CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms

CAG Expansions Are Genetically Stable and Form Nontoxic Aggregates in Cells Lacking Endogenous Polyglutamine Proteins

Proteins containing polyglutamine (polyQ) regions are found in almost all eukaryotes, albeit with various frequencies. In humans, proteins such as huntingtin (Htt) with abnormally expanded polyQ regions cause neurodegenerative diseases such as Huntington’s disease (HD). To study how the presence of endogenous polyQ aggregation modulates polyQ aggregation and toxicity, we expressed polyQ expanded Htt fragments (polyQ Htt) in Schizosaccharomyces pombe. In stark contrast to other unicellular fungi, such as Saccharomyces cerevisiae, S. pombe is uniquely devoid of proteins with more than 10 Q repeats. We found that polyQ Htt forms aggregates within S. pombe cells only with exceedingly long polyQ expansions. Surprisingly, despite the presence of polyQ Htt aggregates in both the cytoplasm and nucleus, no significant growth defect was observed in S. pombe cells. Further, PCR analysis showed that the repetitive polyQ-encoding DNA region remained constant following transformation and after multiple divisions in S. pombe, in contrast to the genetic instability of polyQ DNA sequences in other organisms. These results demonstrate that cells with a low content of polyQ or other aggregation-prone proteins can show a striking resilience with respect to polyQ toxicity and that genetic instability of repetitive DNA sequences may have played an important role in the evolutionary emergence and exclusion of polyQ expansion proteins in different organisms

MEASURING COLLATERAL STATUS IN DIFFERENT VASCULAR BEDS

INTRODUCTION Collateral circulation in the brain is the most effective predictor of clinical outcome in acute ischemic stroke (AIS) patients [2]. Collaterals are vessels in the brain that reroute blood to the affected tissue during AIS. The only available methods of visualizing these vessels are invasive (CT Angiography, DSA) and are only effective if a major artery is occluded. Faber et al. (2010) showed that a correlation exists between collateral status in various body tissues, with the collateral status of the brain [1]. Here we describe a novel, non-invasive method for determining superficial palmar arch status (conduit collateral status), as well as micro vascular collateral status in the human hand.   METHODS The only non-invasive method for determination of the quality of blood flow in the hand is the modified allen’s test (MAT). The current techniques used in this test involve a high level of subjectivity and a low level of accuracy [3]. We improved this test by making the results quantitative, as well as focusing on specific regions of interest (ROI) in the hand. We developed an apparatus for the hand to be placed in, with a mounted research grade camera that would capture the duration of the test. The box was internally illuminated with optimized 740nm LEDs for detection of light intensity changes in the hand during the modified Allen’s test protocol. Our protocol was based upon removing blood from the hand using autonomous compressions, and recording the reperfusion of these vessels from a 1st artery release (radial or ulnar), followed by the 2nd artery release 15 seconds later. The camera recorded the intensity changes in the reflected radiation off the hand, which fluctuated as haemoglobin exited and re-entered the vessels in the hand. RESULTS Pilot data from 10 hands (5 healthy individuals) showed significant variance in both the rate of filling and the time to return to baseline. Our quickest rate of filling was 24 times larger than the slowest rate. Our analyses of the graphs, as well as controlling various confounding variables during assessment, suggest that these results are physiological and reflect differences in micro-vascular and conduit collateral status. DISCUSSION AND CONCLUSIONS This method reliably measures physiological differences in the collateral status of the human hand. These differences are shown in Figure 1, where two separate subjects have different arterial supply to the digits. In the future, we will be correlating hand collateral status with brain collateral status in stroke patients. If these correlations exist, a non-invasive pre-emptive tool would be made available to gain knowledge of brain collateral status before AIS occurs