HIP14 and HIP14L interaction with N-terminal deletion mutants of HTT 1–548.

<p>(<b>A</b>) A diagram of the HTT 1–548 N-terminal deletion mutants used in co-immunoprecipitation experiments with HIP14-GFP and HIP14L-GFP showing the 15Q poly-Q domains, the proline rich region (PRR), and the H1 alpha-rod domain. (<b>B</b>) A representative image (top two panels) of the co-immunoprecipitation between these N-terminal deletion mutants and HIP14-GFP where GFP was immunoprecipitated and the resulting blots were probed for HTT (top panel) and GFP (bottom panel) showing less HTT 88–548, HTT 151–548, and HTT 224–548 co-immunoprecipitated with HIP14-GFP and no HTT 427–548 was co-immunoprecipitated with HIP14. On the right is a beads alone control showing no non-specific binding of the proteins to the beads. The bottom two images show the expression of the HTT deletion mutants (top panel) and of HIP14-GFP (bottom panel). (<b>C</b>) Quantification of three co-immunoprecipitation experiments where the % HTT interaction with HIP14 is the indicated HTT band intensity as a percentage of the HIP14-GFP band intensity from the same sample, normalized to 15Q HTT 1–548. (<b>D</b>) A representative image (top two panels) of the co-immunoprecipitation between the HTT 1–548 N-terminal deletion mutants and HIP14L-GFP where GFP was immunoprecipitated and the resulting blots were probed for HTT (top panel) and GFP (bottom panel). Less HTT 88–548, HTT 151–548, and HTT 224–548 and no HTT 427–548 was co-immunoprecipitated with HIP14L-GFP. On the right is a beads alone control showing no non-specific binding of the proteins to the beads. The bottom two panels show the expression of the HTT deletion mutants (top panel) and of HIP14L-GFP (bottom panel). (<b>E</b>) Quantification of three co-immunoprecipitation experiments where the % HTT interaction with HIP14L-GFP is the indicated HTT band intensity as a percentage of the HIP14L-GFP band intensity from the same sample, normalized to 15Q HTT 1–548.</p

Overview schematics of the domain organization of HTT (A), HIP14 (B), and HIP14L (C).

<p>The domain organization of HTT is shown in (<b>A</b>) with the poly-glutamine domains of WT (15Q) and mutant (128Q) HTT (NP_002102) are shown in grey and black rectangles, respectively, the proline rich repeat is shown in a hatched rectangle, and the H1 alpha-rod domain is shown in a dotted rectangle with the amino acids indicated above. (<b>B</b>) The domain organization of HIP14 (NP_056151) is shown in (<b>B</b>) and of HIP14L (NP_061901) in (<b>C</b>) with the 7 ankyrin repeats making up the ankyrin repeat domain shown in numbered solid grey rectangles, the transmembrane domains shown in hatched rectangles labeled TM1-6, and the DHHC cysteine-rich domain shown in solid black rectangles labeled DHHC. The amino acids corresponding to the appropriate domains are indicated below.</p

A schematic diagram of the two hypothetical binding scenarios of HTT with HIP14 or HIP14L.

<p>In both (<b>A</b>) and (<b>B</b>) for HIP14 or HIP14L the numbered, solid grey boxes are the seven ankyrin repeats that make up the ankyrin repeat domain, the six TMDs are in hatched boxes labeled TM1–TM6, and the DHHC-CR domain is a black box labeled DHHC. (<b>A</b>) In this first scenario, the HIP14 and HIP14L HTT binding site (solid pink box) is between amino acids 224–427 and this binding site interacts with the ankyrin repeat domain of HIP14 or HIP14L. (<b>B</b>) In an alternate scenario there are two binding sites (solid pink boxes), one between amino acids 1–427 and the other between amino acids 224–548, that both interact with the ankyrin repeat domain.</p

Cloning primers used to generate the HTT 1–548 N-terminal deletion mutants.

<p>*Restriction enzyme sites are in italics (EcoRI in forward primers and NotI in the reverse), the start and stop codons are in bold, and the primer binding sequence is underlined.</p

HIP14 and HIP14L interaction with C-terminal deletion mutants of HTT 1-548.

<p>(<b>A</b>) A schematic diagram of the HTT 1–548 C-terminal deletion mutants used in co-immunoprecipitation experiments with HIP14-GFP and HIP14L-GFP showing the 15Q or 128Q poly-Q domains, the proline rich region (PRR), and the H1 alpha-rod domain. (<b>B</b>) A representative image (top two panels) of the co-immunoprecipitation between these C-terminal deletion mutants and HIP14-GFP where GFP was immunoprecipitated and the resulting blots were probed for HTT (top panel) and GFP (bottom panel) showing less 15Q and 128Q HTT 1–427 co-immunoprecipitated with HIP14-GFP. On the right is a beads alone (no antibody) control showing no non-specific binding of the proteins to the beads. The bottom two images show the expression of the HTT deletion mutants (top panel) and of HIP14-GFP (bottom panel). (<b>C</b>) Quantification of three independent co-immunoprecipitation experiments where the % HTT interaction with HIP14 is the indicated HTT band intensity as a percentage of the HIP14-GFP band intensity from the same sample, normalized to 15Q HTT 1–548. (<b>D</b>) A representative image (top two panels) of the co-immunoprecipitation between the HTT 1–548 C-terminal deletion mutants and HIP14L-GFP where GFP was immunoprecipitated and the resulting blots were probed for HTT (top panel) and GFP (bottom panel). Less 15Q and 128Q HTT 1–427 co-immunoprecipitated with HIP14L-GFP. On the right is a beads alone (no antibody) control showing no non-specific binding of the proteins to the beads. The bottom two panels show the expression of the HTT deletion mutants (top panel) and of HIP14L-GFP (bottom panel). (<b>E</b>) Quantification of three independent co-immunoprecipitation experiments where the % HTT interaction with HIP14L-GFP is the indicated HTT band intensity as a percentage of the HIP14L-GFP band intensity from the same sample, normalized to 15Q HTT 1–548.</p

Whole-cell properties of CA1 pyramidal neurons from Hu18/18 and Hu97/18 mice at 9 months of age.

<p>(A–D) Whole-cell patch recordings from CA1 pyramidal neurons revealed no difference in membrane properties including membrane capacitance (Cm, Hu18/18 n = 14, Hu97/18 n = 11), membrane resistance (Rm, Hu18/18 n = 14, Hu97/18 n = 11), membrane tau (ôm, Hu18/18 n = 14, Hu97/18 n = 11) or resting membrane potential (Em, Hu18/18 n = 14, Hu97/18 n = 13). I–V plots (E, Hu18/18 n = 14, Hu97/18 n = 13), mEPSC frequency (F, Hu18/18 n = 13, Hu97/18 n = 13) and mEPSC amplitude (G, Hu18/18 n = 13, Hu97/18 n = 13) were also similar between genotypes.</p

sEPSC amplitude and frequency change in Hu97/18 SPNs.

<p>Cells were patch-clamped with a potassium-based internal solution at V_h = -70 mV and sEPSCs were recorded. (A) Representative sEPSC traces for Hu18/18 (top) and Hu97/18 SPNs (bottom). (B) The average sEPSC amplitude of Hu18/18 and Hu97/18 cells (difference did not reach significance, p = 0.12). (C) Cumulative probability showed a decrease in sEPSC amplitude in Hu97/18 SPNs (significant genotype and amplitude interaction, p<0.0001). (D) Amplitude distribution analysis showed a significant increase in the percentage of small events (<10pA) and a trend towards a decrease in big events (>15pA) in Hu97/18 SPNs. (E) There was no difference in sEPSC decay time between Hu18/18 and Hu97/18 SPNs. (F) Cumulative probability showed an increase in sEPSC inter-event intervals in Hu97/18 SPNs (significant genotype and inter-event intervals interaction, p<0.0001). (G) The average sEPSC frequency was decreased in Hu97/18 SPNs (p = 0.038). For all experiments, Hu18/18 n = 20 and Hu97/18 n = 17. *P<0.05 (two-way ANOVA with Bonferroni correction, D; unpaired Student’s t-test, G).</p

Alterations in basic membrane properties of Hu97/18

<p>SPNs. (A–C): Membrane capacitance (A), membrane resistance (B), and membrane time constant (C) did not differ between Hu18/18 and Hu97/18 SPNs. Membrane capacitance measurement was pooled from the experiments with potassium-based and caesium-based internal solutions (Hu18/18 n = 46, Hu97/18 n = 44), while membrane resistance and tau were measured with potassium-based internal solution only (Hu18/18 n = 21, Hu97/18 n = 17). (D – J): Cells were patch-clamped with potassium-based solution, and membrane voltage changes in response to the injected current were recorded and analysed. (D) Representative I–V response of a Hu18/18 SPN to current injection (50pA increments from -200pA, 1 s each). (E) I–V curves showed no difference between Hu97/18 and Hu18/18 SPNs. (F) Rheobase and (G) rheobase frequency were not different between the genotypes. (H) Hu97/18 had a lower resting membrane potential than Hu18/18 SPNs (p = 0.03). (I) Action potential (AP) threshold was more depolarized in Hu97/18 SPNs (p = 0.02). (J) The change in membrane voltage from resting potential to AP threshold was not different between Hu18/18 and Hu97/18 SPNs. For the experiments in D–J, Hu18/18 n = 11 and Hu97/18 n = 12. *p<0.05 (unpaired Student’s t-test).</p

Biophysical and Biological Characterization of Hairpin and Molecular Beacon RNase H Active Antisense Oligonucleotides

Antisense oligonucleotides (ASOs) are single stranded, backbone modified nucleic acids, which mediate cleavage of complementary RNA by directing RNase H cleavage in cell culture and in animals. It has generally been accepted that the single stranded state in conjunction with the phosphorothioate modified backbone is necessary for cellular uptake and transport to the active compartment. Herein, we examine the effect of using hairpin structured ASOs to (1) determine if an ASO agent requires a single stranded conformation for efficient RNA knock down, (2) use a fluorophore-quencher labeled ASO to evaluate which moieties the ASO interacts with in cells and examine if cellular distribution can be determined with such probes, and (3) evaluate if self-structured ASOs can improve allele selective silencing between closely related huntingtin alleles. We show that hairpin shaped ASOs can efficiently down-regulate RNA <i>in vitro</i>, but potency correlates strongly negatively with increasing stability of the hairpin structure. Furthermore, self-structured ASOs can efficiently reduce huntingtin mRNA in the central nervous system of mice

Long-term potentiation (LTP) is impaired in 9-month old Hu97/18 CA1.

<p>(A) Representative fEPSP traces from hippocampal slices of 9-month old Hu18/18 and Hu97/18 mice in response to two 0.1 ms pulses separated by 100 ms. fEPSPs were recorded in CA1 stratum radiatum during stimulation of the Schaffer collateral pathway. (B) Paired pulse facilitation (PPF) was measured by dividing the slope of the second response to that of the first and was expressed as percent increase. PPF was significantly lower in Hu97/18 CA1 (Hu18/18 n = 8, Hu97/18 n = 6). (C) fEPSPs were normalized to a 10-minute baseline period prior to high-frequency stimulation (HFS; 100 Hz for 1 s×3, 10 s inter-train interval). LTP was easily obtained in hippocampal slices from Hu18/18 mice (n = 6) but showed severe impairment in slices from Hu97/18 mice (n = 6). Representative traces before (black) and 50–60 minutes after (grey) HFS are shown. ***p<0.0001, t-test of average % above baseline 50–60 minutes post-HFS. (D–F) Paired pulse facilitation (D,E) and LTP (F) graphs as above but conducted in slices from 3-month old animals. n = 6 for each genotype for both (E) and (F).</p