Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice

Abstract

The generation and maturation of adult neural stem/progenitor cells are impaired in many neurodegenerative diseases, among them is Parkinson's disease (PD). In mammals, including humans, adult neurogenesis is a lifelong feature of cellular brain plasticity in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ)/olfactory bulb system. Hyposmia, depression, and anxiety are early non-motor symptoms in PD. There are parallels between brain regions associated with non-motor symptoms in PD and neurogenic regions. In autosomal dominant PD, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent. LRRK2 homologs in non-vertebrate systems play an important role in chemotaxis, cell polarity, and neurite arborization.

We investigated adult neurogenesis and the neurite development of new neurons in the DG and SVZ/olfactory bulb system in bacterial artificial chromosome (BAC) human Lrrk2 G2019S transgenic mice. We report that mutant human Lrrk2 is highly expressed in the hippocampus in the DG and the SVZ of adult Lrrk2 G2019S mice. Proliferation of newly generated cells is significantly decreased and survival of newly generated neurons in the DG and olfactory bulb is also severely impaired. In addition, after stereotactic injection of a GFP retrovirus, newly generated neurons in the DG of Lrrk2 G2019S mice exhibited reduced dendritic arborization and fewer spines. This loss in mature, developed spines might point towards a decrease in synaptic connectivity. Interestingly, physical activity partially reverses the decrease in neuroblasts observed in Lrrk2 G2010S mice. These data further support a role for Lrrk2 in neuronal morphogenesis and provide new insights into the role of Lrrk2 in adult neurogenesis.

Introduction

Mutations in leucine-rich repeat kinase 2 (LRRK2) predispose carriers to Parkinson's disease (PD); (Zimprich et al., 2004a, Mata et al., 2006). Lrrk2 G2019S is found in more than 30% of patients of certain ethnicities and has the highest genotype- and population-attributable risk (Hulihan et al., 2008, Healy et al., 2008).

LRRK2 is comprised of 51 exons that encode 2527 amino acids and has a molecular mass of 286 kDa. Lrrk2 is a large, multi-domain protein kinase and consists of an ankyrin repeat region, leucine-rich repeats, a Roc GTPase domain, and a C terminal of Roc (COR) domain. In addition, a serine/threonine protein kinase domain with a high degree of homology with MAPKKK (mitogen-activated protein kinase kinase kinase, where the G2019S mutation is located) and a WD40 domain are present (Mata et al., 2006, Zimprich et al., 2004b). While Lrrk2 transgenic models are yet to recapitulate human PD neuropathology in aspects such as increased cell death of dopaminergic neurons or Lewy bodies, subtle phenotypes including impaired regulation of neurite growth and arborization have been reported. In non-vertebrate systems a role of LRRK2 homologs for chemotaxis, polarization, and regulation of axonal–dendritic polarity of synaptic vesicle proteins was described (van Egmond et al., 2008, Sakaguchi-Nakashima et al., 2007). In primary neuronal cultures, in utero transfection or AAV2 viral injection, mutant Lrrk2 slows axonal outgrowth and reduces neurite length and branching, whereas silencing of LRRK2 leads to the opposite effects (Li et al., 2009, MacLeod et al., 2006, Plowey et al., 2008).

In humans and mice, Lrrk2 is highly expressed in the hippocampus and subventricular zone (SVZ) and colocalizes with the migrating neuroblast marker PSA-NCAM (Melrose et al., 2007). The hippocampal dentate gyrus (DG) and SVZ/olfactory bulb system are the physiological areas of neurogenesis in the adult brain (Altman and Das, 1965, Curtis et al., 2007, Eriksson et al., 1998). In PD and transgenic SNCA mouse models of parkinsonism, the proliferation of dividing cells is impaired in the DG and SVZ/olfactory bulb system (Crews et al., 2008, Hoglinger et al., 2004, Winner et al., 2004). PD affects multiple neurotransmitter systems and includes a variety of non-motor symptoms (Langston, 2006). Frequently observed early symptoms in PD include hyposmia, anxiety, anhedonia, and depression (Tolosa and Poewe, 2009). These symptoms are linked to neuropathological alterations in the olfactory and limbic system, including the olfactory bulb and the hippocampus (Rodriguez-Oroz et al., 2009).

In this study, we take advantage of the hippocampal and SVZ transgene expression in G2019S Lrrk2 mice to study the impact of mutated Lrrk2 on newly generated neurons in the adult brain. With the benefit of inherent Lrrk2 expression, we examine the impact of aberrant human Lrrk2 on adult neurogenesis. We determine neurite outgrowth, and spine numbers in newly generated neurons in the hippocampal DG in vivo and test whether physical activity impacts this system.