- Research article
- Open Access
Loss of leucine-rich repeat kinase 2 causes age-dependent bi-phasic alterations of the autophagy pathway
© Tong et al; licensee BioMed Central Ltd. 2012
- Received: 8 December 2011
- Accepted: 9 January 2012
- Published: 9 January 2012
Dominantly inherited missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson's disease, but its normal physiological function remains unclear. We previously reported that loss of LRRK2 causes impairment of protein degradation pathways as well as increases of apoptotic cell death and inflammatory responses in the kidney of aged mice.
Our analysis of LRRK2-/- kidneys at multiple ages, such as 1, 4, 7, and 20 months, revealed unique age-dependent development of a variety of molecular, cellular, and ultrastructural changes. Gross morphological abnormalities of the kidney, including altered size, weight, texture, and color, are evident in LRRK2-/- mice at 3-4 months of age, along with increased accumulation of autofluorescent granules in proximal renal tubules. The ratio of kidney/body weight in LRRK2-/- mice is increased at 1, 4, and 7 months of age (~10% at 1 month, and ~20% at 4 and 7 months), whereas the ratio is drastically decreased at 20 months of age (~50%). While kidney filtration function evaluated by levels of blood urea nitrogen and serum creatinine is not significantly affected in LRRK2-/- mice at 12-14 months of age, expression of kidney injury molecule-1, a sensitive and specific biomarker for epithelial cell injury of proximal renal tubules, is up-regulated (~10-fold). Surprisingly, loss of LRRK2 causes age-dependent bi-phasic alterations of the autophagic activity in LRRK2-/- kidneys, which is unchanged at 1 month of age, enhanced at 7 months but reduced at 20 months, as evidenced by corresponding changes in the levels of LC3-I/II, a reliable autophagy marker, and p62, an autophagy substrate. Levels of α-synuclein and protein carbonyls, a general oxidative damage marker, are also decreased in LRRK2-/- kidneys at 7 months of age but increased at 20 months. Interestingly, the age-dependent bi-phasic alterations in autophagic activity in LRRK2-/- kidneys is accompanied by increased levels of lysosomal proteins and proteases at 1, 7, and 20 months of age as well as progressive accumulation of autolysosomes and lipofuscin granules at 4, 7-10, and 20 months of age.
LRRK2 is important for the dynamic regulation of autophagy function in vivo.
- Parkinson's disease
- lysosomal proteins
Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmarks of PD are progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta of the brain and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies (LBs), in which α-synuclein aggregates are a major component [1, 2]. Although most PD cases occur sporadically, at least five genes (including α-synuclein, parkin, DJ-1, PINK1, and LRRK2) associated with monogenetic familial forms of the disease mimicking clinical symptoms of sporadic PD have been identified, permitting studies of the pathogenic mechanisms of PD using genetic approaches. Dominantly inherited missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common genetic cause of late-onset PD [3–9], highlighting the importance of LRRK2 in PD pathogenesis. LRRK2 is a large protein of 2527 amino acid residues, consisting of several functional domains, including a Ras-like small GTPase domain, a MAP kinase-like domain, as well as several protein-protein interaction domains, such as the leucine-rich repeat domain [6, 9, 10]. The disease-associated mutations in LRRK2 are present in all functional domains of the protein. Most LRRK2 mutations causes clinically typical PD, but the neuropathological features vary, ranging from pure nigral degeneration without LBs to nigral degeneration with brainstem or widespread LBs, or ubiquitin-positive inclusions, or neurofibrillary tau-positive tangles [9, 11].
Despite the disease relevance of LRRK2, its normal physiological role remains elusive. Elucidation of LRRK2 functions will provide insights into how mutations in LRRK2 lead to dopaminergic dysfunction and degeneration. Although the dominant inheritance of missense mutations and the lack of nonsense or deletion mutations in LRRK2 are consistent with toxic gain-of-function pathogenic mechanisms, we generated LRRK2-/- mouse models to study the normal physiological function of LRRK2 and to determine the consequence of inhibiting LRRK2 function. Similar to other PD genetic mouse models, such as α-synuclein transgenic [12–14], parkin-/- [15, 16], DJ-1-/- [17, 18], PINK1-/- [19, 20], and LRRK2 transgenic and knockin mice [21–25], LRRK2-/- brains did not develop overt dopaminergic degeneration . However, LRRK2-/- kidneys developed striking age-dependent abnormalities that are relevant to PD pathogenesis, such as impairment of protein degradation pathways, apoptotic cell death, oxidative damage, and inflammatory responses . There was striking accumulation and aggregation of α-synuclein and ubiquitinated proteins in the kidneys of LRRK2-/- mice at 20 months of age . The autophagy-lysosomal pathway, which has been implicated in various neurodegenerative diseases with protein aggregation-related pathologies, including Parkinson's disease and Huntington's disease [27–30], was impaired in LRRK2-/- kidneys at 20 months of age, as indicated by impaired conversion of LC3-I to LC3-II, a reliable indicator of the autophagic activity , and accumulation of p62, an autophagy substrate . Although these molecular and cellular changes are observed only in the kidney but not in the brain of LRRK2-/- mice, they are very similar to processes that are thought to be involved in PD pathogenesis, making LRRK2-/- kidneys a relevant and valuable in vivo model to study the physiological function of LRRK2 and to identify the downstream cellular and molecular pathways.
In the current study, our detailed time course study revealed an unexpected finding that loss of LRRK2 dysregulates the autophagy pathway in an age-dependent bi-phasic manner. The autophagic activity is elevated at young ages (e.g., 7 months) but reduced at an old age (e.g., 20 months). Furthermore, this process is accompanied by increased levels of lysosomal proteins and proteases as well as age-dependent, progressive accumulation of autolysosomes and lipofuscin granules. Thus, subsequent impairment of autophagy function in aged LRRK2-/- kidneys may be due to depletion of autophagy machinery and accumulation of subcellular structures containing undigested lysosomal components during aging.
Morphological and histological analyses of LRRK2-/- kidneys at various ages
In addition to the gross morphological abnormalities in LRRK2-/- kidneys beginning at 3-4 months of age, we observed many small autofluorescent puncta in the epithelial cells of the proximal tubules in the deep layer of the renal cortex in LRRK2-/- mice at 4 months of age, which became more evident and distributed more widely at 7 months of age (Figure 1C). By 20 months of age, the cytosolic regions of the epithelial cells of the proximal renal tubules are filled with larger autofluorescent structures (Figure 1C) that are lipofuscin granules . These data suggest that undigested autofluorescent materials accumulate in LRRK2-/- kidneys beginning as early as 4 months of age.
Up-regulation of kidney injury molecule-1 (Kim-1) in LRRK2-/- kidneys
To assess whether the striking abnormalities observed in LRRK2-/- kidneys caused any nephrotoxicity to epithelial cells of proximal renal tubules, we examined the levels of kidney injury molecule-1 (Kim-1 in rodents, KIM-1 in human), a very sensitive and specific biomarker for epithelial cell injury of proximal renal tubules in various settings [33, 34]. An increase in Kim-1 due to kidney injury can occur before any significant increase in serum creatinine . Kim-1 is localized to proximal tubule epithelial cells, and its expression is at a very low level in normal kidneys but increases dramatically after acute kidney injury . Long-term expression of KIM-1 is also observed in patients with chronic kidney disease although its level is lower than that after acute kidney injury . There was already a significant increase in the level of Kim-1 in LRRK2-/- kidneys at one month of age (Figure 2B), which then appeared grossly normal. At 7 months of age, there was approximately 10-fold increase in the level of Kim-1 in LRRK2-/- kidneys compared with wild-type controls (Figure 2B), but this increase was much lower than that in acute kidney injury models (> 500-fold up-regulation), such as those induced by ischemia . The increased expression of Kim-1 in LRRK2-/- kidneys persisted to 20 months of age (Figure 2B). These data suggest that while renal function evaluated by measuring blood urea nitrogen and serum creatinine appears normal, LRRK2-/- mice sustain chronic kidney injury, as indicated by 10-fold up-regulation of kidney injury molecule-1.
Age-dependent bi-phasic alterations of autophagic activity in LRRK2-/- mice
Western blotting also revealed an increased conversion of form I to form II of Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which is a homolog of LC3 and has also been reported to localize to autophagosomal membrane upon form-II formation , in LRRK2-/- kidneys at 7 months of age, further confirming enhanced autophagic activity. By 20 months of age, both forms I and II of GATE-16 were decreased in kidneys of LRRK2-/- mice (Figure 3). These results indicate that loss of LRRK2 in vivo increases autophagic activity initially followed by subsequent decreases of autophagic activity.
Age-dependent bi-phasic alterations of α-synuclein levels in LRRK2-/- kidneys
Age-dependent bi-phasic alterations of oxidation levels in LRRK2-/- kidneys
Accumulation of lysosomal proteins/proteases and autolysosomes in LRRK2-/- mice
Dominantly inherited mutations in LRRK2 are collectively the most common genetic cause of PD, but its normal physiological function remains less clear. We reported previously that loss of LRRK2 causes impairment of the two major protein degradation pathways (i.e., the autophagy-lysosomal pathway and the ubiquitin-proteasome system), accumulation and aggregation of proteins (such as α-synuclein, p62, and ubiquitinated proteins), and increased apoptotic cell death and inflammatory responses in the aged mice, suggesting that LRRK2 plays an essential role in the regulation of protein homeostasis . Although these molecular and cellular changes are observed only in the kidney but not in the brain of LRRK2-/- mice, they bear striking resemblance to processes that are thought to be involved in PD pathogenesis, suggesting that LRRK2 mutations may cause Parkinson's disease and cell death via impairment of protein degradation pathways, leading to protein accumulation and aggregation over time. A recent report shows similar gross morphological abnormalities in the kidneys of an independent line of LRRK2-/- mice as well as a line of kinase-dead mutant mice of LRRK2 . The presence of similar kidney phenotypes in at least four independent lines of LRRK2-/- mice [23, 26, 44] suggests that this is unlikely an artifact and that LRRK2 play an important role in the cell.
In the current study, we performed an age-dependent analysis of LRRK2-/- mice and compared morphological, ultrastructural, and molecular alterations in LRRK2-/- mice from 1 month to 20 months of age. We found that gross morphological abnormalities first become evident in LRRK2-/- kidneys at 3-4 months of age (Figure 1). Surprisingly, more detailed analysis revealed that the autophagic activity appeared enhanced at young age (e.g. 7 months), as evidenced by increased conversion of LC3-I to LC3-II, a reliable marker of autophagosome formation , and increased degradation of p62, one of the best characterized autophagy substrates , as well as increased degradation of α-synuclein (Figures 3 and 4). By 20 months of age, similar analysis showed reduced autophagic activity in LRRK2-/- kidneys (Figures 3 and 4). However, this age-dependent bi-phasic alteration of the autophagic activity is accompanied by progressive accumulation of autolysosomes, reduction of lysosomes, and the ultimate prevalent presence of large lipofuscin granules at 20 months of age (Figure 7).
During the normal process of autophagy (here referred to macroautophagy, the major form of autophagy) [28, 31, 41], a portion of cytoplasm, including damaged proteins and organelles, is first enclosed by isolation membrane (a double membrane-bound structure) to form an autophagosome, the outer membrane of which then fuses with lysosome to form so-called autolysosome. The internal material, including proteins and lipids, is degraded in the autolysosome by acid hydrolases originated from lysosomes, and the degradation products get recycled back to cytoplasm and are to be used as new building blocks and energy for cellular renovation and homeostasis . Any disruption along this process, such as those that affect initiation and elongation of isolation membrane, autophagosome formation, fusion of autophagosomes and lysosomes, and hydrolytic degradation, would alter the autophagic flux . On the one hand, the presence of a large number of autolysosomes is suggestive of enhanced autophagic flux in LRRK2-/- kidneys at young ages (4, 7, and 9-10 months), consistent with increased protein degradation at these ages; On the other hand, the unusual accumulation of such structures may also suggest deficits in turnover and/or recycling of autophagic components, leading to accumulation of autolysosomes, which may evolve into lipofuscin granules through excessive oxidation and crosslinking and eventually result in depletion of autophagic machinery and therefore impaired autophagic activity at old ages (20 months). Deficient regeneration of autophagic lysosomes has been reported to cause accumulation of autolysosomes [45, 46]. Consistent with this interpretation, compared with wild-type controls, normal lysosomes were rarely observed in proximal tubules of LRRK2-/- kidneys, where there were striking accumulation of autolysosomes (at young ages) and lipofuscin granules (at old ages).
In addition to gross morphological abnormalities observed in LRRK2-/- kidneys that first become evident at the age of 3-4 months, the ratio of kidney to body weight in LRRK2-/- mice significantly increased at young ages (~10% at 1 month and ~20% at 4 and 7 months) but dramatically decreased at 20 months of age (~50%, Figure 1). We also observed significantly increased levels of lysosomal proteins and proteases (e.g., LAMP-1, cathepsin B, and cathepsin D, including active forms and proforms) in LRRK2-/- kidneys beginning as early as one month of age throughout all the ages examined (Figure 6). One possibility is that loss of LRRK2 causes induction of autophagy initially via altered kinase signaling. During autophagy induction, synthesis of lysosomal proteins and proteases continues or even up-regulated although other proteins' synthesis is generally down-regulated . At older ages, due to a deficit in clearance or recycling of autolysosomes, the autolysosome-like structures cannot be digested and therefore accumulate and evolve into lipofuscin granules. The increased levels of lysosomal proteins and proteases could be from the accumulated autolysosome-like structures or indigestible lipofuscin granules, both of which contain components originated from lysosomes, including lysosomal proteins and proteases, since the number of lysosomes is not increased, but decreased instead.
Although these molecular and cellular changes are observed only in the kidney but not in the brain of LRRK2-/- mice, they are very similar to processes that have been implicated in pathogenesis of PD and other neurodegenerative diseases, making LRRK2-/- kidneys a relevant and valuable in vivo model, which provides a physiological setting for the studies of LRRK2 function and the identification of the cellular pathways that LRRK2 pathogenic mutations may affect. More questions await further investigation using this unique LRRK2-/- kidney as a model. For example, how does loss of LRRK2 cause bi-phasic alteration of autophagic activity? How does loss of LRRK2 lead to autophagy induction as well as deficits in clearance and/or regeneration of autophagy components? Interestingly, it has recently been reported that siRNA knockdown of LRRK2 increases autophagic activity and the R1441C mutation in LRRK2 induces accumulation of autophagic vacuoles of enlarged size in cultured HEK293 cells . Surprisingly, LRRK2 overexpression in cultured HEK293 cells has also been reported to cause autophagy induction through a calcium-dependent pathway . Although these results may seem contradictory with each other, which may be due to the fact that these studies were performed in cell culture systems using immortalized cell lines, rather than an in vivo physiological setting, they nevertheless implicate that LRRK2 is important for the dynamic regulation of autophagy function. LRRK2 has also been reported to localize to specific membrane subdomains, including autophagosomes and autolysosomes , suggesting that LRRK2 may directly participate in the dynamic process, including formation and clearance, of autophagic vacuoles. What is the role of aging process, which cannot be mimicked in cell culture systems, in this bi-phasic dysregulation of autophagic activity by loss of LRRK2? In addition, LRRK2 has been implicated in both transcriptional and translational regulation [51, 52]. Is protein synthesis besides degradation also affected in the absence of LRRK2? Last but not the least, why are these PD-like cellular changes present only in the kidney but not in the brain of LRRK2-/- mice? One possibility is that LRRK2-/- kidneys suffer the greatest loss of LRRK protein (LRRK1 + LRRK2) because the kidney not only has the highest expression level of LRRK2 compared to other organs (5-fold higher than the brain), but also has the least overlapping expression pattern between LRRK2 and LRRK1 , the other member of the LRRK family. This may explain why LRRK1 does not compensate for the loss of LRRK2 in the kidney, and loss of LRRK2 causes impairment of the protein degradation pathways and striking age-dependent kidney abnormalities. In the brain, LRRK1 may be able to compensate for the loss of LRRK2. This interpretation is supported by the finding that in the developing brain the expression level of LRRK1 is much higher than that of LRRK2, and it is broadly expressed [53, 54]. We are currently in the process of generating LRRK1/LRRK2-deficient mice to determine whether complete loss of LRRK in neurons, especially in dopaminergic neurons where oxidative stress is elevated, results in age-dependent protein aggregation, autophagy alteration, and neurodegeneration. Future studies aimed at addressing these important questions under a physiological setting using our unique LRRK2-/- kidney as a model would no doubt help us better understand the normal physiological function of LRRK2 and its role in PD pathogenesis.
Our latest studies of age-dependent development of phenotypes in LRRK2-/- kidneys show that LRRK2 is required for normal regulation of the autophagy-lysosomal pathway. Loss of LRRK2 causes impairment of the protein degradation pathways and striking age-dependent cellular changes in the kidney, which are similar to PD pathogenesis, making the LRRK2-/- kidney a unique and valuable model for elucidating the normal physiological role of LRRK2 under its physiological settings. LRRK2 mutations may cause Parkinson's disease and cell death by impairing protein degradation pathways, leading to protein accumulation and aggregation over time.
The generation and initial characterization of two independent lines (KO1 and KO2) of LRRK2-/- mice have been described previously . The mice used in this study were obtained by intercrossing heterozygous littermate mice, which were maintained on B6/129 genetic background. All mouse work follows the protocol approved by Harvard Center for Animal Resources and Comparative Medicine.
Histological and immunohistochemical analysis
Each mouse was anesthetized by intraperitoneal injection of sodium pentobarbital 15 min after injection of heparin (500 units in saline). The mouse was then transcardially perfused with 20 ml of Ringer's solution containing 0.25 g/L heparin and 5 g/L procaine followed by 25 ml of ice-cold 4% paraformaldehyde in 1× phosphate buffered saline (pH 7.4). The kidneys were dissected out and post-fixed in 4% paraformalhehyde at 4°C overnight and then processed for paraffin embedding following standard procedures. Kidney sections were cut at 8 μm. For immunohistochemical analysis, some tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 10 or 15 min in 10 mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity was quenched by incubating in 0.3% H2O2 in methanol. After blocking, sections were incubated with primary antibodies overnight at 4°C, followed by 1-h incubation with biotinylated secondary antibodies and 1-h incubation with Vectastain Elite ABC reagent and then developed using chromogenic DAB substrate (Vector Laboratories). For negative controls, primary antibodies alone or together with secondary antibodies were omitted from the incubation buffer.
Transmission electron microscopy analysis
Mice were perfused following a procedure similar to that for histological and immunohistochemical analysis above except a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) was used as the fixative. After overnight post-fixation at 4°C, the dissected tissues were then trimmed to 1-2 mm3 cubes and left in the fixative until processing for embedding in resin. Embedding was performed and ultrathin (60-80 nm) sections were cut by the Harvard Medical School EM facility following a routine protocol. EM images of ultrathin sections were collected on a Tecnai G2 Spirit BioTWIN electron microscope. Some of the tissues from the mice at 9-10 and 20 month of age used for EM analysis were from a third independent line of LRRK2-/- mice described previously , which also show similar kidney morphological phenotypes.
Measurement of blood urea nitrogen and serum creatinine
Approximately 0.3 ml of blood was collected from each mouse as described previously . Briefly, mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was collected into heparinized micro-hematocrit capillary tubes by nicking the tail vein of the anesthetized mice near the tip of the tail. Serum was prepared from the collected blood samples and stored at -80°C. Blood urea nitrogen and serum creatinine were measured following the classical methods for renal chemistry as describe previously .
Preparation of RIPA buffer-soluble and -insoluble fractions
Fresh mouse kidneys were homogenized in an ice-cold stringent RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, supplemented with protease inhibitor mixture and phosphatase inhibitor mixtures), followed by sonication. Homogenates were centrifuged at 14,000 × g for 20 min at 4°C to separate supernatants (RIPA buffer-soluble fractions). The resulting pellets were further lysed with a buffer containing 4% SDS and 20 mM HEPES, pH 7.5, supplemented with protease inhibitor mixture and phosphatase inhibitor mixtures by vortexing and sonication, followed by centrifugation at 19,600 × g for 10 min at room temperature to separate the new supernatants (RIPA buffer-insoluble fractions).
Western blotting and OxyBlot
Equal amount of total proteins from each preparation was loaded and separated in NuPAGE 3-8% Tris-Acetate gels or 4-12% Bis-Tris gels (Invitrogen) and then transferred to nitrocellulose membranes. Oxyblots for detecting protein carbonyls were prepared following the manufacturer's instructions (Millipore). After blocking and overnight incubation with primary antibodies, protein bands of interest were visualized by binding of IRDye-labeled secondary antibodies and band intensity analyzed using Odyssey imaging system (Li-Cor).
Antibodies used in Western blotting and/or immunohistochemical analyses are: goat pAb anti-mouse Kim-1 (R&D Systems), rabbit pAb anti-ERK1/2 (p44/42 MAP kinase; Cell Signaling), rabbit pAb anti-LC3B (Sigma or Cell Signaling), rabbit pAb anti-GATE-16 (MBL), guinea pig pAb anti-p62 (Progen), rabbit pAb anti-p62 (Wako), rabbit pAb anti-α-synuclein (C-20; Santa Cruz), rabbit pAb anti-LAMP-1 (Cell Signaling), rat mAb anti-LAMP-1 (University of Iowa), rat mAb anti-LAMP-2 (University of Iowa), rabbit pAb anti-cathepsin B (Santa Cruz), rabbit pAb anti-cathepsin D (Calbiochem), rabbit pAb anti-Rab7 (Cell Signaling), mouse mAb anti-GAPDH (Millipore), rabbit pAb anti-β-actin (Cell Signaling), mouse mAb anti-β-actin (Sigma).
Statistical analysis was performed using Prism 5 (GraphPad Software) and Excel (Microsoft). Data are presented as means ± SEM. Statistical significance was determined by the P values of Student t test. Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
We thank Huailong Zhao for technical assistance. This work was supported by grants from the National Institute of Neurological Disorders and Stroke (R01 NS071251, R01 NS041779, and R01 NS052745) and the Michael J. Fox Foundation (to J.S.) and grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK039773 and R01 DK072381 to J.V.B.) and by the Intramural Research Program of National Institute on Aging (to H.C.).
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