BackgroundMutations 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.MethodsIn 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.ResultsOur 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.ConclusionsHere 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.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0240-1) contains supplementary material, which is available to authorized users.

The cerebellum has a long history in terms of research on its network structures and motor functions, yet our understanding of them has further advanced in recent years owing to technical developments, such as viral tracers, optogenetic and chemogenetic manipulation, and single cell gene expression analyses. Specifically, it is now widely accepted that the cerebellum is also involved in non-motor functions, such as cognitive and psychological functions, mainly from studies that have clarified neuronal pathways from the cerebellum to other brain regions that are relevant to these functions. The techniques to manipulate specific neuronal pathways were effectively utilized to demonstrate the involvement of the cerebellum and its pathways in specific brain functions, without altering motor activity. In particular, the cerebellar efferent pathways that have recently gained attention are not only monosynaptic connections to other brain regions, including the periaqueductal gray and ventral tegmental area, but also polysynaptic connections to other brain regions, including the non-primary motor cortex and hippocampus. Besides these efferent pathways associated with non-motor functions, recent studies using sophisticated experimental techniques further characterized the historically studied efferent pathways that are primarily associated with motor functions. Nevertheless, to our knowledge, there are no articles that comprehensively describe various cerebellar efferent pathways, although there are many interesting review articles focusing on specific functions or pathways. Here, we summarize the recent findings on neuronal networks projecting from the cerebellum to several brain regions. We also introduce various techniques that have enabled us to advance our understanding of the cerebellar efferent pathways, and further discuss possible directions for future research regarding these efferent pathways and their functions.

The well-organized cerebellar structures and neuronal networks are likely crucial for their functions in motor coordination, motor learning, cognition, and emotion. Such cerebellar structures and neuronal networks are formed during developmental periods through orchestrated mechanisms, which include not only cell-autonomous programs but also interactions between the same or different types of neurons. Cerebellar granule cells (GCs) are the most numerous neurons in the brain and are generated through intensive cell division of GC precursors (GCPs) during postnatal developmental periods. While GCs go through their own developmental processes of proliferation, differentiation, migration, and maturation, they also play a crucial role in cerebellar development. One of the best-characterized contributions is the enlargement and foliation of the cerebellum through massive proliferation of GCPs. In addition to this contribution, studies have shown that immature GCs and GCPs regulate multiple factors in the developing cerebellum, such as the development of other types of cerebellar neurons or the establishment of afferent innervations. These studies have often found impairments of cerebellar development in animals lacking expression of certain molecules in GCs, suggesting that the regulations are mediated by molecules that are secreted from or present in GCs. Given the growing recognition of GCs as regulators of cerebellar development, this review will summarize our current understanding of cerebellar development regulated by GCs and molecules in GCs, based on accumulated studies and recent findings, and will discuss their potential further contributions.