Huntingtin expression stimulates endosomal-lysosomal activity, endosome tubulation, and autophagy

Abstract

An expansion of polyglutamines in the N terminus of huntingtin causes Huntington's disease (HD) and results in the accrual of mutant protein in the nucleus and cytoplasm of affected neurons. How mutant huntingtin causes neurons to die is unclear, but some recent observations suggest that an autophagic process may occur. We showed previously that huntingtin markedly accumulates in endosomal-lysosomal organelles of affected HD neurons and, when exogenously expressed in clonal striatal neurons, huntingtin appears in cytoplasmic vacuoles causing cells to shrink. Here we show that the huntingtin-enriched cytoplasmic vacuoles formed in vitro internalized the lysosomal enzyme cathepsin D in proportion to the polyglutamine-length in huntingtin. Huntingtin-labeled vacuoles displayed the ultrastructural features of early and late autophagosomes (autolysosomes), had little or no overlap with ubiquitin, proteasome, and heat shock protein 70/heat shock cognate 70 immunoreactivities, and altered the arrangement of Golgi membranes, mitochondria, and nuclear membranes. Neurons with excess cytoplasmic huntingtin also exhibited increased tubulation of endosomal membranes. Exogenously expressed human full-length wild-type and mutant huntingtin codistributed with endogenous mouse huntingtin in soluble and membrane fractions, whereas human N-terminal huntingtin products were found only in membrane fractions that contained lysosomal organelles. We speculate that mutant huntingtin accumulation in HD activates the endosomal-lysosomal system, which contributes to huntingtin proteolysis and to an autophagic process of cell death.

Fig. 1.

Overexpressed truncated FLAG-huntingtin in transfected clonal striatal cells, detected with a FLAG antibody, is localized in the cytoplasm in a meshwork of fine tubules (arrows) and punctate structures (a), in dispersed vacuoles throughout cell bodies (b, c) and neurites (c,arrows), and in vacuoles coalesced in the perinuclear region (d, e, arrow). Full-length FLAG-tagged huntingtin with a normal (f) or expanded (g) repeat also produce FLAG-positive vacuoles (arrows). Vacuolar staining can be obtained using huntingtin antibody Ab585 that recognizes an internal epitope of huntingtin (h,arrow). Huntingtin antibody Ab1 made to the N terminus detects vacuoles in cells expressing untagged

Fig. 2.

FLAG-huntingtin-immunoreactive vacuoles contain cathepsin D, and accumulation of cathepsin D is polyglutamine length-dependent. Confocal immunofluorescence microscopy of cells transfected with FH₃₂₂₁-18 or FH₃₂₂₁-100 then double immunostained after 24 hr for cathepsin D (green) and FLAG (red). Top three rows show intact cells, and bottom row shows a cell fragment. Dispersed vacuoles are in the cell in top row. Condensed vacuoles appear in the cells in the two middle rows. Note that FLAG-huntingtin immunoreactivity is present mainly along the periphery of the vacuole, and cathepsin D is inside the vacuole (arrows). The intensity of cathepsin D labeling in FLAG-immunoreactive vacuoles increases with polyglutamine expansion (compare two middle rows). Merged images onright show cathepsin D in green, FLAG inred, and the overlap in yellow.n, Nucleus.

Fig. 3.

Distribution of Golgi labeling (A) and HSP70/HSC70 (B) in clonal striatal cells expressing exogenous huntingtin.A, Confocal immunofluorescence microscopy of cells transfected with FH₃₂₂₁-100 and double immunostained after 20 hr for the Golgi marker GM130 (red) and for huntingtin with Ab1 (green). A cell with diffuse huntingtin expression but lacking vacuoles shows perinuclear position of the Golgi (top row). Cells containing huntingtin-positive vacuoles show the Golgi displaced from the perinuclear region (middle row) or loss of immunoreactivity for GM130 (bottom row,arrows). Cells with reduced Golgi staining were imaged at the cross-sectional plane containing the highest level of GM130 immunoreactivity. B, HSP70/HSC70 localization in cells expressing FH₃₂₂₁-100 and double-labeled after 24 hr for HSP70/HSC70 (red) and for huntingtin with Ab1 (green). HSP70/HSC70 immunoreactivity is not enriched in huntingtin-positive vacuoles (top row). The punctate cytoplasmic staining for HSP70/HSC70, which may be mitochondria, is the same in huntingtin-positive and huntingtin-negative cells. One cell with a large huntingtin-positive vacuole shows increased HSP70/HSC70 immunoreactivity in the cytoplasm surrounding the vacuole (arrows), which may be within clustered mitochondria, but is not within the vacuole (bottom row). Merged images on right show GM130 (A) or HSP70/HSC70 (B) inred, huntingtin in green, and the overlap in yellow. n, Nucleus.

Fig. 4.

Ultrastructure of transfected clonal striatal cells. Transiently transfected cells were immunostained for FLAG and processed using the immunoperoxidase method. a, Electron micrograph of a typical control cell fixed 20 hr after transfection with the expression vector alone shows an unindented nucleus and normal distribution of organelles with no immunoperoxidase label. Electron micrographs from FLAG-positive cells are shown in b–f. Cells were fixed and immunostained 20 hr after transfection with FH₃₂₂₁-100 (b), FH₃₂₂₁-46 (d–f), and FH₃₂₂₁-18 (c). b, Intense immunoreactivity is present throughout the cytoplasm. Large lysosome-like bodies are present in the cytoplasm (open arrow) and outside the cell (long filled arrow). The nucleus is unlabeled and indented (short filled arrow). c, Peripheral portion of a cell body contains intense immunoperoxidase staining and two early autophagic vacuoles (av), which have peroxidase labeling on the double-limiting membranes (arrowheads). The nearby mitochondria (m) is unlabeled, and its inner membranes are disrupted. d, Cell with indented nucleus (short large arrow), numerous early autophagic vacuoles (small arrows), and late autophagic vacuoles (long arrows). This cell had only sparse immunoperoxidase label remaining in scattered regions of the cytoplasm. e, Two late autophagic vacuoles from the boxed region ind are filled with electron-dense vesicles.f, A vacuole containing a membrane whorl (also called myelin body or fingerprint profile) within an immunoperoxidase-labeled cell. Electron-dense tubulovesicular structures are present within the vacuole. Scale bars: a, b,d, 2 μm; c, f, 0.2 μm;e, 0.5 μm.

Fig. 5.

Immunogold labeling of FLAG-huntingtin in clonal striatal cells. Cells were transfected with FH₃₂₂₁-46 and then fixed and stained after 20 hr (a, b,d, f, g, h) or transfected with FH₃₂₂₁-100 and then fixed and stained after 24 hr (c, e, i).a, Gold deposits appear within autophagosomes (top arrows) and in the nearby cytoplasm (bottomright arrow). b, Lysosome-like bodies with radiating filaments (arrowheads) are enveloped with cisternae and limiting membranes (long arrows). Gold particles are scattered within the filamentous matrix of the organelle (short arrows). Mitochondria with disrupted cisternae are to theleft of the organelle. c, Extracellular early autophagosomes with double membranes show gold particles in the lumen (long arrows) and along the cytoplasmic face of the outer limiting membrane (short arrows).d, Gold label (arrows) appears along the cell surface and at clathrin-coated vesicles and invaginations.e, Gold deposits (arrows) appear on the limiting membrane and within the lumen of an early endosomal–multivesicular organelle. f, Tubulovesicular network composed of electron-dense vesicles and tubules appears in the perinuclear region. Nucleus is indented (arrow).g, Higher magnification of boxed region in f. Immunogold is present in the cytoplasm (short arrows), and electron-dense segments of tubules are clearly visible (open arrow). h,i, Gold particles (arrows) are attached or near electron-dense tubulovesicular organelles. h, A tubulovesicular body in the peripheral cytoplasm. i, A tubulovesicular body with ballooned segment located in the extracellular space. Gold particle is on tubule (arrow). Note that some tubules are electron-lucent and electron-dense, whereas the vesicles are electron-dense. Scale bars: a–e,h, i, 0.2 μm; f, 2 μm,g, 0.5 μm.

Fig. 6.

Biochemical analysis of subcellular fractions from cells expressing full-length mutant huntingtin or truncated mutant huntingtin. a, Scheme for subcellular fractionation using differential centrifugation with expected distribution of organelles. CH, Crude homogenate; S1, 2000 × g supernatant; P1, 2000 × g pellet; S2, 100,000 ×g supernatant; P2, 100,000 ×g pellet. b, Western blot shown at different exposures was probed with anti-huntingtin antisera Ab1. Protein fractions were from cells expressing FH₉₇₇₄-100. Cells were collected 24 hr after transfection. Twenty-five micrograms of protein were loaded per lane. Mutant huntingtin (Human) appears slightly above the normal endogenous (Mouse) protein in P1, S2, and P2. Bottom panels show enlargements of the two full-length bands from S2, P1 (longer exposure), and P2. N-terminal fragments are evident at longer exposures. The 90 kDa fragment derived from overexpressed mutant huntingtin is present in P1 and P2 but not S2, and abundantly in cellular debris recovered from the growth medium and washes that were pooled, centrifuged, and resuspended in 100 μl of homogenization buffer, 3 μg loaded. The stacking gel was included in the transfer to nitrocellulose and shows no protein. Apparent molecular weight is indicated in kilodaltons. c, Analysis of truncated huntingtin and its N-terminal huntingtin fragments from cells transfected with plasmid encoding FLAG only (MOCK) or transfected with FH₃₂₂₁-100. Cells were collected 24 hr after transfection. Fifteen micrograms of protein were loaded per lane. The identity and purity of the fractions was assessed with various markers: calnexin for the ER, GM130 for the cis/medial Golgi, transferrin receptor for endosomal–recycling compartments, and cathepsin D for lysosomes. β-tubulin was used to detect the presence of cytosolic constituents. Proteasome and HSP70 distribution are also shown. Apparent molecular weight is indicated in kilodaltons on the left. In blots probed with Ab1, endogenous full-length huntingtin (Mouse) is at the top, and a cleaved endogenous huntingtin fragment migrates at 50 kDa (Mouse). The expressed truncated huntingtin protein runs at the expected size of ∼140 kDa (top right arrow,Human) and as a modified form at ∼175 kDa. The 140 and 175 kDa proteins are present in S1, P1, S2, and P2, whereas the 90 kDa N-terminal product (bottom right arrow,Human) is present in P1 and P2 but not S2.

Fig. 7.

Biochemical analysis of P1 fraction and α-chymotrypsin digestion from cells transfected with FH₃₂₂₁-100. a, Scheme of nuclear isolation. The low speed pellet (P1) was fractionated on a discontinuous iodixanol density gradient as described in Methods and Materials. Fractions are as follows: CH, crude homogenate; S1, 2000 × gsupernatant; P1, 2000 × g pellet; fraction 1, 25% iodixanol step; fraction 2, 30% iodixanol step; fraction 3, 30%/35% interface; fraction 4, 35% iodixanol step. b, Western blots of fractions probed with anti-huntingtin antisera Ab1. Twenty microliters from each fraction was loaded per lane. Nuclear fraction (fraction 3) was identified using histone as a marker and contained the 140 kDa truncated huntingtin but not the N-terminal 90 kDa fragment. Apparent molecular weight is indicated in kilodaltons on theleft. c, Western blots of equal volumes of crude homogenates (CH). The crude homogenate was treated with varied amounts of α-chymotrypsin for 30 min on ice, stopped with 2 μl of 200 mm PMSF, and then analyzed by Western blot.

Fig. 8.

Electron microscopy of P1 pellet. Clonal striatal cells were transfected with FH₃₂₂₁-100. The P1 pellet was prepared 24 hr later and processed for electron microscopy.a, An intact nucleus is isolated with groups of lysosome-like bodies. b, Examples of lysosome-like dense bodies surrounded by electron-dense tubulovesicular structures and ER membranes (right, arrow). The lysosome-like dense bodies were absent from the P1 pellets of mock-transfected cells. Scale bars: a, 2 μm;b, 0.5 μm.