Loss of Cln3 Function in the Social Amoeba Dictyostelium discoideum Causes Pleiotropic Effects That Are Rescued by Human CLN3

The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3− cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3− cells was precocious and cln3− slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3− cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3− cells, strongly supports the use of this new model for JNCL research

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis

Distinct Early Molecular Responses to Mutations Causing vLINCL and JNCL Presage ATP Synthase Subunit C Accumulation in Cerebellar Cells

Variant late-infantile neuronal ceroid lipofuscinosis (vLINCL), caused by CLN6 mutation, and juvenile neuronal ceroid lipofuscinosis (JNCL), caused by CLN3 mutation, share clinical and pathological features, including lysosomal accumulation of mitochondrial ATP synthase subunit c, but the unrelated CLN6 and CLN3 genes may initiate disease via similar or distinct cellular processes. To gain insight into the NCL pathways, we established murine wild-type and CbCln6nclf/nclf cerebellar cells and compared them to wild-type and CbCln3Δex7/8/Δex7/8 cerebellar cells. CbCln6nclf/nclf cells and CbCln3Δex7/8/Δex7/8 cells both displayed abnormally elongated mitochondria and reduced cellular ATP levels and, as cells aged to confluence, exhibited accumulation of subunit c protein in Lamp 1-positive organelles. However, at sub-confluence, endoplasmic reticulum PDI immunostain was decreased only in CbCln6nclf/nclf cells, while fluid-phase endocytosis and LysoTracker® labeled vesicles were decreased in both CbCln6nclf/nclf and CbCln3Δex7/8/Δex7/8 cells, though only the latter cells exhibited abnormal vesicle subcellular distribution. Furthermore, unbiased gene expression analyses revealed only partial overlap in the cerebellar cell genes and pathways that were altered by the Cln3Δex7/8 and Cln6nclf mutations. Thus, these data support the hypothesis that CLN6 and CLN3 mutations trigger distinct processes that converge on a shared pathway, which is responsible for proper subunit c protein turnover and neuronal cell survival

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Effect of <i>cln3</i> knockout on the formation of tipped mounds and slugs.

<p>(A) AX3, <i>cln3⁻</i>, or <i>cln3⁻</i> cells overexpressing GFP-Cln3 or expressing GFP-Cln3 or GFP-CLN3 under the control of the <i>cln3</i> upstream element imaged after 12 and 15 hours of development. Images are a top-view of developing cells. (B) Quantification of the number of tipped mounds observed after 12 hours of development. Data presented as mean % tipped mounds ± s.e.m (n = 10–19). (C) Quantification of the number of fingers and slugs observed after 15 hours of development. Data presented as mean % fingers and slugs ± s.e.m (n = 10–33). Statistical significance was assessed using the Kruskal-Wallis test followed by the Dunn multiple comparison test (***p-value<0.001 vs. AX3). Scale bars = 1 mm. M, mound; TM, tipped-mound; F, finger; S, slug.</p

Effect of <i>cln3</i> knockout on the intra- and extracellular levels of AprA and CfaD.

<p>AX3 and <i>cln3⁻</i> cells grown axenically in HL5 were harvested and lysed after 48 and 72 hours of growth. Whole cell lysates (20 µg) (i.e., intracellular) and samples of conditioned growth media (i.e., extracellular) were separated by SDS-PAGE and analyzed by western blotting with anti-AprA, anti-CfaD, anti-tubulin, and anti-actin. Molecular weight markers (in kDa) are shown to the right of each blot. (A) Intra- and extracellular protein levels of AprA. Immunoblots that were exposed for a longer period of time (i.e., longer exposure) are included to show the 55-kDa and 37-kDa protein bands detected by anti-AprA. Note that the 37-kDa protein was detected in samples of conditioned growth media, but not in whole cell lysates. (B) Intra- and extracellular protein levels of CfaD. Data in all plots presented as mean amount of protein relative to AX3 48 hour sample (%) ± s.e.m (n = 4 independent experimental means, from 2 replicates in each experiment). Statistical significance was determined using a one-sample t-test (mean, 100; two-tailed) vs. the AX3 48 hour sample. *p-value<0.05. **p-value<0.01. (C) Detection of tubulin and actin in whole cell lysates (WC; lanes 1–2), but not in samples of conditioned growth media (lanes 3–6).</p

Intracellular localization of <i>Dictyostelium</i> GFP-Cln3 using epifluorescence microscopy.

<p>(A) AX3 cells overexpressing GFP-Cln3 imaged live in water. Scale bar = 5 µm. (B) AX3 cells overexpressing GFP-Cln3 were fixed in either ultra-cold methanol (for VatM and Rh50 immunostaining) or 4% paraformaldehyde (for p80 immunostaining) and then probed with anti-VatM, anti-Rh50, or anti-p80, followed by the appropriate secondary antibody linked to Alexa 555. Cells were stained with DAPI to reveal nuclei (blue). Images were merged with ImageJ/Fiji. VC, vacuolar-shaped structures; VS, cytoplasmic vesicles; T, tubular-like structures within the cytoplasm; P, punctate distributions within the cytoplasm. Scale bars (B, C) = 2.5 µm.</p

Bioinformatic analysis of <i>Dictyostelium</i> Cln3.

<p>(A) Alignment of human CLN3 and the <i>Dictyostelium</i> ortholog. The following residues are conserved; N-linked glycosylation sites (*), sites of missense point mutations (;), sites of nonsense point mutations (:), target for myristoylation (#), sites that when mutated cause a slower disease progression in compound heterozygosity with the common 1.02 kb deletion mutation (∧), sites that when mutated cause a slower disease progression in homozygosity (<) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-TheInternationalBattenDisease1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Cotman1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Bause1" target="_blank">[85]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110544#pone.0110544-Haskell2" target="_blank">[87]</a>. <i>Dictyostelium</i> Cln3 also contains a putative prenylation motif (i.e., CFIL; underlined). (B) Phylogenetic tree showing the relationship of <i>Dictyostelium</i> Cln3 to CLN3 orthologs from 20 different organisms (i.e., mammals and NIH model systems).</p

Generation of a <i>Dictyostelium cln3</i> knockout mutant.

<p>(A) Creation of a <i>Dictyostelium cln3</i> knockout mutant by homologous recombination. The pLPBLP targeting vector and sites of recombination are shown. (B) Validation of <i>cln3</i> knockout by PCR analysis. Primers are denoted by Roman numerals and arrows. The <i>Dictyostelium</i> gene denoted DDB_G0291155 lies downstream of <i>cln3</i> and was amplified to confirm that the insertion of the <i>bsr</i> cassette did not affect this gene. (C) Validation of <i>cln3</i> knockout by Southern blotting. DNA ladder (in bp) is shown to the left of the blot.</p