Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C

Abstract

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-β-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.

Keywords: Niemann-Pick disease type C; autophagosome; axonal dystrophy; axonal transport; cholesterol; kinesin; lipid; lysosomal storage disorder; lysosome; neurodegeneration.

Figure 1.. Aberrant AV Accumulation in Axons of Npc1^−/− Mice

(A, B) TEM images showing AV accumulation in DRG (A) and cortical (B) axons in Npc1^−/− mice at symptomatic stages (P50–60). Such accumulations were not readily observed in age-matched WT mice. Colored boxes represent four-fold higher magnification of corresponding regions in upper panels (green: WT; orange: Npc1^−/−). (C, D) Images (C) and analysis (D) showing a robust increase in AV density in DRG axons isolated from presymptomatic Npc1^−/− mice (P30–40) compared to WT neurons at the same age. (E, F) Kymographs (E) and analysis (F) showing no significant difference in the motility of axonal AVs between WT and Npc1^−/− neurons. See also Videos S1 and S2. DRG neurons isolated from presymptomatic Npc1^−/− mice (P30–40) were nucleofected with EGFP-LC3 at DIV0, followed by live imaging at DIV3–4. Data were collected from the total number of axons indicated above bars (D) or vesicles (v) from the number of axons (n) indicated in parentheses (F), presented as mean ± SEM with dots representing individual axons, and analyzed by Student’s t test. Scale bars: 800 nm (A, B), 10 μm (C, E). See also Figure S1.

Figure 2.. Reduced Axonal Density of Lysosomes in Npc1^−/− Neurons

(A-D) Images (A, C) and analyses (B, D) showing reduced density of active GCase-labeled degradative lysosomes and LAMP1-labeled endolysosomes in adult DRG neuron axons isolated from presymptomatic Npc1^−/− mice (P30–40). DRG neurons were incubated with MDW933 (500 nM) for 1 hour to label active GCase, followed by immunostaining for β3-tubulin (A, B), or co-immunostained for LAMP1 and β3-tubulin (C, D). (E-H) Images (E, G) and analyses (F, H) showing reduced density of degradative lysosomes and endolysosomes in axons of Npc1^−/− cortical neurons plated in microfluidic devices. At DIV8, neurons were loaded with MDW933 (500 nM) for 1 hour to label active GCase, followed by β3-tubulin staining (E, F), or co-immunostained for LAMP1 and β3-tubulin (G, H). (I-L) Images (I, K) and analyses (J, L) showing lysosomal accumulation in the soma of Npc1^−/− cortical neurons. Neurons at DIV8 were incubated with MDW933 to label degradative lysosomes followed by MAP2 immunostaining, or co-immunostained for MAP2 and LAMP1 to label endolysosomes (I, J), or incubated with BODIPY-FL-pepstatin A to label active cathepsin D prior to live imaging (K, L). Integrated density was measured in thresholded images using ImageJ, and data are presented as mean integrated density normalized to WT neurons. (M, N) Immunoblots (M) and quantitative analysis (N) of LAMP1, GCase, and cathepsins B and D (CTSB and CTSD) in WT and Npc1^−/− cortical neurons at DIV7–8. Equal amounts of neuronal lysates (10 μg) were sequentially immunoblotted. Protein intensities of LAMP1, GCase, and both mature and immature forms of CTSB and CTSD were averaged from three repeats and normalized to WT. Data were collected from the total number of axons (B, D, F, H) or neurons (J, L) indicated below or within bars, presented as mean ± SEM with dots representing individual values, and analyzed by Mann-Whitney test (B) or Student’s t test (D, F, H, J, L, N). Scale bars: 10 μm. See also Figure S2.

Figure 3.. Impaired Axonal Delivery of Degradative Lysosomes in Npc1^−/− Neurons

(A) Schematic diagram of soma-restricted labeling of degradative lysosomes and their delivery to distal axons. Neurons are plated in the soma/dendritic chamber (1) of a microfluidic device that provides physical and fluidic separation of axons from cell bodies and dendrites. Only axons that enter the 450-μm long microgrooves (2) are able to grow into the axon chamber (3). The soma/dendritic chamber is loaded with an activity-based lysosome probe for 30 minutes, followed by washes with imaging buffer and live imaging carried out in the axon chamber for 90 minutes. (B-E) Images (B, D) and analyses (C, E) showing impaired delivery of active GCase- and cathepsin D (CTSD)-labeled degradative lysosomes from the soma chamber to distal axons in Npc1^−/− cortical neurons. At DIV7 and DIV10, soma-restricted labeling of degradative lysosomes was carried out by loading MDW941 (100 nM) (B) or BODIPY-FL-pepstatin A (1 μM) (D) for 30 minutes, followed by washes and live imaging in the axon chamber for 90 minutes. Data were quantified from the number of axons indicated in bars and normalized to WT. (F-J) Kymographs (F, H) and analyses (G, I, J) showing impaired anterograde motility of degradative lysosomes in distal axonal segments of Npc1^−/− cortical neurons. Neurons plated in microfluidic devices were loaded with MDW941 (100 nM) (F, G) or BODIPY-FL-pepstatin A (1 μM) (H, I) for 30 minutes prior to live imaging at DIV8. Time-lapse images were collected every 2 seconds for 90 frames totaling 3 minutes. The percentage of anterograde (antero), retrograde (retro), or stationary (stat) lysosomes was quantified from the total number of vesicles (v) in the total number of axons (n) as follows: (G) WT: n=28 v=344, Npc1^−/−: n=29 v=226; (I) WT: n=36 v=433, Npc1^−/−: n=35 v=271. Active lysosome motility parameters including flux rate, speed, and the velocity in both directions were analyzed in axons of WT and Npc1^−/− cortical neurons (J). See also Videos S3 and S4. Data were analyzed by Student’s t test (C, E, I, J) or Mann-Whitney test (G) and presented as mean ± SEM alone (C, E) or with dots representing individual values (G, I, J). Scale bars: 10 μm. See also Figure S3, Videos S5 and S6.

Figure 4.. Aberrant Sequestration of Kinesin-1 and Arl8 on Lysosome Membranes in Npc1^−/− Neurons

(A-F) STED super-resolution images (A, C, E) and analyses (B, D, F) showing aberrant sequestration of endogenous kinesin-1 and its adaptor Arl8 on the surface of Npc1^−/− lysosome membranes in the soma. Enlarged views of the corresponding boxed regions are shown in (A’), (C’), and (E’). The percentage of LAMP1-labeled areas associated with Arl8 (B), kinesin-1 (D), or SKIP (F) were quantified and normalized to WT. Note that Arl8 and kinesin-1, but not SKIP, display enhanced distribution on the surface of Npc1^−/− lysosomes. (G-J) STED images (G, I) and analyses (H, J) showing non-specific recruitment of truncated KHC-MD (motor domain, G) and KHC-CBD (cargo-binding domain, I) to Npc1^−/− lysosome membranes. WT and Npc1^−/− cortical neurons were transduced with GFP-tagged KHC-MD or KHC-CBD at DIV4, followed by co-immunostaining for GFP and LAMP1 at DIV8. Enlarged views of the corresponding boxed regions are shown in (G’) and (I’). The percentage of LAMP1-labeled areas associated with KHC-MD (H) or KHC-CBD (J) signals were quantified and normalized to WT. Data were collected from the total number of neurons indicated within bars (B, D, F, H, J), presented as mean ± SEM with dots representing individual neurons, and analyzed by Mann-Whitney test. Scale bars: 5 μm.

Figure 5.. Aberrant Accumulation of Kinesin-1 and Arl8 on Npc1^−/− Lysosome Membranes Is Independent of Arl8 GTPase Activity

(A-D) STED images (A, C) and analyses (B, D) showing kinesin-1 sequestration on Npc1^−/− lysosome membranes independent of Arl8 and SKIP. Npc1^−/− cortical neurons at DIV4 were transfected with control scrambled (scr), Arl8-siRNA (A, B), or SKIP-siRNA (C, D), followed by co-immunostaining for LAMP1 and kinesin-1 at DIV8. Pseudocolors were applied: magenta for LAMP1; cyan for kinesin-1. Enlarged views of the boxed regions are shown in (A’) and (C’). The percentage of LAMP1-labeled areas associated with kinesin-1 signals in Npc1^−/− cortical neurons transfected with Arl8-siRNA or SKIP-siRNA were quantified and normalized to Npc1^−/− neurons transfected with scr-siRNA. See also Figures S4A–S4D. (E, F) STED images (E) and analyses (F) showing that kinesin-1 and Arl8 sequestration is not affected by Arl8 GTPase activity in Npc1^−/− neurons. Npc1^−/− cortical neurons were transfected with WT, GTP-locked, or GDP-locked mutant forms of Arl8b-mCh at DIV4, followed by immunostaining for LAMP1, mCherry, and kinesin-1 at DIV8. Pseudocolors were applied. Enlarged views of the boxed regions are shown in (E’). The percentage of LAMP1-labeled areas associated with kinesin-1 or mCherry-tagged Arl8b signals in Npc1^−/− cortical neurons expressing Arl8b mutants were quantified and normalized to Npc1^−/− neurons expressing WT Arl8b. (G, H) Co-immunoprecipitation (G) and analysis (H) showing reduced interaction of Arl8 and SKIP in Npc1^−/− mouse brain homogenates from E18 embryos. Protein band intensities were quantified using ImageJ and normalized to WT. Data were averaged from three repeats and analyzed by Student’s t test. Data were collected from the total number of neurons indicated within bars (B, D, F), presented as mean ± SEM with dots representing individual neurons, and analyzed by Mann-Whitney test (B, D) or one-way ANOVA, where p values represent comparisons of each condition against WT Arl8b (F). Scale bars: 5 μm.

Figure 6.. Reducing Cholesterol Releases Sequestration of Kinesin-1 and Arl8 from Npc1^−/− Lysosome Membranes

(A-C) Images (A) and analyses (B, C) showing elevated membrane and luminal cholesterol in lysosomes of Npc1^−/− neurons at DIV7–8. Colocalization between D4H* and LAMP1 was measured and expressed as the fraction of LAMP1-labeled organelles that were also labeled by D4H* and normalized to Npc1^−/−. To quantify luminal cholesterol levels, the integrated density of filipin was measured in thresholded images and normalized to Npc1^−/−. See also Figures S4E–S4H. (D-F) Images (D) and analyses (E, F) showing reduced cholesterol on endolysosome membranes and the lumen of Npc1^−/− neurons at DIV7–8 following HPCD treatment. (G, H) STED images showing release of sequestrated Arl8 (G) and kinesin-1 (H) from Npc1^−/− endolysosomal membranes by reducing lysosomal cholesterol with HPCD. Pseudocolors were applied: blue for GST-D4H*-mCherry; red for Arl8 (G) or kinesin-1 (H). The edges of cell bodies and proximal processes are outlined with white dashed lines. Enlarged views of the boxed regions are shown in (G’) and (H’). (I, J) Sequential immunoblots (I) and quantitative analysis (J) showing cholesterol-dependent sequestration of kinesin-1 and Arl8 on Npc1^−/− neuronal lysosomes. Cortical neurons were treated with HPCD (100 μM) or H₂O control for 48 hours, followed by magnetic isolation of lysosomes at DIV8. Equal amounts of whole cell lysates (5 μg) and captured endolysosomes (0.8 μg) were loaded and sequentially immunoblotted with antibodies as indicated. Protein band intensities were quantified and averaged from three repeats, and data were normalized to H₂O-treated WT neurons. Data were collected from the total number of neurons indicated below bars (B, C, E, F), presented as mean ± SEM with dots representing individual images (B, E) or neurons (C, F), and analyzed by Mann-Whitney test (B, C), Student’s t test (E, F) or one-way ANOVA (J). ** p < 0.01. Scale bars: 10 μm (A, D), 2 μm (G, H).

Figure 7.. Cholesterol Reduction and Arl8b Expression Rescue Lysosome Transport into Axons of Npc1^−/− Neurons

(A, B) Images (A) and analysis (B) showing rescued axonal lysosome density in presymptomatic Npc1^−/− DRG neurons by reducing membrane cholesterol with HPCD treatment. (C, D) Images (C) and analysis (D) showing enhanced delivery of soma-labeled degradative lysosome to distal axons following HPCD treatment in Npc1^−/− cortical neurons cultured in microfluidic devices. At DIV8, MDW933 (500 nM) was loaded to the soma/dendritic chamber for 15 minutes. Neurons were then washed and fixed after 0, 1, 2, or 3 hours, followed by immunostaining for β3-tubulin. Axonal terminals are outlined with white dashed lines. See also Videos S7 and S8. (E, F) Images (E) and analyses (F) showing reduced density of axonal AVs and increased density of lysosomes with HPCD treatment in DRG axons from P30–40 Npc1^−/− mice. (G, H) Images (G) and analysis (H) showing rescued axonal lysosome density with elevated Arl8b expression in DRG axons from P30–40 Npc1^−/− mice. (I, J) Images (I) and analysis (J) showing reduced axonal autophagic stress with elevated Arl8b expression in DRG neurons from presymptomatic Npc1^−/− mice at P30–40. (K, L) Images (K) and analysis (L) showing ameliorated Npc1^−/− neuron death following Arl8b overexpression. Cortical neurons isolated from presymptomatic mice (P30–40) were transduced at DIV0 with Arl8b-mCh or mCh. TUNEL assays were performed at DIV7 and DIV10, and the percentage of TUNEL-positive cells to total DAPI staining was calculated in thresholded images (705 × 705 μm) using ImageJ. Data were collected from the total number of axons (B, F, H, J) or images (L) as indicated, or from > 30 axon terminals for each time point (D), presented as mean ± SEM with dots representing individual axons, and analyzed by one-way ANOVA (B, H, L) or Student’s t test (D, F, J). ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Scale bars: 10 μm (A, E, G, I), 5 μm (C), and 50 μm (K). See also Figures S5 and S6.