Upregulation of cathepsin D in the caudate nucleus of primates with experimental parkinsonism

Abstract

Background: In Parkinson's disease there is progressive loss of dopamine containing neurons in the substantia nigra pars compacta. The neuronal damage is not limited to the substantia nigra but progresses to other regions of brain, leading to loss of motor control as well as cognitive abnormalities. The purpose of this study was to examine causes of progressive damage in the caudate nucleus, which plays a major role in motor coordination and cognition, in experimental Parkinson's disease.

Results: Using chronic 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine treatment of rhesus monkeys to model Parkinson's disease, we found a upregulation of Cathepsin D, a lysosomal aspartic protease, in the caudate nucleus of treated monkeys. Immunofluorescence analysis of caudate nucleus brain tissue showed that the number of lysosomes increased concurrently with the increase in Cathepsin D in neurons. In vitro overexpression of Cathepsin D in a human neuroblastoma cell line led to a significant increase in the number of the lysosomes. Such expression also resulted in extralysosomal Cathepsin D and was accompanied by significant neuronal death associated with caspase activation. We examined apoptotic markers and found a strong correlation of Cathepsin D overexpression to apoptosis.

Conclusions: Following damage to the substantia nigra resulting in experimental Parkinson's disease, we have identified pathological changes in the caudate nucleus, a likely site of changes leading to the progression of disease. Cathepsin D, implicated in pathogenic mechanisms in other disorders, was increased, and our in vitro studies revealed its overexpression leads to cellular damage and death. This work provides important clues to the progression of Parkinson's, and provides a new target for strategies to ameliorate the progression of this disease.

Figure 1

MPTP treatments and clinical score of monkeys. The rhesus monkeys were treated with MPTP at the time points indicated by the color-coded asterisks. Doses were given on two consecutive days at the amounts (in mg/kg) indicated under the asterisks. The Kurlan rating score (rating the animal's posture, gait, tremor, general mobility, hand movements, climbing, holding food, eating, balance, gross motor skills, and defense reaction) is given for each monkey over time. Untreated control animals and animals before MPTP treatment had values of 0 (normal), higher values indicate increased deficiency. Daggers indicate time of sacrifice of each animal.

Figure 2

Loss of dopaminamergic nerve terminal markers in the striatum of MPTP treated monkeys. (A) Immunohistochemical staining reveals strong staining of striatal sections of control brains (left panel: top and middle) for tyrosine hydroxylase and dopamine transporter; whereas, MPTP administered monkey striatal sections (right panel: top and middle) show a minimal staining. The bottom panels are sections stained with luxol fast blue. The images are unmagnified scans of the slides. (B) Immunohistochemical staining reveals strong staining of striatal sections a control brains (left) for TyH and DAT; whereas, in an MPTP administered monkey, striatal sections (right) show minimal staining, Bar = 20 μm. The images are representative of the monkeys in each group.

Figure 3

Cat D is upregulated in MPTP caudate nucleus. (A) Taqman qRTPCR for Cat D mRNA was performed on RNA from caudate samples. A significant upregulation in Cat D mRNA is seen. The delta Ct (dCt) method was performed to determine relative concentrations, using the average of the Ct of 18S and GAPDH as the normalizing value. Mean and standard error of the mean shown, significance is indicated by *p < 0.05 (Student's t-test). (B) Photomicrographs of representative sections of caudate of two animals (#1, #2) from each group of the control and MPTP monkeys. Caudate sections were immunohistochemically stained with anti-Cat D. Minimal staining is seen in sections from control animals (bar = 50 μm) and in corresponding increased magnification (40×, bar = 10 μm); whereas, in MPTP treated animal, there is increased staining. Increased magnification (40×, bar = 10 μm) reveals that the staining is seen within neurons in the cell body, axon hillock (bottom right).

Figure 4

Double immunofluorescence of CatD with MAP2 and IBA1 in CN od MPTP monkey. (A) Double immunostaining was performed on MPTP CN sections with the neuronal marker anti-MAP2 (red) and anti-Cat D (green). The merged image illustrates a co-localization of Cat D with MAP2, confirming its presence in neurons. Bar = 20 μm. Higher magnification of single neuron is also provided (bottom panel, Mag). Bar = 5 μm. (B) Double immunostaining was performed on MPTP CN sections with the microglial marker anti-IBA1 (red) and anti-Cat D (green). The merged staining of Cat D with IBA1 conforms the absence of Cat D in microglial cells. Bar = 20 μm. Higher magnification (bottom panel, Mag). Bar = 5 μm.

Figure 5

Lysosomal localization of Cat D. (A) Double immunostaining was performed on control and MPTP CN sections with anti-Cat D (green) and the lysosomal marker anti-LAMP2 (red). The merged image (right panel) illustrates a co-localization of Cat D with LAMP2, its increase in sections from MPTP treated animals. Bar = 20 μm. (B) Quantification of lysosomes was performed on sections, n = 10 neurons for each animal were examined, unpaired t-test was performed on the average lysosomes quantified from the three control (Mean ± SEM: 27.8 ± 0.89) and three MPTP (Mean ± SEM: 110.8 ± 4.5) animals, p < 0.001.

Figure 6

Overexpression of Cat D in BE-2 cells. (A) BE-2 neuroblastoma cells were either transfected with GFP (control) or with Cat D-GFP and lysosomes stained with lysotracker red. The GFP expressing BE-2 cells (left panels (LysT-Red, GFP-merge), bar = 5 μm) show few lysosomes localized inside the cell body (see Lysotracker red (LysT-red) panels); whereas, the Cat D-GFP transfected cells (right panels (LysT-Red, CD-merge), bar = 5 μm) show build up of lysosomes (see Lysotracker red (LysT-red) panels). GFP-merge and CD-Merge represent the corresponding merged images with green channel. (B) Quantification of lysosomes was performed on BE-2 cells transfected with either GFP or Cat D (n = 10 cells for each group). Unpaired t-test, p < 0.001 (C) Western blot showing the expression of Cat D (~30 kDa, the mature Cat D heavy chain) in non-transfected (control) and transfected (Cat D) BE-2 cells. β-actin was used as loading control. Lower bar graph is representative of 3 individual experiments. Mean and standard error of the mean shown, significance is indicated by ***p < 0.001.

Figure 7

Cat D is present outside of lysosomes. Fluorometric assay for cathepsin was performed on BE-2 neuroblastoma cell cytosolic extracts (Figure 6A) and on culture supernatants (Figure 6B) transfected with either GFP or Cat D, or non-transfected (NT). Relative fluorescence unit (RFU) per μg protein is calculated by normalizing the fluorescence signal to the amount of protein present in each sample. The experiments are representative of n = 3. Mean and standard error of the mean shown, significance is indicated by ***p < 0.001.

Figure 8

Cat D overexpression leads to release of lactate dehydrogenase (LDH) and caspase activation. (A) LDH assay was performed on the rat striatal neurons transfected with either vehicle alone or Cat D. For positive control, 2% Triton X-100 and staurosporine treatments were performed. Cat D overexpression significantly increased the amount of LDH in media. The experiment is representative of n = 4. Mean and standard error of the mean shown, significance is indicated by ***p < 0.001. (B) Live imaging performed on rat striatal neurons. Neurons were transfected with Cat D and stained for CaspACE FITC-VAD-FMK in situ marker. The green staining represents the activation of caspase and apoptosis. As seen, compared to control the Cat D transfected neurons show a clear activation in FITC caspase levels, including in neurites (boxed area in lower left panel enlarged in lower right, see processes on neuron marked by asterisk). Staurosporine treated neurons were used as a positive control. Bar = 50 μm.

Figure 9

Cat D overexpression leads to apoptosis. (A) Hoechst staining was performed on BE-2 neuroblastoma cells transfected with or without Cat D. Cat D overexpression clearly indicates fragmentation of nucleus in Cat-D GFP cells (see white arrow). The experiment is representative of n = 4. Bar = 5 μm (B) In situ measurement of apoptosis in Cat D transfected BE-2 neuroblastoma cells. As seen, the CY-5 staining in nucleus can be strongly visualized in Cat D transfected cells but not in control cells indicating the presence of fragmented DNA in Cat D. The experiment is representative of n = 4. Bar = 5 μm (C) Protein extracts from mitochondrial and cytosolic fractions, isolated from Cat D transfected and control cells, were blotted for Cyto-C, GAPDH, Tom20 and β-actin. As can be seen from western blot, there is clear increase in cytosolic presence of Cyto-C in Cat D transfected cells compared to controls, whereas another mitochondrial marker (Tom20) as well as a cytoplasmic marker (GAPDH) remain similar, indicating an activation of apoptotic pathway.

Figure 10

Schematic representation of disease progression in the caudate. (A) Depiction of the neuronal pathways involved in normal brain functioning, glutamatergic as well as dopaminergic innervations from cortex and substantia nigra (SN) to the caudate nucleus. (B) MPTP and PD affects the neurons in SN leading to loss of the dopaminergic projections to the caudate, leading to dysregulation of lysosomal pathways, and increasing the number of lysosomes as well as Cat D and extralysosomal Cat D. (C) Slow but persistent damage resulting from upregulation of Cat D expression and leakage leads to neuronal dysfunction and death through apoptosis or other mechanisms.