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 [
26]. 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 [
44]. 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 ). 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 [
31], and increased degradation of p62, one of the best characterized autophagy substrates [
28], as well as increased degradation of α-synuclein (Figures and ). By 20 months of age, similar analysis showed reduced autophagic activity in
LRRK2-/- kidneys (Figures and ). 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 ).
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 [
28]. 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 [
31]. 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 ). 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 ). 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 [
28]. 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.
Deficits in autophagy have been implicated in a variety of neurodegenerative diseases with protein aggregation-related pathologies [
27,
29,
47]. Interestingly, increased accumulation of autophagic vacuoles, including both autophagosomes and autolysosomes, has also been reported in postmortem brains of Alzheimer's and Parkinson's disease patients, with likely reasons of either overproduction of autophagic vacuoles or deficit in clearance or recycling of autolysosomes [
29,
48]. Cathepsin D is also up-regulated in affected neurons. Antibodies to cathepsin D strongly label contents in some of the accumulated autophagic vacuoles, which are identified as autolysosomes, as well as the proteinaceous components of lipofuscins [
48]. Our data demonstrate that the autophagy-lysosomal pathway is dysregulated in the absence of LRRK2. Although loss of LRRK2 may initially cause induction of autophagy, deficient clearance or recycling of autophagic components in the absence of LRRK2 would cause trapping of the components of the autophagy pathway in the forms of autolysosomes and the eventual formation of lipofuscin granules due to excessive oxidation and crosslinking and therefore depletion of autophagy machinery (e.g., autophagic lysosomes cannot be reformed.), which would in turn result in accumulation and aggregation of a large number of autophagy substrate proteins during aging (Figure ). Likely as a consequence or a response to the stresses presumably rendered by the above discussed abnormalities,
LRRK2-/- kidneys sustain chronic injury, indicated by dramatic and persistent up-regulation of kidney injury molecule-1 (Figure ), a very sensitive and specific biomarker for epithelial cell injury of proximal renal tubules in various settings [
33,
34].
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 [
49]. Surprisingly, LRRK2 overexpression in cultured HEK293 cells has also been reported to cause autophagy induction through a calcium-dependent pathway [
50]. 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 [
49], 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 [
53], 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.
1 Emilie Giaime,1 Hiroo Yamaguchi,1 Takaharu Ichimura,2 Yumin Liu,2 Huiqing Si,1 Huaibin Cai,3 Joseph V Bonventre,2 and Jie Shen
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