Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 30;8:e46607.
doi: 10.7554/eLife.46607.

A Drosophila Model of Neuronal Ceroid Lipofuscinosis CLN4 Reveals a Hypermorphic Gain of Function Mechanism

Affiliations
Free PMC article

A Drosophila Model of Neuronal Ceroid Lipofuscinosis CLN4 Reveals a Hypermorphic Gain of Function Mechanism

Elliot Imler et al. Elife. .
Free PMC article

Abstract

The autosomal dominant neuronal ceroid lipofuscinoses (NCL) CLN4 is caused by mutations in the synaptic vesicle (SV) protein CSPα. We developed animal models of CLN4 by expressing CLN4 mutant human CSPα (hCSPα) in Drosophila neurons. Similar to patients, CLN4 mutations induced excessive oligomerization of hCSPα and premature lethality in a dose-dependent manner. Instead of being localized to SVs, most CLN4 mutant hCSPα accumulated abnormally, and co-localized with ubiquitinated proteins and the prelysosomal markers HRS and LAMP1. Ultrastructural examination revealed frequent abnormal membrane structures in axons and neuronal somata. The lethality, oligomerization and prelysosomal accumulation induced by CLN4 mutations was attenuated by reducing endogenous wild type (WT) dCSP levels and enhanced by increasing WT levels. Furthermore, reducing the gene dosage of Hsc70 also attenuated CLN4 phenotypes. Taken together, we suggest that CLN4 alleles resemble dominant hypermorphic gain of function mutations that drive excessive oligomerization and impair membrane trafficking.

Keywords: D. melanogaster; cysteine-string protein; lysosome; neuronal ceroid lipofuscinosis; neuroscience.

Conflict of interest statement

EI, JP, SK, MT, YZ, SC, KZ No competing interests declared

Figures

Figure 1.
Figure 1.. Generation of a Drosophila CLN4 model.
(A) Structure of CSP and position of CLN4 mutations in the cysteine-string (CS) domain of hCSPα and dCSP. CSP’s N-terminal J domain, linker domain and C-terminus are indicated. (B) Larval VNC of animals expressing WT hCSPα in neurons from a single transgene with an elav driver immunostained for hCSPα and endogenous dCSP. Scale bar, 20 μm. (C) Larval NMJs of control and animals expressing WT hCSPα immunostained for hCSPα, dCSP, and HRP marking the presynaptic plasma membrane. Scale bar, 5 μm. (D) Adult lifespan of control (dcspX1/+, black), cspX1/R1 deletion mutants (green) and cspX1/R1 mutants expressing WT hCSPα (blue), hCSP-L115, (orange), hCSPα-L116 (red), or dCSP2 (purple) with an elav driver.
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. Phenotypic effects of CLN4 mutations.
(A) Lipidation of WT hCSPα, hCSP-L115 and -L116. Immunoblot of larval VNC extracts from control (w1118) and animals expressing elav-driven hCSPα, hCSP-L115 or -L116 were probed for hCSPα. Samples were treated overnight with either 0.5 M hydroxylamine (+) or equimolar Tris (-). Asterisk denotes unspecific signal. (B) Adult control (w1118) and animals expressing nSyb-driven hCSP-L115 or -L116 in neurons. (C) Immunoblot probed for dCSP of larval brain extracts from control (w1118) and animals expressing WT hCSPα, hCSPα-L115 or -L116 from either one or two (2x) transgenes with an elav driver. The lipidated monomeric dCSP isoforms (LMs) are indicated. (D) Increase in total protein levels of WT hCSPα, hCSP-L115 and -L116 induced by expressing either one or two transgenes (2x). Signals were normalized to loading control and plotted as n-fold change to respective expression levels from one transgene (mean ± SEM; N = 6, two-tailed unpaired t test; **, p<0.01; ***, p<0.001.
Figure 2.
Figure 2.. CLN4 mutations cause dose-dependent oligomerization of hCSPα in neurons.
WT and mutant hCSPα (L115/L116) were expressed in larval neurons of white1118 animals (control) with an elav driver from one or two transgenes (2x). (A) Western blot of larval brain protein extracts probed for hCSPα. Signals for SDS-resistant hCSPα oligomers (OM), lipidated monomeric hCSP (LM), and non-lipidated hCSP (NLM) are indicated. β-tubulin was used as loading control. (B) Western blots probed for hCSPα or lysine-linked-ubiquitin of hCSPα-immunoprecipitated extracts from adult heads of indicated genotypes. Signals for IgG heavy chain are indicated. (C) Average oligomer/monomer ratios (N = 5). (D–F) Levels of lipidated (D), non-lipidated (E), and total hCSPα (F) normalized to WT hCSPα (N = 6). (G–I) Dosage-dependent increase of hCSPα oligomer (G), lipidated (H), and non-lipidated monomer levels (I). Signals were normalized to loading control and plotted as n-fold change from 1-copy expression of WT hCSPα (N = 6). (J) Levels of monomeric dCSP shown as n-fold change from control (N = 3). Graphs display mean ± SEM. Statistical analysis used one-way ANOVA (C–F, J) or two-tailed unpaired t test (G–I); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 3.
Figure 3.. CLN4 mutations cause dose-dependent lethality and eye degeneration.
(A) Viability of animals expressing WT, L115, or L116 mutant hCSPα pan-neuronally from one or two transgenes (2x) with an elav driver (N ≥ 3, n ≥ 740). (B) Images of adult fly eyes expressing WT hCSPα or hCSPα-L116 with a GMR-Gal4 driver at 23°C and 28°C. (C) Semi-quantitative assessments of CLN4-induced eye phenotypes (N ≥ 9). Graphs display mean ± SEM. Statistical analysis used one-way ANOVA (A) and Kruskal-Wallis test (C); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 4.
Figure 4.. CLN4 mutations reduce synaptic hCSPα levels and cause abnormal accumulations with endogenous dCSP and ubiquitinated proteins in axons and somata.
WT, L115 or L116 mutant hCSPα were expressed in larval neurons with an elav-Gal4 driver from one (D–E, J–L) or two (A, H–I) transgenes. Genotypes are indicated. (A) Larval NMJs immunostained for hCSPα, endogenous dCSP, and the neuronal membrane marker HRP. White lines denote line scans shown in C. (B) Larval VNCs stained for hCSPα. (C) Plots of hCSPα and dCSP fluorescence from single line scans through synaptic boutons (white lines in A). (D) Larval VNC segments stained for hCSPα (red) and lysine-linked-ubiquitin visualizing ubiquitinated proteins (Ubi-protein). (E) Larval brain segments stained for hCSPα and dCSP. (F) Synaptic bouton of larval NMJ stained for hCSPα and Ubi-proteins. (G) Proximal larval segmental nerves stained for hCSPα. (H–I) Average levels of hCSPα (H) and dCSP (I) at synaptic boutons of larval NMJs (N > 4). (J) Cumulative area of abnormal hCSPα accumulations in larval brains (N ≥ 3). (K–L) Average number of accumulations immunopositive for both hCSPα and ubiquitin (K), or only positive for ubiquitin (L) but not hCSPα (N ≥ 4). Scale bars: 5 μm (A), 20 μm (B, G), 15 μm (D), 10 μm (E), 5 μm (F). Graphs display mean ± SEM. Statistical analysis used one-way ANOVA (H–L); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 4—figure supplement 1.
Figure 4—figure supplement 1.. hCSP-L115 and -L116 abnormally accumulate with endogenous dCSP and ubiquitinated proteins.
WT and mutant hCSPα (L115 and L116) were expressed in larval neurons with an elav-Gal4 driver from 1 (A–B) or two (C) transgenes (2x). (A–B) Larval VNCs of indicated genotypes immunostained for hCSPα and dCSP (A) or lysine-linked-ubiquitin (B). (C) Larval NMJ stained for hCSPα and HRP showed occasionally extreme accumulations of hCSP-L115 in synaptic boutons. Scale bar, 20 μm (A–B), 5 μm (C).
Figure 4—figure supplement 2.
Figure 4—figure supplement 2.. Effects of CLN4-analogous mutations in dCSP.
WT dCSP2, dCSP-V117R (analogous to human L115R) and dCSP-I118Δ (analogous to L116Δ) were expressed in larval neurons with an elav-Gal4 driver from one or two transgenes (2x) in otherwise wild type control (w1118) or homozygous dcsp null mutants (dcspX1/R). (A–B) Viability of animals expressing dCSP2, dCSP-V117R, or -I118Δ pan-neuronally from one transgene at 27°C (A) and two transgenes (2X) at 23°C (B, N = 4). (C) Immunoblot of larval VNC extracts of indicated genotypes probed for dCSP. Signals corresponding to lipidated dCSP monomers (LM), dimers (DM), and high-molecular weight oligomers (OM) are indicated. Asterisk denotes partially lipidated dCSP. β-tubulin was used as loading control. (D) Adult lifespan at 27°C of control (w1118; UAS-dCSP2) and mutants expressing WT dCSP2, dCSP-V117, or -I118 from one copy with an elav driver. (E) Larval VNCs of indicated genotypes immunostained for dCSP. (F) Larval NMJs of indicated genotypes stained for dCSP and HRP. Scale bar, 20 μm (A), 10 μm (C). Graphs display mean ± SEM. Statistical analysis used one-way ANOVA (A) or a Kruskal-Wallis test (B); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 5.
Figure 5.. CLN4 mutant hCSPα accumulates on LAMP1- and HRS-positive endosomes.
hCSP-L116 was expressed in larval neurons with an elav-Gal4 driver from one transgene. As indicated, respective reporter transgenes were co-expressed. (A) Neurons of larval VNCs co-immunostained for hCSPα and the ER marker GFP-KDEL, the cis-Golgi marker GMAP, or the trans-Golgi marker Golgin 245. (B) Neurons co-immunostained for hCSPα (red) and co-expressed hLAMP1-GFP, Spinster-GFP or the PI3P marker FYVE-GFP. (C) Fraction of organelle markers colocalizing with hCSPα accumulations (mean ± SEM; n ≥ 65, N ≥ 4). (D–E) Segments of larval brains costained for hCSPα and ATG8/LC3-GFP (D) or HRS (E). (F) Synaptic boutons at larval NMJs co-stained for hCSPα and HRS. (G) Neuron co-immunostained for hCSPα and coexpressed GFP-nSyb. Scale bars: 10 μm (D–E), 5 μm (A–B, F–G).
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. RNAi-mediated KD of TSG101 causes accumulation of dCSP on HRS-positive endosomes.
(A) Larval VNCs immunostained for dCSP and HRS of driverless control (w1118; UAS-TSG101 KD/+) and elav-driven KD of TSG101 using a hairpin transgene (w1118, elav-Gal4; UAS-TSG101 KD/+). Note the mislocalization of endogenous dCSP from its typical diffuse neuropil localization to HRS-positive accumulations in neuropil and neuronal somata. (B) Larval NMJs of indicated genotypes stained for dCSP and HRP. Note the abnormal localization of dCSP from the periphery to more centrally located HRP-positive endosomes. (C) Immunoblot probed for dCSP of larval brain extracts from control and elav-driven TSG101 KD. Note that TSG101 KD does not cause high-molecular weight oligomerization of dCSP despite the induced mislocalization to HRS-positive endosomes.
Figure 6.
Figure 6.. CLN4 mutations cause abnormal endomembrane structures and EM-dense accumulations.
TEM micrographs of ultrathin (70 nm) sections from larval VNCs expressing hCSP-L115 or -L116 with an elav driver from two transgenic copies. Genotypes are indicated. (A–B) Neuronal somata containing electron-dense membrane whirls (black arrowheads), large electron-dense extracellular deposits (black arrow, (B), and occasionally ‘residual lysosomes’ (white arrow, (A), bloated Golgi Apparati (B), and degenerating nuclear membranes (white arrow, (B). (C) Neuropil of larval VNC containing membrane whirls in neuronal processes (black arrowheads). (D) Sagittal section of larval segmental nerve containing membrane whirls and abnormal autophagosome-like structures in axons of sensory and motor neurons. (E–H) High magnification images showing residual lysosome with a diverse variety of intraluminal vesicles (E), various forms of EM-dense membrane whirls (F–G) and autophagosome-like structures (white arrowheads, (G–I) that may interact with EM-dense structures (white arrow, (G), and an electron-dense extracellular deposit (H). Scale bars: 1 μm (A–C), 200 nm (D–E).
Figure 7.
Figure 7.. Altering wild type CSP levels modifies CLN4 phenotypes.
WT hCSPα, hCSP-L115 or -L116 were expressed in neurons from one or two transgenes (2x) with an elav driver in control (w1118), heterozygous cspX1/+, and homozygous cspX1/R1 deletion mutants, or co-expressed with WT hCSPα or dCSP. Genotypes are indicated. (A–C) Effects of reducing endogenous dCSP (A) or co-expressing WT hCSPα (B–C) on the viability of hCSP-L115 and -L116 mutant flies (N > 3; n > 144). (D–E) Immunoblots of protein extracts from larval VNC of indicated genotype probed for hCSPα and β-tubulin (loading control). hCSPα oligomers (OM), lipidated (LM), non-lipidated hCSPα monomers (NLM), and unspecific signals (*) are denoted. (F–N) Effects of reduced endogenous dCSP (F–H), increased dCSP (I–K), and increased WT hCSPα (L–N) levels on hCSPα oligomers (F, I, L), lipidated monomers (G, J, M), and non-lipidated monomers (H, K, N). Signals were normalized to loading control and plotted as n-fold change of L115 or L116 levels when expressed in a WT background (N = 5). Graphs display mean ± SEM. Statistical analysis used unpaired t test (A, I–L) or one-way ANOVA (B–C, F–H, M–N); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 8.
Figure 8.. Effects of reduced dCSP levels on CLN4 mutant hCSPα localization and protein ubiquitination.
WT hCSPα, hCSP-L115 or -L116 were expressed in neurons with an elav driver in control (w1118), heterozygous cspX1/+, or homozygous cspX1/R1 deletion mutants. (A) Larval VNC stained for hCSPα and dCSP. Genotypes are indicated. Scale bar, 20 μm. (B–D) Effects of reduced dcsp gene dosage on the accumulation of hCSP-L115 and -L116 in larval VNC (B, N ≥ 3), ubiquitinated protein levels in larval VNCs (C, N ≥ 4), and synaptic levels of hCSP-L115 and -L116 at larval NMJs (D, N ≥ 4). Signals were normalized and plotted as n-fold change to levels of elav-L115 and -L116 expression in WT control background. Graphs display mean ± SEM. Statistical analysis used a paired (D) and unpaired t test (B–C); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 9.
Figure 9.. Reducing the gene dosage of Hsc4 attenuates CLN4 phenotypes.
(A) Adult eyes of flies expressing WT hCSPα (control) or hCSP-L116 with a GMR-Gal4 driver in the absence or presence of a heterozygous Hsc4+/- (hsc4∆356) deletion, a Hsc4 KD (34836), OE of Syx1A or Syb. (B–D) Immunoblots of extracts from larval VNCs of indicated genotypes were probed for hCSPα. Lipidated monomeric hCSP (LM), non-lipidated hCSP (NLM) and hCSPα oligomers (OM) are indicated. Respective transgenes were expressed with an elav-driver; control was w1118. β-tubulin was used as loading control.( E) Semi-quantitative assessments of genetic modifier effects on L116-induced eye phenotypes (N ≥ 12).( F) Viability of animals expressing hCSPα-L115 or -L116 from two transgenes driven by elav-Gal4 in a control (w1118) or a heterozygous hsc4∆356 deletion background (N ≥ 3; n > 74). (G-K) Effects of reduced hsc4 gene dosage on levels of hCSPα oligomers (G), lipidated monomers (H), non-lipidated monomers (I) and total protein levels (J). Signals were normalized to loading control and plotted as n-fold change to levels induced by elav-driven expression of hCSP-L115 or -L116 in a WT control background (N > 6). (K) Effect of reduced hsc4 gene dosage on lipidated WT hCSPα monomer levels (N = 4). (L) Effects of reduced hsc4 gene dosage on endosomal accumulations of hCSP-L115/L116 in larval VNC (N ≥ 4). (M–N) Effects of reduced hsc4 gene dosage on ubiquitinated protein levels in larval VNCs (M, N = 5) and synaptic hCSP-L115/L116 expression levels at larval NMJs (N, N = 6). Signals were normalized and plotted as n-fold change from levels of elav-L115/L116 expression in WT control background. (O) Motor neuron somata of larval VNCs co-expressing HA-tagged Hsc4 with mutant hCSPα-L115 or -L116 stained for HA and hCSPα. Scale bars: 20 μm (K), 15 μm (M). Graphs display mean ± SEM. Statistical analysis used an unpaired t test (F, M), paired t test (L, N), or one-way ANOVA (E, G–J); *, p<0.05; **, p<0.01; ***, p<0.001.
Figure 9—figure supplement 1.
Figure 9—figure supplement 1.. Effects of altered hsc4 gene dosage on CLN4 mutant hCSPα protein expression and localization.
(A-D) Effects of Hsc4 overexpression (OE) on hCSP-L115 and -L116 oligomers (A, N ≥ 8), lipidated monomers (B, N ≥ 6), non-lipidated monomers (C, N = 8), and overall protein levels (D, N = 8). Signals were normalized to loading control and plotted as n-fold change from L115/L116 levels (control) when expressed in a WT background (N = 5). (E) Larval VNCs for indicated genotypes immunostained for dCSP (green) and hCSPα (red). hCSP-L115 and -L116 were expressed with an elav driver from one transgene in WT control (white1118) or heterozygous hsc4∆356 deletion mutants. Scale bar, 20 μm. Graphs display mean ± SEM. Statistical analysis used RM one-way ANOVA (A–D); *, p<0.05; **, p<0.01; ***, p<0.001.

Comment in

Similar articles

See all similar articles

Cited by 1 article

References

    1. Abramov E, Dolev I, Fogel H, Ciccotosto GD, Ruff E, Slutsky I. Amyloid-beta as a positive endogenous regulator of release probability at hippocampal synapses. Nature Neuroscience. 2009;12:1567–1576. doi: 10.1038/nn.2433. - DOI - PubMed
    1. Anderson GW, Goebel HH, Simonati A. Human pathology in NCL. Biochimica Et Biophysica Acta (BBA) - Molecular Basis of Disease. 2013;1832:1807–1826. doi: 10.1016/j.bbadis.2012.11.014. - DOI - PubMed
    1. Arnold C, Reisch N, Leibold C, Becker S, Prüfert K, Sautter K, Palm D, Jatzke S, Buchner S, Buchner E. Structure-function analysis of the cysteine string protein in Drosophila: cysteine string, Linker and C terminus. Journal of Experimental Biology. 2004;207:1323–1334. doi: 10.1242/jeb.00898. - DOI - PubMed
    1. Benitez BA, Alvarado D, Cai Y, Mayo K, Chakraverty S, Norton J, Morris JC, Sands MS, Goate A, Cruchaga C. Exome-sequencing confirms DNAJC5 mutations as cause of adult neuronal ceroid-lipofuscinosis. PLOS ONE. 2011;6:e26741 doi: 10.1371/journal.pone.0026741. - DOI - PMC - PubMed
    1. Benitez BA, Cairns NJ, Schmidt RE, Morris JC, Norton JB, Cruchaga C, Sands MS. Clinically early-stage cspα mutation carrier exhibits remarkable terminal stage neuronal pathology with minimal evidence of synaptic loss. Acta Neuropathologica Communications. 2015;3:73 doi: 10.1186/s40478-015-0256-5. - DOI - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback