Pathological Functions of LRRK2 in Parkinson's Disease.

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are common genetic risk factors for both familial and sporadic Parkinson's disease (PD). Pathogenic mutations in LRRK2 have been shown to induce changes in its activity, and abnormal increase in LRRK2 kinase activity is thought to contribute to PD pathology. The precise molecular mechanisms underlying LRRK2-associated PD pathology are far from clear, however the identification of LRRK2 substrates and the elucidation of cellular pathways involved suggest a role of LRRK2 in microtubule dynamics, vesicular trafficking, and synaptic transmission. Moreover, LRRK2 is associated with pathologies of α-synuclein, a major component of Lewy bodies (LBs). Evidence from various cellular and animal models supports a role of LRRK2 in the regulation of aggregation and propagation of α-synuclein. Here, we summarize our current understanding of how pathogenic mutations dysregulate LRRK2 and discuss the possible mechanisms leading to neurodegeneration.

LRRK2 and membrane trafficking: nexus of Parkinson's disease.

Recent evidence from genetics, animal model systems and biochemical studies suggests that defects in membrane trafficking play an important part in the pathophysiology of Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) constitute the most frequent genetic cause of both familial and sporadic PD, and LRRK2 has been suggested as a druggable target for PD. Although the precise physiological function of LRRK2 remains largely unknown, mounting evidence suggests that LRRK2 controls membrane trafficking by interacting with key regulators of the endosomal-lysosomal pathway and synaptic recycling. In this review, we discuss the genetic, biochemical and functional links between LRRK2 and membrane trafficking. Understanding the mechanism by which LRRK2 influences such processes may contribute to the development of disease-modifying therapies for PD. [BMB Reports 2019; 52(9): 533-539].

LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation.

Propagation of α-synuclein aggregates has been suggested as a contributing factor in Parkinson's disease (PD) progression. However, the molecular mechanisms underlying α-synuclein aggregation are not fully understood. Here, we demonstrate in cell culture, nematode, and rodent models of PD that leucine-rich repeat kinase 2 (LRRK2), a PD-linked kinase, modulates α-synuclein propagation in a kinase activity-dependent manner. The PD-linked G2019S mutation in LRRK2, which increases kinase activity, enhances propagation efficiency. Furthermore, we show that the role of LRRK2 in α-synuclein propagation is mediated by RAB35 phosphorylation. Constitutive activation of RAB35 overrides the reduced α-synuclein propagation phenotype in lrk-1 mutant C. elegans. Finally, in a mouse model of synucleinopathy, administration of an LRRK2 kinase inhibitor reduced α-synuclein aggregation via enhanced interaction of α-synuclein with the lysosomal degradation pathway. These results suggest that LRRK2-mediated RAB35 phosphorylation is a potential therapeutic target for modifying disease progression.

Dysregulated phosphorylation of Rab GTPases by LRRK2 induces neurodegeneration.

BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease (PD). Elevated kinase activity is associated with LRRK2 toxicity, but the substrates that mediate neurodegeneration remain poorly defined. Given the increasing evidence suggesting a role of LRRK2 in membrane and vesicle trafficking, here we systemically screened Rab GTPases, core regulators of vesicular dynamics, as potential substrates of LRRK2 and investigated the functional consequence of such phosphorylation in cells and in vivo. METHODS: In vitro LRRK2 kinase assay with forty-five purified human Rab GTPases was performed to identify Rab family proteins as substrates of LRRK2. We identified the phosphorylation site by tandem mass-spectrometry and confirmed it by assessing phosphorylation in the in vitro LRRK2 kinase assay and in cells. Effects of Rab phosphorylation on neurodegeneration were examined in primary cultures and in vivo by intracranial injection of adeno-associated viral vectors (AAV) expressing wild-type or phosphomutants of Rab35. RESULTS: Our screening revealed that LRRK2 phosphorylated several Rab GTPases at a conserved threonine residue in the switch II region, and by using the kinase-inactive LRRK2-D1994A and the pathogenic LRRK2-G2019S along with Rab proteins in which the LRRK2 site was mutated, we verified that a subset of Rab proteins, including Rab35, were authentic substrates of LRRK2 both in vitro and in cells. We also showed that phosphorylation of Rab regulated GDP/GTP-binding property in cells. Moreover, in primary cortical neurons, mutation of the LRRK2 site in several Rabs caused neurotoxicity, which was most severely induced by phosphomutants of Rab35. Furthermore, intracranial injection of the AAV-Rab35 -T72A or AAV-Rab35-T72D into the substantia nigra substantially induced degeneration of dopaminergic neurons in vivo. CONCLUSIONS: Here we show that a subset of Rab GTPases are authentic substrates of LRRK2 both in vitro and in cells. We also provide evidence that dysregulation of Rab phosphorylation in the LRRK2 site induces neurotoxicity in primary neurons and degeneration of dopaminergic neurons in vivo. Our study suggests that Rab GTPases might mediate LRRK2 toxicity in the progression of PD.

Activation of the ATF2/CREB-PGC-1α pathway by metformin leads to dopaminergic neuroprotection.

Progressive dopaminergic neurodegeneration is responsible for the canonical motor deficits in Parkinson's disease (PD). The widely prescribed anti-diabetic medicine metformin is effective in preventing neurodegeneration in animal models; however, despite the significant potential of metformin for treating PD, the therapeutic effects and molecular mechanisms underlying dopaminergic neuroprotection by metformin are largely unknown.In this study, we found that metformin induced substantial proteomic changes, especially in metabolic and mitochondrial pathways in the substantia nigra (SN). Consistent with this data, metformin increased mitochondrial marker proteins in SH-SY5Y neuroblastoma cells. Mitochondrial protein expression by metformin was found to be brain region specific, with metformin increasing mitochondrial proteins in the SN and the striatum, but not the cortex. As a potential upstream regulator of mitochondria gene transcription by metformin, PGC-1α promoter activity was stimulated by metformin via CREB and ATF2 pathways. PGC-1α and phosphorylation of ATF2 and CREB by metformin were selectively increased in the SN and the striatum, but not the cortex. Finally, we showed that metformin protected dopaminergic neurons and improved dopamine-sensitive motor performance in an MPTP-induced PD animal model. Together these results suggest that the metformin-ATF2/CREB-PGC-1α pathway might be promising therapeutic target for PD.

Inflammatory signals induce the expression of tonicity-responsive enhancer binding protein (TonEBP) in microglia.

Tonicity-responsive enhancer (TonE) binding protein (TonEBP) is known as an osmosensitive transcription factor that regulates cellular homeostasis during states of hypo- and hypertonic stress. In addition to its role in osmoadaptation, growing lines of evidence suggest that TonEBP might have tonicity-independent functions. In particular, a number of studies suggest that inflammatory stimuli induce the expression and activation of TonEBP in peripheral immune cells. However, whether TonEBP is expressed in microglia, resident immune cells of the central nervous system, is unknown. Here we show that inflammatory signals induce the expression of TonEBP in microglia both in vitro and in vitro. In cultured primary microglia, treatment with lipopolysaccharide (LPS), interferon-γ, and interleukin 4 increased the expression of TonEBP. Moreover, we found that stereotaxic injection of LPS into the substantia nigra region of rat brain increased TonEBP expression in OX-42-positive cells. Furthermore, expression of TonEBP was induced in OX-42-positive cells in a rat model of transient middle cerebral artery occlusion. Together these results show that the expression of TonEBP is regulated by inflammatory signals in mammalian brain, suggesting that TonEBP might play a part during neuroinflammation.