Analysis of macroautophagy related proteins in G2019S LRRK2 Parkinson’s disease brains with Lewy body pathology

Abstract

LRRK2, the gene encoding the multidomain kinase Leucine-Rich Repeat Kinase 2 (LRRK2), has been linked to familial and sporadic forms of Parkinson's disease (PD), as well as cancer, leprosy and Crohn's disease, establishing it as a target for discovery therapeutics. LRRK2 has been associated with a range of cellular processes, however its physiological and pathological functions remain unclear. The most prevalent LRRK2 mutations in PD have been shown to affect macroautophagy in various cellular models while a role in autophagy signalling has been recapitulated in vivo. Dysregulation of autophagy has been implicated in PD pathology, and this raises the possibility that differential autophagic activity is relevant to disease progression in PD patients carrying LRRK2 mutations. To examine the relevance of LRRK2 to the regulation of macroautophagy in a disease setting we examined the levels of autophagic markers in the basal ganglia of G2019S LRRK2 PD post-mortem tissue, in comparison to pathology-matched idiopathic PD (iPD), using immunoblotting (IB). Significantly lower levels of p62 and LAMP1 were observed in G2019S LRRK2 PD compared to iPD cases. Similarly, an increase in ULK1 was observed in iPD but was not reflected in G2019S LRRK2 PD cases. Furthermore, examination of p62 by immunohistochemistry (IH) recapitulated a distinct signature for G2019S PD. IH of LAMP1, LC3 and ULK1 broadly correlated with the IB results. Our data from a small but pathologically well-characterized cases highlights a divergence of G2019S PD carriers in terms of autophagic response in alpha-synuclein pathology affected brain regions compared to iPD.

Pathogenic mutations in LRRK2 sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia

“…Lysosomal pH regulates iron release and impaired lysosomal acidification has been reported to trigger iron deficiency and inflammation in vivo as well as mitochondrial defects (30,31). As LRRK2 mutations have been reported to alter the autophagic lysosomal pathway in carriers (32,33), as well as to perturb lysosomal acidification in knock-in mouse models (34), it is plausible that LRRK2 mutations affect Tf-mediated iron uptake by modulating lysosomal acidification. In our experiments we observe sequestration of TfR and accumulation to lysosomes driven by mutant LRRK2, while no significant difference was found on total TfR levels in these cells (data not shown) or in G2019S knock-in mice that exhibited iron deposition and ferritin upregulation following LPS administration.…”

What Have We Learned from Cerebrospinal Fluid Studies about Biomarkers for Detecting LRRK2 Parkinson’s Disease Patients and Healthy Subjects with Parkinson’s-Associated LRRK2 Mutations?

“…There is evidence for impaired autophagic mechanisms in PD [135][136][137][138]. Notably, LRRK2 is thought to play an important role in the regulation of autophagy [139]. Although wildtype LRRK2 is a substrate of (i.e., can be degraded by) CMA, G2019S LRRK2 is poorly degraded by this pathway [49,140].…”

Autophagy and LRRK2 in the Aging Brain

“…Interestingly, G2019S PD patients did not show the increase of LC3II, p62 and ULK-1 levels observed in idiopathic PD patients, but instead a significant reduction of LAMP1 levels. This was interpreted as being due to a different pathobiology associated with idiopathic and G2019S LRRK2 PD (Mamais et al, 2018). Consistent with this view, G2019S LRRK2 cases had lower insoluble α-syn levels compared to idiopathic PD patients, suggesting differences in the biochemical properties of aggregated α-syn (Mamais et al, 2013).…”

“…Moreover, LC3II has been shown to colocalize with α-syn in LBs (Alvarez-Erviti et al, 2010;Dehay et al, 2010). The patterns of ALP markers were compared in the brains of idiopathic vs. G2019S LRRK2 PD patients (Mamais et al, 2018). Interestingly, G2019S PD patients did not show the increase of LC3II, p62 and ULK-1 levels observed in idiopathic PD patients, but instead a significant reduction of LAMP1 levels.…”

“…Changes in ALP markers were reported in the human brain which are consistent with an impairment of autophagic machinery. A significant reduction of lysosomal and CMA markers, such as LAMP1, LAMP2A, Hsc70 or Cathepsin D, associated with an increase of autophagic markers LC3II and p62 levels, was reported in the whole-brain (Mamais et al, 2018) or SNpc (Chu et al, 2009;Dehay et al, 2010) of idiopathic PD patients (Xilouri and Stefanis, 2015). Moreover, LC3II has been shown to colocalize with α-syn in LBs (Alvarez-Erviti et al, 2010;Dehay et al, 2010).…”