Proposed pathway of kalirin-7-mediated synphilin-1 aggresome formation.

<p>(A) Under normal conditions, misfolded synphilin-1 is mainly accumulated in cytoplasmic small aggregates. (B) When kalirin-7 is overexpressed, it facilitates the recruitment of HDAC6 and the dynein motor complex and acts on microtubule dynamics by stimulating the deacetylase activity of HDAC6, thereby increasing the transportation of synphilin-1 into aggresomes.</p

Characterization of synphilin-1-containing aggregates as aggresomes.

<p>HEK293 cells coexpressing HcRed-synphilin-1 and FLAG-kalirin-7 were fixed 48 h post-transfection and subsequently stained with the indicated antibodies. Arrows indicate the colocalization between synphilin-1 inclusions and γ-tubulin, ubiquitin and Hsp27 while the intermediate filament protein vimentin forms a cage surrounding a pericentriolar core of aggregates. Merged images are shown to the right. <i>Blue</i>, DAPI. <i>Scale bar</i>, 10 µm.</p

Kalirin-7 mediates perinuclear synphilin-1 inclusion formation in a microtubule-dependent manner.

<p>(A) HEK293 cells were cotransfected with HcRed-synphilin-1 and FLAG-kalirin-7. After 36 h cells were incubated with DMSO, 5 µM nocodazole or 10 µM colchicine for 12 h before being subjected to immunofluorescence with anti-FLAG and anti- β-tubulin antibodies. Cells expressing HcRed-synphilin-1 alone served as controls (arrowhead). In cells treated with nocodazole or colchicine, more cytoplasmic small aggregates (arrows) were formed. (B) Quantification (n >250 cells per group) shows that nocodazole and colchicine inhibited the kalirin-7-mediated formation of synphilin-1-containing perinuclear inclusions. P, perinuclear aggregates; C, cytoplasmic small aggregates. The asterisks indicate statistical significance (^**<i>P</i>≤0.005). <i>Error bars</i>, S.E.</p

Reduced Motivation in the BACHD Rat Model of Huntington Disease Is Dependent on the Choice of Food Deprivation Strategy

<div><p>Huntington disease (HD) is an inherited neurodegenerative disease characterized by motor, cognitive, psychiatric and metabolic symptoms. Animal models of HD show phenotypes that can be divided into similar categories, with the metabolic phenotype of certain models being characterized by obesity. Although interesting in terms of modeling metabolic symptoms of HD, the obesity phenotype can be problematic as it might confound the results of certain behavioral tests. This concerns the assessment of cognitive function in particular, as tests for such phenotypes are often based on food depriving the animals and having them perform tasks for food rewards. The BACHD rat is a recently established animal model of HD, and in order to ensure that behavioral characterization of these rats is done in a reliable way, a basic understanding of their physiology is needed. Here, we show that BACHD rats are obese and suffer from discrete developmental deficits. When assessing the motivation to lever push for a food reward, BACHD rats were found to be less motivated than wild type rats, although this phenotype was dependent on the food deprivation strategy. Specifically, the phenotype was present when rats of both genotypes were deprived to 85% of their respective free-feeding body weight, but not when deprivation levels were adjusted in order to match the rats' apparent hunger levels. The study emphasizes the importance of considering metabolic abnormalities as a confounding factor when performing behavioral characterization of HD animal models.</p></div

Decreased tubulin acetylation in kalirin-7 expressing cells under TSA treatment.

<p>(A, B) An interaction of FLAG-kalirin-7, V5-synphilin-1 and HA-HDAC6 was examined by co-immunoprecipitation experiments. 24 h after transfection, HEK293 cells were lysed and 500 µg of protein lysates were subjected to immunoprecipitation with anti-FLAG or anti-V5 conjugated agarose beads, respectively. The precipitates were probed with HA antibodies to detect HDAC6 and revealed an interaction of HDAC6 with both kalirin-7 and synphilin-1. 30 µg of protein lysates were visualized as input control. The asterisk indicates a non-specific band observed in all raw lysates detected with anti-V5. (C) Cells transiently overexpressing FLAG-kalirin-7 (c, g), FLAG-kalirin-7 plus HcRed-synphilin-1 (d, h), or empty vectors (a, b, e, f) were immunostained for acetylated tubulin (a-d, green) and kalirin-7 (g, h light blue) after DMSO or 1 µM TSA treatment. While TSA treatment resulted in higher acetylation levels in comparison to controls (arrowheads), the overexpression of kalirin-7 led to a significant decrease of the α-tubulin acetylation levels (arrows). Blue, DAPI. Scale bar, 10 µm. (D) Acetylated tubulin levels were quantified by the fluorescence signal of individual cells, as described in Materials and Methods. Kalirin-7 transfected cells treated with TSA were compared to untransfected cells in the same cell population. Comparably, kalirin-7/synphilin-1 doubly transfected cells treated with TSA were quantified relative to untransfected cells in the same population. The asterisks indicate statistical significance (^**<i>P</i>≤0.005). <i>Error bars</i>, S.E., n = 100.</p

Startle testing.

<p>Results are expressed as Mean ± SEM. (a) Prepulse inhibition. A 2 way-ANOVA revealed a GENOTYPE difference in 9 months old BACHD rats especially at PP 6 and PP 12; however any significant differences were found in 1, 4 and 12 months old rats. (b) Startle habituation amplitude in 6 months old rats. Each trial consisted of 10 blocks of 120 dB startle stimuli. WT and TG rats presented a normal startle habituation. (c) Startle threshold. Amplitude to varying startling stimulus intensities in 9 months old rats. WT and TG response amplitude increased with higher stimulus intensities. No GENOTYPE effect was observed. However, a significant difference was detected at 120 dB. Asterisks indicate statistical significance in BACHD rats (*p < 0.05).</p

Kalirin-7-mediated recruitment of synphilin-1 inclusions into aggresome is blocked by the HDAC inhibitor trichostatin A.

<p>(A) HEK293 cells expressing HcRed-synphilin-1 (a,b,c) or co-expressing HcRed-synphilin-1 and FLAG-kalirin-7 (d,e,f) were incubated in the presence of DMSO (a,d), 1 µM TSA (b,e) or 5 mM NaBu (c,f) for 18 h before being fixed and immunostained with anti-FLAG antibodies. The arrow indicates synphilin-1 cytoplasmic small aggregates. <i>Blue</i>, DAPI. <i>Scale bar</i>, 10 µm. (B) Quantification shows that treatment with the HDAC6 inhibitor TSA counteracts the recruitment of synphilin-1 into aggresomes mediated by kalirin-7, whereas the broad deacetylase inhibitor NaBu does not exert such an effect. The asterisks indicate statistical significance (**<i>P</i>≤0.005). Error bars, S.E.</p

Flinch-jump test.

<p>Sensitivity of 6-shocks for [<b>a</b>] flinch and [<b>b</b>] jump (WT n = 15, TG n = 15). Individual intensity response is plotted and bars indicate median values for each genotype. There was no difference between WT and TG rats in current intensities that elicited a flinch or a jump response.</p

Delay and Trace fear conditioning.

<p>[<b>a</b>] Schematic illustration of the delay fear conditioning paradigm. [<b>b</b>] Results in 9 months old BACHD rats are expressed as Mean ± SEM (WT n = 13, TG n = 15). TG rats presented a significant lower freezing response to the context and to the tone. [<b>c</b>] Schematic illustration of trace fear conditioning paradigm. [<b>d</b>] Results in 3 months old BACHD rats are expressed as Mean ± SEM (WT n = 13, TG n = 15). TG rats, compared to WT rats, showed a significantly lower freezing response to the context, to the tone and trace period during retention tests. Asterisks indicate significant differences between WT and TG rats (**p<0.01 and ***p<0.001).</p

Strategy shifting.

<p>Number of rats that exhibited Place (P) or Response (R) learning strategy during each Probe trial P1, P2 and P3 (days 8, 16 and 23 respectively) are represented for WT and TG cohorts of 2 and 6 months of age. The size corresponds to animals that made (1) correct arm choices with a learning index greater than 0.5 during each training session, and (2) were successful for the two last trials prior to the probe trials in the cross maze.</p