The function of LRRK remains to be fully

The function of LRRK2 remains to be fully elucidated, but studies have demonstrated a role for LRRK2 in neurite elongation and arborization. Cultured dopaminergic neurons from human LRRK2-G2019S-expressing transgenic mice and patient-specific induced pluripotent stem cetrizine (iPSCs) display shortened neurites and reduced neurite complexity (Ramonet et al., 2011; Cooper et al., 2012; Sánchez-Danés et al., 2012; Reinhardt et al., 2013). The molecular basis underlying the effects of LRRK2 on neurite growth and integrity is not known, though putative LRRK2 effectors have been implicated in the regulation of neurite outgrowth including ezrin, radixin, and moesin (ERM), which play roles in cytoskeletal dynamics. Increased LRRK2 activity has been correlated with increased ERM phosphorylation and decreased axon extension (Parisiadou et al., 2009). Although sensory nerve disruption in PD has been reported by functional assessments of cutaneous sensory nerve endings and post-mortem analysis (Dabby et al., 2006; Ikemura et al., 2008; Nolano et al., 2008; Shishido et al., 2010; Toth et al., 2010), there are limited data on the effect of LRRK2 on sensory neuron structure and function. We sought to investigate whether PD mutations confer intrinsic defects in sensory neuron structure and function using iPSCs. We compared sensory neurons derived from two PD patients with homozygous LRRK2 G2019S mutations and one asymptomatic patient heterozygous for LRRK2 G2019S to one PD patient with α-synuclein (SNCA) triplication and three unaffected individuals. Dopamine neurons derived from the mutant LRRK2 lines showed shortened neurites and reduced neurite branching, consistent with other well-characterized models of LRRK2 PD. In contrast, LRRK2 G2019S iPSC-derived sensory neurons exhibited normal neurite outgrowth but increased cytoskeletal aggregations and altered calcium dynamics compared to control or SNCA iPSC-derived sensory neurons. Treatment with LRRK2 kinase inhibitors resulted in significant but incomplete morphological and functional rescue. Together, these data indicate that excessive LRRK2 kinase activity can negatively impact sensory neuron structure and function and may play a role in the development of sensory dysfunction in PD.
Results
Discussion PD patients suffer from both motor and non-motor symptoms, and due to the differentiation capacity of iPSCs, we can take advantage of this system to study multiple cell types that may be impacted by disease processes. Consistent with other reports (Cooper et al., 2012; Sánchez-Danés et al., 2012; Reinhardt et al., 2013), we show that LRRK2 G2019S iPSC-derived dopamine neurons display shortened neurites and reduced branching compared to controls. However, the same LRRK2 G2019S mutation creates a different phenotype in peripheral sensory neurons. Specifically, we find neurite length is comparable to control cells, but the neurites harbor large cytoskeletal aggregates. Reinhardt and colleagues (2013) had previously found that Brn3a-positive sensory neurons derived from heterozygous LRRK2 G2019S iPSCs did not exhibit increased cell death following treatment with rotenone, but no other aspects of sensory neuron structure or function have previously been studied. Although very little is known about the cause of somatosensory symptoms in PD, the observation of neurite aggregates within sensory neurons aligns with PD patients exhibiting peripheral nervous system impairment and may be a pathological feature of PD (Nolano et al., 2008; Donadio et al., 2014; Doppler et al., 2014). Additionally, intermediate filament- and peripherin-containing aggregates are commonly found in the peripheral axon of motor neurons in amyotrophic lateral sclerosis (Xiao et al., 2006), suggesting peripheral neurons and central neurons with peripheral targets may undergo common alterations in neurodegenerative diseases. Using immunocytochemical analysis, we found that LRRK2 G2019S iPSC-derived sensory neurons displayed neurite aggregations comprised of PD-associated proteins β-tubulin, MAP2, Tau, LRRK2, and SNCA. β-tubulin, a component of Lewy bodies, has been shown to directly interact with LRRK2, which regulates tubulin phosphorylation and acetylation (Law et al., 2014). Moreover, tubulin interacts with a number of other PD-related proteins including SNCA, parkin, and tau (Alim et al., 2002; Yang et al., 2005; Gillardon, 2009; Kawakami et al., 2012; Law et al., 2014), which likely implicates microtubule dysfunction as a common mechanism leading to the clinical and pathological hallmarks of PD (Cartelli et al., 2012). Consistent with this, MAP2-positive neurite aggregates have been identified in dopaminergic and non-dopaminergic neurons in post-mortem PD brain (D’Andrea et al., 2001). Tau has been implicated in the pathogenesis of PD in recent genome-wide screens (Simón-Sánchez et al., 2009; Edwards et al., 2010) and is seen in animal models expressing LRRK2 mutations (Li et al., 2009b; Lin et al., 2010; Melrose et al., 2010). LRRK2 has been shown to facilitate tau phosphorylation in a kinase-independent manner (Shanley et al., 2015), and our tau data align with previously reported data in LRRK2 G2019S and I2020T iPSC-derived neurons (Reinhardt et al., 2013; Ohta et al., 2015). Tau hyperphosphorylation has been shown to favor tau detachment from microtubules (Lindwall and Cole, 1984) that can lead to neurofibrillary tangle formation. Moreover, in a hLRRK2 (R1441G) BAC transgenic mouse model, fragmented axons, axonal spheroids, and dystrophic neurites were observed and found to be associated with abnormally phosphorylated tau (Li et al., 2009a). Together with our data, these observations suggest a role of LRRK2 in tau-related neuronal pathology, which may be contributing to the increased neurite aggregate formation in iPSC-derived sensory neurons.