Characterizing pathogenic processes in Batten disease: use of small eukaryotic model systems.
ABSTRACT The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders. Nevertheless, small model organisms, including those lacking a nervous system, have proven invaluable in the study of mechanisms that underlie the disease and in studying the functions of the conserved proteins associated to each disease. From the single-celled yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to the worm, Caenorhabditis elegans and the fruitfly, Drosophila melanogaster, biochemical and, in particular, genetic studies on these organisms have provided insight into the NCLs.
- SourceAvailable from: kelly-landes.orgFly 03/2007; 1(2):118-22. · 1.11 Impact Factor
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ABSTRACT: Neuronal ceroid lipofuscinosis (NCL) comprises ∼13 genetically distinct lysosomal disorders primarily affecting the central nervous system. Here we report successful reprogramming of patient fibroblasts into induced pluripotent stem cells (iPSCs) for the two most common NCL subtypes: classic late-infantile NCL, caused by TPP1(CLN2) mutation, and juvenile NCL, caused by CLN3 mutation. CLN2/TPP1- and CLN3-iPSCs displayed overlapping but distinct biochemical and morphological abnormalities within the endosomal-lysosomal system. In neuronal derivatives, further abnormalities were observed in mitochondria, Golgi, and endoplasmic reticulum. While lysosomal storage was undetectable in iPSCs, progressive disease subtype-specific storage material was evident upon neural differentiation and was rescued by reintroducing the non-mutated NCL proteins. In proof-of-concept studies, we further documented differential effects of potential small molecule TPP1 activity inducers. Fenofibrate and gemfibrozil, previously reported to induce TPP1 activity in control cells, failed to increase TPP1 activity in patient iPSC-derived neural progenitor cells. Conversely, nonsense suppression by PTC124 resulted in both an increase of TPP1 activity and attenuation of neuropathology in patient iPSC-derived neural progenitor cells. This study therefore documents the high value of this powerful new set of tools for improved drug screening and for investigating early mechanisms driving NCL pathogenesis.Human Molecular Genetics 11/2013; · 7.69 Impact Factor
- Regenerative Medicine 09/2013; 8(5):527-9. · 3.87 Impact Factor
Characterizing pathogenic processes in Batten disease:
Use of small eukaryotic model systems
Seasson N. Phillipsa, Neda Muzaffara, Sandra Codlinc,1, Christopher A. Koreyd,1,
Peter E.M. Taschnere,1, Gert de Voerf,1, Sara E. Molec, David A. Pearcea,b,g,⁎
aCenter for Aging and Developmental Biology, Aab Institute of Biomedical Science, University of Rochester School of Medicine and Dentistry,
Rochester, NY 14642, USA
bDepartment of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
cMRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, UK
dDepartment of Biology College of Charleston 66 George Street, Charleston, SC 29424, USA
eDepartment of Human Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
fThe Cell Microscopy Center, Department of Cell Biology and Institute of Biomembranes, University Medical Center Utrecht,
Utrecht, The Netherlands
gDepartment of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
Received 11 May 2006; received in revised form 8 August 2006; accepted 27 August 2006
Available online 1 September 2006
The neuronal ceroid lipofuscinoses (NCLs) are neurodegenerative disorders. Nevertheless, small model organisms, including those lacking a
nervous system, have proven invaluable in the study of mechanisms that underlie the disease and in studying the functions of the conserved
proteins associated to each disease. From the single-celled yeast, Saccharomyces cerevisiae and Schizosaccharomyces pombe, to the worm,
Caenorhabditis elegans and the fruitfly, Drosophila melanogaster, biochemical and, in particular, genetic studies on these organisms have
provided insight into the NCLs.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Batten disease; Model system; NCL
Several genes that cause different NCLs have been
identified. Congenital NCL (CNCL), infantile NCL (INCL),
late-infantile NCL, juvenile NCL (JNCL), adult NCL (Kuf's
disease), Finnish variant late-infantile NCL, variant late-
infantile Gypsy/Indian NCL, Turkish variant late-infantile
NCL/Northern epilepsy harbor mutations in Cathepsin D,
PPT1 (formerly CLN1), CLN2, 3, 4, 5, 6, and 8, respectively
(see http://www.ucl.ac.uk/ncl/;  reviewed in [2,3]). However,
for most of these proteins, the biological function and
mechanism of disease development remains unclear. Evolu-
tionary conservation of each NCL gene allows for their
functional analysis in different model organisms. The lesser
complexity of model organisms enables researchers to obtain
information about a certain protein, the processes it is
involved in, and to extrapolate that knowledge with the aim
of unraveling the more complex systems in higher organisms.
As such, combining the data from the different models will
lead to an increased understanding of the pathways affected in
Known homologs to NCL proteins in Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Caenorhabditis ele-
gans and Drosophila melanogaster and their high amino acid
sequence similarity are shown by Multalin sequence alignment
(Figs. 1 2 and 3) [4,5]. The homolog to CLN3 in the budding
yeast Saccharomyces cerevisiae is Btn1p. Homologs of CLN3
and PPT1 in fission yeast Schizosaccharomyces pombe are
Biochimica et Biophysica Acta 1762 (2006) 906–919
⁎Corresponding author. Center for Aging and Developmental Biology, Box
645, University of Rochester School of Medicine and Dentistry, Rochester, NY
14642, USA. Fax: +1 585 506 1972.
E-mail address: David_Pearce@urmc.rochester.edu (D.A. Pearce).
1Contributed equally to this work.
0925-4439/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
Btn1p and Pdf1p, respectively. The ease of using yeast for
genetic and biochemical studies has made it a powerful model
organism for characterizing these disorders. Similarly, the
nematode Caenorhabditis elegans has proven useful in the
study of both INCL and JNCL, which are studied using the
worm homologs PPT-1 and CLN-3, respectively. Along with its
many other advantages, C. elegans has a well-studied nervous
system making it useful in characterizing the disease mecha-
nism and protein function in the nervous system. Like C.
elegans, neuronal development and function has been well
characterized in the fly. This, along with its sophisticated
genetics, makes Drosophila melanogaster a valuable model
system in the study of human disease, including CNCL, JNCL
and INCL where Cathepsin D, CLN3 and PPT1 are studied. In
this review, the advantages of each model will be outlined and
current research using genetic and biochemical techniques, as
well as advances in the characterization of lysosomal storage
material, will be discussed.
2. Genetic and biochemical studies
2.1. Saccharomyces cerevisiae
The budding yeast S. cerevisiae has been used in scientific
research for over half a century, resulting in a highly annotated
genome and many optimized approaches and tools, coined
“the awesome power of yeast genetics”. S. cerevisiae has
approximately 6000 Open Reading Frames (ORFs) spread
across 16 fully-sequenced chromosomes. It is a single-celled
eukaryote that stably exists in both the haploid and diploid
state with a fast generation time. Two haploid strains of
opposite mating type can fuse to form a diploid strain,
allowing for complementation assays, haplo-insufficiency
analysis, and essential gene deletion. Likewise, a diploid
strain can sporulate into four haploid spores, allowing for
genotypic analysis. Yeast readily takes up, recombines, and
expresses foreign DNA, further providing relative ease in
genetic manipulation. For example, a gene of interest can be
intra-chromosomally tagged, allowing for endogenous expres-
sion of the tagged protein, which eliminates the need for
specific antibodies and some of the artifacts resulting from
overexpression. Entire ORFs can be replaced by reporter
genes for promoter and expression analysis, or conversely, an
inducible promoter can be inserted before a gene of interest to
facilitate the study of essential genes. Making a deletion strain
by introduction of a disruption cassette with a selectable
marker by homologous recombination is straightforward and
rapid. In fact, a deletion library of all nonessential yeast genes
was recently made commercially available [6,7]. Likewise,
libraries of almost all genes with green fluorescent protein
(GFP), glutathione-S-transferase (GST), or tandem affinity
purification (TAP) tags are widely used [8–11]. There are
techniques accessible in yeast that are not yet optimized, or
even available, for higher eukaryotes, such as suppressor
screening and synthetic genetic arrays for identification of
genetic interactions, demonstrating the power of yeast as a
model for NCL .
S. cerevisiae is used to study the juvenile form of NCL in
which CLN3, encoding a yet uncharacterized protein, is
mutated . Btn1p, also designated Yhc3p, is 39% identical
and 59% similar in amino acid sequence to human CLN3 (Fig.
1) . Btn1p is a 46kDa integral membrane protein, and like
CLN3, has several predicted phosphorylation, myristoylation,
and glycosylation sites and is predicted to be farnesylated
[4,15–20]. Furthermore, Btn1p and CLN3 are functional
homologs, in that plasmid-derived CLN3 can complement for
the absence of Btn1p in the BTN1 deletion strain, btn1-Δ,
suggesting that the primordial function of CLN3 is conserved
in yeast [21,22]. This property of the yeast model has the
potential as a powerful tool to test the functionality of CLN3
and Btn1p constructs. For example, it is unclear if GFP fusions
of CLN3 are functional , and a simple complementation
assay in the yeast model could test this. Importantly, the
residues that are mutated in Batten disease patients are
conserved in Btn1p, suggesting that the primary activity of
the protein is conserved. Moreover, the yeast model has been
used to test the CLN3 point mutants' ability to complement
BTN1 and it was found that disease severity correlated with
the degree of complementation [22,24]. Human CLN3 has
been localized to the lysosome in various studies and cell types
[15,25] (reviewed in [26,27]). Similarly, Btn1p has been
localized to the vacuole, the analogous structure to the higher
eukaryotic lysosome, further emphasizing the relevance of the
yeast model [28,29].
To date, Btn1p has been implicated in three main cellular
processes, two of which have been validated in mammalian
models. Although not completely independent, Btn1p has been
linked to regulation of cellular pH, basic amino acid
homeostasis, and nitric oxide production [29–32]. Using pH
sensitive dyes, Pearce and colleagues first reported that the
vacuolar pH in btn1-Δ cells was decreased as compared to
BTN1+vacuoles at early growth and continued to increase
throughout log and stationary phases . More recently, it has
been shown that as btn1-Δ cells grow, vacuolar pH will rise
above that of normal . Moreover, vacuolar pH in both
normal and btn1-Δ strains was shown to be altered by
extracellular pH. In btn1-Δ cells, activity of the plasma
membrane H+-ATPase is increased, likely acting to buffer
altered vacuolar pH. Importantly, this led to the discovery of a
plate phenotype for the btn1-Δ strain, which due to the
increased plasma membrane H+-ATPase activity has an
elevated rate of media acidification. This elevated media
acidification allows btn1-Δ cells to grow in the presence of D-
as the increased acidity of the medium results in ANP being
non toxic to btn1-Δ cells whereas ANP is toxic to BTN1+cells
[22,32] . In addition, when the vacuolar pH of btn1-Δ cells was
artificially increased using chloroquine, the plasma membrane
ATPase activity decreased strengthening the link between
external and vacuolar pH [29,31]. Importantly, these studies
demonstrated that human CLN3 could complement for the ANP
phenotype in btn1-Δ yeast. The pH and ANP results are in
contrast to the Schizosaccharomyces pombe model, where an
increased vacuolar pH and sensitivity to ANP are observed in
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908S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
the deletion  (see below). Subsequent follow up studies on
the pH of lysosomes from human fibroblasts indicated that pH
was slightly elevated in JNCL, suggesting that indeed defects in
CLN3 result in a disruption in the regulation of this organelle's
pH [22,35]. However, limitations in what can be explored in cell
culture means that it is not feasible to explore the possibility of a
correlation between the dynamic change from lower to higher
pH of the lysosome and the disease. Nevertheless, it is clear that
the processes that govern vacuolar/lysosomal pH are disrupted
in the absence of a functional Btn1p/CLN3 ultimately leading to
an elevated pH of this organelle.
and lysine levels and ATP-dependent vacuolar arginine uptake
are significantly decreased in btn1-Δ . This is interesting
since the vacuole acts as a storage organelle for sequestration of
transport returns to normal when human CLN3 is expressed in
btn1-Δ. Moreover, lysosomes isolated from JNCL patient
human lymphoblasts cell lines demonstrate decreased arginine
transport [21,38]. The role of Btn1p in both regulating vacuolar
pH and arginine transport has recently been clarified. In a recent
Fig. 2. Human PPT1 aligned with corresponding model organism homologs. Sequences were aligned usingthe multalin program.(A) Human PPT1 with fly PPT1; (B)
Human PPT1 with worm PPT1; (C) Human PPT1 with yeast Ppt1p.
Fig. 1. Human CLN3 aligned with corresponding model organism homologs. Sequences were aligned using the multalin program. (A) Human CLN3 with fly CLN3;
(B) Human CLN3 with the three worm CLN3 sequences; (C) Human CLN3 with the yeast Btn1p sequences.
909S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
publication, the coupling of proton pumping and the activity of
the vATPase was found to be dependent on extracellular pH
. Importantly, btn1-Δ, results in an alteration in the
coupling of proton pumping and the activity of the vATPase
. Thus, defective arginine transport in btn1-Δ could result
from an alteration in the regulation of the electrochemical
gradient driving this transport . Interestingly, subsequent
studies aimed at dissecting out whether alterations in intracel-
that overexpression of Can1p, the plasma membrane basic
Fig. 3. Human CTSD aligned with corresponding model organism homologs. Sequences were aligned using the multalin program. (A) Human CTSD with fly
CATHD; (B) Human CTSD with asp-4; (C) Human CTSD with the yeast Pep4p and Sxa1p sequences.
910S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
amino acid transporter, is lethal [39,40]. It is tempting to
speculate that the decrease in intracellular arginine and lysine
levels may result from a buffering mechanism against arginine
and lysine being toxic to cells lacking Btn1p [39,40].
Alternatively, it should be noted that BTN1+ cells with either
endogenous or overexpressed levels of Can1p have similar rates
of plasma membrane arginine uptake, suggesting that btn1-Δ
cells may lack the ability to regulate the amount or activity of
Can1p at the plasma membrane, resulting in arginine and lysine
toxicity.It is pertinent to point out that as amino acid levels,
and possibly metabolism, are clearly affected by btn1-Δ, the
studies described have avoided the typical use of auxotrophic
markers for gene deletion and genetic studies . In other
words, btn1-Δ strains have identical amino acid growth
requirements to that of wild type to avoid studying artifacts
of altered amino acid metabolism.
A recent study has highlighted involvement of a third
pathway in the absence of Btn1p, namely nitric oxide (NO)
synthesis . As arginine serves as the substrate for NO
synthesis this is also likely linked to the aforementioned
alterations in arginine levels. Specifically, a new publication by
Osorio and colleagues demonstrated that btn1-Δ cells are more
resistant to menadione due to defective synthesis of NO and
consequent decreased levels of reactive oxygen and nitrogen
species. If btn1-Δ cells are preincubated with arginine before
menadione exposure, the phenotype is lost, suggesting that the
decrease in nitric oxide results from the decrease in cellular
arginine . Although this phenotype may result as a
consequence of the primary defect associated to lacking
Btn1p, it could underlie pathophysiological aspects of the
disease. It will be important to recapitulate these observations in
human cell lines.
Microarray analysis of btn1-Δ strains revealed that BTN2
mRNA levels were increased in a btn1-Δ background .
Btn2p interacts with Yif1p, Rhb1p, and Ist2p, with deletion of
BTN2 resulting in an altered localization of these proteins
[30,43,44]. Yif1p is involved in ER to Golgi transport, Rhb1p is
a small GTPase that has been implicated in plasma membrane
arginine transport regulation, and Ist2p is a putative ion channel
at the plasma membrane [45–48]. Taken together, these
observations would suggest that Btn2p is involved in
trafficking. Btn2p does not have a true mammalian homolog,
however, as a cytosolic coiled-coil protein, it shows very
specific domain similarity to the higher eukaryotic protein
Hook1. Hook1 is a microtubule binding protein involved in
trafficking to the late endosome, multivesicular body formation,
and endosomal fusion [49–53]. Up-regulation of Btn2p may be
explained by the pH alterations as it has been shown that
decreases in vATPase activity can have an effect on protein
trafficking and degradation [54–60]. Therefore, up-regulation
of Btn2p may result in a compensatory response to minimize
disruptions in protein trafficking in btn1-Δ cells due to altered
In summary, it appears that the primary defect in btn1-Δ
involves a disruption in regulation of vacuolar pH, suggesting
that Btn1p functions in a pH regulatory pathway. However, at
this point a direct role for Btn1p in arginine homeostasis, nitric
oxide production or protein trafficking cannot be ruled out. It is
important to note that studies thus far have focused on
determining the role for Btn1p through the use of btn1-Δcells
that lack Btn1p and are therefore based on a loss-of-function
model, where the cell may be altering these pathways to correct
for the loss of Btn1p. It is possible that pH alterations are a
secondary consequence of the loss of Btn1p, and the primary
defect is yet to be elucidated. Future studies are focused on
looking on the direct function of Btn1p, especially its role in
coupling transport mechanisms with vATPase activity. S.
cerevisiae has played and will continue to play a strong part
in the efforts to understand JNCL.
Recently, Cathepsin D mutations have been implicated in
CNCL . Cathepsin D, a lysosomal aspartic protease, is highly
conserved throughout lower and higher eukaryotes. S. cerevi-
siae Pep4p shares 43% identity and 58% similarity with human
Cathepsin D (Fig. 3) . Pep4p is a nonessential enzyme
required for activation of other vacuolar proteases and glucose-
induced vacuolar degradation of peroxisomes, with deletion of
PEP4, pep4-Δ, resulting in accumulation of vacuolar protease
precursors and aberrant vacuolar morphology [62–69]. Because
Pep4p and pep4-Δ are well characterized, S. cerevisiae could
become a useful CNCL model.
2.2. Schizosaccharomyces pombe
Fission yeast, Schizosaccharomyces pombe, has recently
been developed as a model system to study Batten disease. Itis a
popular model organism that over the past 50years has greatly
influenced the understanding of cell cycle control and cell
division. It is a rod-shaped eukaryote that grows in a highly
polarized manner and divides by medial fission. Fission yeast
have 4912 genes organized onto three chromosomes, which is
the smallest number of protein-coding genes yet recorded for a
eukaryote. Like S. cerevisiae, fission yeast has a fast cell cycle
and exists in both the haploid and diploid states. Genes can be
readily deleted, mutated and tagged allowing for deletion/
mutation characterization and localization studies. Inhibition of
gene expression by RNAi is also under development. A deletion
library of all nonessential genes is near completion, as is a
library containing almost all the genes tagged with green
fluorescent protein (GFP). cDNA and genomic libraries exist
that allow for powerful suppressor screen studies. Importantly
for the study of lysosomal storage disorders, fission yeast cells
have large numbers of small vacuoles (50–80 per cell), the yeast
equivalent of the lysosome . Two NCL genes are conserved
in S. pombe, namely CLN1 and CLN3. The fission yeast
homolog of human CLN3, termed Btn1p, is a predicted
transmembrane protein of 396 amino acids that is 30% identical
and 48% similar to its human counterpart (Fig. 1). Importantly,
the same residues that are mutated in Batten disease patients are
conserved in Btn1p, suggesting that they are important for the
function of both CLN3 and Btn1p.
Recent work has focused on cell morphology and vacuole
function in a strain deleted for the btn1 gene, btn1Δ. This strain
is viable, but shows subtle and reproducible defects in cell cycle
progression, with an increased cell size and an increase in the
911 S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
number of mitotic and dividing cells . Significantly,
alterations in vacuole dynamics are evident. First, vacuoles
from btn1Δ cells are larger than those of wild type cells with a
mean size of 1.3μm compared to 0.9μm, and show a broader
vacuole size distribution, suggesting an inherent defect in
vacuole size regulation. Second, btn1Δ cells have an elevation
in vacuole pH of 1 pH unit, (pH 5.1 compared to pH 4.1 for wild
type cells) . A correlation is known to exist between
increased vacuole size and increased pH in fission yeast ,
and growth ofthe btn1Δstrainin acidic media (pH 4) was found
to restore the vacuolar size defect to wild type levels . Thus,
vacuole size is a reflection of the increased pH of the vacuole in
btn1Δ cells. btn1Δ cells were also found to be sensitive for
growth in media containing 1mM ANP. Although the
mechanism for this sensitivity in fission yeast remains to be
determined, growth of btn1Δ on plates containing 1mM ANP
was restored when this media was at pH 4 (S. Codlin,
unpublished). Thus, sensitivity of btn1Δ cell appears to be
related to pH homeostatic mechanisms. The increased vacuolar
pH and ANP sensitivity in S. pombe are in contrast to S.
cerevisiae btn1-Δ cells which have alterations in vacuolar pH
that change through growth, and is resistant to ANP. Although
intracellular pH in many yeast species including S. cerevisiae
and S. pombe has reported to be similar [72,73], these studies
indicate a clear difference in wild type vacuolar pH between
other yeast and S. pombe, with S. pombe having a vacuolar pH
near 4 and S. cerevisiae a pH around 6.2. Although, vacuolar pH
forS. pombewas previously reported tobe intherange of pH6–
of vacuolar pH in growing cells. In the btn1Δ S. pombe studies
the fluorophore lysosensor green D189 which vividly fluoresces
at the vacuole in wild type S. pombe was used. This compound
fails to fluoresce above pH 6, supporting the results that the
vacuole pH of wildtype S. pombe is less than pH 6 . This, in
vacuole pH of BTN1 deletions of the two different yeast models.
The vacuolar pH of S. pombe is similar to that of mammalian
cells which are also acidic .Expression of btn1and CLN3in
fission yeast deleted for btn1 causes a decrease in pH of the
vacuoles . These results are in contrast to overexpression
studies of CLN3 in HEK293 cells , where lysosomal pH is
increasedbutinagreementwithstudies onJNCLfibroblasts that
had a slightly elevated lysosomal pH . Importantly, a clear
genetic interaction has been established between btn1 and the
vATPase, the major complex associated with vacuolar acidifi-
cation. Cells deleted for both vma1 and btn1 exhibited slow
growth andsynthetic lethality at30°C .Whilethemolecular
basis of this interaction has not been determined, previously
described studies in S. cerevisiae implicating Btn1p in the
coupling of proton pumping and the activity of the vATPase
, may provide a clue.
Overexpression of N-terminally fused GFP-Btn1p and GFP-
CLN3 constructs in btn1Δ cells complemented the vacuolar
size and pH defects as well as the subtle cell growth defects,
proving that Btn1p and CLN3 are functional homologs. In
fission yeast, both Btn1p and CLN3 traffic to the vacuolar
membrane via FM4–64 stained pre-vacuolar compartments,
suggesting an endocytic trafficking route for Btn1p. Localiza-
tion of Btn1p using a functional GFP-tagged Btn1p to the
vacuole membrane was Ras GTPase Ypt7p dependent, with
Btn1p being excluded from the vacuole and held in prevacuolar
compartments in ypt7Δ cells . Cells deleted for both ypt7
and btn1 showing synthetic lethality at 36 °C and vacuoles in
these cells were larger than those of cells deleted for ypt7 alone
and again showed reduced pH. Btn1p must therefore have a
functional role prior to reaching the vacuole, and this function
impacts on vacuolar function.
In addition to the subtle growth defects in cell cycle
progression of btn1Δ cells at 25 °C, btn1Δ cells are also
temperature sensitive for growth at 37 °C (S. Codlin,
unpublished). btn1Δ cells passed through no more than three
cell cycles at this temperature and subsequently lost rod-shaped
morphology, resulting in swollen and rounded cells. Cell lysis
was found to be the subsequent cause of cell death. Electron
microscopy reveals grossly thickened cell walls and septum
regions, suggesting defects in the development of the main cell
wall components, the ά- and β-glucans. Indeed, btn1Δ cells
were found to be highly sensitive to zymolase, a β-glucanase,
but not to novozyme, an ά-glucanase. Also, the swelling
of 1 M sorbitol, an osmolyte, to the media, suggesting that the
swelling and lysis may be caused by aberrant osmoregulation in
btn1Δ cells .
S. pombe also has a homolog PPT1, which encodes a
lysosomal palmitoyl protein thioesterase 1 (Fig. 2) . The
PPT1 gene product is mutated in INCL patients . The S.
pombe ORF SPBC530.12c encodes the homolog to human
PPT1, denoted Ppt1p, fused to dolichol pyrophosphate phos-
phatase 1, Dolpp1p. The entire coding region is denoted pdf1
and the resulting propeptide is cleaved into its two distinct
proteins most likely by a kex-related protease . Through
deletion and complementation of pdf1, Cho and Hofmann
observed that Ppt1p is not required for viability, but Dolpp1p is.
However, Ppt1p is required for growth in sodium orthovanadate
and basic pH, a phenotype that indicates lysosomal dysfunction
in S. pombe, and, importantly, expression of human PPT1 could
complement for this phenotype suggesting that the primordial
function of the protein is conserved . This study again links
the NCLs to pH alteration phenotypes and supports the use of
fission yeast to study NCLs.
Fission yeast is also a potential model for CNCL as it has a
homolog to Cathepsin D, which is implicated in CNCL . The
S. pombe protein Sxa1p is 22% similar and 40% identical in
amino acid sequence to human Cathepsin D (Fig. 3). Sxa1p is
an aspartyl protease involved in the mating pathway . Thus,
fission yeast has proved to be a valid model for several NCLs
and will be valuable tool in the understanding of the molecular
basis and progression of CNCL.
2.3. Caenorhabditis elegans
The nematode Caenorhabditis elegans is a relatively simple
multicellular eukaryotic organism of approximately one mm in
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length, which normally lives in soil . These worms have a
reproduction cycle of approximately 3 days and a life span of
about two to 3 weeks. The C. elegans genome, divided over six
chromosomes, has been completely sequenced, and is predicted
to contain about 19,000 genes . The existence of the
hermaphrodite sex, which by self fertilization after a cross of
two different mutant strains easily yields homozygote offspring
and the possibility to grow large quantities for large genetic
screens, has made this animal a popular model for genetic
research . Moreover, extensive research into the nematode
anatomy and development has lead to a complete map of all 959
somatic cells and a full understanding of their cell lineage. The
nervous system of the worm comprises of 302 neuronal cells
with approximately 7600 synaptic junctions and is invariably
wired . The broad knowledge of the worm's neuronal
anatomy and wiring combined with its genetic power make this
organism very suitable to investigate the molecular mechanisms
underlying neuronal disorders.
Homologs of the CLN1 and CLN3 genes involved in the
infantile and the juvenile forms of NCL, respectively, have been
identified in C. elegans (Figs. 1 and 2). Models for both diseases
have been generated using worms with deletion mutations
identified by the C. elegans knock-out consortium and other
laboratories. The ppt-1 mutant, PPT-1 being the homolog to the
mutantlibrarythat wasgeneratedby chemical mutagenesis .
Two different ppt-1 mutant strains were isolated, both carrying a
deletion of at least two exons predicted to lead to truncation of
the protein . Although ppt-1 mutants displayed no
morphological, locomotion or neuronal defects, the onset of
egg-laying was delayed for approximately 4 h. The ppt-1
mutants appeared to carry more embryos than wild type, and
17.8% of them displayed “bagging”, a reproductive phenotype
found in only 1.6% of the wild type worms, where eggs hatch
inside the parent. Since not all of the mutant worms that failed to
lay them on time displayed the “bagging” phenotype, the effect
of the mutation varies inseverity.Lifespan and brood size of the
ppt-1 mutants were similar to wild type, when worms with the
bagging phenotype were not taken into account. Furthermore,
ppt-1 mutants were suggested to have a decreased “health
than wild type. In electron micrographs of ppt-1 mutant
nematodes, neuronal cells presented many enlarged mitochon-
dria containing less cristae and whorling inner membranes. The
ppt-1 mutants contained 26–29% more mitochondria than wild
type, and in six-day old adult worms the average size of ppt-1
mutant mitochondria was severely decreased. Currently, it is
unclear what caused this phenotype, but it is too mild to be used
in genetic screens for genes that may enhance or suppress the
mitochondrial effect. The ppt-1 mutants, however, did not
display two of the main characteristics of INCL, neuronal
degeneration or accumulation of storage material. Nonetheless,
the mitochondrial abnormalities of the ppt-1 mutants suggest
that these organelles are involved in NCL pathogenesis, as
previously suggested [84–97] and have prompted further
investigations into the integrity of mitochondria in INCL
patients as well as other INCL model organisms.
The generation of worm models for juvenile NCL turned out
to be more complicated: C. elegans is the only model organism
with three CLN3 homologs, designated cln-3.1, cln-3.2, and
cln-3.3. The presence of three CLN3 homologs in Caenorhab-
ditis briggsae, a nematode species closely related to C. elegans
suggests that the three genes have evolved before the separation
of the two species, some 100million years ago . Assuming
that the genes result from ancient duplications of a common
ancestor, their genomic sequences have diverged beyond
recognition, but the encoded protein sequences show consid-
erable homology . The degree of conservation across their
complete protein sequences suggests that none of the genes is a
pseudogene, which is expressed but has lost most of its original
function, as was shown for the elt-4 gene . The biological
reason for the existence of multiple CLN3 genes in the worm is
unknown, but expression analysis of the cln-3 genes using
promoter–GFP fusion constructs indicated that these genes
differ in their temporal and spatial expression patterns.
Expression of cln-3.1 was restricted to cells of the intestine
and was observed in transgenic hermaphrodite and male worm
embryos, larvae and adults . The cln-3.2 gene is expressed
in cells of the hypoderm only in adult worms of both sexes.
Expression of cln-3.3 was detected in the intestinal muscle cells
and hypoderm of adult hermaphrodite and male worms and also
in posterior diagonal muscle cells of males. Except for co-
expression of the cln-3.2 and cln-3.3 genes in hypodermal cells
of adult worms, each of the cln-3 genes is not expressed at
detectable levels in all cells. It should be noted that this does
not indicate that the cln3-1 genes are not expressed, but
merely that expression levels in other cell types (neurons) are
likely to be below the detection threshold of GFP by fluo-
Apart from the number of CLN3 homologs, the nematode
also differs from other model organisms in the organization and
regulation of some of its genes. C. elegans is one of the few
multi-cellular eukaryotic organisms in which several genes are
organized in operons . In C. elegans the question, whether
the gene products of operons are functionally related or just
linked to ensure coordinated temporal expression, cannot be
answered unequivocally in all cases and remains a topic of
investigation and discussion. The cln-3.2 and cln-3.3 genes
have closely located upstream genes and may therefore be in an
operon. In accordance with this, cln-3.2 was found to be trans-
spliced to a SL2 spliced leader, but cln-3.3 is associated to an
SL1 spliced leader. However, we cannot completely exclude
that cln-3.3 and its upstream gene, ZC190.2, are members of an
operon, as the putative cln-3.3 promoter–GFP fusion construct
failed to cause GFP fluorescence in transgenic nematodes (De
Voer et al., unpublished results), whereas transgenic worms
containing a larger upstream sequence including the ZC190.2
promoter and gene in front of the cln-3.3 promoter–GFP fusion
did show GFP fluorescence . Since both reporter
constructs contain an in-frame fusion of GFP to the first three
exons of cln-3.3 and thus are partial translational reporter
constructs , the GFP fluorescence from the longer one can
only be caused by the presence of additional cis-acting
elements. Currently, it remains unclear whether the ZC190.2
913 S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
promotor drives also the cln3.3 expression or whether cis-acting
elements overlapping the ZC190.2 coding region are respon-
sible for the observed expression pattern.
The cln-3.2 gene is the fourth in an operon also containing
erm-1, dnj-4, and dhs-1. The first gene, erm-1, encodes a
protein with homology to ezrin, radixin, and moesin proteins of
the ERM family of cytoskeletal linkers, and is involved in
organism development and positioning of cell–cell contacts
. ERM proteins have diverse roles in cell architecture, cell
signaling and membrane trafficking , and have recently
been shown to be important for actin assembly by phagosomes,
which may facilitate their fusion with lysosomes . This
gene is expressed from the two-cell stage onward throughout
the entire life of the worm in epithelial cells lining the luminal
surfaces of intestine, excretory canal, and gonad, whereas the
cln-3.2 gene was expressed in the hypoderm of adult worms
. This difference in expression between genes in the same
operon could be caused by common errors in operon
transcription, of which the probability decreases with increasing
distance between the operon genes, or their mRNAs may be
subject to differential mRNA destabilization . Expression
patterns of the other operonic genes, dnj-4, dhs-1, have not
been reported, and RNAi knockdown of these genes did not
result in obvious phenotypes (Wormbase website). Therefore,
the function of these genes can only be derived from protein
sequence homology. The DHS-1 protein has both chaperone
and heat shock protein domains, which could indicate a role in
protein folding. The dhs-1 protein has dehydrogenase and
reductase domains and may have a function in metabolism of
short chain alcohols. Although a role of ERM proteins in
lysosome–phagosome fusion potentially connects it function-
ally with cln-3.2, it is unclear whether a functional relationship
exists between cln-3.2 and the other genes in this operon.
JNCL worm models with single cln-3.1, cln-3.2, or cln-3.3
deletions were generated from the original mutants isolated
from the deletion mutant libraries by out-crossing six times into
wild type background to remove additional mutations .
Since the cln-3 single mutant models had a wild type
appearance, which might be caused by redundancy, they were
crossed to generate three double and one triple cln-3 mutant
models. The cln-3 triple mutant model was viable and
superficially displayed wild type behavior and normal mor-
phology, indicating that the cln-3 genes are not essential.
Comparison of the life span of the different models to wild type
worms suggested the cln-3.1 mutant has a shorter life span than
wild type worms, while cln-3.2 and cln-3.3 single mutants have
a normal life span. This effect becomes more prominent in the
cln-3 triple mutant when cln-3.2 and cln-3.3 are also deleted.
The cln-3.2 single mutant has a decreased brood size compared
to wild type. The brood size of the cln-3 triple mutant is
decreased more prominently than that of the cln-3.2 single
mutant, even though the other single mutants do not have a
significantly decreased brood size. To detect functional
aberrations, the cln-3 triple mutants have been investigated
extensively, using assays for correct neuronal function and
response to a diversity of external cues, such as temperature,
touch, presence of other worms, mating behavior (De Voer et
al., unpublished results). The integrity of the cln-3 triple mutant
nervous system was investigated using GFP which was
expressed from the unc-119 promoter in neuronal cells and
was similar to wild type in all of the tests. Electron micrographs
of cln-3 triple mutant neurons did not reveal altered morphology
or the presence of lysosomal storage material. The cln-3 triple
mutants could not be distinguished from wild type worms after
staining with organelle or compound specific fluorescent dyes,
Lysotracker Red, Acridine Orange, and Nile Red to assess
whether lysosomes, acidic organelles and lipid content,
respectively, were altered.
Human Cathepsin D, the protein mutated in CNCL patients,
and the worm homolog asp-4 share 58% identity and 73%
similarity (Fig. 3) . Worm asp-4 is an aspartyl protease that
mediates necrotic cell death and is required for neurodegenera-
tion [107–109]. Like the other model organisms, CNCL
research could benefit from work on the worm homolog of
2.4. Drosophila melanogaster
The evolutionary conservation of gene function between
humans and Drosophila make it an ideal model system for the
study of common eukaryotic cell biological mechanisms.
Furthermore, the sophisticated cell biological and genetic
reagents available in the fly provide the experimental tools for
elucidating the biological function of any gene that may play a
role in human disease processes. In regards to neurological
disorders, a recent study has shown that of the molecularly
defined human genetic disorders that produce mental retarda-
tion, which includes the NCLs, approximately 87% have a
homolog in the Drosophila genome . The success of using
Drosophila as an important model of neurological disease is
obvious in the contributions the fly has made to the
understanding of several kinds of neuronal degeneration
including Huntington's, Parkinson's, Alzheimer's, and several
Spinocerebellar Ataxias (reviewed in [111,112]).
As with the other small eukaryotic model systems, Dro-
sophila has clear homologs of the CNCL, JNCL, and INCL
disease genes (Figs. 1–3). The analysis of several genes,
including the INCL homolog Drosophila ppt1, suggests that
the fruit fly will also be an extremely valuable model system
to study lysosomal storage disorders and the NCLs in parti-
cular. For example, while the human homolog has yet to be
implicated in an NCL disorder, mutations in a predicted
lysosomal sugar carrier benchwarmer (bnch) produce both
autofluorescent lipopigments and neural degeneration charac-
teristic of NCLs [113,114]. Analysis of bnch mutations
demonstrated endo-lysosomal trafficking defects, defective
larval synapse development, impaired synaptic vesicle recy-
cling, and age-dependent synaptic dysfunction [113,114].
Mutations in the fly homolog of the lysosomal aspartyl protease
cathepsinD (Fig. 3), known to produce an ovine NCL and the
recently identified congenital NCL, also produce autofluores-
cent storage material and a low level of age-dependent
neurodegeneration in the adult Drosophila brain . These
fly mutants, along with the phenotypes of ppt1−flies described
914 S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
below, suggests an evolutionary conservation of cell biological
mechanisms in the fly that will be useful in our understanding of
human NCL phenotypes [115,116].
The Drosophila PPT1 homolog is ∼55% identical and
∼72% similar to the human protein at the amino acid level (Fig.
2) . The ppt1 transcript appears to be expressed
ubiquitously, although at different levels during all stages of
fly development . Consistent with the levels of mRNA,
PPT1 enzymatic activity is present at varying levels in all
tissues that have been tested . The development of a
Drosophila human disease model can be approached in two
distinct, yet complementary ways by producing a loss-of-
function mutation in the fly homolog or by using a gene-
overexpression approach to dissect gene function . In the
first approach a disease model can be developed to observe
whether aspects of the INCL phenotype are recapitulated in
ppt1−flies. The second approach makes use of a modular
misexpression system to overexpress ppt1 in order to produce
an in vivo assay of the protein's function. The results of both
approaches for INCL in Drosophila have proven to be fruitful.
Loss of function analysis of the ppt1 gene has been
performed through the use of a small deficiency, RNA
interference and several point mutations in the gene
[116,117]. Ppt1−flies are viable, although with a reduced life
span, and have storage material but no visible signs of
neurodegeneration . Targeted over-expression of ppt1 in
the developing Drosophila visual system using GMR-Gal4
leads to the loss of cells, including neurons, through apoptotic
cell death both early in eye development and also after
ommatidial differentiation has finished, yielding black omma-
tidial spots . To determine whether the abnormal eye
phenotypes were the result of increased levels of ppt1
enzymatic activity or are due to an ectopic, non-wildtype
function of the protein, an enzyme dead form of ppt1 was also
misexpressed. Expression of a ppt1 serine 123 to alanine
(S123A) catalytic mutant with GMR-Gal4 yielded no observ-
able abnormal phenotypes when analyzed with SEM .
Further quantification of semi-thin retinal sections demonstrat-
ed a significant reduction in loss of rhabdomeres in ppt1-S123A
over-expressing eyes when compare to overexpression of the
wild type enzyme indicating that a majority of the ppt1 over-
expression phenotypes observed are due to increased levels of
the wildtype protein activity . These findings demonstrate
that, while recessive mutations that severely decrease PPT1
activity cause neuronal cell death in INCL patients, increased
levels of PPT1 activity can also lead to neurodegeneration,
revealing that the precise level of PPT1 activity is important for
neuronal cell survival . The development of both loss- of-
function and gain-of-function models for ppt1 in Drosophila
provides a firm base for the future examination of the cellular
basis of neuronal dysfunction and degeneration in INCL
The next step in NCL research is the elucidation of what
cellular processes are being disrupted in the patient's neuronal
cells that ultimately lead to dysfunction and degeneration.
Towards this end, work in cultured ppt1−/−mouse neuronal
cells demonstrated a reduction in synaptic vesicle pool size in
the absence of large amounts of storage material suggesting that
synaptic abnormalities may contribute to the early progression
of INCL . The Drosophila model system will also play an
important role in this endeavor. The power of the Drosophila
system lies in the ability to perform large-scale second site
genetic modifier screens once a fly disease model has been
produced . These genetic screens allow the unbiased
identification of the cellular pathways that are relevant to
protein function and disease progression. For other neurode-
generative disorders, such as Huntington's disease, the results
of Drosophila modifier screens have led to the identification of
genes that may be therapeutic targets for the treatment of human
patients . Modifier screens focused on ppt1 are presently
underway in Drosophila the results of which will facilitate the
identification of in vivo substrates and signaling pathways that
are regulated by ppt1 activity. While only ppt1 has been
extensively studied in Drosophila, the success of this work
bodes well for future genetic analysis and functional character-
ization of the Drosophila JNCL gene, cln3.
Human cathepsin D (CTSD) and fly cathepsin D (CATHD)
are 53% identical and 68% similar in amino acid sequence (Fig.
3). A Cathepsin D-deficient fly contains storage material that at
the ultrastructural level is similar to the storage material in
CNCL and INCL patients, indicating that the fly will be an
appropriate organism to study the storage material and NCL-
3. Autofluorescent storage material
The NCLs belong to a large group of disorders commonly
termed lysosomal storage disorders. For a majority of these
disorders, the substrates that accumulate and the mutated gene
products have been identified. What remains unclear is why
the build-up of these particular substrates produces the ob-
served disease pathophysiology . Is the substrate
accumulation the primary cause of the disorders or rather a
secondary symptom of the disease? In some cases it is
becoming clear that the storage phenotype underlies more
significant changes in cell physiology produced by the loss of
a particular protein. The loss of the protein may have a
primary effect on cellular processes such as cellular trafficking
or signal transduction, which on their own, or through se-
condary effects, leads to the disease pathology . Recent
work in mice, sheep and humans has thoroughly described the
cellular storage materials and progressive cellular pathology in
affected NCL brains (reviewed in ). This work suggests
that there is no clear connection between the amount of
storage material and the observed neurodegeneration in these
systems [122,123]. The analysis of the cathepsinD and ppt1
fly mutants further supports this hypothesis [115,116].
The ppt1 mutant flies show CNS-specific accumulation of
autofluorescent storage material characteristic of the NCLs and
abnormal cytoplasmic inclusions although they are different in
morphology to the granular osmiophilic deposits typical of
INCL patients . Analysis of the adult nervous systems of
the ppt1−flies by transmission electron microscopy reveals the
presence of cytoplasmic deposits with some similarities to the
915 S.N. Phillips et al. / Biochimica et Biophysica Acta 1762 (2006) 906–919
multi-laminate deposits present in Tay–Sachs patients .
Expression of a ppt1 cDNA in the nervous systems of ppt1−
flies using the Gal4/UAS system rescues the laminar deposit
phenotype and the autoflouresence in ppt1−males was rescued
by a genetic duplication of the ppt1 chromosomal region
located on the Y chromosome . Taken together, these data
suggest that the NCL-like phenotypes observed are due to the
loss of ppt1.
In C. elegans ppt-1 and cln-3 triple mutant worms, storage
of autofluorescent lipopigments could not be detected. There-
fore, an attempt was made to increase the amount of
lipopigments in the cln-3 triple mutant worm model by
overexpressing the main component of the storage material
found in Batten disease patients, the hydrophobic subunit c of
the mitochondrial ATP synthase [124–127]. The only homolog
to the human ATP5G1 gene encoding subunit c in C. elegans is
atp-9. Overexpression of this worm homolog was deleterious to
wild type animals, causing overall structural impairment,
increased transparency, and near paralysis (De Voer et al.,
manuscript in preparation). On electron micrographs of worms
overexpressing subunit c, damaged mitochondria could be
observed, which is in accordance to the loss of mitochondrial
staining with Mitotracker Red seen in these animals. A mild
subunit c overexpression in a cln-3 triple mutant background
allowed the worms to survive, but did not result in an obviously
different phenotype compared to mild subunit c overexpression
in a wild type background.
It has been assumed that the use of single-celled organisms
like yeast in the study of lipopigment was futile due to their
short life-spans. Not only did they not live long enough to
accumulate the storage material to a detectable level, but normal
budding yeast have increased total cellular fluorescence as they
age, making it difficult to detect the vacuolar storage material
fluorescence above normal background fluorescence. However,
with new, more sensitive techniques, yeast model organisms are
likely to become increasingly an asset in the assessment of the
processes that lead to storage material. For example, both yeast
models do have detectable storage material. Autofluorescence
in the Schizosaccharomyces pombe JNCL model has been
observed upon growth at 37 °C in that cell swelling was
accompanied by an increase in vacuolar autofluorescence and
the accumulation of neutral lipids, both characteristic hallmarks
of Batten disease (S. Codlin and S.E. Mole, unpublished).
Furthermore, the Saccharomyces cerevisiae JNCL model
shows a small amount of fluorescent material co-localizing
with the vacuolar marker FM4-64 after entering the stationary
phase of growth (N. Muzaffar and D. A. Pearce, unpublished).
However, caution in interpreting subtle differences in the accu-
mulation of storage material in yeast will be necessary as the
time-frame for this accumulation is beyond the growth phases
that are characteristically studied.
The model organisms S. cerevisiae, S. pombe, C. elegans,
and D. melanogaster have pushed NCL research toward areas
that, without them, probably never would have been uncovered.
Most of what has been shown in the model organisms can be
recapitulated in mice or humans making these small eukaryotes
invaluable. In JNCL, they are responsible for uncovering the pH
maintenance pathway for future studies. Research into INCL
using the model organisms has brought us further in
understanding the activity of PPT1 and what can occur in its
absence. Model organisms will continue to be a cornerstone in
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