LRRK2 Is Recruited to Phagosomes and Co-recruits RAB8 and RAB10 in Human Pluripotent Stem Cell-Derived Macrophages.

The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ "maturing" phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes.

LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.

Excess α-synuclein compromises phagocytosis in iPSC-derived macrophages.

To examine the pathogenic role of α-synuclein (αS) in Parkinson's Disease, we have generated induced Pluripotent Stem Cell lines from early onset Parkinson's Disease patients with SNCA A53T and SNCA Triplication mutations, and in this study have differentiated them to PSC-macrophages (pMac), which recapitulate many features of their brain-resident cousins, microglia. We show that SNCA Triplication pMac, but not A53T pMac, have significantly increased intracellular αS versus controls and release significantly more αS to the medium. SNCA Triplication pMac, but not A53T pMac, show significantly reduced phagocytosis capability and this can be phenocopied by adding monomeric αS to the cell culture medium of control pMac. Fibrillar αS is taken up by pMac by actin-rearrangement-dependent pathways, and monomeric αS by actin-independent pathways. Finally, pMac degrade αS and this can be arrested by blocking lysosomal and proteasomal pathways. Together, these results show that macrophages are capable of clearing αS, but that high levels of exogenous or endogenous αS compromise this ability, likely a vicious cycle scenario faced by microglia in Parkinson's disease.

LRRK2 in peripheral and central nervous system innate immunity: its link to Parkinson's disease.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in familial and idiopathic cases of Parkinson's disease (PD), but are also associated with immune-related disorders, notably Crohn's disease and leprosy. Although the physiological function of LRRK2 protein remains largely elusive, increasing evidence suggests that it plays a role in innate immunity, a process that also has been implicated in neurodegenerative diseases, including PD. Innate immunity involves macrophages and microglia, in which endogenous LRRK2 expression is precisely regulated and expression is strongly up-regulated upon cell activation. This brief report discusses the current understanding of the involvement of LRRK2 in innate immunity particularly in relation to PD, critically examining its role in myeloid cells, particularly macrophages and microglia.

A novel real time imaging platform to quantify macrophage phagocytosis.

Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.

Disruption of the blood-brain barrier in Parkinson's disease: curse or route to a cure?

The vertebrate blood-brain barrier (BBB) is critical for ensuring the maintenance of brain homeostasis, whilst protecting the brain against toxic insults. Various pathological events disrupt BBB integrity, holding several important clinical implications. In instances where the normal mechanisms controlling passage of substances into the brain are compromised, these could sensitize or even worsen endogenous pathological conditions. Recognition has grown recently that patients diagnosed with Parkinson's disease (PD) present with concurrent medical problems, including cerebrovascular lesions. However, cerebrovascular disturbances may also result from PD-related disease processes; the pathological mechanisms which could entail interaction between environment-derived and genetic factors. The current review addresses the accumulation of studies aimed at better understanding the series of processes affecting the neurovascular unit in human Parkinsonism, due in part to the BBB presenting as a formidable opponent in the effective delivery of therapeutics that have shown promise as therapeutic strategies for treating aspects of PD when tested in vitro.

Enhanced central nervous system transduction with lentiviral vectors pseudotyped with RVG/HIV-1gp41 chimeric envelope glycoproteins.

UNLABELLED: To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons. IMPORTANCE: In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.

Dopaminergic denervation and spine loss in the striatum of MPTP-treated monkeys.

Striatal spine loss is a key pathological feature of human Parkinson's disease (PD) that can be induced after complete degeneration of the nigrostriatal dopaminergic system in rodent models of parkinsonism. In line with these observations, our findings reveal a significant (30-50%) reduction in spine density in both the caudate nucleus and putamen of severely DA-depleted striata of MPTP-treated monkeys; the sensorimotor post-commissural putamen being the most severely affected region for both dopamine depletion and spine loss. Using MPTP-treated monkeys with complete or partial striatal dopamine (DA) denervation, we also demonstrate that striatal spine loss is an early pathological feature of parkinsonism, which progresses along a positive rostrocaudal and mediolateral gradient in parallel with the extent of striatal dopamine denervation. Quantitative electron microscopy immunocytochemistry for D1 dopamine receptor (D1) in the striatum of control and severely DA-depleted animals revealed that both D1-immunoreactive and immunonegative spines are lost in the putamen of MPTP-treated monkeys. These data demonstrate that striatal spine loss in MPTP-treated monkeys is an early pathological event of parkinsonism, tightly correlated with the degree of nigrostriatal dopamine denervation that likely affects both direct and indirect striatofugal pathways.