Intrabody Gene Therapy Ameliorates Motor, Cognitive, and Neuropathological Symptoms in Multiple Mouse Models of Huntington's Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease resulting from the expansion of a glutamine repeat in the huntingtin (Htt) protein. Current therapies are directed at managing symptoms such as chorea and psychiatric disturbances. In an effort to develop a therapy directed at disease prevention we investigated the utility of highly specific, anti-Htt intracellular antibodies (intrabodies). We previously showed that V_(L)12.3, an intrabody recognizing the N terminus of Htt, and Happ1, an intrabody recognizing the proline-rich domain of Htt, both reduce mHtt-induced toxicity and aggregation in cell culture and brain slice models of HD. Due to the different mechanisms of action of these two intrabodies, we then tested both in the brains of five mouse models of HD using a chimeric adeno-associated virus 2/1 (AAV2/1) vector with a modified CMV enhancer/chicken β-actin promoter. V_(L)12.3 treatment, while beneficial in a lentiviral model of HD, has no effect on the YAC128 HD model and actually increases severity of phenotype and mortality in the R6/2 HD model. In contrast, Happ1 treatment confers significant beneficial effects in a variety of assays of motor and cognitive deficits. Happ1 also strongly ameliorates the neuropathology found in the lentiviral, R6/2, N171-82Q, YAC128, and BACHD models of HD. Moreover, Happ1 significantly prolongs the life span of N171-82Q mice. These results indicate that increasing the turnover of mHtt using AAV-Happ1 gene therapy represents a highly specific and effective treatment in diverse mouse models of HD

Antibody therapy in Neurodegenerative Disease

Advances in medical science have led to increased life expectancy and increased median age in the population. Because the symptoms of neurodegenerative diseases generally onset in mid- to late-life, a concomitant increase in the number of persons afflicted with these devastating diseases has occurred. Developing therapies for neurodegenerative diseases is of the highest priority due to the enormous cost of medical care required, as well as for the human suffering involved. Although caused by a variety of genetic and environmental insults, such diseases share commonalities. Many of these diseases are proteinopathies-diseases caused by misfolded, aggregating proteins. Antibodies that can recognize and remove misfolded proteins are ideally suited for proteinopathy therapeutics. The numerous intriguing advances in antibody-based therapies for neurodegenerative diseases are discussed in this review

Inclusion of seasonal variation in river system microbial communities and phototroph activity increases environmental relevance of laboratory chemical persistence tests

Regulatory tests assess crop protection product environmental fate and toxicity before approval for commercial use. Although globally applied laboratory tests can assess biodegradation, they lack environmental complexity. Microbial communities are subject to temporal and spatial variation, but there is little consideration of these microbial dynamics in the laboratory. Here, we investigated seasonal variation in the microbial composition of water and sediment from a UK river across a two-year time course and determined its effect on the outcome of water-sediment (OECD 308) and water-only (OECD 309) biodegradation tests, using the fungicide isopyrazam. These OECD tests are performed under dark conditions, so test systems incubated under non-UV light:dark cycles were also included to determine the impact on both inoculum characteristics and biodegradation. Isopyrazam degradation was faster when incubated under non-UV light at all collection times in water-sediment microcosms, suggesting that phototrophic communities can metabolise isopyrazam throughout the year. Degradation rate varied seasonally between inoculum collection times only in microcosms incubated in the light, but isopyrazam mineralisation to 14CO2 varied seasonally under both light and dark conditions, suggesting that heterotrophic communities may also play a role in degradation. Bacterial and phototroph communities varied across time, but there was no clear link between water or sediment microbial composition and variation in degradation rate. During the test period, inoculum microbial community composition changed, particularly in non-UV light incubated microcosms. Overall, we show that regulatory test outcome is not influenced by temporal variation in microbial community structure; however, biodegradation rates from higher tier studies with improved environmental realism, e.g. through addition of non-UV light, may be more variable. These data suggest that standardised OECD tests can provide a conservative estimate of pesticide persistence end points and that additional tests including non-UV light could help bridge the gap between standard tests and field studies

Intrabodies Binding the Proline-Rich Domains of Mutant Huntingtin Increase Its Turnover and Reduce Neurotoxicity

Although expanded polyglutamine (polyQ) repeats are inherently toxic, causing at least nine neurodegenerative diseases, the protein context determines which neurons are affected. The polyQ expansion that causes Huntington's disease (HD) is in the first exon (HDx-1) of huntingtin (Htt). However, other parts of the protein, including the 17 N-terminal amino acids and two proline (polyP) repeat domains, regulate the toxicity of mutant Htt. The role of the P-rich domain that is flanked by the polyP domains has not been explored. Using highly specific intracellular antibodies (intrabodies), we tested various epitopes for their roles in HDx-1 toxicity, aggregation, localization, and turnover. Three domains in the P-rich region (PRR) of HDx-1 are defined by intrabodies: MW7 binds the two polyP domains, and Happ1 and Happ3, two new intrabodies, bind the unique, P-rich epitope located between the two polyP epitopes. We find that the PRR-binding intrabodies, as well as VL12.3, which binds the N-terminal 17 aa, decrease the toxicity and aggregation of HDx-1, but they do so by different mechanisms. The PRR-binding intrabodies have no effect on Htt localization, but they cause a significant increase in the turnover rate of mutant Htt, which VL12.3 does not change. In contrast, expression of VL12.3 increases nuclear Htt. We propose that the PRR of mutant Htt regulates its stability, and that compromising this pathogenic epitope by intrabody binding represents a novel therapeutic strategy for treating HD. We also note that intrabody binding represents a powerful tool for determining the function of protein epitopes in living cells

Perturbation with Intrabodies Reveals That Calpain Cleavage Is Required for Degradation of Huntingtin Exon 1

Background: Proteolytic processing of mutant huntingtin (mHtt), the protein that causes Huntington's disease (HD), is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD. Methodology/Principal Findings: We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1) clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA) 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V_L12.3, turnover of soluble mHDx-1 in living cells is blocked. Conclusions/Significance: These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications

Aqueous extraction of residual oil from sunflower press cake using a twin-screw extruder: Feasibility study

The objective of this study was to evaluate the feasibility of an aqueous process to extract the residual oil from sunflower press cakes using a co-rotating twin-screw extruder. Two different configurations were tested: the expression from whole seeds followed by the aqueous extraction, in two successive apparatus or in the same one. For the aqueous extraction stage, the oil yield depended on the operating conditions including screw rotation speed, screw profile, and inlet flow rates of press cakes and water. Liquid/solid separation required the addition of a lignocellulosic residue (wheat straw), upstream from the filtration zone. However, even with maximum fiber inlet flow (around 20% of the inlet flow rate of the solid matters for the highest amount of wheat straw), drying of the cake meal did not improve. The lixiviation of the material was also incomplete. Oil yield was better when the expression and the aqueous extraction were conducted in the same extruder. For all the trials carried out using such a configuration, the corresponding cake meal contained less than 10% residual oil, and the total oil yield was 78% in the best operating conditions. Nevertheless, the contribution of the aqueous extraction stage was extremely limited, less than 5% in the best trial, partly due to a ratio of the water to the press cake too low. For the aqueous extraction stage, the oil was extracted in the form of an oil-in-water emulsion whose stability was minimized because of its low proteins content due to their thermomechanical denaturation during the expression stage

Direct extraction of oil from sunflower seeds by twin-screw extruder according to an aqueous extraction process: Feasibility study and influence of operating conditions

The objective of this study was to evaluate the feasibility of an aqueous process to extract sunflower seed oil using a co-rotating twin-screw extruder. Aqueous extraction was carried out using whole seeds and the influence of the operating conditions on oil yield was examined. Operating conditions included screw profile, screw rotation speed, and input flow rates of sunflower seeds and water. Liquid/solid separation required the addition of a lignocellulosic residue upstream from the filtration zone. However, even with maximum fiber input flow, drying of the cake meal did not improve. The lixiviation of the sunflower seeds was also incomplete. The aqueous extraction of the oil was more efficient in the twin-screw extruder than the reference trial conducted in a batch reactor. The best oil extraction yield obtained was approximately 55% and the residual oil content of the cake meal was approximately 30%. The hydrophobic phases produced were oil-in-water emulsions. These emulsions were stabilized by phospholipids and proteins at the interface, which are natural surface-active agents co-extracted during the process