Desorción de paraquat con cloruro de amonio

Conditions were as described (section 2.4.5) using Ipg DNA template with an annealing temperature of 60°C and 35 cycles of PCR reaction. Products were visualised on a 3% metaphor gel (section 2.4.9). The mutant HD gene amplification yielded a fragment of 535bp and the wild-type a 397bp fragment. The DNA products were extracted from the gel (section 2.4.10), purified (section 2.4.3) and then amplified further using the PCR primers G24 (forward) and G25 (reverse) as illustrated in Figure 5.3. An annealing temperature of 67°C was used for this reaction with the G24 and G25 primers and 35 cycles of PCR reaction. The mutant HD gene fragment resulted in a 577bp product and the wild-type in a 439bp product. These PCR products were demonstrated using a 3% metaphor gel then extracted from the agarose gel, cleaned up (section 2.4.6) and restricted prior to sticky-end ligation into pIND (section 2.4.11).

Restriction enzyme digest of amplified HD gene fragments and plND

This was performed exactly as described for the a-synuclein constructs (section 6.2.9). Both mutant and wild-type amplified HD gene fragments were restricted with Kpn-1 and Bcl-1 and cloned into the Kpn-1 and BamHI sites of pIND vector.

sticky-end ligation and propagation through E.coli

The restricted products were ligated overnight at 14°C. These ligated products were then used to transform E coli (section 2.4.13). Transformed E coli were plated out onto agar plates (section 2.4.12) and incubated overnight at 37°C.

Screening piND for the presence of the HD gene construct

The was performed in two ways. Initially this was achieved by randomly picking off transformed E coli colonies from the agar plates and then isolating plasmid from each of them using the Wizard™ mini-prep kit (section 2.4.14). The plasmid preparations were then all run on a 0.8% agarose gel to see which pIND preparations contained an insert. pIND alone (5kB) was run as a control; with pIND plus mutant HD gene insert running at approx.5.6kB and wild-type running at approx. 5.4kB. Those plasmid preparations that clearly contained an HD gene insert were then sequenced as described (section 2.4.15). The second method of screening was developed because it was more rapid and efficient. Colony screening was performed exactly as described for the a-synuclein constructs (section 6.2.11). The resultant PCR products were visualised on a 1.2% agarose gel (section 2.4.9). Those colonies that contained the mutant HD gene insert (61 repeats) gave a 777bp product and those that contained the wild-type HD gene insert (15 repeats) gave a 639bp product. pIND alone gave a 210bp product. In this way it was possible to rapidly screen tens of clones for the presence of construct. Those colonies that were positive for the inserts were then removed from the agar plate and the E coli grown up to isolate the plasmid (section 2.4.14).

Sequencing of Group IV constructs in pIND

The resultant purified plasmid was sequenced to check if the constructs were in the correct orientation and the coding sequence was correct. This was performed using both manual and automated sequencing (section 2.4.15) using the pIND sequencing primers (Forward G49: 5’ CTC TGA AT A CTT TCA ACA AGT TAC 3’; and Reverse G50: 5' TAG AAG GCA CAG TCG AGG 3') as described (section 2.4.15).

5.2.4.4 Cell lines and transfections

HEK293 cells stably expressing pVgRXR were purchased from Invitrogen (EcR293 cells). These were transfected with 5|ig each of both mutant and wild-

type Group III HD gene constructs in pIND using the Escort (Sigma) lipofection method (section 2.3). 24 hours post transfection stable clones were selected with Zeocin (400|ig/ml) and G418 (400|ig/ml). After approx. 15 days stable clones were ring-cloned, and individual clones analysed for expression.

NT2 cells that had been stably transfected with pVgRXR (section 5.3.4.1) were tested for pVgRXR expression by transfecting with pIND lacZ as described (section 2.3) and then treated with Ponasterone A (lO^M) for 24 hours post transfection. These were then stained for p-galactosidase expression as described (section 2.3.3). The clone with the highest expression of p- galactosidase was chosen for further transfection experiments. This

NT2/pVgRXR clone was then transfected with Group I, II and III HD constructs as described above for the EcR293 cells.

5.2.4.5 Detection of HD gene expression in EcR293 and NT2 cells This was performed using immunofluorescence (IF) as described (section 2.3.4) using the antibodies in Table 5.5 at the dilutions given. Cells were pre-treated for 48 hours with lOpM Ponasterone A to induce protein expression.

TABLE 5.5

Antibodies and dilutions used for IF detection of HD gene expression

HD antibodies Dilutions

102 mouse monoclonal 1/200

675 rabbit polyclonal** 1/100

HA-Fluorescein tagged mouse monoclonal*** 1/100

* a kind gift from Dr Yvon Trottier, against the expanded polyglutamine stretch ** a kind gift from Dr Lesley Jones, against the N-terminus of huntingtin (both wild-type and mutant); ***from Boehringer Mannheim, against the HA tag

5.3 Results

5.3.1 HD fibroblast analysis

Mitochondrial fractions were prepared from HD and control fibroblasts (n=5) and assayed for complex II and III and CS activity. There was no significant difference in the activities of complex II, complex III, or CS activities in fibroblast mitochondria from HD patients and controls (Table 5.6 and Fig 5.4). The complex II and III activities were also corrected for 08 activities to adjust for any difference in mitochondrial content between samples but again showed no significant difference. Aconitase activity was measured in whole cell homogenates from HD fibroblasts and controls (n=5) and there was no difference in aconitase activity between the two groups (Table 5.6 and Fig. 5.4).

Mutant huntingtin expression in human fibroblasts was confirmed in our patients by immunoblotting using the 102 antibody which specifically recognises the expanded polyglutamine stretch in mutant huntingtin (data not shown).

Table 5.6 Enzyme activities in control and HD fibroblasts (n=5)

CONTROLS HD

Complex ll/OS ratio 0.30±0.10 0.30±0.09

Complex lll/CS ratio(xlO) 0.64±0.31 0.59±0.22

Aconitase (nmol/min/mg protein] 0.46±0.26 0.79±0.36

5.3.2 HD Brain analysis

Oomplex I I/Ill, OS, aconitase and G A P D H enzyme activities were assessed in homogenates from four brain regions from control and HD brains. When expressed per unit protein, there were highly significant decreases in mean complex I I/I 11 activity in H D putamen (to 40% of control mean) whilst activity in the H D cortex and cerebellum was within control values (Table 5.7 and Fig 5.4). OS activity was significantly decreased in HD putamen and cortex to 59% and

73% of the control mean respectively. When expressed as a ratio with OS activities, the complex I I/I 11 activities were significantly reduced in putamen to 70% of control values but were not significantly different from controls in cerebral cortex.

C om plex IV a c tiv ity in HD p u tam en w as assayed by Dr Paul H a rt, D e p t, of Clinical N eurosciences, R F H S M and w a s not significantly d iffe re n t from control p u ta m e n (Fig 5 .4 ).

Aconitase activities were significantly decreased in HD caudate (to 8% of control), putamen (to 27% of control), and frontal/temporal cortex (to 52% of control) but was unaffected in cerebellum (Table 5.7 and Fig 5.4). GAPDH levels were comparable in controls and HD samples in all four brain regions studied. Fig 5.4 graphically illustrates the significant enzyme defects found in HD brain and it can be seen that the defects in aconitase, complex I I/I 11 and IV parallel the neuropathological severity of the disease with caudate and putamen being most affected.