Download Slide 1

Role of cathepsin D in U18666A-induced cell death in mouse primary hippocampal neurons
A.
1
AMRITRAJ ,
1Dept
Background: Cathepsin D
D.
2
VERGOTE ,
T.
2
REVETT ,
M.
1
SONG ,
D.
2
WESTAWAY ,
S.
3
KAR
of Psychiatry, 2Dept of Med., 3Depts of Psychiatry and Med., Univ. of Alberta, Edmonton, AB, Canada
Cholesterol accumulation alters primary neurons viability
Cholesterol accumulation alters cathepsin D level/activity
Cathepsin D is a soluble lysosomal aspartic protease of the pepsin
superfamily, distributed ubiquitously in almost all tissues including the brain.
Functionally, the enzyme has been involved in a variety of biological
activities including metabolic degradation of intracellular proteins, activation
of some hormones and growth factors, brain antigen processing and
regulation of cell death mechanisms (Chwieralski et al., 2006; Benes et al.,
2008; Boya and Kroemer, 2008).
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mcb&part=A4816
In certain physiological and pathological conditions, cathepsin D escapes
normal targeting mechanism and is secreted from the cells (Benes et al.,
2008). Also, partial lysosomal permeabilization with subsequent release of
cathepsin D can trigger apoptosis or apoptosis-like death, whereas
generalized rupture results in rapid cellular necrosis. Cathepsin D may thus
have an important role in a variety of lysosomal storage disorders that are
associated with extensive neurodegeneration and progressive cognitive
decline. However, at present the significance of cathepsin D either in the
degeneration of neurons and/or development of pathological features
associated with any of these diseases remains unclear.
Background: Niemann-Pick disease type C
Niemann-Pick disease type C (NPC):
 autosomal recessive neurovisceral disorder,
 abnormal accumulation of unesterified cholesterol and glycosphingolipids
within the endosomal-lysosomal (EL) system of many tissues including the
brain.
 widespread neurological deficits such as ataxia, dystonia, seizures and
dementia that eventually lead to premature death,
 common neuropathological features with Alzheimer’s disease (AD)
including the formation of neurofibrillary tangles, increased levels of
intracellular amyloid b peptide and the loss of neurons.
EL system is altered in “at risk” neurons of both AD and NPC brains, which
are reflected by an increased volume of early endosomes and lysosomes and
enhanced synthesis of all classes of lysosomal hydrolases including cathepsin
D.
Mice recapitulating NPC pathology have also shown an up-regulation of
cathepsin D level/activity in selected brain regions, but their significance, if
any, in the development of pathology and/or degeneration of neurons has
yet to be established (Liao et al., 2007; Amritraj et al., 2009).
The class 2 amphiphile U18666A has been used in
cellular models of NPC pathology (Cheung et al.,
2004). U18666A alters the trafficking of cholesterol by inhibiting its transfer between the plasma
membrane, the ER and the lysosomes. U18666A
induces the dysfunction of lipid storage under in
vitro paradigm resulting in the accumulation of
cholesterol that can be visualized by filippin
staining (right panel). This U18666-induced
cholesterol accumulation can be associated with
cell death as observed in NPC pathology.
This work was supported by:
Aim of the study
To explore, in a cellular model, the influence of cholesterol
accumulation on cathepsin D level/activity and the potential role
of this lysosomal enzyme in NPC-associated neurodegeneration
U18666A-mediated cholesterol accumulation induced neuronal cell
death in primary hippocampal cultures in a concentration and timedependent manner as evidenced by MTT assay (A-B), Hoechst 33258
staining (C-F) and Live-dead assay (G-H). The Live-dead assay uses
Calcein AM staining to show the intracellular esterase activity in living
neurons (green; arrow heads), while EthD-1 depicts dead neurons with
disintegrated plasma membrane (red; arrows). The cell death induced
by 5 μg/ml U18666A is accompanied by caspases-9 and -3 activation as
evidenced by immunoblots (I). All results are presented as means
±SEM and were obtained from three separate experiments performed
in triplicate. Scale bar = 25 µm. *p < 0.05, **p < 0.01, ***p < 0.001.
U18666A-treated hippocampal cultured neurons
showed larger EL vesicles
labeled with LysoSensor
dye DND-160 in treated
neurons (arrows)
compared to untreated
control (Ctrl) cultures (AC). This treatment also
decreased the level of cathepsin D (D and E; immunoblots and quantification, respectively) but increased its activity (F) and
mRNA levels (G) compared to control, untreated cultures (Ctrl). Cytosolic levels of cathepsin D and cytochrome C was higher in
the treated hippocampal neurons compared to controls (H). All results are presented as means ± SEM and were obtained from
three separate experiments. Scale bar = 40 µm. *p < 0.05, **p < 0.01, ***p < 0.001.
Cathepsin D mediates cholesterol accumulation-induced neuronal death
Summary of the results
 U18666A-mediated toxicity in hippocampal primary neurons is accompanied
by increased levels of cathepsin D mRNA, enzyme activity and cytosolic levels
along with caspases-9 and -3 activation
 Cathepsin D inhibitor pepstatin A can partly prevent the toxicity by
attenuating the activation of caspase-dependent pathways
Cathepsin D inhibitor pepstatin A protects against U18666A-mediated toxicity in hippocampal neurons as
measured using the MTT assay (A), Hoechst 33258-labelling (B) and Live-dead assay (C-E). Pepstatin A treatment
can partially reverse the relative increase in the cytosolic cathepsin D and cytochrome C levels in U18666A-treated
cultured neurons (F) together with the activation of caspases-9 (G) and -3 (H). All results, which are presented as
means ± SEM, were obtained from three separate experiments. Ctrl, control; UA, U18666A; CYTO, cytoplasmic;
MEMB, membrane; Pep A, pepstatin A; NUC, nuclear. Scale bar = 25 µm. *P < 0.05; **P < 0.01; ***P < 0.001.
 Down-regulation of cathepsin D level by siRNA treatment rendered N2a cells
resistant to U18666A-induced toxicity
 Cathepsin D released from U18666A-treated neurons or application of
exogenous enzyme can induce toxicity to hippocampal neurons
Discussion/Conclusion
Increase of cathepsin D level/activity in affected regions may be involved in
the degeneration of neurons following lysosomal destabilization and enzyme
leakage into the cell cytoplasm
Like primary cultured neurons, U18666A treatment induced cholesterol accumulation in N2a neuronal cells (A and B; arrows). N2a cells are susceptible to 3 μg/ml U18666A-induced
neurotoxicity as evidenced by MTT assay (C). Cathepsin D-targeting siRNA efficiently decrease the levels of both pro- and active-cathepsin D in N2a cells compared to scrambled
siRNA (D and E). Knocking down cathepsin D using siRNA in N2a cells prevents U18666A-induced toxicity compared to cells treated with scrambled siRNA as detected using the MTT
assay (F). All results are presented as means ± SEM and were obtained from, at least, three separate experiments. Scale bar = 25 µm ***p < 0.001.
 Release of cathepsin D both intracellularly into the cytosol and extracellularly
into the conditioned media following treatment with U18666A can trigger
degeneration of neurons
 Underlying mechanisms by which extracellular cathepsin D induce toxicity in
primary neurons remains unclear
 The neuronal vulnerability to cholesterol accumulation may be conditioned
by the cytosolic level/activity of cathepsin D. However, this needs to be
explored further under in vivo conditions.
Cholesterol accumulation altered cathepsin D release impacts neuronal survival
References
U18666A treatment increases the release of cathepsin D from neurons as detected in the
conditioned media of the hippocampal neurons (A and B). Exposure of the hippocampal neurons to
conditioned media from U18666A-treated neurons induces loss of viability (C). This toxicity is
prevented following removal of cathepsin D using cathepsin D antibody tagged beads (D). Exogenous
cathepsin D is toxic to primary hippocampal neurons as detected by MTT (E) and EthD-1 (F and G)
assays.
 Amritraj A., Peake K., Kodam A., Salio C., Merighi A., Vance J.E. and Kar S.
Increased activity and altered subcellular distribution of lysosomal enzymes
determine neuronal vulnerability in Niemann-Pick type C1-deficient mice. Am. J.
Pathol. 2009, 175:2540-2556.
 Benes P., Vetvicka V. and Fusek M. Cathepsin D - many functions of one aspartic
protease. Crit. Rev. Oncol. Hematol. 2008, 68:12-28.
 Boya P. and Kroemer G. Lysosomal membrane permeabilization in cell death.
Oncogene. 2008, 27: 6434-6451.
 Cheung N.S., Koh C.H., Bay B.H., Qi R.Z., Choy M.S., Li Q.T., Wong K.P. and
Whiteman M. Chronic exposure to U18666A induces apoptosis in cultured murine
cortical neurons. Biochem. Biophys. Res. Commun. 2004, 315:408-417.
 Chwieralski C., Welte T. and Buhling F. Cathepsin-regulated apoptosis. Apoptosis
2006, 11:143-149.
 Liao G., Yao Y., Liu J., Yu Z., Cheung S., Xie A., Liang X. and Bi X. Cholesterol
accumulation is associated with lysosomal dysfunction and autophagic stress in
Npc1-/- mouse brain. Am. J. Pathol. 2007, 171:962-975.