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Предложен молекулярный механизм, объясняющий причину возникновения болезни Альцгеймера (АD) в позднем возрасте (50-80 лет). Cohen, E. et al. в своей работе, опубликованной в журнале Science, показали, что транскрипционные факторы HSF-1 и DAF-16, регулируемые в процессе старения (central ageing pathway), имеют противоположную дисагрегационную и агрегационную активность и осуществляют свои функции совместно для предупреждения АD.

AD, как и многие другие нейродегенеративные заболевания с поздним началом, связаны с токсической аберрантной белковой агрегацией – амилоидный белок-предшественник распадается на A&betha; 1-42 пептиды, которые способны агрегировать. Но почему агрегационно-опосредованная токсичность связана с возрастом, остается неясным.

Авторы изучили вопрос о зависимости задержки начала агрегации у Caenorhabditis elegans от увеличения продолжительности жизни (или замедления старения). Если это так, то позднее начало AD может быть обусловлено детоксифицирующей активностью, которая становится «компромиссной» (подвергается риску) с возрастом. Если это не так, то позднее начало AD можно объяснить стохастическим, связанным со временем, накоплением токсических агрегатов.

Чтобы разделить эти два варианта, авторы нарушили инсулиновый сигнальный путь, который является основным в регуляции старения у червей, мух и млекопитающих. У C. elegans единственный инсулиновый рецептор – DAF-2 - трансдуцирует сигнал, который редуцирует экспрессию генов, регулируемых транскрипционными факторами DAF-16 и HSF-1, приводя к уменьшению продолжительности жизни. Нокдаун daf-2 in C. elegans, которые экспрессировали A&betha; 1-42 показал, что черви с более высокой продолжительностью жизни имели сниженную токсическую агрегацию. Позднее начало агрегации, следовательно, было обусловлено у них «компромиссной» детоксифицирующей активностью, а не случайным накоплением агрегатов. Двойной нокаут daf-2 c daf-16 или с hsf-1 реверсировал этот эффект.

Каким же образом DAF-16 и HSF-1 ингибируют токсичность белковой агрегации? Исследуя количества A&betha; 1-42 агрегатов с высоким молекулярным весом и небольших A&betha; 1-42 агрегатов, авторы обнаружили несколько интересных находок. Во-первых, HSF-1 регулирует дезсагрегацию A&betha; 1-42 агрегатов, а DAF-16 - нет. Но, как оказалось, DAF-16 опосредует формирование высокомолекулярных A&betha; 1-42 агрегатов, но эти агрегаты не коррелируют с токсичностью. И, наконец, было обнаружено, что именно небольшие A&betha; 1-42 агрегаты коррелируют с токсичностью.

Эти данные указывают на механизм, связывающий старение с поздним началом AD. При образовании агрегатов HSF-1 активность опосредует их дезагрегацию. DAF-16 активность поддерживает альтернативный путь (который, вероятно, функционирует как back-up путь), который опосредует формирование низко токсичных, высокомолекулярных агрегатов из высокотоксичных мелких агрегатов. Поскольку оба детоксикационных пути опосредуются инсулиновым сигнальным путем, то оба могут стать компромиссными (подвергаться риску) при старении, приводя к накоплению агрегатов.

Представляет интерес и то, что инсулиновый сигнальный путь также ассоциируется с формированием других токсических агрегатов, таких, например, как при болезни Гентингтона. Дальнейшие исследования в этой области позволят выявить терапевтические мишени для того, чтобы предупредить развитие нейродегенеративных заболеваний с поздним образование агрегатов.

Fig.1 IIS regulates proteotoxicity of A&betha;1-42 expressed in C. elegans. (A) daf-2 RNAi extends life span of A?1-42 worms. A&betha;1-42 worms were grown on bacteria expressing either EV or daf-2 RNAi during development and adulthood. daf-2 RNAi worms lived significantly longer, P 0.0001 [squares, mean life span (LS) = 28.6 days], than their EV-grown counterparts (triangles, mean LS = 17.8 days). (B) daf-2 RNAi reduces A&betha;1-42-mediated toxicity. A&betha;1-42 worms were grown as in (A). Numbers of paralyzed worms were scored daily for 12 days of adulthood. daf-2 RNAi (squares) reduced the number of paralyzed worms compared with the EV (triangles). (C) Both daf-16 and hsf-1 RNAi abolished the protective effect of daf-2 RNAi toward A?1-42 toxicity. A&betha;1-42 worms were grown during development and adulthood on EV bacteria (triangles) or on dilutions of equal amounts of bacteria expressing the following RNAi species: daf-2 and EV (solidsquares), daf-2 and daf-16 (open squares), or daf-2 and hsf-1 (diamonds). (D) daf-16 or hsf-1RNAi during adulthood results in an elevated rate of paralysis late in life. A&betha;1-42 worms were developed on EV and were transferred at day 1 of adulthood to bacteria expressing either EV (triangles), daf-2 RNAi (solid squares), daf-16 RNAi (open squares), or hsf-1 RNAi (diamonds). daf-2 RNAi reduced the number of paralyzed animals, whereas both daf-16 and hsf-1 RNAi increased the number of paralyzed worms late in life compared with the EV. (E) Reduced expression of hsf-1 during development (dev) and adulthood (ad) further accelerates the rate of paralysis. A&betha;1-42 worms were grown during development and adulthood on bacteria expressing either EV (triangles), daf-2 RNAi (squares) or hsf-1 RNAi (open diamonds) or were developed on EV and transferred to hsf-1 RNAi bacteria on day 1 of adulthood (solid diamonds). Error bars indicate standard deviations.

Fig.2. Lack of correlation between Ahsf-1 is required for efficient disaggregation of A&betha;1-42 aggregates. (A) Pre-aggregated, ThT-labeled A&betha;1-40 fibrils were incubated with either buffer (green), A?1-42 worm PDS (black), or heat-inactivated PDS (red) in the presence of epoxomicin (10 µM). ThT fluorescence emission declined in the presence of worm PDS, indicating disaggregation activity. The A&betha;1-40 fibrils were stable in both buffer and heat-inactivated (HI) PDS. (B) Pre- and postdisaggregation samples were loaded onto 10% PAA gel. A?1-40 was visualized by WB using 6E10 before the reaction (lane 1) and after 96 hours in the presence of worm PDS (lane 2) or in the presence of heat-inactivated PDS (lane 3). Less A&betha;1-40 was observed after incubation with worm PDS but not after incubation with heat-inactivated PDS. No proteasome inhibitors were used in this experiment. (C) Disaggregation reaction was performed in the absence and the presence of a protease inhibitor cocktail (PI) (lanes 2 and 3, respectively). WB analysis indicated that in the presence of PI, the total quantity of A&betha;1-40 did not change compared to the buffer-incubated fibrils, despite the disaggregation. (D) In vitro aggregated A&betha;1-40 fibrils were visualized by using AFM with no treatment (i), after a 36-hour incubation with EV-grown A?1-42 worm PDS (ii), and after a 36-hour incubation with buffer only (iii). No large fibrils were detected after incubation with worm PDS. All large horizontal bars represent 1 µm; inset bar, 200 nm; and height scale bar, 20 nm. (E) Worm disaggregation activity reduces the A&betha;1-40 fibril-mediated cytotoxicity in cell-based assays. By using the disaggregation assay and conditions as in (B) and 72-hour incubation, we added A&betha;1-40 disaggregation samples (500 nM) to PC12 cell culture medium for 3 days. Cell viability was assayed by MTT metabolic activity . A?1-40 toxicity was reduced in samples incubated with worm PDS in the presence (green) or absence (blue) of epoxomicin (10 µM). Samples incubated without worm PDS (red) showed similar toxicity to the starting material (purple). Monomeric A&betha;1-40 peptide did not exhibit toxicity under the assay conditions (black). Similar results were found with the use of a resazurin-based assay (fig. S12). (F) hsf-1 is required for efficient disaggregation of pre-formed A&betha;1-40 fibrils. RNAi of A&betha;1-42 worms as in Fig. 2A. PDS of hsf-1 RNAi worms (black) exhibited 20 to 50% decline in disaggregation activity compared with PDS of EV worms (green). There was no significant change in disaggregation activities in PDS of daf-2 (blue) or daf-16 (red) RNAi worms. (Inset) Statistical analysis of disaggregation results shown in (F) indicate that EV and hsf-1 RNAi worms are significantly (P < 0.03) different (n = 3).

Fig.3. hsf-1 is required for efficient disaggregation of A&betha;1-42 aggregates. (A) Pre-aggregated, ThT-labeled A&betha;1-40 fibrils were incubated with either buffer (green), A&betha;1-42 worm PDS (black), or heat-inactivated PDS (red) in the presence of epoxomicin (10 µM). ThT fluorescence emission declined in the presence of worm PDS, indicating disaggregation activity. The A&betha;1-40 fibrils were stable in both buffer and heat-inactivated (HI) PDS. (B) Pre- and postdisaggregation samples were loaded onto 10% PAA gel. A&betha;1-40 was visualized by WB using 6E10 before the reaction (lane 1) and after 96 hours in the presence of worm PDS (lane 2) or in the presence of heat-inactivated PDS (lane 3). Less A&betha;1-40 was observed after incubation with worm PDS but not after incubation with heat-inactivated PDS. No proteasome inhibitors were used in this experiment. (C) Disaggregation reaction was performed in the absence and the presence of a protease inhibitor cocktail (PI) (lanes 2 and 3, respectively). WB analysis indicated that in the presence of PI, the total quantity of A&betha;1-40 did not change compared to the buffer-incubated fibrils, despite the disaggregation. (D) In vitro aggregated A&betha;1-40 fibrils were visualized by using AFM with no treatment (i), after a 36-hour incubation with EV-grown A&betha;1-42 worm PDS (ii), and after a 36-hour incubation with buffer only (iii). No large fibrils were detected after incubation with worm PDS. All large horizontal bars represent 1 µm; inset bar, 200 nm; and height scale bar, 20 nm. (E) Worm disaggregation activity reduces the A&betha;1-40 fibril-mediated cytotoxicity in cell-based assays. By using the disaggregation assay and conditions as in (B) and 72-hour incubation, we added A?1-40 disaggregation samples (500 nM) to PC12 cell culture medium for 3 days. Cell viability was assayed by MTT metabolic activity . A?1-40 toxicity was reduced in samples incubated with worm PDS in the presence (green) or absence (blue) of epoxomicin (10 µM). Samples incubated without worm PDS (red) showed similar toxicity to the starting material (purple). Monomeric A&betha;1-40 peptide did not exhibit toxicity under the assay conditions (black). Similar results were found with the use of a resazurin-based assay (fig. S12). (F) hsf-1 is required for efficient disaggregation of pre-formed A&betha;1-40 fibrils. RNAi of A&betha;1-42 worms as in Fig. 2A. PDS of hsf-1 RNAi worms (black) exhibited 20 to 50% decline in disaggregation activity compared with PDS of EV worms (green). There was no significant change in disaggregation activities in PDS of daf-2 (blue) or daf-16 (red) RNAi worms. (Inset) Statistical analysis of disaggregation results shown in (F) indicate that EV and hsf-1 RNAi worms are significantly (P < 0.03) different (n = 3

Fig.4. Intensity of an A? immunoreactive 16-kD band correlates with toxicity. (A) Worm PDS supernatants and pellets were prepared as in Fig. 2A, except the PDS was incubated for 10 min on ice in 1% sarkosyl to maintain more proteins in their membrane-associated state. 16-kD A&betha; bands were detected in all pellets (lanes 2, 4, 6 and 8, solid arrow). The most intense 16-kD bands were seen in the pellet of daf-16 and hsf-1 RNAi worms (lanes 6 and 8, respectively); the least amount was found in the pellets of daf-2 RNAi worms (lane 4). A&betha; monomers could be seen in supernatants of EV and daf-2 RNAi worms (lanes 1 and 3, respectively) but not in supernatants of daf-16 or hsf-1 RNAi worms (lanes 5 and 7, respectively). (B) High resolution IF microscopy using 4G8 A&betha; mAb indicates that muscles are labeled in hsf-1 RNAi A&betha;1-42 worms (image ii, arrows) but not in wild-type worms (image i). Small A&betha;1-42 aggregates were detected along the muscular fibers of worms grown on hsf-1.

Fig.5. Model of age-regulated HSF-1 and DAF-16 opposing anti-proteotoxicity activities. Aggregation-prone peptides spontaneously form small toxic aggregates (stage 5-I). Specialized cellular machinery identifies toxic aggregates and rapidly disaggregates and prepares them for degradation (stage 5-II). The products of this machinery are rapidly degraded (stage 5-V). This preferred mechanism is positively regulated by HSF-1 (stage 5-A) and negatively regulated by DAF-2 (stage 5-C). (Stage 5-III) When the HSF-1–regulated disaggregation machinery is overloaded, a secondary machinery that mediates aggregation is activated (stage 5-III), forming less-toxic high-MW aggregates. This machinery is positively regulated by DAF-16 (stage 5-B) and negatively by DAF-2 (stage 5-D). The high-MW aggregates, which accumulate as a result of the DAF-16–regulated mechanism, undergo either slow disaggregation and degradation by the HSF-1–regulated mechanism (stages 5-IV and 5V) or possibly secretion to the extracellular matrix (5-VI).

См. методы и рисунки в Supplement