A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants

Abstract

Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.

Keywords: AAV-MNM008; Cell replacement; Parkinson's disease; animal model; capcid engineering; circuit mapping; dopamine neurons; human embryonic stem cells; monosynaptic tracing; retrograde transport.

Graphical abstract

Figure 1

Experimental design for retrograde induction of rabies tracing and the principle behind the AAV-based TRIO approach (A) Project timeline. For details, see the Experimental design section. (B–E) Injection paradigms to restrict retrograde tracing to either endogenous A9 (SNpc) or A9-type graft neurons (B and C) or to endogenous A10 (VTA) or A10-type graft neurons (D and E). (F) Vector constructs used in the study. (G) Schematics of the selective retrograde tracing in grafted animals achieved through the Cre-restricted AAV-based TRIO approach. The schema shows the group in (B and C) targeting the A9 (SNpc-type) grafted neurons with axonal projections to the Str. The retrogradely transported MNM008 | DIO-Flp vector is injected into the Str. The Flp will only be activated in the Cre⁺ neurons (large + in cells). fDIO genes will then turn on and allow for the synthesis of trans-synaptically-competent rabies virus, which is transported to all afferent neurons within the midbrain and in input regions. The end state is that the starter (A9-type) neurons are YFP⁺ (pseudo-colored green) and mCherry⁺ (Red), while connected afferent neurons will only be mCherry⁺. (H) An hESC graft ectopically placed in the Str, visualized using hNCAM DAB (brown) immunohistochemistry (IHC), specific to the human cell origin. (H′) mCherry DAB IHC expressed from the SADΔG-mCherry rabies virus injected into the TVA⁺ and oG⁺ graft. (I and J) Immunohistochemical characterization of the phenotype and maturation of an hESC-derived Syn1-Cre graft placed in the Str. Scale bar in (H′) represents 200 μm in (H and H′), and in J represents 100 μm in (I and J).

Figure 2

AAV and monosynaptic rabies-based tracing from host DA neurons (A–B‴) Validation of the Cre-restricted AAV-based TRIO approach in intact TH-Cre rats. The MNM008 | DIO-Flp vector was injected into the dorsolateral Str (needle tract shown as a dashed line in (A). Starter cells and their projections are YFP⁺ (A′ and B′) and are all TH⁺ (A‴ and B‴). Both starter cells and traced cells are mCherry⁺ (A′ and B″). (C–F) In vivo generated rabies virus in the SN is transported to afferent neurons. Afferent neurons are found in other regions as well, including the anterior cingulate cortex (ACC [C], the STN [D], hypothalamus [Hyp] [E] and the DRN [F]). Scale bar in (A‴) represents 200 μm in (A–A‴), in (B‴) represents 200 μm in (B–B‴), in (C) represents 200 μm, in (F) represents 200 μm in (D–F).

Figure 3

AAV-based TRIO from nigral hESC-derived DA transplants (A) Innervation of the ipsilateral Str from DA graft transplanted to the SN (A). (B and C) comparison between the axonal re-innervation of the PFC (A, C) between hESC grafts placed in the Str (A) and in the SN (SN, C) 26 weeks after grafting. (D–E‴) Representative section from the PFC in SN grafted rats where afferents to A10 (VTA-type) neurons are traced. The AAV-MNM008 based DIO-Flp vector was injected into the PFC (along the dashed line in D), and the fDIO-TVA and fDIO-oG AAV vectors were injected into the graft. Both starter cells and traced cells are mCherry⁺ (D–E′), starter cells and their projections are YFP⁺ (D and E″), and all afferents from the transplant are hNCAM⁺ (D and E‴). (F–H‴) A large fraction of the starter cells was confirmed to be TH + DA neurons in the graft (F–G‴), and in the case of the PFC-injected animals, some starter cells are Calbindin⁺ (H–H‴). (I) The approach successfully labeled neurons with mCherry in regions expected to have monosynaptic contacts to the VTA, e.g., the PFC (I). (J–L) In the second group of transplanted animals, we instead injected the AAV-MNM008 | DIO-Flp vector into the Str to map the afferents to the A9 type DA neurons of the transplant. In this case, we observed labeled neurons in the different regions, e.g., the motor cortex (MCx) (J), the Str (K), and the STN (L). Scale bar in C represents 50 μm in (A–C), in (D) represents 200 μm, in (E‴) represents 50 μm in (E–E‴), in (F‴) represents 200 μm in (F–F‴), in (G‴) represents 25 μm in (G–H‴), in (L) represents 200 μm in (I–L).

Figure 4

Regional mapping and quantification of two-neuron tracing to nigral transplants (A) Correlation between host input to the SN and the A9-type neurons in the hESC-derived transplant (TX), log-log transformed plot (R = 0.7; p < 0.01). (B) Corresponding correlation between the input to the host VTA and A10-type neurons in the TX (R = 0.4; p = 0.07). (C) Plotting of the number of labeled afferent neurons in each region compared between the two tracing groups and the intact dopamine system of TH-Cre⁺ rats. In each brain structure (one line), the top bars (darker red and blue) represent the TH-Cre⁺ intact rats, and the lower bars (lighter red and blue) represent the hESC transplanted animals. The centerline's left side contains the animals where tracing was initiated from the PFC (A10-type neurons) and the right side with tracing initiated from the Str (A9-type neurons). ^∗A significant difference from A9-type neurons. (D–F) Mapping of all traced neurons into a 3D brain atlas. (D) Individual monosynaptic afferent neurons to A9-type (SN) neurons. (E) Individual monosynaptic afferent neurons to A10-type (VTA) neurons. (F) The traced cells are aggregated into 3D heat maps for A9-type neurons, compared with A10-type neurons and connectivity traced from an ectopically placed transplant in the Str.