The Journal of Neuroscience, April 26, 2006, ():
CA150 Expression Delays Striatal Cell Death in Overexpression and Knock-In Conditions for Mutant Huntingtin Neurotoxicity
J. Neurosci. Arango et al.
26: 4649
Supplemental data
Files in this Data Supplement:
- supplemental material -
Supplemental figure 1. CA150 reduces the striatal shrinkage induced by mutant htt expression in a (Gln-ala)38-dependent manner. Brain sections were treated with cresyl violet and percent areas of the bundles of fibers quantified around the injection point. The shrinkage of striatal tissues translates into an increase of the percent area of fibers. A, Percent areas of the bundles of fibers at 8 and 12 weeks. At 8 weeks, htt171-82Q expression produced striatal shrinkage (***P < 0.0001, compared to htt171-19Q expression). The co-expression of CA150 fully rescued striatal shrinkage, and CA150∆QA co-expression showed partial (62%) rescue (***P < 0.0001 and **P < 0.001, compared to htt171-82Q alone). At 12 weeks, rescue by CA150 co-expression was limited 47% (**P < 0.001, compared to htt171-82Q alone), and CA150∆QA co-expression showed no rescue. Data are meanąSD from 7-8 animals. B, Examples of cresyl violet staining. Black arrrowheads indicate picnotic nuclei, and white arrowheads indicate the injection point. In non toxic conditions (upper panels), only the cells positioned along the needle track and near the injection point show picnotic nuclei.
- supplemental material -
Supplemental figure 2. CA150 overexpression increases the proportion of htt-positive neuritic aggregates. Eight weeks after injection, brain sections were stained using the htt antibody 2B4 and analyzed as described (see Materials and Methods). A shift in the distribution towards small objects corresponding to neuritic aggregates was observed when htt171-82Q was co-expressed with CA150. Data are meanąSD from 6-8 animals. ***P < 0.0001, **P < 0.005, and *P < 0.05 compared to htt171-82Q
- supplemental material -
Supplemental figure 3. CA150∆QA and full-length CA150 equally repress a4-integrin promoter activity. A, HEK293T cells were transfected with CA150 and CA150∆QA expressing constructs, the localization of exogenous CA150 and CA150∆QA was revealed using HA epitope staining. Nuclei were stained using acridine orange. The upper panel show images of HA epitope staining (red) and nuclear counterstaining (green). White arrow heads show nuclear CA150 and CA150∆QA.. The lower panels show the quantification of cytoplasmic and nuclear signals for CA150 compared to CA150∆QA.. Shown are the intensity and area of nuclear and cytoplasmic HA signals, indicating no difference between CA150 and CA150∆QA localization. Data are meanąSD of three independent experiments (greater than 30 cells analyzed per experiment). B, The CA150 responsive 42a4CAT reporter construct was co-transfected with empty vector (250 ng) or CA150 or CA150∆QA (three doses) expression plasmids. The upper pannel shows the relative CAT activity, indicating no difference between CA150 and CA150∆QA transcriptional repression ability at all doses tested. Data are meanąSD from three different experiments. The lower panel shows cellular lysates from one representative experiment immunobloted with T7 antibody to detect the expression of CA150 species. Polypyrimidine tract binding protein (PTB) was used as a loading control.
