Tissue-specific proteolysis of Huntingtin (htt) in human brain: evidence of enhanced levels of N- and C-terminal htt fragments in Huntington's disease striatum - PubMed (original) (raw)

Tissue-specific proteolysis of Huntingtin (htt) in human brain: evidence of enhanced levels of N- and C-terminal htt fragments in Huntington's disease striatum

L M Mende-Mueller et al. J Neurosci. 2001.

Abstract

Proteolysis of mutant huntingtin (htt) has been hypothesized to occur in Huntington's disease (HD) brains. Therefore, this in vivo study examined htt fragments in cortex and striatum of adult HD and control human brains by Western blots, using domain-specific anti-htt antibodies that recognize N- and C-terminal domains of htt (residues 181-810 and 2146-2541, respectively), as well as the 17 residues at the N terminus of htt. On the basis of the patterns of htt fragments observed, different "protease-susceptible domains" were identified for proteolysis of htt in cortex compared with striatum, suggesting that htt undergoes tissue-specific proteolysis. In cortex, htt proteolysis occurs within two different N-terminal domains, termed protease-susceptible domains "A" and "B." However, in striatum, a different pattern of fragments indicated that proteolysis of striatal htt occurred within a C-terminal domain termed "C," as well as within the N-terminal domain region designated "A". Importantly, striatum from HD brains showed elevated levels of 40-50 kDa N-terminal and 30-50 kDa C-terminal fragments compared with that of controls. Increased levels of these htt fragments may occur from a combination of enhanced production or retarded degradation of fragments. Results also demonstrated tissue-specific ubiquitination of certain htt N-terminal fragments in striatum compared with cortex. Moreover, expansions of the triplet-repeat domain of the IT15 gene encoding htt was confirmed for the HD tissue samples studied. Thus, regulated tissue-specific proteolysis and ubiquitination of htt occur in human HD brains. These results suggest that the role of huntingtin proteolysis should be explored in the pathogenic mechanisms of HD.

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Figures

Fig. 1.

Fig. 1.

Domain-specific antibodies of htt. Htt in brain samples was analyzed by Western blots that used monoclonal antibodies recognizing an N-terminal domain (residues 181–810) and a C-terminal domain (residues 2146–2541). Antiserum (rabbit) was also generated against residues 1–17 of htt that recognize the N terminus.

Fig. 2.

Fig. 2.

Proteolysis of huntingtin in cortex: htt fragments detected with N- and C-terminal domain antibodies. a, Brodmann area 4 of cortex from HD and control brains. Tissue homogenates from control (lanes 1, 3) and HD (lanes 2, 4) cortex corresponding to Brodmann area 4 were subjected to Western blots with N-terminal domain (lanes 1, 2) and C-terminal domain (lanes 3, 4) antibodies. Identical amounts of homogenate protein (70 μg) were applied to each lane of the SDS-PAGE gel (4–20% polyacrylamide gradient gel). b, Brodmann area 6 of cortex from HD and control brains. Tissue homogenates from control (lanes 1, 3) and HD (lanes 2, 4) cortex corresponding to Brodmann area 6 were subjected to Western blots with N-terminal domain (lanes 1, 2) and C-terminal domain (lanes 3, 4) antibodies. Identical amounts of homogenate protein (70 μg) were applied to each lane of the SDS-PAGE gel (4–20% polyacrylamide gradient gel).C, Control.

Fig. 3.

Fig. 3.

N-terminal fragments of huntingtin in cortex. Tissue homogenates from Brodmann area 4 (lanes 1, 2) and area 6 (lanes 3, 4) of cortex, from control (lanes 1, 3) and HD (lanes 2, 4) brains, were subjected to Western blots with anti-(1–17) serum that recognizes the N terminal of htt. All lanes of the gel (4–20% polyacrylamide gradient SDS-PAGE gel) contained equal amounts of homogenate protein (70 μg).

Fig. 4.

Fig. 4.

Protease-susceptible domains of huntingtin in cortex. Protease-susceptible domains indicated as A and_B_ illustrate the predicted regions of htt that undergo proteolysis. Proteolysis within the A domain, or within both A and B domains, would generate low-M_r N-terminal fragments and high-M_r C-terminal fragments that are consistent with those detected in cortex by Western blots with N- and C-terminal domain antibodies (N-Ab and_C-Ab, respectively), as well as by anti-(1–17) serum [(1–17)Ab_]. Recognition of each predicted htt fragment (shown by horizontal bars) by the three different antibodies is indicated by antibody-specific_patterns_ that fill the horizontal bars.aa, Amino acids.

Fig. 5.

Fig. 5.

Striatum (putamen): huntingtin fragments detected with antibodies recognizing the N terminus, N-terminal domain, and C-terminal domain of htt. Striatum (putamen) tissue homogenates from control (lanes 1, 3, 5) and HD (lanes 2, 4, 6) brains were analyzed by Western blots with anti-(1–17) serum (lanes 1, 2), N-terminal domain antibody (lanes 3, 4), and C-terminal domain antibody (lanes 5, 6). _Each lane_of the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (18 μg).

Fig. 6.

Fig. 6.

Protease-susceptible domains of huntingtin in striatum. Protease-susceptible domains indicated as _A_and C illustrate the predicted domains of htt proteolysis in striatum (putamen). Proteolysis within both “_A_” and “_C_” domains would generate low-_M_r N-terminal fragments of 20–50 kDa, concomitantly with 35–50 kDa low-_M_r and 100–250 high-M_r C-terminal fragments. These htt fragments are consistent with those detected in striatum (putamen) by Western blots with N-Ab and C-Ab, as well as by (1–17)Ab. Recognition of the predicted htt fragments (indicated by horizontal bars) by each of the three anti-htt antibodies is illustrated by antibody-specific_patterns that fill the horizontal bars.

Fig. 7.

Fig. 7.

Huntingtin fragments in cerebellum. Cerebellum tissue homogenates from control (lanes 1, 3) and HD (lanes 2, 4) brains were analyzed by Western blots with the N-terminal domain antibody (lanes 1, 2) and the C-terminal domain antibody (lanes 3, 4).Each lane of the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (18 μg).

Fig. 8.

Fig. 8.

Analysis of ubiquitination of N-terminal fragments of huntingtin by Western blots. a, Striatum. Homogenate samples of striatum (putamen) from control (lanes 1, 3) and HD (lanes 2, 4) brains were subjected to parallel Western blots with anti-(1–17) serum (lanes 1, 2) and anti-Ub serum (lanes 3, 4). Each lane of the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (18 μg).b, Cortex area 4. Homogenate samples of cortex, from Brodmann area 4, from control (lanes 1, 3) and HD (lanes 2, 4) brains were subjected to parallel Western blots with anti-(1–17) serum (lanes 1, 2) and anti-Ub serum (lanes 3, 4). Each lane of the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (70 μg). c, Cortex area 6. Homogenate samples of cortex, from Brodmann area 6, from control (lanes 1, 3) and HD (lanes 2, 4) brains were subjected to parallel Western blots with anti-(1–17) serum (lanes 1, 2) and anti-Ub serum (lanes 3, 4). Each lane of the gel (12% SDS-PAGE) contained identical amounts of homogenate protein (70 μg).

Fig. 9.

Fig. 9.

PCR amplification of the triplet-repeat domain of the IT15 gene from control and Huntington's disease brain samples. PCR amplification of genomic DNA used primers flanking the triplet-repeat domain of the IT15 gene encoding huntingtin. PCR used DNA isolated from four control and four Huntington's disease brains. Two control samples showed a single band of ∼250 bp generated by PCR (lane 1); two other controls showed lower and upper bands of ∼250 and 300 bp, respectively (lane 2). All four Huntington's samples showed lower and upper DNA bands of ∼250 and 320–330 bp, respectively (lane 3). The lower and upper bands from each PCR reaction were subjected to DNA sequencing to determine the number of CAG repeats (see Table 1).

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