In traditional molecular biology technology genes are expressed from cDNAs driven by strong heterologous promoters. It is increasingly clear, however, that non-coding DNA, including promoters, introns, upstream and downstream sequences all contain essential information for the appropriate expression and function of genes (34
). We therefore expressed WT and mutant human LRRK2
from a complete genomic DNA locus in human cells. Since LRRK2 protein has been reported to be difficult to detect in some applications (8
), we decided to fuse a fluorescent tag to LRRK2 with a minimum alteration of potential regulatory sequences. However, this kind of seamless genetic manipulation of genomic DNA material contained in vectors such as BACs has proven to be difficult and inefficient (35
). In this article, we report the development of STEP, a novel strategy based on recombineering (19
) which obtains high recombination efficiency without leaving any unwanted sequences behind. Our constructs expressed full-length LRRK2 in human cells which could be easily detected in real-time fluorescence live-cell microscopy, IF, immunoblotting and IEM.
Despite the absence of a known membrane-targeting signal we have found a robust association of LRRK2 with both the plasma membrane and intracellular membranes. Interestingly, we report for the first time that LRRK2 was recruited to specific membranous microdomains such as the neck of caveolae, microvilli/filopodia and ILVs of MVBs. These three microdomains share important topological and biochemical characteristics. Topologically, they form when cellular membranes modify their curvature so that the cytosolic side protrudes on the extracellular or intravesicular side (36
), a process that likely involves the rich underlying actin cytoskeleton network whose arrangement is regulated by the ERM family of proteins (37
). ERM proteins are enriched precisely in the neck of caveolae, microvilli and ILVs of MVBs (21
) and are regulated though phosphorylation. Since LRRK2 has been shown to phosphorylate ERM proteins (15
), the specific presence of LRRK2 in the neck of caveolae, microvilli and ILVs of MVBs suggests a role for LRRK2 in regulating actin cytoskeleton reorganization and membrane curvature dynamics.
Our proposal that LRRK2 is involved in the formation of these membranous structures is further supported by two experiments. First, upon induction of formation of microvilli/filopodia LRRK2 was recruited to the plasma membrane, increasing its presence in these microdomains (Fig. A and B). Second, ILVs formation is the key event leading to the formation of MVBs (36
) and the R1441C mutation, which seems to induce a constitutively active form of LRRK2 (11
), increased the appearance of MVBs and led to the formation of skein-like structures which likely represented abnormal MVBs (Fig. F, H1–3 and G). Evidence of the role of such a pathway in neurodegenerative disease mechanisms is supported by mutations inducing malfunction of MVB formation that cause frontotemporal dementia and amyotrophic lateral sclerosis (40
). Moreover, LRRK2 has been recently found to directly interact with the Alix-related protein His domain phosphotyrosine phosphatase/His-Domain/Type N23 protein tyrosine phosphatase (41
), which is essential for endosomal cargo sorting and MVB morphogenesis (42
In addition to localizing to the neck of caveolae, microvilli and MVBs, LRRK2 localized to AVs, where it partially colocalized with p62 and LC3. Mono-ubiquitination or Lys63-linked di-ubiquitination are the most common signals in the endocytic pathway (43
) and the fact that most anti-ubiquitin sera have a bias towards K48 poly-ubiquitination (44
) explains the lack of ubiquitin staining of LRRK2 puncta. LRRK2 also localized to morphologically undefined smaller vesicles. Double labelling experiments suggest that these vesicles may be part of the ER (Fig. B3). Unlike previous studies which mainly relied on subcellular fractionation (45
), we did not find any association of LRRK2 with mitochondria (Supplementary Material, Figure S2E
). Nevertheless it may be possible that LRRK2 associated with the outer membrane of engulfed mitochondria within AVs.
Our data provide for the first time direct evidence of a functional relationship between LRRK2 and autophagy. Using a physiologically-relevant human genomic DNA expression system we have shown that the R1441C LRRK2 mutation recapitulates a key and early pathological feature of neurodegenerative disorders, including PD, such as the accumulation of AVs (46
). We found an accumulation of multivesicular structures and AVs with ‘late’ characteristics filled with incompletely degraded material (52
) distinct from the smaller and uniformly dense mature lysosomal dense bodies (53
). This observation suggests that the R1441C LRRK2 mutation induces an imbalance between macroautophagy induction and maturation of AVs to lysosomes, further supported by the accumulation of p62 within AVs (Supplementary Material, Figure S2B
). Interestingly, LRRK2 has been found to accumulate in neuritic varicosities and globular lesions in the brainstem of PD in association with p62 positive aggregation centres and early stage aggregation forms of alpha-synuclein (6
), linking our present findings to the reported accumulation of MVBs and AVs as the main component of neuritic varicosities (54
). Furthermore, tyrosine hydroxylase-positive neuritic varicosities and globular lesions appeared in R1441G LRRK2 BAC transgenic mice suffering from levodopa-responsive akinesia, similar to sporadic PD (56
). Our finding that LRRK2 puncta are transported towards the cellular soma, possibly by dynein, may indicate that axonal transport deficits could further contribute to the imbalanced maturation of AVs in neurons. Conversely, LRRK2 knockdown increased the autophagic activity and counterbalanced the effect on cell death of the autophagy inhibitor BFA under starvation conditions. Therefore our study suggests that modulating LRRK2 function could help restore the altered autophagic equilibrium of many neurodegenerative disorders and lead to new treatments and neuroprotection strategies.