To address this issue we examined

To address this issue, we examined striatal neurons innervated by nigral DA neurons using the plant lectin wheat-germ agglutinin (WGA). WGA can be efficiently transferred between neurons via synapses and has been already used as a transsynaptic tracer (Fabian and Coulter, 1985; Broadwell and Balin, 1985). Classically, WGA was used to visualize specific neural pathways by microinjection into a target region (Peschanski and Ralston, 1985; Buttry and Goshgarian, 2014). A more recent technique for tracing induces WGA cDNA via adeno-associated virus-mediated gene transduction (Yoshihara et al., 1999; Ohashi et al., 2011). Using this technique and histological analysis, we show here that integrin α5 is selectively expressed in the striatal neurons innervated by nigral DA neurons. Furthermore, we show that activation of integrin α5β1 by systemic administration of estradiol-2-benzoate (E2B), an E2 derivative, can promote early improvement in the rotational behavior of hemi-parkinsonian rat models that received iPSC-derived DA neuron transplantation. Our findings provide a strategy that takes pharmacotherapeutic advantage of clinically approved drugs to promote efficacious cell transplantation therapy for PD.
Results
Discussion Robust and reproducible protocols for the induction of midbrain DA neurons from human ESCs/iPSCs have been reported by several groups (Kriks et al., 2011; Denham et al., 2012; Kirkeby et al., 2012; Sundberg et al., 2013; Doi et al., 2014). To understand the therapeutic mechanisms of grafted DA neurons, it is necessary to investigate how the grafted wst-1 integrate into the host neuronal circuit. Recently, several technologies such as diphtheria toxin-based neuronal ablation, rabies virus-mediated monosynaptic tracing, and optogenetics have been used for this purpose (Abematsu et al., 2010; Grealish et al., 2015; Steinbeck et al., 2015). In the present study we describe another experimental method, WGA-based transsynaptic tracing, to observe transsynaptic connection. Using this technique, we demonstrated that integrin α5β1 is highly expressed in DARPP32+ striatal neurons innervated by nigral DA neurons. The integrin superfamily consists of cell adhesion molecules that bind to the extracellular matrix and form αβ heterodimers, and includes at least 18 α and 8 β subunits in humans (Takada et al., 2007). Integrin α5 is broadly distributed in the body, but at low levels in the brain (Pinkstaff et al., 1999). According to experiments on cortical and hippocampal neurons, integrin α5β1 interacts with FN and regulates cellular migration, synaptogenesis, and the maintenance of synaptic plasticity (Chan et al., 2003; Webb et al., 2007; Gardiner et al., 2007; Hu and Strittmatter, 2008; Sekine et al., 2012). These findings are consistent with our in vitro data showing that human iPSC-derived DA neurons efficiently form synapses with integrin α5β1-overexpressing HEK293 cells (Figure 4). Systemic administration of E2B induced activation of integrin α5β1 in rat striatum (Figure 3). Since the activation of integrin α5β1 could facilitate attachment to FN, it is possible that the affinity between graft-derived neurites and target neurons is enhanced. Accordingly, the transmission of WGA from the grafted DA neurons to DARPP32+ striatal neurons was promoted by the administration of E2B (Figure 6). E2B did not directly promote the expression of several midbrain DA markers (NURR1, FOXA2, TH, SLC18A2, and SLC6A3), synaptic markers (SYNAPSIN and DREBRIN), or estradiol receptors (ESR1 and ESR2) by iPSC-derived DA progenitors (day 28), whereas a combination of glial cell-derived neurotrophic factor (GDNF), ascorbic acid (AA), brain-derived neurotrophic factor (BDNF), and dibutyryl cyclic AMP (dbcAMP) promoted the expression of NURR1, TH, SLC18A2, and SYNAPSIN in vitro (Figures S4D–S4N). These findings suggested that the enhanced synapse formation and function of the grafted DA neurons was mediated by E2B-induced activation of integrin α5β1 of host striatal neurons.