the synthetic lethality of Deltatig DeltadnaK in cells lacking TF and DnaK, prevented protein aggregation in these cells, and cross-linked to nascent polypeptides in vitro. However, DeltatigDeltadnaK cells expressing the N domain alone grew more slowly and showed less viability than DeltatigDeltadnaK cells synthesizing either NP, NC, or full-length TF, indicating beneficial contributions of the P and C domains to TF's chaperone activity. In an in vitro system with purified components, none of the TF fragments assisted the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase in a manner comparable to that of wild-type TF, suggesting that the observed chaperone activity of TF fragments in vivo is dependent on their localization at the ribosome. These results indicate that the N domain, in addition to its function to promote binding to the ribosome, has a chaperone activity per se and is sufficient to substitute for TF in vivo.     

The optimal eukaryotic signal for translation initiation from non-AUG codons,present upstream of bacteriophage lambda P cistron,is inactive in Escherichia coli

Expression of the replication genes of bacteriophage lambda, O and P, is believed to be translationally coupled. However, it was previously noted that, under conditions of amino acid starvation, when O is not synthesized, P continues to be expressed at a relatively high level. The results presented in this report, contrary to the previously presented hypothesis, suggest that an AGACUGGAU sequence (an optimal context for translation initiation from non-AUG codons in eukaryotes, and present upstream the P cistron) is inactive in Escherichia coli. Comparative sequence analysis confirms that such a signal is unlikely to be important for P synthesis. Instead, a weak Shine-Dalgarno sequence may be present upstream the P cistron, and be active in the absence of O gene expression.     

Apple beta-galactosidase. Activity against cell wall polysaccharides and characterization of a related cDNA clone.

A beta-galactosidase was purified from cortical tissue of ripe apples (Malus domestica Borkh. cv Granny Smith) using a procedure involving affinity chromatography on lactosyl-Sepharose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that two polypeptides of 44 and 32 kD were present in the fraction that showed activity against the synthetic substrate p-nitrophenol-beta-D-galactopyranoside. The enzyme preparation was incubated with polysaccharide extracts from apple cell walls containing beta-(1-->4)-linked galactans, and products of digestion were analyzed by gas chromatography. Small amounts of monomeric galactose were released during incubation, showing that the enzyme was active against native substrates. Amino acid sequence information was obtained from the purified protein, and this showed high homology with the anticipated polypeptide coded by the ethylene-regulated SR12 gene in carnation (K.G. Raghothama, K.A. Lawton, P.B. Goldborough, W.R. Woodson [1991] Plant Mol Biol 17: 61-71) and a harvest-related pTIP31 cDNA from asparagus (G. King, personal communication). Using the asparagus cDNA clone as a probe, an apple homolog (pABG1) was isolated. This clone contains a 2637-bp insert, including an open reading frame that codes for a polypeptide of 731 amino acids. Cleavage of an N-terminal signal sequence would leave a predicted polypeptide of 78.5 kD. Genomic DNA analysis and the isolation of other homologous apple clones suggest that pABG1 represents one member of an apple beta-galactosidase gene family. Northern analysis during fruit development and ripening showed accumulation of pABG1-homologous RNA during fruit ripening. Enzyme activity as measured in crude extracts increased during fruit development to a level that was maintained during ripening.     

Rapid ephemeral cell sensitization as the mechanism of histone-induced and protamine-induced enhancement of transfection by poliovirus RNA

Summary The mechanism of transfection enhancement by protamine and histone was investigated, using poliovirus RNA and sheet cultures of the CLI line of chimpanzee liver cells. The transfection obtained using inocula prepared by premixing the viral RNA with a large excess of basic protein could be quantitatively accounted for by direct sensitization of the cells by the excess free basic protein postinoculation. Such direct sensitization to transfection was very rapid; at 25 °C with protamine chloride or histone at 1 mg/ml peak sensitivity was reached in about 16 and 25 seconds, respectively. Sensitivity then rapidly decreased, but could be partially restored later by a second pretreatment with fresh basic protein. The original sensitization, desensitization, and resensitization phases of the cell sensitivity curves are transfectionspecific, since ordinary infection with intact poliovirions was not influenced by the pretreatments with basic protein, except that prolonged pretreatment with histone decreased sensitivity to intact virus. The viral RNA transfectivity in inocula premixed with protamine chloride at 1 mg/ml and held at 0 °C was labile, with an initial half-life of only 5 minutes; the transfectivity of RNA similarly held in buffer alone or with histone was completely stable at 0 °C for at least 1.5 to 2 hours. With the best basic protein transfection method (cell sensitization by protamine), the specific transfectivity of RNA isolated from purified poliovirions was 130,000 plaque-forming units/g RNA.