Primary fibroblasts from CSPα mutation carriers recapitulate hallmarks of the adult onset neuronal ceroid lipofuscinosis

Abstract

Mutations in the co- chaperone protein, CSPα, cause an autosomal dominant, adult-neuronal ceroid lipofuscinosis (AD-ANCL). The current understanding of CSPα function exclusively at the synapse fails to explain the autophagy-lysosome pathway (ALP) dysfunction in cells from AD-ANCL patients. Here, we demonstrate unexpectedly that primary dermal fibroblasts from pre-symptomatic mutation carriers recapitulate in vitro features found in the brains of AD-ANCL patients including auto-fluorescent storage material (AFSM) accumulation, CSPα aggregates, increased levels of lysosomal proteins and lysosome enzyme activities. AFSM accumulation correlates with CSPα aggregation and both are susceptible to pharmacological modulation of ALP function. In addition, we demonstrate that endogenous CSPα is present in the lysosome-enriched fractions and co-localizes with lysosome markers in soma, neurites and synaptic boutons. Overexpression of CSPα wild-type (WT) decreases lysotracker signal, secreted lysosomal enzymes and SNAP23-mediated lysosome exocytosis. CSPα WT, mutant and aggregated CSPα are degraded mainly by the ALP but this disease-causing mutation exhibits a faster rate of degradation. Co-expression of both WT and mutant CSPα cause a block in the fusion of autophagosomes/lysosomes. Our data suggest that aggregation-dependent perturbation of ALP function is a relevant pathogenic mechanism for AD-ANCL and supports the use of AFSM or CSPα aggregation as biomarkers for drug screening purposes.

Figure 1

Lysosome dysfunction in AD-ANCL in vivo and in vitro. (A) Representative images of AFSM in cortical pyramidal neurons from an AD-ANCL patient (right panel) and control (left panel). (B) Graph shows the lysosomal enzyme activities of PPT-1, β-gluc and β-Hexa measured in the parietal lobe and occipital lobe from three AD-ANCL patients compared to the same brain regions from three controls. Enzymatic activity was normalized to the total protein and pooled by genotype. (Mean ± SEM of quantification by triplicate in each individual). **p ≤ 0.01; ***p ≤ 0.001. (C) Representative Western blots showing the expression of LAMP-1, LAMP-2 and V-ATPase B1/2 in occipital lobe from three controls and three AD-ANCL patients. Transmembrane proteins are normalized to Flotillin. The histogram shows the quantification of LAMP-1, LAMP-2 and V-ATPase B1/2 detected by immunoblot relative to control levels. (D) Representative Western blots illustrate the expression of LAMP-1, V-ATPase B1/2, Rab7 and SNAP23 in fibroblasts from controls and asymptomatic CSPα mutation carriers. Transmembrane proteins are normalized to Flotillin. The histogram shows the quantification of LAMP-1, V-ATPase B1/2, Rab7 and SNAP23 detected by immunoblot relative to control levels. (E) Graph shows the lysosomal enzyme activities of PPT-1, β-gluc and β-Hexa in the culture medium (right panel) or in the cell homogenates (left panel) of two CSPα mutation carriers relative to cells from two age-matched control individuals. Enzymatic activity was normalized to the total intracellular protein and pooled by genotype. (Mean ± SEM of quantification by triplicate in each individual). **p ≤ 0.01; ***p ≤ 0.001.

Figure 2

Time course of AFSM and CSPα.pL115R aggregate accumulation in vitro. (A) Representative images of AFSM in primary dermal fibroblasts from an asymptomatic CSPα mutation carrier (top, right panel) and control (top, left panel). The histogram shows the quantitative analysis of AFSM by flow cytometry. (B) The rate of AFSM accumulation (calculated as the slope of the percentage of cells with autofluorescence). The data points were fitted using linear regression analysis. (C) Representative Western blot showing the expression of CSPα monomers (M-CSPα) and its aggregates (Aggregates) in human fibroblasts from CSPα mutation carriers and controls. The histogram shows the quantification of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to control levels. Proteins are normalized to β-actin. (D) Representative Western blot showing the levels of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) after 8 days in culture normalized to β-actin. The histogram shows the quantification of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels on day zero. Proteins are normalized to β-actin.

Figure 3

CSPα and its aggregates localize to the lysosome. (A) Representative high magnification merged (overlap, yellow) pictures of normal human primary fibroblasts stained for endogenous CSPα (CSPα, red) and LAMP-2 (LAMP-2, green). Graph shows the co-localization index (Pearson correlation) between the green (LAMP-2) and the red (CSPα) channels within the boxed area. The graph also shows the Pearson correlation between (CSPα, red) and Calnexin (green) and Giantin-1 (green) (See Supplemental Fig. 1C,D). (B) Representative Western blot of LAMP-1 and CSPα from the total cell homogenates (T) and lysosome enriched (L) fractions of normal human fibroblasts. (C) Representative high magnification merged (overlap, yellow) pictures of murine primary fibroblasts stained for endogenous CSPα (CSPα, red) and LAMP-2 (LAMP-2, green). Graph shows the co-localization index (Pearson correlation) between the green (LAMP-2) and the red (CSPα) channels within the boxed area. The graph also shows the Pearson correlation between (CSPα, red) and Calnexin (green) and Giantin-1 (green) (See Supplemental Fig. 1C,D). (D) Representative Western blot of LAMP-1 and CSPα of the total cell homogenates (T) and lysosome enriched (L) fractions of murine fibroblasts. (E) Representative high magnification merged (overlap, yellow) pictures of N2A cells stained for endogenous CSPα (CSPα, red) and LAMP-2 (LAMP-2, green). Graph shows the co-localization index (Pearson correlation) between the green (LAMP-2) and the red (CSPα) channels both in the soma and neurites (See Supplemental Fig. 1E) within the boxed area. (F) Representative Western blot of LAMP-1 and CSPα of the total cell homogenates (T) and lysosome enriched (L) fractions of N2A cells. (G) Representative high magnification merged (overlap, yellow) pictures of primary cortical neurons stained for endogenous CSPα (CSPα, red) and LAMP-2 (LAMP-2, green). Graph shows the co-localization index (Pearson correlation) between the green (LAMP-2) and the red (CSPα) channels both in the soma and neurites (See Supplemental Fig. 1F) within the boxed area. (H) Representative Western blot of LAMP-1 and CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) from the total cell homogenates (T) and the lysosome-enriched fraction (L) of primary fibroblasts from two age-matched controls, two asymptomatic CSPα mutation carriers and from CSPα-deficient fibroblasts stably expressing CSPα-p.L115R (CSPα−/− p.L115R) or both CSPα-WT plus CSPα-p.L115R (CSPα−/− p.L115R + WT).

Figure 4

Lysosome dysfunction depends on the CSPα.pL115R mutation. (A) Representative Western blots illustrate the expression of LAMP-1, V-ATPase B1/2, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in fibroblasts from a wild-type mouse stably expressing nothing (Empty), CSPα-WT (WT) or CSPα-p.L115R (p.L115R). The histogram shows the quantification of LAMP-1, V-ATPase B1/2, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels in cells expressing the Empty vector. Proteins are normalized to β-actin. (B) The histogram shows the quantification of the transcript levels of LAMP-1 and LAMP-2 in fibroblasts from a wild-type mouse stably expressing CSPα-WT (WT) or CSPα-p.L115R (p.L115R) normalized to levels found in cells expressing nothing (Empty). Values represent the mean ± S.E. of three independent experiments *p = 0.02; **p = 0.01. (C) Lysotracker signal in primary fibroblasts from a wild-type mouse stably expressing nothing (Empty), CSPα-WT (WT) or CSPα-p.L115R (p.L115R). Values represent the mean ± S.E. of three independent experiments *p = 0.03; **p = 0.01. (D) Graph shows the activities of the lysosomal enzymes, β-gluc, β-Hexa and PPT-1 measured in cell homogenates of a wild-type mouse stably expressing nothing (Empty), CSPα-WT (WT) or CSPα-p.L115R (p.L115R). Enzymatic activity was normalized to the total intracellular protein. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector ***, p ≤ 0.001 . (E) Graph shows the lysosomal enzyme activities of β-gluc, β-Hexa and PPT-1 measured in the culture medium. Enzymatic activity was normalized to the total intracellular protein. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector ***, p ≤ 0.001. (F) Non-permeabilized surface LAMP-1 levels analyzed by flow cytometry using LAMP1-1DB4 (anti-intraluminal) in primary fibroblasts from a wild-type mouse stably expressing nothing (Empty), CSPα-WT (WT) or CSPα-p.L115R (p.L115R). Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector *, p = 0.02; **, p = 0.0047.

Figure 5

CSPα mutation p.L115R alone failed to cause lysosome dysfunction. (A) Representative Western blots illustrate the expression of LAMP-1, V-ATPase B1/2, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in fibroblasts from a CSPα-deficient mouse stably expressing nothing (Empty), CSPα-WT (WT), CSPα-p.L115R (p.L115R) or both CSPα-WT plus CSPα-p.L115R (WT+p.L115R). The histogram shows the quantification of LAMP-1, V-ATPase B1/2, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels in cells expressing the Empty vector. Proteins are normalized to β-actin. (B) The histogram shows the quantification of the transcript levels of LAMP-1 and LAMP-2 in fibroblasts from a CSPα-deficient mouse stably expressing CSPα-WT (WT), CSPα-p.L115R (p.L115R) or both CSPα-WT plus CSPα-p.L115R (WT+p.L115R) normalized to levels found in cells expressing the Empty vector. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector **, p ≤ 0.01. (C) Lysotracker signal in primary fibroblasts from a CSPα-deficient mouse stably expressing nothing (Empty), CSPα-WT (WT), CSPα-p.L115R (p.L115R) or both CSPα-WT plus hCSPα-p.L115R (WT+p.L115R). Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector **, p = 0.01; ***, p = 0.001. (D) Graph shows the activity of the lysosomal enzymes, β-gluc, β-Hexa and PPT-1 measured in cell homogenates. Enzymatic activity was normalized to the total intracellular protein. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector *, p < 0.05; **, p < 0.01. (E) Graph shows the activity of the lysosomal enzymes, β-gluc, β-Hexa and PPT-1 measured in the culture medium. Enzymatic activity was normalized to the total intracellular protein. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells transduced with empty vector *, p < 0.05; **, p < 0.01.

Figure 6

Autophagy-Lysosomal degradation of CSPα and CSPα.pL115R aggregates. (A) Representative Western blot displaying the levels of endogenous CSPα monomers (M-CSPα) in N2A cells treated with CHX, CHX plus Lactacystin or CHX plus NH4 + E64d + Leupeptin for 6, 12 and 24 hours. The graph shows the quantification of CSPα monomers detected by immunoblot. Values represent the mean ± S.E. of three independent experiments compared to the levels of cells treated with CHX alone *, p = 0.05; **, p = 0.01. (B) Representative Western blot of CSPα monomers (CSPα) in the membrane-enriched fraction (Membrane) in N2A cells treated with CHX, CHX plus Lactacystin or CHX plus NH4 + E64d + Leupeptin for 24 hours. The histogram shows the quantification of CSPα monomers detected by immunoblot relative to protein levels in untreated N2A cells. Proteins are normalized to Flotillin. (C) Representative Western blot of CSPα monomers (CSPα) in the cytosolic/soluble (Soluble) fraction in N2A cells treated with CHX, CHX plus Lactacystin or CHX plus NH4 + E64d + Leupeptin for 24 hours. The histogram shows the quantification of CSPα monomers detected by immunoblot relative to protein levels in untreated N2A cells. Proteins are normalized to HSC70. (D) Representative Western blot displaying the levels of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in fibroblasts from a CSPα-deficient mouse stably expressing CSPα-p.L115R treated with CHX, CHX plus Lactacystin, CHX plus NH4 + E64d + Leupeptin or CHX plus Bafilomycin A1 for 24 hours. The histogram shows the quantification of p62, LC3-II, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels in untreated cells. Proteins are normalized to β-actin. (E) Representative Western blot showing the levels of CSPα monomers (M-CSPα) in primary fibroblasts from a CSPα-deficient mouse stably expressing hCSPα-WT or hCSPα-p.L115R under serum withdrawal (SW) for 3, 6, 12 and 24 hours. The graph shows the quantification of CSPα monomers detected by immunoblot relative to protein levels in untreated cells at time = 0 h. Proteins are normalized to β-actin. (F) Representative Western blot showing the levels of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in fibroblasts from a CSPα-deficient mouse stably expressing CSPα-p.L115R (CSPα−/− p.L115R) under Serum withdrawal (SW) or SW + E64d + Leupeptin (SW + E64d/Leup) for 2, 4 and 6 hours. The graph shows the quantification of CSPα aggregates detected by immunoblot relative to protein levels in untreated cells at time = 0 h. Proteins are normalized to β-actin.

Figure 7

AFSM and CSPα aggregate accumulation are modulated by the autophagy-lysosome pathway. (A) Baseline Western blot of p62 and LC3-I/II in primary fibroblasts from an age-matched control and an asymptomatic CSPα mutation carrier. The histogram shows the quantification of p62 and LC3-II detected by immunoblot relative to protein levels in control cells. There are no changes in HSC70 levels. Proteins are normalized to β-actin. (B) Representative Western blot of Lamp1, CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates), HSC70, p62, LC3-I/II and SNAP23 in untransduced (UT) and stably expressing CSPα-p.L115R (p.L115R) N2A cells. The histogram shows the quantification of p62, HSC70, LAMP1, SNAP23, LC3-I/II, Aggregates and CSPα monomers detected by immunoblot relative to protein levels in untrandusced (UT) N2A cells. Proteins are normalized to β-actin. (C) Quantitative analysis by flow cytometry of AFSM in primary fibroblasts from an asymptomatic CSPα mutation carrier maintained at 2% FBS for 6 days in culture and treated with NH4 + E64d + Leupeptin (NH4 + E64 + Leup) or Bafilomycin A1 relative to untreated cells. Values represent the mean ± S.E. of three independent experiments compared to the levels of untreated cells **, p < 0.01. (D) Representative Western blot of CSPα monomers (M-CSPα), CSPα aggregates (Aggregates), p62 and LC3-I/II in primary fibroblasts from an asymptomatic CSPα mutation carrier treated with NH4 + E64d + Leupeptin (NH4 + E64 + Leup) or Bafilomycin A1 (Baf A1) relative to untreated cells. Upper bands represent a longer exposure of the aggregates (Aggregates*). The histogram shows the quantification of CSPα monomers (M-CSPα), CSPα aggregates (Aggregates), p62 and LC3-I/II detected by immunoblot relative to protein levels in untreated cells. Proteins are normalized to β-actin. (E) Quantitative analysis by flow cytometry of AFSM in primary fibroblasts from an asymptomatic CSPα mutation carrier maintained at 10% FBS for 6 days in culture and treated with 0% serum (SW), Torin-1 or SW plus Baf A1 for 24 hours compared to the levels of untreated cells *, p < 0.05; **, p < 0.01. (F) Representative Western blot of CSPα monomers (M-CSPα-), CSPα aggregates (Aggregates), p62 and LC3-I/II in primary fibroblasts from asymptomatic CSPα mutation carrier treated with 0% FBS, Torin-1 or SW plus Baf A1 for 24 hours. Upper bands represent a longer exposure of the aggregates (Aggregates*). The histogram shows the quantification of CSPα monomers, CSPα aggregates (Aggregates), p62 and LC3-I/II detected by immunoblot relative to protein levels in untreated cells *, p < 0.05; **, p < 0.01. Proteins are normalized to β-actin.

Figure 8

NtBuHA enhances the reduction of CSPα aggregates and AFSM induced by serum withdrawal. (A) Quantitative analysis by flow cytometry of AFSM in primary fibroblasts from an asymptomatic CSPα mutation carrier treated with increasing doses of N-(tert-Butyl)hydroxylamine (NtBuHA) for 24 hours in the absence of serum compared to the levels of untreated cells **, p < 0.01. (B) Representative Western blot of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in cultured fibroblasts from an asymptomatic CSPα mutation carrier treated with increasing doses of NtBuHA for 24 hours in absence of serum. Upper bands represent a longer exposure of the aggregates (Aggregates*). The histogram shows the quantification of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels in untreated cells. Proteins are normalized to β-actin. (C) Representative Western blot of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) in homogenates from CSPα-deficient mouse fibroblasts stably expressing both CSPα-WT plus CSPα-p.L115R (CSPα−/− p.L115R + WT) treated with increasing doses of NtBuHA for 24 hours in the absence of serum. The histogram shows the quantification of CSPα monomers (M-CSPα) and CSPα aggregates (Aggregates) detected by immunoblot relative to protein levels in untreated cells. Proteins are normalized to β-actin.